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J . TOXIC0L.-TOXIN REVIEWS. 7(2). 121-209 (1988-89)
Hemorrhagic Toxins from Snake Venoms
Jon Bragi Bjnrnason and Jay William Fox*.
Science Institute. University of Iceland. Reykjavik. Iceland (JBB) and Department of
Microbiology. University of Virginia Medical School. Charlottesville. VA 22908 (JWF)
Table of Contents
1 . 0
2.0
2.1
2.2
2.3
2.4
3.0
3.1
3.2
3.3
3.4
3.5
Introduction ................................................... 123
General Background on Snake Venoms ............................. 123
Classification of Poisonous Snakes ................................. 124
Distribution of Venomous Snakes ................................. 125
Composition of Snake Venoms .................................... 126
127 Biological Effects of Snake Envenomation ........................... Biochemistry of Hemorrhagic Toxins .............................. 130
Agkistrodon ................................................... 131
Bothrops ..................................................... 1 4 1
Crotalus ..................................................... 146
Trimerisurus .................................................. 1 6 1
Vipera ....................................................... 170
*TO whom request for reprints should be addressed . This work was supported by grants to the University of Virginia from the National Institutes of Health (JWF) (GM31289) and the North Atlantic Treaty Organization (JBB) (RG- 104.82)
121
Copyright 0 1989 by Marcel Dekker. Inc .
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1 2 2 BJARNASON AND FOX
4.0
4.1
4.2
4.3
4.4
5.0
5.1
5.2
6.0
Detection, Pathology, and Biochemical
Mechanism of Hemorrhage Activity .............................. 1 7 1
Observation of Venom Induced Hemorrhage ......................... 1 7 2
Evaluation of Hemorrhagic Activity ................................ 176
Assays of Hemorrhagic Toxins for
Proteolytic Activity ........................................... 179
Biochemical Mechanism of Hemorrhage ............................. 182
Inhibitors of Hemorrhagic Proteinases .............................. 184
Naturally Occurring Inhibitors ..................................... 184
Synthetic Inhibitors of Hemorrhagic
Proteinases .................................................. 191
Summary ...................................................... 193
Abstract
One of the more dramatic consequences of envenomation by crotalid and viperid
snakes is the occurrence of hemorrage. In cases where the envenomation is less severe,
the hemorrhagic is generally observed to be localized at the site of the bite. However,
hemorrhage can be found disseminated through a substantial area of the involved
extremity. In cases where the envenomation is severe, bleeding in organs such as heart,
lungs, kidneys and brain may also occur. From the biochemical investigations on these
toxins over the past 30 years, the nature of the venom toxins and their mechanism of
activity are now becoming clear. Virtually all of the hemorrhagic toxins isolated and
characterized thus far have been determined to be metalloproteinases. In this review we
discuss the history of the isolation and characterization of these toxins in an attempt to
clarify some of the confusion surrounding these toxins and their biochemical activities.
We also survey the data available on the natural and synthetic inhibitors against the
toxins. Finally, based upon the literature, we propose possible biochemical mechanisms
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 123
which may give rise to the hemorrhagic pathology associated with crotalid/viperid
envenomation.
1. INTRODUCTION
The general population, as well as the scientific community, have long been
fascinated with venomous snakes and the pathologies associated with snake
envenomation. Virtually since the beginning of modern chemical analysis of
biomolecules snake venoms have been the subject of investigation. Unfortunately, due
to the initially crude level of analysis and the rather complex nature of venoms, some of
the scientific literature on snake venom research is unclear, confusing, and in some
instances misinterpreted. This is particularly the case in the field of hemorrhage-
producing toxins.
The object of this review is to critically discuss the literature available on
hemorrhagic toxins in order to attempt to clarify the literature and to describe the
current state of the field. The review begins with a general background on venomous
snakes and their venoms. This is followed by an indepth discussion of the various
hemorrhagic toxins that have been isolated and characterized. The last portion of the
review deals with the methods of hemorrhagic toxin characterization and some of the
inhibition mechanisms that have been studied for these toxins. Finally, an attempt is
made to categorize the toxins based upon their biochemical and biological properties and
to assess the future directions of the field.
2.0 General Backaround on Snake Venoms
In this section we wish to present a brief overview of the major families of
This section should poisonous snakes and the primary characteristics of their venoms.
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124 BJARNASON AND FOX
give the reader a reasonable foundation for understanding the differences as well as the
similarities among the venoms of the poisonous snakes. Hopefully, the detailed
discussion of the hemorrhagic toxins which follows will then be more easily assimilated
into the understanding of the complex mechanisms involved in snake envenomation.
2.1 Classification of Poisonous Snakes
There are an estimated 2000 to 2500 species of snakes inhabiting the earth.
These snakes are classified into the fourteen families listed below: Acrochordidne;
Aniliidae; Anomalepidae; Boidae; Bolyeridae; Leptotyphlopidae; Typhlopidae;
Uropeltidae; Xenopeltidae; Colubridae; Crotalidae; Elapidae; Hydrophidae; and
Viperidae. Approximately fifteen percent of these snakes are poisonous. All of the
poisonous snakes are members of the last five families (Colubridae, Crotalidae, Elapidae,
Hydrophidae, Viperidae).
The snakes of the family Colubridae comprise the largest family, with two thirds
of all the snakes belonging to this group. Members of this fmii ly generally have either
posterior, grooved fangs (Opisthoglypha) or solid teeth (Aglypha). Not all members of
this family are poisonous. The most well known venomous member of the Colubridae is
the Boomslang (Dispholiduc fypus) which is found in the rain forests of Africa.
The family Crotnlidae are also known as the pit vipers due to the temperature-
differential receptors located in a pit on both sides of the head between the nostril and
the eye. Six genera make this family: Agkistrodon; Bothrops; Crolalus; Lnchesis;
Trimcrcsurus, and Sisturus.
The Elapidae is the rather large family containing nearly half of the known
venomous snakes. In this family are the kraits, coral snakes, and cobras among others.
There are thirty genera in this family.
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 12 5
Sea snakes are in the Hydrophidae family. Many of these sea dwelling snakes are
still the subject of controversy with respect to their precise taxonomical classification.
However, there appear to be fifteen genera with approximately fifty species distributed
among them.
The Viperidae family is commonly called the true vipers or Old World vipers.
This family is most closely related to the Crotalidae. There are ten genera in the
Viperidae family.
2.2 Distribution of Venomous Snakes
Members of the Colubridae family are found throughout the world; however,
only two genera of the sub-family Opisthoglypha are venomous. The genera Dispholidus
and Tldoforrzis each contain only one species, both of which are poisonous and both are
found only in Africa.
Most of the members of the six genera of the Crotalidae family inhabit North,
Central and South America. The genera Bofhrops, Crotnlus, Sistrus and Lachesis are
found only on the American continent. The genus Agkisfrodorz is found both in North
and Central America as well as some species inhabiting in Asia. Snakes of the genus
Trinzeresurus are found only in Southeast Asia and certain islands in the Pacific ocean.
The snakes of Elapidae are found primarily in the Orient, Australia, and Africa.
The coral snakes (genera Lepfonzicrurus, Micrurus, and Micruroides) range throughout
North, Central, and South America.
The sea snakes (Hydrophidae) are generally found from around the coasts of Asia
to the Arabian Sea and the Persian Gulf as well as in the China Sea, and in the waters
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126 BJARNASON AND FOX
of New Guinea, Australia, lndonesia and off the east coast of Africa. One species,
Pelamis platurus (Pelagic sea snake), is found along the western coasts of the Pacific and
Indian oceans.
Members of the family Viperidae are found in Africa, Asia, Europe, and some
Pacific islands. No snakes of the Viperidae family inhabit the American or Australian
continent.
2.3 ComDosition of Snake Venoms
The venoms of snakes are usually composed of a complex mixture of organic and
Insoluble tissue debris is also often noted in the venom from inorganic components.
milked snakes.
The inorganic constituents of the venoms include: Ca, Cu, Fe, K, Mg, Mn, Na,
P, Co, and Zn (1). Not all of these metals are found in every type of venom and the
amounts of each metal varies with the species of snake. The biological role for each of
the metals is not clear, however, it is likely that some of them are quite important for
the stabilization of certain venom proteins’ structures as well as being involved in the
mechanism of catalysis for certain enzymatic reactions.
It is convenient to divide the organic compounds of the venom into the protein
and non-protein components. The majority of the crude venom is composed of proteins.
The other compounds include: carbohydrate (in the form of glycoproteins); lipids
(primarily phospholipids); biogenic amines (particularly abundant in Viperidae and
Crotalidae venoms); nucleotides; amino acids; and peptides.
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS
2.4 Biological Effects of Snake Envenomation
12 7
The effects of snake envenomation are quite varied and dependent upon many
factors, some of which include type, age, health and size of the snake, amount of venom
injected, and biological condition of the prey. Due to the complexity of most snake
venoms, several different biological effects resulting from envenomation may be
observed. Venoms may contain a panel of toxic factors which may act individually,
each producing a particular biological effect, or they may act synergistically. Some of
the biological effects of snake envenomation which will be briefly discussed below
include coagulation, cytotoxicity, hemolysis, hemorrhage, hypotension, necrosis, and
neurotoxicity.
i) Coagulation.
Affects upon the blood coagulation system have been observed for many
snake venoms. The venoms are sometimes classified as either anti-coagulant or pro-
coagulant: however this is an oversimplification in that some venoms contain both anti-
and pro-coagulation factors. In general, the venom factors which affect the blood
coagulation system are proteinases.
The venoms of the Hydrophidae typically do not give rise to any
significant perturbation of the blood coagulation system. This perhaps reflects the lack
of significant proteolytic activity in their venoms.
The Elapidae venoms have been reported to affect the blood coagulation
system primarily in a n anti-coagulant manner (2,3) whereas both anti- and pro-
coagulation activities have been observed in Viperidae venoms (43). Crotalidae venoms
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BJARNASON AND FOX 120
also demonstrate profound affects upon the coagulation system in both pro- and anti-
coagulant fashions (6-8).
Although only a few venoms from the Colubridae family have been
examined they do appear to affect coagulation (9).
ii) Cytotoxic affects.
Many venoms give rise to the lysis of various cell types. The primary
cytotoxic factors in the venoms t h a t have thus far been identified are small, basic, non-
neurotoxic proteins (10). The Elapidae venoms are particularly rich in these toxins ( I I ) .
toxins have also been isolated from the venom of members of the Crotalidae and
Viperidae families.
iii) Hemolysis.
Hemolysis is typically described as the disruption of the erythrocyte membrane
allowing the escape of hemoglobin from the cell. Hemolysis, due to snake venom, can
be the result of either direct or indirect hemolytic agents. Phospholipase A2 action on
phosphatidylcholine produces lysolecithin. Lysolecithin can then in turn lyse the red cell
membrane (12). Most snake venoms from all the poisonous families have phospholipase
A2 present in their venoms and are therefore potentially capable of producing red cell
lysis via the indirect mechanism (10).
Some snakes have proteins present in their venoms which are capable of directly
lysing the red cell membrane. These factors have been identified in the venoms of
certain snakes from the Elapidae family (13).
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS
iv) Hemorrhage.
129
Venoms from the families Crotalidae and Viperidae are strongly hemorrhagic
whereas most of the venoms of Elapidae and Hydrophidae do not give rise to significant
hemorrhage. It is the hemorrhagic toxins of Crotalidae and Viperidae snakes which will
be discussed in detail later in this review.
v) Hypotensive Effects.
Hypotension of varying degrees of severity and duration have been
observed with venoms from the Crotalidae, Viperidae, and Elapidae families. The
hypotensive effect has been shown to be due to one or more factors present in the
venom. In some cases, hypotension has been attributed to venom proteolytic enzymes
with kallikrein-like activity (14). These enzymes act by releasing kinin, a hypotensive
peptide, from its precursor kininogen. Also, some venoms contain small peptides which
are, in effect, hypotensive due to their inhibitory effect on angiotensin converting
enzyme (15). Certain venoms can also give rise to endogenous tissue histamine release
with subsequent vasodilation (16,17).
vi) Tissue Necrosis.
Local tissue necrosis is often a consequence of snake envenomation. In
some instances, the necrosis can be directly attributed to individual factors in the venom.
Necrosis can also be observed as a secondary effect stemming from the biological action
on the tissues by other venom factors such as hemorrhagic proteases (18).
Tissue necrosis has been observed upon envenomation by members of the
Crotalidae, Elapidae, and Viperidae families (19-21). Hydrophidae venoms do not
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130 BJARNASCN AND FOX
appear to be significantly necrotic. Direct-acting necrotic toxins have been isolated
from the venoms of Notechis scufu~us and Crofalus viridis viridis, among others (22,23).
vii) Neurotoxic Effects.
Typically, neurotoxins' sites of action are at the level of axonic
transmission or synaptic transmission. The inhibition of synaptic transmission can be
further considered as either presynaptic or postsynaptic. With regard to axonic
transmission, snake venoms have not been observed to perturb the action potentials of
axonic transmission (10).
Several of the Elapidae venoms contain presynaptic toxins. Examples of
these toxins are found in the venom of the snakes Burigarus multicitilus and Notechis
scutatas scufalus (24,25). One presynaptic toxin has been isolated from the venom of the
Bulgarian viper, Vipcra ammodyfes amnzodyfes (26) . Presynaptic toxins also have been
isolated from the venoms of certain Crotalidae snakes (27,28).
Due to the paucity of data on Colubridae venoms, it is not known for
certain whether they are neurotoxic. However, the rather high toxicity of the venoms of
this family suggests that neurotoxins may be present.
3. Biochemistrv of Hemorrhaaic Toxins
In this section, a review will be given of the biochemical properties of the
hemorrhagic snake venom components which have been isolated and purified to such a
degree as to allow characterization of their properties to be made. This review will be
made on the basis of snake genera and in alphabetical order of the Latin names of the
snakes.
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 131
Early investigations of the mechanism of snake venom induced hemorrhage were
greatly hampered by the lack of sufficiently purified hemorrhagic venom components,
which was due to the complexity of the venoms, coupled with the primitive state of
protein purification techniques. The past decade however, has seen a proliferation in
the numbers of purified hemorrhagic venom components such that today they number in
the forties. Furthermore, it is evident that the degree of purity of the hemorrhagic
components isolated in the sixties and early seventies is in many cases suspect, as
exemplified by the 20 year development of the purification of HR-IA and HR-lB , the
hemorrhagic components from the venom of Trinieresurus /lavoviridis.
The hemorrhagic components from the various snake venoms have been assigned
a multitude of different names by the researchers that purified them, such as
hemorrhagic toxins, hemorrhagic proteases, hemorrhagins, hemorrhagic principles,
hemorrhagic factors or simply proteases, along with designating numbers or letters.
Since hemorrhage is one of the most pronounced, basic effects of crotalid snake
envenomation, we have chosen to refer to the venom components responsible for these
effects as hemorrhagic toxins. It would probably be beneficial if a consensus could be
reached on this issue of nomenclature by the researchers in the field. We would thus
suggest that they be termed hemorrhagic toxins along with designating letters and the
species name of the snake that produced the venom.
3.1 ,4e kistrodon
ArkiPtrodort acufuv. A total of nine hemorrhagic toxins have been isolated from
the venoms of Agkistrodorz acutus from Taiwan and China (Table 1). Mori ef al. (29)
reported in 1984 on the purification of a hemorrhagic proteinase (Acs-Proteinase) and
the characterization of this enzyme as well as the characterization of four other
hemorrhagic proteinases, which had previously been purified from A. acufus (Taiwan)
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TAB
LE 1
Prop
ertie
s of
Hcm
orrh
agic
Tox
ins f
rom
the
Ven
om of
Ack
istro
don m
.
Prop
ertie
s A
cl
Ad.
A
d
Ac4
A
d
AaH
l A
aHll
AaH
III
FP
Mol
ecul
ar w
t. 24
Mo
Isoe
ledr
ic pt
. 4.7
MH
D(u
g)
022
Met
al c
onte
nt
1 Zn
Hem
orrh
agic
Prot
eoly
tic
Oth
er
Act
iviti
es
Inhi
bito
rs
Furth
er
Com
men
ts
Rcf
ercn
ee
+ + Leth
al
EDTA
JJO
- ph
e n a n
. &
cystc
ine
Sim
ilar
to A
c2,
AaH
I, &r
n
29
3
wxw)
4.9
0.43
+ + Leth
al
EDTA
,l,lO
- ph
cnan
. &
eyste
inc
Acl
& A
c2
are
prob
ably
al
lylic
im
nzym
es
29
5700
0
4.1
0.95
+ + Leth
al
EDTA
,lJO
- ph
e n a n
. &
eys
tein
c
Cle
aves
ox
Insu
lin B
C
hain
at
Hisl
O -
Leul
l A
la14
- Leu
l5
TF1
6 - L
eu17
Ph
e24
- Phc
Z
3300
0
4.4
0.31
+ + EDTA
,l,lO
- ph
enan
. &
eyst
einc
29
29
24000
2200
0
6.7
4.6
0.37
0.4
12
2
ca
+ + Leth
al
t t Leth
al
&
libri
nol.
EDTA
,l,lO
- ED
TA,l,
lO-
phen
an.
&ey
stci
ne
eyste
ine,
&
snak
e serum
Sim
ilar t
o
the
appa
rmt
isom
zym
es
Acl
& A
d
22oo
o 22
ooo
2400
0
5.3
>9
3.8
15
10
+ +
t
+ +
t
Leth
al
Leth
al
Fibr
inol
ytic
&
&
&
Gbr
inog
enol
G
brin
ol.
Gbr
inol.
EDTA
ED
TA
EDTA
E
yste
im&
cy
stei
ne&
&
sn
ake serum
snak
c Serum
cystc
ine
38
38
W 4
Sim
ilar t
o ?-
A
cl, A
c2
E &
AaH
l v)
Did
not
sho
w
z
colla
geno
lytic
?-
activ
ity
3 ?- 0
-a
31.32
0
x
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 133
TABLE 2
Amino Acid Compositions of Hemorrhagic Toxins from Aekistrodon venom.
Amino acid F.P. AC 1 Ac2 Aa-HI Ac5 Ac3
Asx 25 25 28 23 39 66 Thr I1 12 12 9 16 20 Ser 23 24 23 16 12 32 Glx 21 20 21 18 17 51 G ~ Y 12 12 19 17 9 53 Ala 12 11 13 11 12 26
cmCys 8 7 7 8 10 49 Val 10 15 14 12 10 20 Met 6 6 6 04 7 13 Ile 15 22 19 14 12 21
Leu 12 14 13 10 20 20 TYr 10 12 12 5 27 Phe 5 5 4 8 7 15 LYS I1 11 10 5 19 32 His 7 7 7 9 5 15 Arg 7 8 I 5 5 10 Pro 8 7 8 6 5 26 Tro 5 9 6 3 2 I I
Total 208 227 229 178 212 507
Reference 36 29 29 39 29 29
(30-33). All five toxins were shown to be proteinases using casein as substrate and were
designated Acl-, Acz-, Ac3-. Ac4- and Acg- proteinase. Their purity was demonstrated
by disc gel polyacrylamide electrophoresis as well as by SDS polyacrylamide electro-
phoresis. All the proteinases were reported to have lethal activities except Ac4-
proteinase and all their assigned activities (proteolytic, hemorrhagic and lethal) were
inhibited by EDTA, cysteine and ],lo-phenanthroline. The hemorrhagic potencies,
isoelectric points and molecular weights of all the toxins were reported (Table 1) as well
as the amino acid compositions of all toxins except Ac4-proteinase (Table 2).
In a report from 1982, Nikai et al. (34) determined the zinc content of Acl-
proteinase and compared it’s characteristics with hemorrhagic toxin e from Gotalus
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134 BJARNASQN AND FOX
atrox venom, a proteolytic toxin whose zinc content had previously been determined.
The zinc content of Acl-proteinase as well as its hemorrhagic and proteolytic activities
were compared before and after the removal of zinc. It was found that both
hemorrhagic and proteolytic activities disappeared upon removal of zinc, as had
previously been demonstrated for hemorrhagic toxin e from C. atrox venom (35). The
authors stated that it might be logical to conclude that all hemorrhagic toxins possess
proteolytic activities and zinc regardless of the geographical origins of the venoms.
In 1976, Ouyang and Huang reported isolating a fibrinolytic principle from the
venom of Agki~lrodot i acutus (36). It had a molecular weight of 24,100 daltons and an
isoelectric point of 3.8. It also had caseinolytic and fibrinolytic activities cleaving the
A a chain of fibrinogen leaving the BP chain and the 7 chain unaffected. The same
authors reported a year later that the fibrinolytic principle possessed hemorrhagic
activity (37). Also, both EDTA and cysteine completely inhibited the fibrinolytic,
fibrinogenolytic, hemorrhagic and caseinolytic activities of the principle.
Xu and co-workers (38) purified three hemorrhagic toxins with proteolytic
activity from the venom of Agkislrodotz acutus (China) and designated them Aa-
hemorrhagin I, I1 and Ill. Their purity was demonstrated by single bands on
polyacrylamide gel electrophoresis and as single precipitin lines upon
immunoelectrophoresis. The three toxins were determined to be immunologically
distinct fr6m each other. Aa-hemorrhagin I and I1 were shown to be acidic proteins,
while Aa-hemorrhagin 111 was basic. All have a molecular weight of about 22,000
daltons. Their hemorrhagic and caseinolytic activities were inhibited by EDTA and
cysteine. In their discussion, the authors compared the hemorrhagins to the Acl-
proteinase and the fibrinolytic principle from the same venom.
Recently, Zhang and co-workers (39) reported carrying out further
characterizations on Aa Hemorrhagic toxin I (AaHI), previously designated Aa
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 135
Heniorrhagin I. They found that AaHI contained one mole of zinc and two moles of
calcium per mole of protein. When dialyzed against EDTA, AaHI was completely
inactivated although only zinc was partially removed under some of the experimental
conditions while calcium was for some reason not removed at all. When dialyzed against
1 ,lo-phenanthroline only, both calcium and zinc were removed in approximately
equimolar amounts and in concert with decreasing activities of hemorrhage and
proteolysis. However, when AaI-11 was dialyzed against 1,lO-phenanthroline containing
5mM Ca2' approximately 75% of the zinc was removed while apparently only 10% of
the proteolytic and hemorrhagic activities was lost. The inactivation of AaHI could in
no instance be reversed by readdition of the metal ions.
Concomitant conformational changes of AaHI were observed by circular
dichroism spectroscopy when the metal ions were removed. Large conformational
changes were observed when 80% of both metals were removed with 5mM 1,lO-
phenanthroline. Likewise, almost the same conformational changes were observed when
only the zinc was removed to the same extent using 5mM I,lO-phenanthroline
containing 5mM calcium. Oddly, these large conformational changes following zinc
removal were accompanied by only slight activity decreases, while total activity losses
were associated with the zinc removal from hemorrhagic toxin e from Crotalus atrox
venom (35) and from Acl-proteinase from A . acutus venom (34), an enzyme that appears
to be homologous if not identical to AaHI. The authors do not explain this apparent
discrepancy. They conclude that calcium is probably essential for the activity of AaHI
and that there is likely a correlation between the binding of zinc and calcium to AaHI.
Finally, they state that it is not clear whether zinc is necessary for any functional
activity, but that a role for zinc could not be excluded since zinc is slightly bound to
AaHI. This is confusing in view of the large conformational changes observed with the
removal of zinc but not calcium from AaHI using a solution of 1,lO-phenanthroline
containing calcium.
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136 BJARNASON AND FOX
Possibly AaHI is an enzyme containing two or three binding sites for calcium,
which, when filled, confer the most active conformation upon the enzyme, as well as a
binding site for zinc in the active center of the enzyme where i t probably acts as a
Lewis acid as well as conferring stability to the enzyme in the native conformation.
When the properties of the hemorrhagic toxins from Agkistrodori aculus are
compared (Tables 1 and 2), it appears quite probable that some of them are homologous
enzymes. Careful scrutiny of the data suggests that the Acl- and Ac,- proteinases are
isoenzymes, like hemorrhagic toxins c and d from C. atrox venom (40). Furthermore,
AaHI and the fibriiiolytic enzyme appear to be identical to Acl- or Acz-proteinase or
additional allyllic isoenzynies of these. This would reduce the number of unrelated
hemorrhagic toxins isolated so far from Agkis frodon acutus to a total of six, all of which
have been demonstrated to be metal dependent proteolytic enzymes. One of these
apparently unrelated six enzymes (the homologs Ac- 1, and AaHI) has been demonstrated
to contain zinc and calcium. It would be of interest to learn more about the proteolytic
substrate specificities and metal content of the other toxins from this venom.
APkistrodort hnli~s hlonihoffii
Early investigations of the venom of A . halys blonrhojfii revealed two
hemorrhagic fractions designated HR-I and HR-11, both which also contained lethal
activities (41). The lethal and hemorrhagic activities in crude venom were completely
inactivated by treatment with EDTA while DFP had no effect on these activities.
Previously, three proteinases, termed proteinase a, b and c, had been found in this
venom by Satake e t a1 (42). In 1965, the same group of investigators came to the
conclusion that proteinase b was identical to the hemorrhagic factor HR-I1 (43). The
purified enzyme showed some heterogeneity which the authors thought might be due to
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 137
autodigestion. An improved method of purifying HR-I1 with DEAE cellulose and gel
filtration chromatography was described in 1968 (44). Additionally, HR-I1 was shown to
be a glycoprotein with a molecular weight of 95,000 daltons and an isoelectric point of
8.51. Carbohydrate content was determined and found to be 8% neutral sugar (galactose,
niannose and trace of fucose) 6.5% glucosamine and 3% sialic acid. The amino acid
composition of HR-I1 was determined and Cound to contain a high ratio of acidic amino
acids and a rather high amount of cysteine. Thus, HR-I1 was the first hemorrhagic
toxin to be purified to a high degree of homogeneity and characterized to some extent.
Purification and characterization of the non-hemorrhagic proteinases a and c
were also described by the same authors in 1968, see Table 3 (45).
Further characterization of HR-I1 by the same group of scientists followed in
1971 (46). They found that HR-I1 or proteinase b contained 2 moles of calcium per
mole of enzyme. The removal of the metal resulted in protein conformational changes
as judged by the blue shift in its ultraviolet difference spectra, with concomitant loss of
caseinolytic and hemorrhagic activities, which was not regenerated by addition of
calcium ions. Thus, they concluded that the calcium ions seem very important in
maintaining the nntive tertiary structure associated with the caseinolytic and hemorrhagic
activities of proteinase b or HR-11. Unfortunately, the zinc content of HR-I1 was not
measured.
The following year, these researchers described the isolation and properties of
hemorrhagic factor I or HR-I (47). They found HR-I to be an acidic glycoprotein
having an isoelectric point of 4.7 and a molecular weight of 85,000 daltons with no
detectable protease activity or other enzymatic activity associated with the crude venom
(see Table 3). The amino acid composition of HR-I showed high concentrations of
aspartic and glutamic acids as well as lysine, arginine, tyrosine and cysteine.
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TAB
LE 3
Prop
ertie
s of
Hem
orrh
agic
Tox
ins a
nd o
ther
Pro
teas
es fr
om th
c V
enom
of A
dist
rodo
n ha
lvs b
lom
hoff
ii
Prop
ertie
s H
R-I
H
R-I
1 Pr
otei
nase
a
1
Prot
eina
se c
Mol
ecul
ar w
eigh
t 85
000
9500
0 5o
ooo
7ooo
o
Isoe
lect
ric p
oint
MH
D dus.,
4.18
6.0
3.85
4.7
0.00
12
0.03
1 0.1
9
Met
al c
onte
nt
Hem
orrh
agic
Prot
eoly
tic
Oth
er a
ctiv
ities
Inhi
bito
rs
2 C
a
t +
+ 4
+ t
Leth
al
Leth
al
EDTA
, Th
iol r
eage
nts
EDTA
, cy
stei
ne,
1,lO
- ph
enan
thro
line
Gly
copr
otei
n C
leav
es
Insu
lin B
ch
ain
at
Ala
14-L
eufi t
TylG
-Leu
17
Hfi
10-k
u11,
ED
TA
ED
TA
Furt
her c
omm
ents
G
lyco
prot
ein
Sam
e as
Pr
otei
nase
b
Und
erw
ent
conf
orm
atio
nal
chan
ges w
ith
calc
ium
rem
oval
by
ED
TA
41,44
Gly
copr
otei
n C
leav
es 1n
s.B
chai
n at
H
is5-L
eu6,
Se
r9-H
is10 t
Alal
4-LC
ulS
Gly
copr
otei
n C
leav
es 1
ns.B
ch
ain
at H
isl0-
Leull, Al
a14-
Leuf
i t G
lyB
-Phe
24
Ref
eren
ce
45
45
41,4
7,48
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HEMORRHAGIC T O X I N S FROM S N A K E VENOMS 139
The hemorrhagic activity of purified HR-I was very sensitive to chelating
reagents such as EDTA and cysteine. Even at relatively low concentrations, these
reagents completely inactivated HR-I. Re-addition of the metal ions, calcium,
magnesium or manganese did not restore the activity of the toxin. Thus, they concluded
that some metal ions might be essential for maintaining the conformation required for
the activity of HR-I. Unfortunately, they did not measure the metal content of HR-I,
perhaps due to lack of material.
The hemorrhagic toxins HR-I and HR-I1 from A . hnlys blonzhofjii venom are
similar in many respects, both having high molecular weights, and both being acidic
glycoproteins with similar amino acid compositions (see Tables 3 and 4). Both have high
contents of aspartic and glutamic acids, lysine, arginine and cysteine. There are,
however, clear differences in their tyrosine, valine and proline contents, as well as
differences in activity, indicating that they are not isozymes (Tables 3 and 4).
A distinct difference between HR-I and HR-I1 is based on the observation that
HR-I1 has strong proteolytic activity while the investigators concluded that HR-I had
none. HR-I was also considerably more hemorrhagic and more lethal than HR-11. The
authors of this review have repeatedly cautioned, that the lack of detection of
proteolytic activity using only one substrate or one assay method does not constitute a
proof that proteolytic activity does not reside with the proteins in question (45). The
conclusion that HR-I is not proteolytic is the only major misinterpretation of the
otherwise sound work done by these authors on the hemorrhagic toxins from A . halys
blomhoffii venom. This point was reaffirmed by Nikai and co-workers in a recent
publication where they demonstrated that although HR-I showed no activity on casein as
substrate, it did cleave azocasein, azoalbumin, dimethylcasein and hide powder azure
(48). They also state that their recent work indicated that both HR2a and 2b from T.
/Iavoviridis have proteolytic activities. The proteolytic, hemorrhagic toxin HR-I was
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140 BJARNASON AND FOX
TABLE 4
Amino Acid Compositions of Hemorrhagic Toxins and Related Protenses from the Venom of A. hn(vs blomhoffii.
Amino acid
Asx Thr Ser Glu G ~ Y Ala c y s Val Met Ile Leu TYr Phe LYS His A rg Pro Trp
Nearest integer
HR-I
79 30 1 3 62 86 44 21 35
9 38 52 26 24 29 17 21 28 10
Grams of amino acid residues per lOOg protein
HR-I HR-I1 Proteinnse Proteinase a C
10.76 2.58 2.94 7.58 2.68 3.06 4.67 3.23 0.96 3.54 4.40 5.15 2.07 4.61 2.50 5.02 2.54 2.60
11.79 3.95 2.74 8.43 2.58 2.82 4.54 4.10 2.60 4.55 5.44 3.86 2.67 3.99 2.37 4.12 3.25 1.68
12.00 4.82 4.21 8.32 2.88 3.15 5.09 3.91 1.62 3.96 6.01 4.05 3.18 6.36 2.01 4.73 2.77 1.84
11.11 3.10 3.41 8.67 3.16 2.86 5.38 3.75 2.39 3.62 4.2 1 6.06 2.59 5.01 3.58 2.34 3.36 1.90
Total 696 12.56 77.54 82.54 77.79
Reference 48 47 47 45 45
found to cleave the oxidized B chain of insulin at the Hislo-Leull, Ala14-Leu16 and
TyrlG-Leu17 bonds as well as the A a and BP chains of fibrinogen. The proteolytic
activity of HR-I was inhibited by various chelating agents such as EDTA, 1,lO-phenan-
throline and others.
Thus, we reach the conclusion that four distinct proteases have been isolated
from the venom of A . hulys blonihoffii, two of which are hemorrhagic while the other
two are nonhemorrhagic proteases. Both hemorrhagic toxins appear to be metal
dependent acidic glycoproteins, of a high molecular weight, with unique proteolytic
specificities.
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS
3.2 Bothroos
141
Bothroos iararaca, mooieni and neuwiedi. Five hemorrhagic toxins from the
venoms of snakes of the genus Bothroos have been purified and partially characterized
(Table 5).
In 1976, Mandelbaum and co-workers reported on some physical and biochemical
characteristics of HF-2 one of the hemorrhagic toxins of Bolhrops jararuca venom (49).
They isolated HF-2 from a protein fraction of the crude venom that precipitated at 4OVo
- 50% ammonium sulfate saturation. HF-2 was reported to have a molecular weight of
50,000 daltons and possess low proteolytic activity toward casein as substrate. Glucagon
and the p'-chain of oxidized insulin were also cleaved by HF-2, and the identification of
cleavage products demonstrated that the peptide bonds Hislo-Leull, Ala14-Leu15, TYT16-
Leul7 and Phez4-Phez5 of the insulin /+-chain were hydrolyzed by HF-2.
One of the proteases active on casein from B. jararaca venom termed
Bothropasin was isolated and characterized by the same group of researchers (SO).
Bothropasin was isolated by ammonium sulfate precipitation, DEAE-cellulose and
DEAE-Sephadex A-SO chromatographies and Sephadex G-100 gel filtration.
Homogeneity was demonstrated by polyacrylamide gel electrophoresis. The authors
reported that the preparation was free of hemorrhagic activity and possessed no other
detectable activities which were present in the crude venom. However, in more recent
reports, the same group of researchers concede that bothropasin does cause hemorrhage
when injected in doses of 1 fig (51,52). Bothropasin was reported to have a molecular
weight of 48,000 daltons and its proteolytic activity was inhibited by EDTA and EGTA.
The enzyme hydrolyzed the HiS6-LeU6, His,o-Leull, Alal4-Leul6. Tyr16-Leu17 and
Phez4-Phe2s bonds of the p-chain of oxidized insulin.
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142 BJARNASON AND FOX
These same authors observed a time dependent disappearance of the 48,000
molecular weight band on SDS gels after incubation in a lOmM EDTA or EGTA
solution at 4°C. Aliquots were withdrawn at various times for up to 30 days and
submitted to denaturation and reduction followed by SDS-gel electrophoresis. Besides
the molecular weight band of 48,000 daltons, another band of 38,000 daltons appeared.
This latter band increased with time of incubation, along with a concomitant
disappearance of the 48,000 daltons molecular weight band. After 12-15 days, only a
band of 38,000 daltons and two minor bands were observed, one of 20,000 daltons and
another, migrating with the tracking dye, of about 10,000 daltons. If EDTA was added
only a few minutes before denaturation and reduction, a band representing a molecular
weight of 48,000 daltons is obtained, but none of the lower molecular weight bands.
These results suggested to the authors that bothropasin is composed of two polypeptide
chains, one heavy chain of 38,000 daltons and another of 10,000 daltons, which are
bound by disulfide and mete1 bonds, the 10,000 dalton peptide chain being present as a
monomer and in higher percentage, as a dimer. Another plausible interpretation of these
results, and one that we consider more reasonable, is that a very low residual proteolytic
activity in bothropasin causes autolysis by hydrolyzing the inactive, denatured portion of
the sample during the long incubation period of 12-15 days at 4OC. It is most unlikely
that metal bond disruption occurs during a two day incubation period with lOmM EDTA
at 4OC presumably at neutral pH, which would not occur during an incubation of the
protein for 5-10 minutes at boiling temperatures with EDTA, P-mercaptoethanol and
SDS.
In a recent communication, the same group of researchers state that HF-2,
bothropasin and crude B. jararaca venom cause hemorrhage, myonecrosis and arterial
necrosis (51).
Two hemorrhagic toxins have been isolated from the venom of Boihrops rieuiviedi
termed neuwiedi hemorrhagic factors NHFa and NHFb (53). Purification was achieved
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 143
by ion exchange and gel filtration chromatographies followed by a final step of disc
electrophoresis on polyacrylamide gels. The toxins apparently have identical isoelectric
points of pH 4.2-4.3, while their molecular weight differs somewhat; NHFa being 46,000
daltons while NHFb was 58,000 daltons. The toxins are immunologically related
antigens. They are not distinguished by horse serum antivenom of Bofhrops tzeuwiedi
venom. However, with specific rabbit anti-hemorrhagic serum, it is possible to
recognize these two toxins as distinct entities.
Both toxins hydrolyzed casein although NHFa was about 20 times more active
than NHFb, while the hemorrhagic activity of HNFb was about 23 times greater than
that of NHFa, thus giving one more example of the lack of a direct relationship between
hemorrhagic activity and the hydrolytic action on casein as substrate for two or more
toxins. Actually, as previously observed, there is no reason to expect such a relationship.
On the contrary, since the peptide bonds and structure of casein are unlikely to
represent the features of the natural, in vivo, substrate of hemorrhagic toxins, such a
relationship is most unlikely (3554).
Recently, Assakura and co-workers isolated and characterized the major
proteolytic enzyme from the venom of Bofhrops nioojetii (52). This enzyme, termed
moojeni protease A or MPA, was purified by chromatography on Sephadex G-100,
DEAE Sephadex A-50 and rechromatography on Sephadex G-100. The protease is a
weak hemorrhagic toxin, possessing 10 times less hemorrhagic activity than bothropasin
from Bofhrops jararaca and half of the activity of crude B. nioojetzi venom. According
to these authors, Moojeni protease A is inhibited by EDTA and 1,lO-phenathroline in
the sense that treatment with these metal chelating compounds results in a complex
which has undergone a severe conformational change, causing denaturation and
precipitation of the enzyme. However, no evidence of conformational change is
presented in this paper. The authors also state that the metal ion plays an essential role
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144 BJARNASON AND FOX
in the structure of the enzyme, although no metal analysis of the enzyme is presented.
The isoelectric point of MPA was reported to be approximately 7.7 and the molecular
weight from 20,400 dnltons using SDS gel electrophoresis to 22,800 daltons by
sedimentation equilibrium measurements. The authors state that the minimum molecular
weight of MPA corresponds to 17,000 daltons. However, judging from the amino acid
composition, that minimum molecular weight is incorrect. The correct minimum
molecular weight is approximately half of that or 8,000 to 8,500 daltons. The authors
base the amino acid composition on a molecular weight of approximately 17,000 daltons
or a two fold minimum molecular weight from our estimate, while a three fold minimum
molecular weight appears more appropriate in light of the results of the molecular
weight measurements. Thus in Table 6 , we show the amino acid composition of MPA as
published by the authors as well as a corrected composition based on a threefold
minimum molecular weight resulting in a molecular weight of approximately 24,000
daltons, similar to the results of the sedimentation equilibrium measurements. When the
corrected amino acid composition of MPA is compared to that of proteinase I or I1 from
C. adaniarzteus or the isoenzymes Ht-c and Ht-d from C. afrox, it becomes apparent that
MPA is possibly homologous to the aforementioned low activity hemorrhagic toxins.
Further characterization of these toxins is however, necessary to confirm this.
Five hemorrhagic toxins have been isolated from venoms of Boihrops snakes and
characterized to some degree. All five toxins appear to be proteolytic enzymes and all,
except HF-2, have been shown, by metal chelating experiments, to be metal dependent.
Unfortunately, the metal composition of these toxins have not been determined. The
toxins with high hemorrhagic potency, such as HF-2 and NHFb, demonstrate low
caseinolytic activity while the reverse is true for the low activity toxins such as
bothropasin and MPA, although only a small difference was found for the cleavage
specificities of the oxidized p-chain of insulin by HF-2 and bothropasin (see Tables 5
and 6 ) .
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TAB
LE 5
Prop
ertie
s of H
emor
rhag
ic T
oxin
s fro
m th
c Ven
om of B
othr
oos i
arar
aca,
an
d ne
uwie
di
Prop
ertie
s H
F2
Bot
hrop
asin
N
HF-
a N
HF-
b M
Pr
otea
se A
Mol
ecul
ar w
eigh
t m
4
m
urn
m
2n-23,m
Isoe
lcdr
ic p
int
1.1
MH
DW
Met
al w
nten
t
Hem
orrh
agic
Prot
eoly
tic
Oth
er ac
tivili
es
Inhi
bito
rs
Furth
er w
mm
cnts
Rcf
crcn
ce
+ 4 Fibr
ino-
ge
noly
tic
Myo
necr
osis
&
arte
rial
necr
osis
+ + Myo
neao
sis
& a
rteria
l ne
cros
is
EDTA
EG
TA
Oxa
late
Cle
aves
B
Cle
avcs
B
chai
n of
ch
ain
of In
sulin
at
Insu
lin at
H
islO
-Len
ll,
His5
-Leu
(j,
Ala
14-L
CU1S
> H
isln-
LCul
l, Tp
16-L
eu17
8L
Ala
14
-~~
1~
Ph
e24-
Ph%
Tp
16-L
cu17
Ph
c24-
Phc2
5
49
w,s
2
+ A ED
TA, &
1.
10-p
hcna
n-
thro
line
20 fo
ld m
ore
activ
e on
case
in th
an
NH
F-b.
53
+ +
+ +
EDTA
&
Oxa
late
1,
lO-p
hena
n-
EDTA
&
thro
line
1,lO
-phc
nan
thro
line
53
Am
ino
term
i- 23
fold
mor
e he
mor
rhag
ic
ndle
u. U
nder
th
an N
HF-
a go
es d
enat
ur-
atio
n &
aut
o-
lysis
in lo
w
salt
solu
- tio
ns.
Wea
k hc
mor
rhag
ic
resp
onse
. A
ctiv
ated
by
cal
cium
52
2
m B z X
Y- o
U
n
rj
0
X
H f
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146 BJARNASON AND FOX
TABLE 6
Amino Acid Compositions of Hemorrhagic Toxins from the Venom of BothroDs jnraroca and Bothrovs mooieni as well Proteinase I1 from C. adamanteus
Amino Bothro- acid pasin
Asx 51 Thr 14 Ser 20 Glx 36 Gly 30 Ala 22 CYS 32 Val 20 Met 1 1 Ile 20
Leu 20 T Y ~ 20 Phe 12 LYS 26 His 14 Arg 9 Pro 22 Tw 4
MPA
18 7 11 14 8 7 4 10 4 6 12 4 5 6 8 6 5 2
MPA (corrected)
27 1 1 17 21 12 1 1 6 15 6 9 18 6 1 13 12 10 7 3
Prot.11 c. &.
27 8 16 21 13 8 4 13 6 15 21 8 1 9 6 16 7 2
Total 389 140 211 207
Reference 50 52 52 56
3.3 Crotalus
Crotalus adamanteus and Crotalus horridus horridus. A hemorrhagic toxin designated
protease H has been purified and partially characterized from the venom of Crotalus
adanzarzteus, the eastern diamondback rattlesnake (55). This enzyme is active on casein
and hide powder azure, but does not digest benzoyl-L-arginine ethyl ester or benzoyl-L-
tyrosine ethyl ester. The proteolytic and hemorrhagic activities were both abolished by
treatment with EDTA and neither activity was restored by prolonged dialysis against
Zn2' or Ca2+. It is noteworthy that the caseinolytic activity of proteinase H was not
inhibited during incubation with human at-macroglobulin nor was the inhibitor
inactivated by proteinase H. Proteinase H is a single chain glycoprotein with a
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 147
molecular weight of 85,700 daltons, an isoelectric point of 6.1 and a minimum
hemorrhagic dose of 0.02 ug (Table 7). In many respects, protease H resembles
hemorrhagic toxin a (Ht-a) from the venom of Crofalus afrox, a species closely related
to Crofalus adaniarzfeus. When the amino acid composition of Ht-a is adjusted for the
difference in molecular weights and the total sum of amino acids for the two toxins (see
Table 8) and then compared to proteinase H, it becomes apparent that they me probably
homologous enzymes from the two venoms. However, further characterization is needed
to establish this definitely.
Protease H is the major hemorrhagic toxin in C. ndaniarzteus venom although
apparently not the only one. Two proteases, designated protease I and 11, that inactivate
human a-proteinase inhibitor, have been isolated from this venom by Kurecki el a/ .
(56). They had previously been termed collagenase I and I1 in an abstract by Kurecki
and Laskowski, due to their activity on powder azure collagen (57). They were also
reported to cause hemorrhagic spots on rabbit skin (57), but in a more recent report, it
was stated that they lacked hemorrhagic activity in rabbits (121). Judging from their
amino acid compositions, protease I and I1 appear to be isoenzymes as well as homologs
of the weakly hemorrhagic isoenzymes Ht-c and Ht-d from the venom of Crofalus afrox
(see Table 8). We thus conclude from the available data that protease I and I1 are
probably hemorrhagic toxins of low potency, homologous to Ht-c and Ht-d although
further investigations will be needed to establish this.
Recently, one hemorrhagic toxin was isolated by ion exchange and high pressure
liquid chromatography from the venom of the timber rattlesnake, Crofalus horridus
horridus (58). The toxin also contained proteolytic and lethal activities and was
designated hemorrhagic protease IV, meaning the hemorrhagic proteinase isolated from
fraction 1V. HP-IV is a metalloprotease containing 1 mole of zinc per mole of enzyme
as well as 2.5 moles of calcium in the isolated form. The proteinase activity is destroyed
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TAB
LE I
I.. c.
OD
Prop
ertie
s of
Hem
orrh
agic
Tox
ins a
nd o
ther
Pro
teas
es fr
om th
e V
enom
of C
rota
lus a
dam
ante
us an
d C.
hor
ridus
hor
ridus
~~
Prop
ertie
s Pr
otei
nase
H
Prot
eina
se I
Prot
eina
se I1
H
emor
rh. p
rote
ase
1V
Mol
ecul
ar w
t 85
700
2m
23
800
5700
0
Isoe
lect
ric p
t
MH
D 0%)
Met
al c
onte
nt
Hem
orrh
agic
Prot
eollt
ic
Oth
er
activ
ities
Inhi
bito
rs
Furt
her
com
mcn
ts
Ref
eren
ce
6.1
0.02
+ + EDTA
Not
inhi
bite
d by
4-m
acro
- gl
obdi
n.
Res
embl
es
Ht-
a fr
om
-_
_
C. a
trox
55
+ Inac
tivat
es
U2-
mac
ro-
glob
ulin
Sam
e as
Col
lage
nase
I pr
evio
usly
de
scrib
ed (9
0).
Sim
ilar t
o
from
C. a
trox
Ht-
c & H
t-d
56,5
1
+ Inac
tivat
es
*,-m
acro
- gi
obul
in
Sam
e as
colla
gena
se I1
pr
evio
usly
de
scrib
ed (90).
Sim
ilar t
o
from
C. a
trox
Isoe
nzym
e of
Prot
ease
I
Ht-
c &
Ht-d
56,51
5.1
4 1 Zn
2.
3 C
a + t Le
thal
, Col
lage
nase
, Fi
brin
ogen
ase.
D
iges
ts b
asem
ent
mem
bran
e
EDTA
Ala1
4-Le
u15
of I
nsul
in
B c
hain
Se
rI2-
Leul
3 of
Ins
ulin
A
chai
n an
d
Ser
1~-?
~19
of
mel
littin
5s59
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HEMORRHAGIC T O X I N S FROM S N A K E VENOMS
TABLE 8
Amino Acid Compositions of Hemorrhagic Toxins and Related Proteases from the Venom CrotaluS adamanteus and 6 horriduq horridus and Ht-a and Ht-d from C. atrox
Amino acid
Asx Thr Ser Glx GlY Ala CYS Val Met Ile Leu TYr Phe Lys His Arg Pro Trp
Ht-d
29 9
15 23 9
11 4
10 6
16 26
8 7
10 10 13 8 4
Prot. I
30 8
18 21 14 9 4
13 6
15 21
8 I 9 6
15 7 3
Prot. I1
27 8
16 21 13 8 4
13 6
15 21
8 7 9 6
16 7 2
Prot. H
98 47 44 66 50 36 50 31 16 67 49 35 22 27 23 41 32 8
Ht-a adjusted
99 42 41 64 50 42 77 41 13 42 57 25 20 32 21 39 32 7
Ht-a
85 36 35 55 43 36 66 35 11 36 49 21 17 27 18 33 27
6
149
HP-IV
68 21 29 49 43 27 41 27 13 20 27 28 14 31 18 14 30 7
Total 218 214 207 742 744 636 507
Reference 40 56 56 55 35 35 58
by incubation with EDTA as well as with disulfide-reducing agents. Coincident with
the loss of proteinase activity was a corresponding loss of lethal and hemorrhagic activi-
ties, suggesting that all three are related. Restoration of activity by readdition of metals
was unsuccessful. Reduction of one disulfide bond per molecule decreases proteinase
activity by 50% while reduction of eight disulfide bonds decreases activity by 8OYo. Loss
of hemorrhagic activity parallels the decrease in proteinase activity, further confirming
the correspondence between the proteolytic and hemorrhagic activities of hemorrhagic
toxins.
Hemorrhagic protease IV is a large acidic proteolytic enzyme having a molecular
weight of 57,000 daltons and an isoelectric point of 5.1. Amino acid analysis revealed a
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150 BJARNASON AND FOX
composition of 507 amino acids and an unusually high cysteine content forming
approximately 20 disulfide bonds. The protease exhibited little activity towards most
protein substrates but totally solubilized cow hide powder azure. Cow hide that did not
contain covalently bound dye was also hydrolyzed by HP-IV although at only 20% of the
rate at which hide powder azure was hydrolyzed. HP-IV exhibited activity on type I
collagen from bovine achilles tendon at 37'C, pH 7.4, albeit much slower and more
limited in quantity than bacterial collagenase (59). The authors state that only a small
amount of the collagen appeared susceptible to hydrolysis by the proteinase. It is
unclear whether this means that only a small portion of the collagen molecules are
susceptible to cleavage or whether only one or a few bonds in each collagen molecule are
hydrolysed. When coniparing HP-IV to bacterial collagenase it must be kept in mind
that the latter cleaves many bonds in the collagen triple helix, while, for instance, a
mammalian collagenase, which HP-IV might resemble, cleaves only one bond of the
native collagen helix.
Only one peptide bond was cleaved by HP-IV in each of the oxidized A and B
chains of insulin, the Ala14-Leu15 bond of the B chain and the Serlz-Leu13 bond of the
A chain. Bee venom melittin was cleaved at the Ile2-Gly3, Prol+-Ala16 and Serla-Trp19
bond (59). Dansylation of the hydrolysis fragments of cowhide showed the formation of
six new N-terminal residues, namely, Tyr, Leu, Met, Trp, Pro and hydroxylysine.
Various unblocked dipeptides and the doubly blocked dipeptides N-Cbz-Ser-Leu-NHz,
N-Cbz-Ala-Leu-NHz and N-Cbz-Ile-Gly-NH2 were not cleaved. The peptides used
corresponded to known cleavage sites in the insulin chains and melittin. HP-IV
catalyzed the complete solubilization of glomerular basement membrane in the presence
of lOmM Caz+ at a rate 60% as fast as an equal amount by weight of bacterial
collagenase.
Incubation of HP-IV with fibrinogen solutions caused rapid digestion of the Aa
This was accompanied by chain followed by a slower degradation of the Bp chain.
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 151
formation of a precipitate, which could easily be removed from solution by
centrifugation. No f i rm clot formed even with incubation up to 20 hours, nor would
thrombin induce a clot with HP-IV treated fibrinogen. Finally, the authors concluded
that results obtained from the hydrolysis of the various substrates by HP-IV suggested
that cleavage points are determined by the size and conformation of the substrate, not
just by recognition of the amino acids comprising the cleaved peptide bond.
Croialuy airox. To date seven hemorrhagic toxins have been reported from the
venom of the western diamondback rattlesnake, Crotalus airox (Table 9). In 1978,
Bjarnason and Tu reported on the isolation and characterization of f ive hemorrhagic
toxins and the role of zinc in hemorrhagic toxin e (35). The toxins were purified by
anion and cation exchange chromatographies as well as gel filtration and termed
hemorrhagic toxins a, b, c, d and e. Their n~olecular weights were determined to be
68,000, 24,000, 24,000, 24,000 and 25,700 daltons, respectively (see Table 9). From the
amino acid compositions it was evident that Ht-c and Ht-d were very similar proteins.
This was also apparent from the isolation procedure and electrophoresis of Ht-c and Ht-
d as well as their proteolytic cleavage progression curves. The high content of cysteine
in Ht-a was also noteworthy.
All five hemorrhagic toxins were found to lose their hemorrhagic activities after
treatment with the metal chelators EDTA and 1,lO-phenanthroline. When analyzed for
metals, all five were found to contain approximately 1 mole of zinc per mole of enzyme.
Hemorrhagic toxin e was also measured for other metals and found to have some calcium
associated with it (35). The toxins were analyzed for proteolytic activity using
dimethylcasein and dimethylhemoglobin as substrates and then reacted with
trinitrobenzenesulfonic acid to detect the newly formed amino groups. With this
sensitive assay, all five toxins showed cleavage of both substrates. The proteolytic
progression curves were clearly different for the different hemorrhagic toxins,
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TAB
LE 9
Prop
ertie
s of
Hem
orrh
agic
Tox
ins f
rom
the
Ven
om of
Cro
talu
s atro
x
Prop
er tie
s H
I-a
Ht-b
H
t-c
Ht-d
H
te
Ht-f
H
t-g
Mol
ecul
arw
t 68
000
2400
0 24
ooo
24oM
) 25
700
64ooo
m
lsoe
lcct
ric p
t
MH
D(%
)
Met
al co
nten
t
Hem
orrh
agic
Prot
coly
tic
Oth
cr
advi
ties
Inhi
bito
rs
Furth
er
com
mcn
ts
Rcf
crcn
cc
weakly a
cidi
c
0.04
1%
t
t
Fibr
inog
enas
e La
min
inol
ytic
EDTA
, 1.1
0-
phen
an-
thro
line
HTI
from
--
C.a
dam
ante
us
Cle
aves
In
sulin
B-
chai
n at
A
sn3-
Gln
4,
Hisy
Leus
.
Ala
l4-L
e~1-
3 H
~lo
-Leu
ll,
Tr16
-Leu
17.
35,s
basi
c
3 1 Zn
t
t
Fibr
inog
enas
e M
yoto
xic
Lam
inin
olyt
ic
EDTA
, lJ
0-
phen
an-
thro
line,
Opr
in
& H
TI fr
om
C.a
dam
antc
us
Clc
aves
ox
Insu
lin B
- ch
ain
at
His5
-Leu
6,
Hisl
O-L
cull,
A
la14-
Leu1
5,
Tr16
-Lcu
17(
Gly
B-P
hc2@
35.5
4
6.0
8 1 Zn
t
t
Fibr
inog
enas
e La
min
inol
ytic
EDTA
, 1,
10-
phcn
an-
thro
line
amin
o ac
id
hydr
oxam
ate
Cle
avcs
ox
Insu
lin B
- ch
ain
at
His5
-Leu
6,
Hisl
O-L
eull,
A
la14-
Leu1
5 Tr
lC;L
eu17
, G
ly23
-Ph3
4-
35.4
0
6.1
11
1Z
n
t
t Fibr
inog
enas
e La
min
inol
ytic
EDTA
, 1,
lO-
phen
an-
thro
line
amin
o ac
id
h ydr
oxam
ate
Clc
aves
ox
Insu
lin B
- ch
ain
at
His5
-Leu
6,
Hisl
O-L
cull,
A
la14
-Leu
5
Tr](j-L
cuD
, G
lyB
-Phe
24
35.40
5.6
1
1 Zn
t
t
Fibr
inog
enas
e La
min
inol
ytic
EDTA
, 1,
10-
phcn
an-
thro
line
Cle
aves
ox
Insu
lin B
- ch
ain
at
Asn
3-G
ln4,
Se
r9-H
is10,
A
la14-
Leu1
5.
35
7.7
0.5 1 Zn
t
t
Leth
al &
fib
rinog
enax
. ac
tivity
on
theA
-&
B
-cha
ins
EDTA
, 1.10
- ph
cnan
- th
rolin
e
Cle
avcs
ox
Insu
lin B
- ch
ain
at
VaI2-
A:ri3
, G
ln4-
Hisg
.
Hisl
O-L
eull,
~u
6-cy
S7S
O3,
Ala
l4-L
eulg
T
Pl6
4-9
7
61
6.8
1.4 + t
Leth
al &
fib
rinog
enas
e ac
tivity
on
theA
-&
B
-cha
ins
EDTA
, EG
TA &
1,
lO p
hcna
n-
thro
line
62
-Y 0
x
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 153
suggesting different proteolytic specificities by these toxins. Furthermore, the toxin
with the greatest hemorrhagic activity, hemorrhagic toxin a, showed the lowest activity
toward dimethylcasein as substrate. Thus, there does not seem to be a direct relationship
between the hemorrhagic activities of the different toxins and their proteolytic activities
on dimethylcasein. These observations suggested a highly selective specificity of the
toxins, especially hemorrhagic toxin a, the most potent one.
The hemorrhagic toxins from C . alrox venom were the first hemorrhagic toxins
assayed for zinc and found to contain approximately one mole of zinc per mole of toxin.
Since the report of this finding, at least five other hemorrhagic toxins from three
different genera of snakes have been assayed for zinc and found to contain this metal
ion. This is especially noteworthy since zinc is a common prosthetic group for
proteolytic enzymes, such as many neutral proteases and collagenases. Hemorrhagic
toxin e did not show collagenase activity using native bovine achilles tendon collagen as
substrate, nor did it cleave Furylacryloylglycyl-L-leucinamide, a substrate for neutral
proteases.
When zinc was removed from hemorrhagic toxin e with dialysis against 10 mM
Hepes buffer, pH 7.2 containing 2mM 1,lO-phenanthroline, 10 m M CaClz and O.1M
NaCl for 24 hours, less than 10% of the original zinc content was found associated with
the apotoxin according to atomic absorption measurements. Less than 5% of the original
proteolytic activity remained with the apotoxin and approximately 59/0 of the original
hemorrhagic activity was still associated with the apotoxin. When the apotoxin was
incubated with zinc ion using the dialysis method, 20% of the original proteolytic
activity was regenerated and approximately 25% of the hemorrhagic activity was found
associated with the regenerated toxin. Thus, a direct relationship between the
hemorrhagic and proteolytic activities of hemorrhagic toxin e was demonstrated, the first
time such a relationship was firmly established for a hemorrhagic toxin.
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154 BJARNASON AND FOX
Spectroscopic methods were used to study the structure of native hemorrhagic
toxin e as well as structural changes caused by zinc removal and readdition. With C D
spectroscopy, the native toxin Ht-e was estimated to consist of 23% a-helix and 6% 8-
structure. When over 9OYo of the zinc was removed, the a-helix content dropped from
23% to 7qo. When the apotoxin was incubated with zinc, an increase in a-helix content
was observed from the peptide region of the CD spectrum. Parallel changes could be
seen in the aromatic regions of the CD and UV spectra which are indicative of changes
in the tryptophan environments. These interpretations were also supported by
appearances of peaks in the Raman spectrum of the apotoxin attributable to tryptophan,
which were not visible in the Raman spectrum of the holotoxin. Thus, there appear to
be significant conformational changes in hemorrhagic toxin e with the removal of zinc
from the toxin (35).
In 1983, Bjarnason and Fox described the exchange of cobalt for zinc in Ht-e
and the properties of the cobalt containing toxin as well as the proteolytic specificity of
hemorrhagic toxin e and cobalt hemorrhagic toxin e (60). Cobaltous ion was introduced
into hemorrhagic toxin e by the method of direct exchange with dialysis. An 80 mM
€It-e solution in 5mM Tris-Cl buffer pH 8.5 containing 0.1M NaCl and 2 mM CaCl,
was dialyzed against a fifty-fold volume of 0.2 M cobaltous ion in 0.1M sodium acetate
pH 5.5 or 36 hours at 2-4'C. Excess
cobalt was removed by extensive dialysis. The cobalt hemorrhagic toxin e thus formed
contained 1.1 moles of cobalt per niole of toxin and no measurable zinc. Previous
attempts to incorporate cobalt into the apotoxin were unsuccessful. The cobalt
containing toxin was found to possess both hemorrhagic and proteolytic activities to a
similar or slightly lesser degree as the native toxin. The minimum hemorrhagic dose of
Co(I1)HT-e was found to be 1.2 mg as compared to 1.0 mg for the native toxin. The
cobalt-containing toxin was estimated to have a turnover number for the native toxin of
10 min-l whereas the turnover number for the native toxin is 13 min-1.
The solution was constantly purged with Nt.
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HEMORRHAGIC Toxxns FROM SNAKE VENOMS 155
The UV spectra of hemorrhagic toxin e and cobalt hemorrhagic toxin e appeared
to be identical, with shoulders at 291 and 284 nm and a peak at 278 nm. Likewise, the
CD spectra of the cobalt containing toxin in the aromatic and peptide regions appeared
totally identical to the previously published spectra of the native toxin. There thus
appears to be no change in structure of hemorrhagic toxin e accompanying the metal
exchange. This was in stark contrast to the considerable structural changes observed
with the same spectroscopic methods accompanying production of the apotoxin by zinc
removal. Thus, zinc removal causes large structural perturbations, whereas exchanging
zinc with cobalt is accomplished without observable structural changes.
The absorption and CD spectra of the complex of cobaltous ion and hemorrhagic
toxin e in the visible region were also measured. The absorption spectrum has a peak at
505 nm with a molar absorptivity value of 170 M-1 cm-1. There also appears to be a
shoulder a t 550 nm and another peak at about 600 nm. The CD spectrum shows minima
at 480 nm and 520 nm. These spectra resemble those of certain other cobalt enzyme
complexes thought to possess distorted tetrahedral structures. However, we have not
observed in the literature spectra with striking similarities to those of cobalt Ht-e.
The proteolytic specificity of Ht-e and cobalt Ht-e was investigated by using the
oxidized A and B chains of bovine insulin. The most rapid cleavage on the two insulin
chains occurred at the Ala14-Leu15 bond on the insulin B-chain. A secondary site of
cleavage on the B-chain was observed at Serg-Hislo, and a tertiary and very slow
cleavage occurred at Asn,-Gln4. The primary site of cleavage on the insulin A chain is
the Tyr,,-Gln15 bond with a secondary site of cleavage at the Alas-Sers bond. The
difference in the rates of cleavage at the different sites was estimated to be
approximately one order of magnitude.
An effort was made to quantitate the rates of the various proteolytic activities of
hemorrhagic toxin e and cobalt hemorrhagic toxin e. The turnover of cleavage for the
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156 BJARNASON AND FOX
primary site in the A chain of oxidized insulin, the Tyr14-Gln15 bond, was estimated to
be 3.6 min-1 for Ht-e and 2.6 niin-1 for cobaltous Ht-e, but for the secondary site, the
Ala8-Ser9 bond, the turnover of cleavage was estimated to be 0.46 min-1 for both forms
of the toxin (Table 11). It was thus demonstrated in this investigation that cobaltous ion
can be exchanged for zinc in the native toxin without detectable changes in the protein
conformation and with only minor changes in function (60).
Nikai and co-workers isolated hemorrhagic toxin f from Crofalus afrox venom in
a five step isolation scheme, three of which are identical to the previously published
first three steps of Hb-a isolation from C. afrox venom. It is odd and inappropriate that
three authors republish this isolation scheme without any reference to the previous work
(61). Ht-f has a molecular weight of 64,000 daltons and contains one mole of zinc per
mole of protein. Zinc is essential for both the toxin’s hemorrhagic and proteolytic
activities. When Ht-f was incubated with the B-chain of oxidized insulin it was found
to cleave at the Va12-Asn3, Gln4-His5, Leu6-Cysv, Hislo-Leull, Ala14-Leu15 and TYT16-
Leul7 bonds. Ht-f was also found to hydrolyze the 7 chain of fibrinogen without
affecting either the ALY or BP chains. This was the first time that a hemorrhagic toxin
was shown to have fibrinogenase activity although the reverse was demonstrated seven
years earlier: that is a fibrinogenase and fibrinolytic principle was shown to possess
hemorrhagic activity (37). Ht-f was also shown to differ immunologically from Ht-a
and Ht-c from C. airox venom. The Ht-f had a lethal toxicity (LD50) of 9.8 mg/g
compared to 3.3 mg/g for the crude venom. When zinc was removed, Ht-f lost its lethal
toxicity. From the amino acid composition it is noteworthy that Ht-f, like Ht-a and
other high molecular weight hemorrhagic toxins, contains an unusually high amount of
cysteine of approximately 10% of the total number of amino acid residues (see Table 10).
In 1985, Nikai and co-workers reported on the isolation and characterization of
hemorrhagic toxin g from Crofalus afrox venom (62). A five step purification, of which
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157 HEMORRHAGIC T O X I N S FROM SNAKE VENOMS
TABLE 10
Amino Acid Compositions of Hemorrhagic Toxins from the Venom Crotalus atrox
Amino Ht-a Ht-b Ht-c Ht-d Ht-e Ht-f Ht-g acid
Asx 85 26 27 29 30 75 66 Thr 36 7 10 9 10 33 33 Ser 35 14 15 15 14 32 42 Glx 55 16 22 23 26 50 51 G ~ Y 43 13 9 9 14 49 48 Ala 36 8 12 11 7 41 33 CYS 66 4 4 4 8 45 48 Val 35 11 10. 10 12 29 29 Met 1 1 6 6 6 8 11 4 Ile 36 12 15 16 21 23 21 Leu 49 19 30 26 14 37 31 TY r 21 7 7 8 11 22 18 Phe 17 7 6 7 6 18 15 LYS 27 10 9 10 9 31 20 His 18 8 10 10 9 14 12 Arg 33 12 12 13 8 27 22 Pro 27 6 8 8 8 26 19 TrP 6 4 4 4 4 10 4
Total 636 200 216 218 209 572 516
Reference 35 35 40 40 35 61 62
TABLE 11
Activities of the Two Forms of Hemorrhagic Toxin g
Form of Toxin
Holo HT-e Cobaltous Ht-e Activity
Turnover of hydrolysis of dimethylcasein 13 rnin-' 10 min-l
Minimum hemorrhagic dose (pg toxin)
Turnover of cleavage of the Tyr14-Gln15 bond
Turnover of cleavage of the Ala8-Ser9 bond 0.46 min-l 0.46 min-l
Ref. 60
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158 BJARNASON AND FOX
the first three were again identical to the final three steps in the Ht-a isolation, yielded
5.9 mg of purified Ht-g from 2 grams of crude venom. Again the authors made no
mention of the previous work. Amino acid analysis gave a composition of 516 amino
acids based on a molecular weight of 60,000 daltons in a single polypeptide chain. The
most outstanding feature of the amino acid composition of this toxin is once more the
relatively high cysteine content of 9.30/0.
Hemorrhagic toxin g was found to possess proteolytic and lethal activities, which,
in addition to the hemorrhagic activity were inhibited by EDTA and 1.10-
phenanthroline, suggesting that the activities are metal ion dependent. However, no
metal analysis was performed on the toxin. Hemorrhagic toxin g also showed
fibrinogenase activity, cleaving the Aa- and BP-chains of fibrinogen, in contrast to Ht-f
which only digested the 7-chain. Komori and co-workers showed that Ht-b also
contains fibrinogenase activity, hydrolyzing the Aa-chain of fibrinogen first, followed
by cleavage of the BP-chain ( 6 3 ) . The degradation products of fibrinogen were found to
be different from those of thrombin, indicating that the cleavage sites in the Aa- and
BP chains are different from those of thrombin. Also, Ht-b did not produce a fibrin
clot or hydrolyze N-Benzoyl-Phe-Val-Arg-p-nitroanilide, a substrate hydrolyzed by
thrombin and reptilase.
Recently, Bjarnason and Fox reported on further characterization of hemorrhagic
toxins c and d (40), as well as on the substrate specificities and inhibition of these toxins
(64). The two toxins were characterized and compared to one another. Their isoelectric
points are slightly acidic, Ht-c being the more basic of the two with a n isoelectric point
of 6.2, whereas Ht-d has an isoelectric point of 6.1. The pH optimum of proteolysis by
Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The
circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to
minima positions and elipticities, indicative of very similar solution structures for the
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 159
two enzymes. They also appeared to share identical antigenic structures. These results
were corroborated by tryptic mapping of the two toxins. Only one major difference was
observed from the maps. In the case of Ht-c, it was determined that an aspartate was
substituted by an alanine when compared to Ht-d. From these characterization studies,
the authors concluded that Ht-c and Ht-d are isoenzymes with only very minor
differences in their structures. It is of interest to note that the amino acid compositions
of Ht-c and Ht-d are very similar to those of protease I and protease I1 from C.
adunianleus suggesting that these might be homologous enzymes from the two species of
snakes (40).
The proteolytic specificities of Ht-c and Ht-d were investigated by using the
oxidized B-chain of bovine insulin and synthetic peptide substrates (64). The enzymes
cleaved the Ala14-Leuls bond of the insulin B-chain most rapidly and the Tyr16-Leu17
bond slightly more slowly. The His5-Leue, Hislo-Leull and Gly2,-Phez4 bonds were
also cleaved but at a considerably slower rate. In order to assess the substrate length
preferences of the enzymes, peptide analogs of the B-chain about the A1aI4-Leul5 bond
were synthesized ranging in length from four to seven residues. The heptapeptide NHz-
Leu-Val-Glu-Ala-Leu-Tyr-Leu-COOH was the best peptide substrate tested in this
series with the other peptides having decreasing kcat/Km values with decreasing length.
The tetrapeptide NH2-Ala-Leu-Tyr-Leu-COOH was not cleaved by the enzymes. On
the contrary, this peptide was shown to be a competitive inhibitor of the toxins. A
series of N-acetylated pentapeptides and one hexapeptide, synthesized to probe the
active site environment of the enzymes, were significantly better substrates than their
unacetylated counterparts by approximately two orders of 3 magnitude in kcat/Km
values.
The N-acetylated peptide Ac-Val-Ala-Leu-Leu-Ah-COOH gave the largest
kcat/Km value of all the N-acetylated peptides assayed with both Ht-c and Ht-d in this
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160 BJARNASON AND FOX
TABLE 12
Kinetic Parameters of Cleavage of Synthetic Peptides by Ht-c and Ht-d
Peptide Substrate site K, x 104 kcat k,,t/Km X
P P P P 'P 'P ' Ht-c Ht-d Ht-C Ht-d Ht-C Ht-d
M -- min-1 - mi n- 'h.~- I-
1 Ac-V-A-L-L-A 6.2 6.3 a s 8.3 137 132 2 Ac-V-A-L-G-A 4.5 3.1 0.46 0.5 10.2 16.1 3 Ac-V-A-L-G-A 3.1 3.1 0.47 0.6 15.2 19.4 4 Ac-A-A-G-L-A 7.1 7.1 0.30 0.3 4.23 4.23 5 Ac-V-A-A-L-G-A 6.2 6.9 5.9 5.9 95.2 85.5 6 Ac- A-V-L-G-A nd nd I Ac-V-G-L-G-A 2.1 0.46 21.9 a Ac-V-A-F-G-A 5.0 1.1 22.0 9 Ac-E-A-L-G-A 1.6 1 .o 13.8 10 AC-V-E-L-G-A~ nd nd 11 AC-V-K-L-G-A~ nd nd 12 AC-K-A-L-G-A? nd nd 13 AC-V-A-I-G-A" nd nd 14 Az-A-G-L-A-Nb 3.1 3.5 145 127 4680 3629
a Inhibition assayed for but not detected.
(nd, No Detectable Cleavage) (55).
Reference 64.
series. The peptides were sensitive to substitutions at the peptide sites from position P'3
to PZ and possibly P3 (see Table 12). A generalized view of the enzymes' substrate-
binding sites (S3 through S3) can be constructed from the cleavage data of the N-
acetylated synthetic peptides. The enzymes seem to prefer hydrophobic residues in their
S1 and sites. However, an interesting exception is seen with peptide 13 which has an
isoleucyl residue at its F1 site. Apparently, the enzymes cannot accept an isoleucyl
residue at S'1 sites since peptide 13 does not act as a substrate or a competitive inhibitor
toward the enzymes. The S1 sites in the enzymes are rather selective in that they do not
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 161
cleave peptides containing charged residues (glutamyl, lysyl) or bulky aliphatic side-
chains a t that position. This is in contrast to thermolysin. The S2 sites of the enzymes
seem to prefer a bulky aliphatic side-chain, like valine, over a smaller side-chain ( A h )
and the enzymes will not cleave a peptide with a positively charged lysyl residue at that
position. Finally, it appears that the enzymes have an extended substrate-binding site in
that the placement of large, bulky groups at the P3 position of a substrate enhances
catalysis compared to similar but truncated peptides.
The hemorrhagic toxins of C. alros have been used as model proteolytic enzymes
for the design and synthesis of inhibitors against these toxins. The results of this work
are discussed in Section 5.
3.4 Trimerisurus
Trinwrirurur f~mov i r id i r . In 1960 Ohsaka and coworkers separated T.
flailoviridis (Habu) venom by zone electrophoresis and demonstrated the presence of two
separate fractions containing hemorrhagic activity (65). These fractions were termed
hemorrhagic principles 1 and 2 or HR-1 and HR-2, and were found to contain minor
proteolytic activities on casein as substrate. It thus appeared to the authors that a
correlation existed between the hemorrhagic activity and proteolytic activity on casein.
The HR-I fraction overlapped with the main lethal toxicity peak whereas the HR-2
fraction overlapped with the minor lethal toxicity peak. Also proteolytic activity was
not detected in the fraction containing HR-I after re-electrophoresis of the previous
fraction HR-I . This was the first report of hemorrhagic activity being fractionated
away from proteolytic activity on casein as substrate and measured by the trichloroacetic
acid precipitation method of Kunitz (66). In 1967, these same researchers reported on
the separation of HR-I and HR-2 using molecular sieving chromatography using a
column of Sephadex G-100 resin (67).
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162 BJARNASON AND FOX
In 1970 Takahashi and Ohsaka reported purifying the hemorrhagic fraction HR-2
further into two parts, HR-2a and HR-2b, by column chromatography on Bio-Rex 70
(68) . Homogeneity was examined by ultracentrifugation and electrophoresis. A single
symmetrical boundary was obtained with HR-2a, while an asymmetrical boundary was
observed with HR-2b on the ultracentrifuge. Upon electrophoresis on Separax, a
cellulose acetate membrane, the HR-2a preparation gave a single protein band migrating
toward the cathode. The HR-2b preparation gave two bands, of which the major one
migrated toward the cathode slightly faster than the minor one which had a mobility
identical to that of HR-2a. Thus HR-2a was considered homogeneous, while HR-2b
appeared to be contaminated with HR-2a. The authors stated that complete elimination
of proteolytic activity from HR-2 had been accomplished by chromatography on Bio-
Rex 70 with resolution of the hemorrhagic activity into two parts, HR-2a and HR-2b,
which in their opinion clearly demonstrated that the hemorrhagic principle and the
proteolytic enzyme are separate entities. Thus, they conclude without reservations that
the hemorrhagic toxins HR-2a and HR-2b do not contain proteolytic activity, using only
casein as substrate and an insensitive method of detecting cleavage products. Apparently
they also implied that the venom contains only one proteolytic enzyme.
Interestingly, in a recent report, Nikai and coworkers reported that both HR-2a
and 2b have very high protease activity when the B-chain of oxidized insulin was used
as a substrate, giving evidence for the proteolytic character of these toxins (48).
Also in 1970, Omori-Satoh and Ohsaka reported on the purification and some
properties of hemorrhagic principle 1 (69). The preparation was homogeneous as judged
by electrophoresis on cellulose acetate membrane, isoelectric focusing and
immunodiffusion, but not homogeneous by ultracentrifugation. The toxin had a
molecular weight of 100,000 daltons, an isoelectric point of 4.3 and its hemorrhagic
activity was inhibited by EDTA, cysteine and formaldehyde but not by DFP or soybean
trypsin inhibitor. The inhibition by EDTA could not be reversed by an excess amount
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 163
of Ca2'. The hemorrhagic principle contained 7% of the lethal toxicity and 0.6% of the
proteolytic activity of the crude venom, both of which the authors considered to be due
to impurities. Ohsaka and coworkers studied the action of the isolated hemorrhagic
toxins HR-1, HR-2a and HR-2b, as well as bacterial collagenase, known to induce
hemorrhage, on isolated glonierular basement membrane (70). Upon incubation of the
basement membrane preparation with any of the four agents, the supernatant gave
positive reactions for soluble proteins and carbohydrates. Liberation of protein and
carbohydrate containing fragments from basement membrane by HR- 1, HR-2a and HR-
2b was inhibited by EDTA, cysteine, antivenom and antihemorrhagic factor from snake
serum, all of which inhibit the hemorrhagic activity of these principles. The authors
concluded that the hemorrhagic effect of HR-I , HR-2a, HR-2b and collagenase is
attributed to enzymatic destruction of the basement membrane with consequent lowering
of the stability of the vessel wall.
In 1979 Omori-Satoh and Sadahiro described the resolution of the major
hemorrhagic component of T. jlavovir-idis into two parts, 1A and l B , by further
purification with gel filtration on Sephadex G-200 superfine (71). The purified
preparations of HR-IA and HR-IB were homogeneous as judged by several criteria (see
Table 13). The molecular weights of the purified principles determined by SDS gel
electrophoresis were approximately 60,000 daltons. Toxin HR-1A showed anomalous
behavior on ultracentrifugation and gel filtration owing to concentration-dependent
polymeric interaction. The purified components were acidic glycoproteins with
isoelectric points of 4.4 and they contained neutral sugars, amino sugars, and sialic acid,
altogether amounting to 17-18% on a total weight basis. From the amino acid
composition, it is noteworthy that both toxins have a relatively high cysteine content,
similar to other high molecular weight hemorrhagic toxins (see Table 14).
Proteolytic activity of the purified hemorrhagic toxins HR-1A and HR- 1B was
The assayed with casein as substrate, measuring trichloroacetic acid-soluble materials.
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164 BSARNASON AND FOX
authors state that purified HR-IB had threshold detectable proteolytic activity. From
this result and the fact that fraction HR-IB contained about 60% of the hemorrhagic
activity and lethal toxicity, the authors conclude that the main parts of the hemorrhagic
activity and lethal toxicity (HR-IB) were almost free from proteolytic activity. On the
other hand, purified HR- IA had appreciable proteolytic activity. They thus concluded
that their results strongly suggested that HR-IA is a proteolytic enzyme.
The purified components, HR-IA and HR-IB, are closely related immuno-
logically. Antisera to each hemorrhagic component neutralized both components to the
same extent. However, the formation of a coalescing precipitin line with a single spur
between the two purified components and antiserum to partially purified HR-1 on
immunodiffusion suggests some immunological difference between them (7 1).
We thus conclude that four proteolytic hemorrhagic toxins from the venom of
Trinwisurus f laiwiridis have been purified and characterized to some extent (see Tables
12 and 13). The twenty year development of the purification of these toxins exemplifies
the difficulty in studying hemorrhagic toxins from snake venom. Furthermore, the use
by these researchers of a very insensitive and limited assay method for proteolytic
activities clearly demonstrates the pitfalls of such procedures.
Trimerisurus arnmitreus and muoosaunmatus
Huang and coworkers reported in 1984 on the purification and characterization
of two hemorrhagic toxins from the venom of Trimerisurus gramineus (72). The toxins
were termed hemorrhagin I and I1 or HRI and HR2. They were homogeneous as judged
by SDS-polyacrylamide gel electrophoresis. Their molecular weights were estimated to
be 23,500 daltons for HRI and 81,500 daltons for HR2. They were single polypeptide
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TAB
LE 13
Prop
ertie
s of
Hem
orrh
agic
Tox
ins f
rom
the
Vcn
om of T
rimcr
csur
us fl
avov
iridi
s
Prop
ertie
s H
R-I
A
HR
-1B
H
R-2
a H
R-Zb
Mol
ecul
ar w
eigh
t m
m
n.d.
lsoe
lcdr
ic p
oint
MH
DW
Met
al c
onte
nt
Hem
orrh
agic
Prot
eoly
tic
Oth
er a
ctiv
ities
Inhi
bito
rs
Furth
er c
omm
ents
Ref
eren
ce
4.4
0.016
n.d.
+ + Base
men
t m
embr
ane
lysis
. Le
thal
EDTA
, cy
stein
e,
snak
e ser
um
antih
emor
r-
hagi
c fa
dor
HR
-1, W
ore
sepa
ratio
n in
to H
R-I
A &
IB
, was
foun
d to
caus
e ba
sem
ent
mem
bran
e ly
sis.
71
4.4
0.010
n.d.
+ + Bas
emen
t m
embr
ane
lysis
. Le
thal
EDTA,
cystc
ine,
sn
ake s
erum
an
tihem
orr-
ha
gic
fact
or
Con
tain
ed lo
w
casc
inol
ytic
ac
tivity
. O
thcr
pro
teas
e su
bstra
tes w
ere
not t
este
d.
71
basi
c
0.0GG
n.d.
+ + Bas
emen
t m
embr
ane
lysis
.
EDTA
, cy
stcin
e,
snak
e ser
um
antih
emor
r-
hagi
c fac
tor
Foun
d by
Nik
ai
& c
owor
kers
to
cle
ave t
he
ox.in
sulin
B
chai
n,al
thou
gh
prev
ious
ly
foun
d no
t to
bc w
sein
o-
lytic
.
*,a
n.d.
basi
c
0.066
n.d.
+ + Bas
emen
t m
embr
ane
lysis
.
EDTA
, cy
stei
ne,
snak
e se
rum
an
tighe
mor
r-
hagi
c fac
tor
Foun
d by
Nik
ai
& co
wor
kers
to
clea
ve th
e ox
.insu
lin B
ch
ain
alth
ough
pr
evio
usly
fo
und
not t
o be
cas
cino
- ly
tic.
‘%a
2
I
0
P P 2
>
CJ
H
n
n
cj
0
x H z 1
P
0 I
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166 BJARNASON AND FOX
TABLE 14
Amino Acid Compositions of Hemorrhagic Toxins from the Venoms of Tinieresurus flavoviridis, warnineus and mucrosauamatus
Grams of residues per lOOa orotein Nearest Integer
Mucrotoxin
Amino HR-1A HR-1B HRI HR2 A HR-a HR-b acid
Asx 12.09 10.86 Thr 3.53 2.98 Se r 3.62 3.62 Glx 10.86 10.55
Ala 3.24 2.67 CYS 6.1 1 4.77 Val 4.45 3.58 Met 1.46 1.34 Ile 3.04 3.57 Leu 4.77 4.22 Tyr 5.36 4.82 Phe 2.97 3.24 LYS 3.36 5.23 His 2.64 2.82 Arg 6.69 4.22 Pro 2.97 3.4 1 TrP
GlY 2.97 2.75
26 96 86 13 37 36 1 1 42 94 16 66 110 10 56 56 9 37 48 8 45 39
16 35 69 7 1 1 15
10 35 29 17 46 42 7 23 41 7 22 42
19 33 62 8 19 20 8 27 30 9 39 27 2 16
12 4
14 19 10 12 6 8 2 3 6 6 8 7 4 5 2 3
25 I 1 28 33 25 12 15
1 5 ti
15 8
12 16 6
10 7 2
Total 73.77 74.66 203 669 862 131 239
Reference 71 71 72 72 73 75 75
chains and their hemorrhagic activities were heat-labile, while their optimal pH for
hemorrhagic activity was broad, pH 5.0-10.0. The relative potency ratio of hemorrhagic
activity for the crude venom, HRI and HR2 was about 8:1:2 respectively when compared
by minimum hemorrhagic dose (MHD). The amino acid composition of HR2 yielded
669 amino acids, not counting tryptophan, of which 45 were cysteines, while HRI
contained only 203 amino acids (Table 14). The carbohydrate content of HRI and HR2
were 4% and 10% respectively.
Hemorrhagic toxin HR1 showed strong proteolytic activity toward fibrinogen,
casein and azocoll, whereas HR2 showed a rather weak proteolytic activity on these
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 167
substrates. Toxin HR1 selectively cleaved the Ao! chain of fibrinogen, whereas HR2
partially cleaved both the Aa and BB chains simultaneously, indicating that the
proteolytic specificities of HR1 and HR, are different. Both EDTA and 1 , l O -
phenanthroline almost completely inhibited the hemorrhagic, fibrinolytic, caseinolytic
and azocollolytic activities of HR1. The subsequent addition of Co2+ or Zn2+ reversed
the EDTA-inhibitory effect on the hemorrhagic activity. On the other hand, EDTA
only partially inhibited the hemorrhagic activity of HR2 (about 30%), but completely
inhibited the fibrinolytic and caseinolytic activities. The authors did not attempt to
explain this apparent discrepancy and they did not measure the zinc content of the
EDTA treated toxins. Possibly the residual activity left with HR2, due to incomplete
zinc removal, was enough to give a substantial positive response in the 24 hour
hemorrhagic assay of complex biological events, while no proteolytic response was
observed with their 60 minute protease assay of HR2 which in the native form (prior to
EDTA treatment) showed only a weak proteolytic response. The authors concluded that
the proteolytic activity of HR1 was related to its hemorrhagic activity, but that the
proteolytic activity of HR2 may be unrelated to its hemorrhagic activity. Treatment
with Zn2+ and Co2+ did not reverse the inhibitory effect of EDTA on the hemorrhagic
activity of HR2. Atomic absorption data indicated that HRI and HR2 contained
approximately 0.5 and 1 mole of Zn2+ per mole of toxin, respectively. However, Co2+
was not detected in HRI or HR2. Thus the authors concluded that the reversal effect of
CO" on the EDTA inhibitory activity might be due to its high binding toward EDTA.
A more probable explanation is that the Co2+ ion binds to the zinc site of HRl as
previously demonstrated by Bjarnason and Fox for hemorrhagic toxin e from C. 4lrO.X
venom (58) .
Mucrotoxin A was purified from the venom of T. niucrosquamalus using gel
filtration on a Sephadex G- 100 column, followed by chromatography on CM-Sephadex
C-50 and DEAE-Sephadex A-50 (73). The purified toxin was homogeneous by disc
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168 BJARNASON AND FOX
electrophoresis on polyacrylamide gel at pH 8.5, isoelectric focusing and by the presence
of one precipitin line on immunodiffusion. Mucrotoxin A possessed both lethal and
hemorrhagic activities, but did not show caseinolytic activity. It had a molecular weight
of approximately 94,000 daltons and an isoelectric point of 4.3. Metal analysis yielded a
composition of approximately 3 moles of calcium and 2 moles of zinc per mole of toxin.
From the amino acid composition it was determined that Mucrotoxin A contained 862
amino acids, and a lower cysteine content than the other high molecular weight
hemorrhagic toxins (see Table 14). The hemorrhagic and lethal activities were inhibited
by chelating agents, such as EDTA and 1,lO-phenanthroline indicating that calcium and
zinc ions play an important role in the activity of this toxin.
More recently Kishida el al. (74) have shown that, although Mucrotoxin A does
not hydrolyze casein, it does however cleave dimethylcasein as well as the oxidized B
chain of insulin and fibrinogen. The sites of cleavage in the oxidized B chain of insulin
were identified as Serg-Hislo, HislO-Leull, Ala14-Leu16, Leul6-Tyrl6 and Tyr16-Leu17.
Recently, Nikai and co-workers reported on the isolation and characterization of
two additional hemorrhagic toxins from the venom of T. nzucrosquanzalus (75). The
toxins were termed hemorrhagic factors a and b and designated HR-a and HR-b. The
toxin HR-b is a basic glycoprotein with a molecular weight of 27,000 daltons and an
isoelectric point of 8.9 whereas HR-a is acidic with an isoelectric point of 4.72 and a
molecular weight of 15,000 daltons and no carbohydrate (Table 15). Both toxins
possessed proteolytic activity with fibrinogen and the oxidized B chain of insulin as
substrates but neither toxins cleaved casein. The toxin HR-a hydrolyzed the Hislo-
Leull, Tyrl~-LeUl, and Arg22-Cly23 bond of the oxidized insulin B chain whereas HR-b
hydrolyzed the A1al4-LeuI6 bond. Thus HR-a is unique among the hemorrhagic toxins
in that it is the only one so far studied for insulin B chain specificity which does not
cleave the Ala14-LeulS bond, while HR-b is only the second example of a hemorrhagic
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TAB
LE 1
5
Prop
crtic
s of
Hcm
orrh
agic
Tox
ins l
rom
the Venom
of T
rimer
esur
us m
amin
eus a
nd m
ucro
soua
mat
us
X m
P
a
X r 0
H 0
rj
0
x H
z
01
.sl a 0 z
rn
x
c z
0
m
m 5
Muc
roto
xin A
H
R-a
H
R-b
Pr
oper
ties
HR
1 H
R2
Mol
ecul
ar w
eigh
t 23
500
81500
91OOo
1500
0 27
00
8.9
2.3
4.3
2.31
2 Z
n 3
Ca
+ + Leth
al
4.7
1.7
Isoe
lect
ric p
oint
MH
D 6G)
M
etal
cont
ent
0.5 Z
n 1 Z
n
Hcm
orrh
agic
Prot
eoly
tic
Oth
er a
ctiv
ities
+ +
+ +
+ +
t
Fibr
inog
cnas
e ac
tivity
on A
ch
ain.
Inh
ibits
pl
atel
et
aggr
egat
ion
EDTA
, ,4
-mer
capt
octh
anol
, an
tiven
in &
1,lO
- ph
enan
thro
line
Subs
eque
nt
addi
tion
of
Zn2
+ rc
vcrs
cd
EDTA
inhi
bitio
n
Fibr
inog
enas
e ac
tivity
on A
an
dB c
hain
s
Nec
rosi
s Fi
brin
ogcn
ase
activ
ity on
B ch
ain
Nec
rosis
Fi
brin
ogcn
ase
activ
ity o
n A
ch
ain
Inhi
bito
rs
Furt
her c
omm
ents
EDTA
, P-
mer
wpt
octh
anol
, an
tiven
in &
1,lO
- ph
enan
thro
line
Gly
copr
otei
n.
Hcm
orrh
agic
ac
tivity
was
on
ly p
artia
lly
inhi
bite
d by
ED
TA &
1,lO-
phcn
anth
rolin
e
72
EDTA
, 1,IO
- ph
enan
thro
line
and
antiv
cnin
EDTA
, 1,lO
- ph
enan
anth
rolin
e &
p-c
hlor
omeu
ri-
benz
oate
EDTA
, 1,lO
- ph
enan
thro
line
& p
-chl
orom
euri-
be
nzoa
te
Ser,
- Hisl
O
Hisl
O -
Leul
l A
hl4
- Leu
l5
Leu1
5 - T
Y~I
C,
of th
e ox. i
nsul
in
B-c
hain
cle
aved
.
'yr16
- L
eu17
Hisl
O-L
eull,
Ty
16-L
eu17
? &.
the
ox. i
nsul
in
B-c
hain
cle
aved
.
A'S
229Y
23 o
f
Gly
copr
otei
n A
la14
-Leu
lS o
f th
e ox
. ins
ulin
B
-cha
in c
leav
ed.
Ref
eren
ce
72
73,7
4 75
75
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170 BJARNASON AND FOX
toxin that cleaves only at the Ala,l-Leuls bond. The hemorrhagic activity of these
toxins was inhibited by EDTA, 1 ,lo-phenanthroline and p-chloromercuribenzoate, but
not by soybean trypsin inhibitor or diisopropyl fluorophosphate. Unfortunately, the
metal content of these toxins was not determined, but they do appear to be metallo-
proteases like most if not all the hemorrhagic toxins.
Eight hemorrhagic toxins have thus been purified from three species of snakes of
the genus Trinzeresurus. They all appear to be metal dependent toxins and all have been
reported to possess some proteolytic activity, although four of these did not hydrolyze
casein but did cleave fibrinogen or the B chain of oxidized insulin or both.
Vinem nnlesririne. Three hemorrhagic toxins have been purified from the venom
of V . palasefiiiae using Sephadex G-25 gel filtration, ammonium sulfate precipitation
and DEAE cellulose chromatography (76). The toxins were designated HR-1, HR-2 and
HR-3. All these toxins were found to have a similar molecular weight of 60,000
daltons. HR-I was found to be a basic glycoprotein having a minimum hemorrhagic
dose of 0.2 kg, HR-2 a weakly acidic protein also with MHD of 0.2 fig, whereas HR-3
was strongly acidic with a MHD of 0.4 fig. The toxins HR-1 and HR-2 show both
gelatinase and caseinolytic activity while HR-3 had no detectable activity towards these
substrates. The weakly acidic protein HR-2 is the fraction most similar to the
hemorrhagin previously isolated by the same group of researchers f rom the venom but
was first reported as having a molecular weight of 44,000 daltons (77).
ViDera aminodvtes amrnodvteg.
In summary it seems that relatively little is known about the viperid hemorrhagic
toxins when compared to the crotalid family. Fox and colleagues (78) have isolated five
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 171
hemorrhagic toxins from the venom of Vipera ammodvtes ammodvtes using ion-
exchange, gel filtration, and hydrophobic interaction chromatographies. One of the
toxins is basic (VA-I) and the other four toxins are acidic (VA-2,3,4). All have
proteolytic activity on azocasein and this activity, as well as their hemorrhagic activity,
are inhibited by treatment with EDTA. Their apparent molecular weights, as
determined by SDS electrophoresis, are between 64,000 and 66,000 daltons. The toxins
cleave the oxidized B chain of insulin in a manner similar to the hemorrhagic toxins
from C. afros. The amino terminals of these toxins are currently being sequenced in
order to understand the relationships among the VA toxins and their homology with
other hemorrhagic toxins. In light of the many similarities between the two crotalid and
viperid families, we would anticipate that the venoms would share many toxic
components and that with further characterization strong similarities will be noted
between the hemorrhagic toxins of the viperid and crotalid snake venoms.
4.0 Detection. Patholoav. and Biochemical Mechanism of Hemorrhage Production.
Bleeding is a common phenomenon in the victims of Crotalidae envenomation
(79). This is likely the result of one or more factors in the venom. The main factors
responsible are the hemorrhagic toxins and the other secondary factors; those being the
venom components which produce a non-clotting blood as well as the enzymes which
release kinins from kininogen (80,81).
The smaller blood vessels appear to be particularly susceptible to the affects of
the venom, the result being an alteration of the vessel permeability. The consequences
of this altered capillary permeability include the escape of plasma and red blood cells
into the surrounding tissues giving rise to ecchymosis, blistering, cyanosis and edema
(81).
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172
4.1 Observations of Venom Induced Hemorrhagg
BJARNASON AND FOX
One of the first groups of investigators to microscopically examine the effects of
a venom hemorrhagic toxin was McKay et al. They compared the effects of a
partially purified hemorrhagic toxin, (hemorrhagin) from the venom of Viperu palastime
with phospholipase isolated from the same venom. The previously characterized
hemorrhagic toxin had an estimated molecular weight of 44,000 daltons. It exhibited
gelatinase activity, which was inhibited by DFP as well as soybean trypsin inhibitor but
did not affect the hemorrhagic activity (77). The hemorrhagic toxin was injected
subcutaneously into the backs of rabbits and samples for microscopic examination were
taken over various time periods. Initially, they noted swelling of the cytoplasm of the
endothelial cells followed by damage of the capillaries and endothelium and leakage of
plasma into the connective tissue around the capillary. There was some development of
pseudopods of endothelial cytoplasm while the disruption of basement membrane was
occurring. The capillaries eventually became occluded with platelets and at 8 minutes
following injection the cytoplasmic organelles of many of the endothelial cells were
absent and the extravasation of red blood cells was maximal. Throughout the course of
observation, no disruption of the intercellular junctions was noted. Intravenous injection
of the hemorrhagic toxin gave rise to visible hemorrhage in the lungs and gastrointestinal
tract as well as damage in the heart, liver, lungs, and intestinal tract as seen under the
light microscope. Subcutaneous injection of venom phospholipase did not appear to
cause hemorrhage although interstitial edema was observed as well as perturbation of the
red blood cell membrane.
(82).
From the results of these experiments, the authors concluded that the
hemorrhagic toxin acted to disrupt the basement membrane and cause endothelial cell
lysis allowing red blood cells and plasma to escape from the capillaries at those sites.
The hemorrhagic toxin also appeared to cause platelet aggregation at the site of damage.
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 173
However, since the state of purity of this toxin is, in the opinion of these authors as
well as the investigators who originally isolated the fraction (77), in question,
interpretation of the data from these experiments must be made with caution.
Ohsaka e t al. (70) examined the effects of several hemorrhagic toxins isolated
from the venom of Trinieresurus flavoviridis on glomerular basement membranes. The
three hemorrhagic toxins which they studied were called hemorrhagic principles HRI ,
HR2a, and HR2b (65). At the time of these experiments, HRI was not pure and has
since been further fractionated into two components H R l a and H R l b (71). When H R I ,
HR2a, and HR2b hemorrhagic toxins were incubated with glomerular basement
membrane preparations, both soluble peptides/proteins and carbohydrates were released.
They also studied the size of the protein and carbohydrate containing fragments released
and observed that HR2a and HR2b released similar size fragments which were different
in size from the fragments released by HRI . From this, they concluded that they HR2a
and HR2b hemorrhagic toxins have a different affect on basement membrane than HRI.
They attributed the hemorrhagic effect of the toxins to be due to enzymatic disruption
of the basement membrane of capillary walls.
The results of this study, with regard to the action of the HRI toxin, must also
be considered with the caveat that this toxic fraction was not homogeneous. Ohsaka et
al. (69) stated at the time that HRI could be resolved into at least four components by
polyacrylamide gel electrophoresis. Nevertheless, the data clearly demonstrated that all
of the toxins tested were capable of enzymatically disrupting basement membrane.
In 1974, Tsuchiya et al. (83) reported on a cinematographic and electron
microscopic study of the affect of the hemorrhagic toxin HRI (from Trinicresurus
flavoviridis) on capillaries from rat mesentery tissue. The studies revealed that the toxin
caused a partial disappearance of basement membrane followed by red blood cells
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174 BJARNASON AND FOX
"oozing" out through inter-endothelial gaps. The endothelial cells and pericytes did
appear to undergo some functional and organic changes, however, the cells were not
lysed. The destruction of the basement membrane by the toxin as observed
microscopically is in agreement with the results of McKay ef al. (82) and Ohsaka ef al.
(70). However, in the case of the Vipera palnesfiriae hemorrhagic toxin, the red blood
cells (RBC) were noted passing from the capillaries into the surrounding tissue through
an endothelial cell space. With the H R l toxin, red blood cells were observed escaping
through widened gap junctions. Although it is quite possible that the biochemical
mechanism of these hemorrhagic toxins may indeed be different which, in turn, could
result in the observed differences in the pathway of RBC escape from the capillaries, it
must again be mentioned that the HRI preparation used in these experiments was not
homogenous.
Ownby ef al. ( 8 4 ) in 1974 reported on the effects of crude Crofaltu afrox venom
when injected in mouse muscle tissue. From their electron microscopic observations
they noted that the crude venom initially caused the endoplasmic reticulum and the
perinuclear space of the endothelial cells to dilate along with a swelling of the
cytoplasm. The endothelial cells eventually began to bleb into the capillary lumen
followed by rupture of the endothelial cell membrane and the extravasation of RBCs
into the surrounding tissue. Disruption of the basement membrane of the capillary was
also noted. This sequence of events preceding hemorrhage is similar to that
demonstrated by the Vipera palasfirrue hemorrhagic toxin isolated by Grotto et al. (77).
The pathological manifestations of the Crotnlus afrox crude venom are obviously
difficult to attribute to any one particular venom component in light of the wide variety
of toxic components present in this venom. However, this study clearly demonstrated a t
the microscopic level the hemorrhagic capability of the crude venom.
In 1978, Ownby et al. el al. ( 8 5 ) investigated the effects of three different
hemorrhagic toxins isolated from the venom of Crofalus afrox on mouse muscle tissue.
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 175
The purified toxins, Ht-a, Ht-b, and Ht-e were each injected into the muscle tissue and
then the mice were biopsied at various times for microscopic observation of hemorrhage.
Hemorrhagic toxins Ht-a and Ht-e both produced hemorrhage within 5 minutes of
injection whereas Ht-b induced hemorrhage appeared after much longer incubation
times ( 5 hours). Tissue necrosis was also noted in the Ht-b injected samples. As was
seen for all three toxins, the endothelial cells became thin and formed vesicles prior to
rupturing. The gap junctions of the endothelium did not appear to be disturbed. The
basement membrane around the capillaries as well as the surrounding connective tissue
was also disrupted. The RBCs escaped from the capillary through the lysed endothelial
cells and basement membrane into the surrounding tissue. These results are in fact
identical to those found by the same authors for the crude venom.
The literature on the microscopic examination of the hemorrhagic effects of
either crude venom or purified hemorrhagic toxins leaves one somewhat unclear as to
whether there is only one or several mechanisms in which the hemorrhagic toxins can
induce hemorrhage from capillaries. In the case of the toxins and crude venom of
Trinieresurus flavoviridis, it seems that the capillary basement membrane is disrupted,
probably in an enzymatic fashion, followed by a widening of the endothelial gap
junctions allowing for the escape of RBCs into the surrounding tissue. The studies on
Crofnlus afrox crude venom and purified hemorrhagic toxins indicated that basement
membrane is also disrupted by the venom and toxins, not unlike that observed with T.
flavoviridis: however, the major difference between the two venoms or their
hemorrhagic toxins is that the C . alrox venom and toxins also cause rupture of the
endothelial cells with RBCs then passing through the sites of lysis. It is difficult to
explain these differences. Perhaps the two venoms do indeed have different mechanisms
of hemorrhage or perhaps it is the results of different experimental methodologies. The
data currently available in the literature do not allow definitive conclusions to be made
at this time.
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i 76
4.2 Evaluation of Hemorrhagic Activity
BJARNASON AND FOX
Ever since it became obvious that one of the major symptoms of Crotalidae
envenomation was local hemorrhage, several methods have been developed for the
qualitative and quantitative evaluation of venom and hemorrhagic toxin induced
hemorrhage.
One of the earliest methods employed as an intradermal injection of the venom
into the shaved skin of rabbits or guinea pigs followed by observation of hemorrhagic
necrosis on the outer surface of the skin (86,87). Several limitations of this method were
soon realized in that somewhat larger doses of the material were necessary to clearly
visualize the effect and there also appeared to be a wide variability in the size and shape
of the necrotic lesion as seen from the outer side of the skin. This method is generally
not in use today.
In 1960, Kondo ef al. (88) reported on a method which allowed for a statistical
quantitation of hemorrhage induced by snake venoms and toxins. In their procedure, the
backs of rabbits were dipiliated and injections were made intradermally at evenly spaced
intervals across the back. One advantage of this method and the experimental animal
used is that as many as 60 to 70 injections can be made on one rabbit. Following
twenty-four hours, after which time the hemorrhagic response is maximal, the rabbit is
killed and skinned. The skin is then placed between two glass plates and the
hemorrhagic spots visualized from the visceral rather than the outer side. They
recommended measuring the cross-diameters of the hemorrhages and using the mean
value for quantitation. From these measurements a minimum hemorrhagic dose can be
estimated (89) which is the amount of protein in the injection solution which caused a
hemorrhagic spot with a defined area.
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS I77
Many modifications of this technique have been made since the original report.
These include varying the incubation time following the injection, the experimental
animal, the route of toxin administration and the method of estimating hemorrhagic size
(35,90). Although all of these modification seem to be useful in their own right, they
may complicate matters when minimum hemorrhagic doses for different venoms and
toxins are to be compared since the modifications listed above may affect the minimum
hemorrhagic dose.
Rather than visually estimating the hemorrhagic response, Miles and Wilhelm (91)
used mice which, prior to the intradermal injection of the toxin, had been intravenously
infused with Evans Blue dye. After a short incubation period, the animals were killed
and the dye from the blue "hemorrhagic spots" was extracted and spectrophotonietrically
quantitated. Although the method superficially appears adequate, the actual procedure is
rather awkward and gives a measure of capillary permeability to serum and possibly
erythrocytes rather than a direct measurement of the hemorrhagic response (appearance
of erythrocytes).
The method of Ownby Ual. (90) circumvented the above problem of a lack of
differentiation between capillary permeability and extravasation of erythrocytes. In their
assay they used all intramuscular injection of venom in the thighs of mice. The mice
were killed three hours after injection and a portion of the muscle removed and assayed
for hemoglobin content. The principle being that the hemoglobin present was a direct
result of the escape of erythrocytes into the tissue at the site of capillary damage. Their
data correlated well with their modification of the method of Kondo el al. (88).
However, this hemoglobin assay technique appears somewhat troublesome to perform as
compared to the skin response method. Furthermore, i t would seem that the amount of
hemoglobin detected in the tissue would be very dependent upon the precise site from
which the tissue sample was taken in relationship to the injection site. The authors did
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I 78 BJARNASON AND FOX
not address this possible complication. They did state that although the dose-response
curves for the assay are rather good from the statistical standpoint, the method is
somewhat less sensitive than their modification of the method of Kondo el al.
6 1 In lieu of a dye, Just el al. (92) administered to rabbits Cr-labeled erythrocytes
and I-labeled albumin intravenously followed by intradermal injections of the
hemorrhagic compounds. After 24 hours, the animals were killed and the skin at the
injection site assayed for radioactivity. This method is advantageous when one wishes to
differentiate the effects of various agents as to their ability to cause varying degrees of
capillary permeability.
126
Another method for the assay of hemorrhage is via the topical application of
venom/toxins to surface of dog lungs (93). In this procedure, the chest cavity of a dog
is opened allowing for direct application on the exposed lungs of filter disks soaked in
the venom/toxin solution. After an arbitrary length of time (usually 3 minutes), the
disks are removed and the time of the appearance of hemorrhage is recorded. This
method allows for actual determination of the onset of hemorrhage as well as the ability
to observe differences in the appearance of the hemorrhagic spot.
There are several difficulties associated with this technique including the
somewhat high concentration of toxin solution necessary, the surgical requirements for
the assay, and the variations that are observed with different dogs. These problems
make the assay somewhat problematic to perform and not particularly useful for
quantitation and standardization.
Of all the methods discussed above, the method of Kondo ef al. is, in some form
of modification which suits the individual investigator’s particular needs and resources,
most commonly used. This is likely due to the high sensitivity of the assay as well as
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 179
the high correlation coefficient of t h e dose-response curve and the relative ease of
performance of the assay.
4.3 Assavs of Hemorrhaaic Toxins for Proteolvtic Activity
As mentioned earlier, the venoms of both Crotalidae and Viperidae are notable
for their proteolytic activities, particularly when compared to the venoms of the
Elapidae and Hydrophidae (94.95). Since hemorrhage is one of the most obvious
symptoms of crotalid and viperid envenomation, the outcome of hemorrhage has long
been thought to be associated with proteolytic activities in the venom. Some of the
specific proteolytic activities which have been identified in crotalid venoms include
esterolytic, collagenolytic, caseinolytic, fibrinolytic, fibrinogenolytic, thrombin-like, and
kallikrein-like (95-97).
The quest to couple the hemorrhagic manifestation with purified hemorrhagic
toxins and their proteolytic potencies requires the use of well established, sensitive
proteolytic activity assays. The typical proteolytic assay used in the early period of
hemorrhagic toxin research was the method developed by Kunitz using the milk protein
casein as the protein substrate (98) and detection of trichloroacetic acid soluble peptide
products after timed incubation.
In this assay, the peptidase is incubated with the casein and af ter a set reaction
time the mixture is subjected to acid precipitation with trichloroacetic acid. The
resultant digestion fragments which remained in solution were then quantitated and the
value used as a measure of the proteolytic action of the enzyme on the substrate.
Unfortunately, there are two important limitations of this technique as it affects the
hemorrhagic peptidases from snake venoms. In our experience, the hemorrhagic toxins
are rather specific with respect to their sites of action and substrate preferences and
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180 BJARNASON AND FOX
consequently few peptide bonds of casein may be cleaved. Furthermore, the bonds that
are cleaved may not give rise to soluble peptides, particularly if only a few bonds of
casein are cleaved.
The second limitation with the Kunitz method is that if one is monitoring the
soluble peptides with absorbance at 280nm, the sensitivity is somewhat reduced and
dependent upon the amino acid composition of the peptides released. For enzymes with
relatively weak activity on casein, TCA precipitation followed by 280 nm absorbance
monitoring may, in fact, be so insensitive that the enzyme many not appear to be active
on this substrate and lead to the conclusion that it is not proteolytic.
Several methods have since been used with success for the detection of the
proteolytic activity of hemorrhagic toxins. These can generally be divided into two
classes: protein substrates and peptide substrates. The commonly used protein substrates
which give a more qualitative assay of proteolysis include azocoll, azoalbumin, azocasein,
and hide powder azure. Various hemorrhagic toxins have been shown to be relatively
active on these substrates (48,64). The assays are simple to perform and sensitive due to
the release of a strongly absorbing dye upon proteolysis of the substrate. Unfortunately,
due to the nature of the binding of the dye to the substrate, and also to the insolubility
of some of these substrates, precise kinetic analyses of these digestions cannot be
performed.
Lin et al. (99) introduced a very sensitive assay for proteolytic enzymes which
used N,N-dimethylhemoglobin or N,N-dimethylcasein. They estimated an approximate
hundred-fold increase in the sensitivity of this assay over that of Kunitz. The
sensitivity increase is based on the use of trinitrobenzenesulfonic acid to detect the
appearance of new terminal amino groups. This assay was used with success in the
isolation and kinetic characterization of five hemorrhagic toxins f rom Qofalus afrox
venom (35,60).
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 181
Another interesting approach for the qualitative determination of proteolytic
activity of enzymes recently used with snake venoms is the fibrinogen gel method of
Lachs and Springhorn (100) as modified by Markland and Perdon (101). Essentially, the
assay involves the electrophoresis of venoms in fibrinogen-polyacrylamide gels followed
by an incubation period prior to staining. Electrophoresed proteins which are
proteolytically active against fibrinogen will digest the surrounding fibrinogen in the gel,
thereby eliminating the ability of coomassie to stain that area of the gel. This technique
is interesting in that specific inhibitors of the serine or metalloprotease enzyme family
can be used to distinguish those particular enzyme types within the gel. However,
according to Markland and Perdon, this method was not as sensitive as the proteolytic
assays of snake venoms which employ small chromogenic substrates. yet, the
chromogenic substrates used by Meier et al. (102) may not be suitable for the assay of
hemorrhagic proteases since most are substrates for serine proteases and not
metalloproteases.
The use of peptides and small chromogenic substrates has proven advantageous
with the hemorrhagic toxins in certain instances, particularly where substrate specificity
and kinetic data are required. The oxidized B chain of insulin has been used with many
of the hemorrhagic toxins for analysis of specificity of peptide bond hydrolysis. I f
properly applied, this protocol can yield kinetic data (60,59). The use of the oxidized B
chain of insulin has, likely due to traditional reasons, been the initial choice of peptide
substrate. To date, many of the hemorrhagic toxins have been assayed for proteolysis of
the B chain and useful comparisons of the cleavage sites with other hemorrhagic toxin
can be made (see section 3 in this review).
Other small peptides and chromogenic substrates have been tested for use in
proteolytic assays of hemorrhagic toxins (35,48,59,). In general, due to the apparent
high degree of substrate specificity of the hemorrhagic toxins and the length
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182 BJARNASON AND FOX
requirements that they demonstrate for their substrates, most hemorrhagic toxins have
not shown significant activity on these small substrates. These requirements have
recently been determined by the authors of this review with C. atrox hemorrhagic toxins
c and d (60). These toxins were demonstrated to have both specific size and substrate
specificity for hydrolysis of small peptides.
One small substrate which has been useful in certain applications is the
fluorogenic peptide 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzyl amide upon which
five of the hemorrhagic toxins from C. alrox are proteolytically active (see section 3 and
ref. 64). All of these hemorrhagic toxins cleave the -Gly-Leu- bond to yield a change
in fluorescence in the reaction mixture. This peptide has proven to be extremely useful
to these authors for the routine quantitative analysis of the proteolytic activity of the C.
atmx hemorrhagic toxins as well as in the assays of inhibitors to these enzymes.
As more specificity data is gained on the various hemorrhagic toxins development
of better substrates, perhaps similar to the one mentioned above, can be initiated.
Obviously, to obtain kinetic data similar to that for the hemorrhagic toxins c and d from
C. atrox, a wide variety of specifically designed peptide substrates of varying lengths
and sequences are necessary in order to develop the optimal peptide substrate for a
particular hemorrhagic toxin (64).
4.4 Biochemical Mechanisms of Hemorrhaee
As mentioned in section 4.1, a primary site of action of the various hemorrhagic
toxins appears to be at the basement membrane surrounding capillaries. Capillary
basement membrane (basal lamina) is a thin layer of extracellular matrix which separates
the capillary endothelial cells from surrounding tissues. Some of the proteins which
comprise the basal lamina include laminin, heparin sulfate proteoglycans, fibronectin,
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 183
type IV collagen, nidogen/entactin, small quantities of type V collagen and other
proteins as yet uncharacterized (103,104). In order for a hemorrhagic toxin to disrupt
such a matrix, one or more of these membrane proteins must be cleaved by the toxin.
Bjarnason and colleagues have examined the ability of five hemorrhagic toxins
isolated from C . alrox venom (Ht-a,b,c,d, and e) to proteolytically cleave various
proteins present in basement membranes (105). All five of the C. atros hemorrhagic
proteinases that we examined were capable of releasing soluble peptides from a mouse
basement membrane preparation. Hemorrhagic toxins a and e were demonstrated to be
the most active in production of soluble peptides from the basement membrane
preparation. These two toxins are also the most hemorrhagic compared to the other
three toxins. This suggests a correlation between the hemorrhagic toxins’ minimum
hemorrhagic dose and the ability to proteolytic disrupt basement membranes.
These basement membrane preparations were subjected to analysis by SDS-PAGE
following digestion by the C . alrox hemorrhagic toxins so that the various components of
the preparation which were cleaved could be identified. All of the toxins appeared to
digest the laminin A chain as well as nidogen. Fibronectin did not appear to be
digested. This result was confirmed by the incubation of fibronectin alone with the
hemorrhagic toxins. Type IV procollagen when incubated with hemorrhagic toxins Ht-a
and e was digested to discrete fragments.
Nidogen, type IV procollagen, and laminin are all interrelated in their roles to
maintain the integrity of the basement membrane (106,107). Disruption of these matrix
components could then expose the capillary endothelial cells to the surrounding
environment. Consequently, leakage of capillary contents into the surrounding tissues
could result. These hypotheses are currently being examined in our laboratory.
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184 BJARNASON AND FOX
5.0 Inhibitors of Hemorrhnaic Proteinases
As discussed in Section 3, the venoms of the Crotalidae and Viperidae families
demonstrate profound proteolytic activities (108,109), many of which have been
associated with certain pathologies of envenomation (1 10-1 11). Most organisms have a
natural defense system of proteinase inhibitors poised for the control of endogenous as
well as exogenous proteinases (112,113). In this section we will discuss the action of
certain of the hemorrhagic toxins on the endogenous proteinase inhibitor systems the
factors responsible for the resistance of certain organisms against venom induced
hemorrhage and the development of synthetic inhibitors against the hemorrhagic toxins
of crotalid venoms.
5.1 Naturallv Occurring Inhibitors
Approximately 10% of the protein content of human plasma is comprised of
inhibitors of proteinases ( 1 14). The prevalent plasma inhibitor is a2-macroglobulin.
This inhibitor is a large tetrameric protein with a rather broad specificity towards
various classes of proteinases (115). Another important class of inhibitors found in the
plasma is the serpins (serine proteinase inhibitors) (116). As discussed above, the
hemorrhagic proteinases are metalloproteinases, however, the venoms of most crotalid
and viperid snakes are also rich in serine proteinases, many of which may give rise to
the particular pathologies associated with envenomation.
The potency of cr2-macroglobulin as an effective inhibitor of the hemorrhagic
metalloproteinases of snake venoms is somewhat uncertain. Werb et AI. (1 17) reported
that a2-macroglobulin was capable of inhibiting all of the metalloproteinase activity in
the venom of C. nfrox. Kress and Kurecki (118) reported that proteinase I1 from C.
adnmmifeus forms a complex with human a2-macroglobulin in an approximately 1.7/ 1
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 185
ratio of enzyme to inhibitor. Interestingly, in the presence of a high molecular weight
proteinase from the venom of the same snake, the enzyme was released from the
inhibitor. They also reported that the enzyme-inhibitor complex was unstable in serum
and that the released enzyme was capable of catalytically inactivating other serum
proteinase inhibitors. Our investigations on the interaction of the hemorrhagic
proteinases from C. alros venom with a2-macroglobulin reveal similar results (1 19). The
ratio of enzyme to inhibitor in the complex was in the range of 1.3 - 1.6 to 1. All of
the C. afrox toxins, upon incubation with a2-macroglobulin, were able to cleave the
inhibitor such that upon SDS PAGE a new 90kd band appeared, suggesting the digestion
of the 180kd subunit. This form of enzymatic cleavage of the a2-macroglobulin
inhibitor has been noted in the interaction of several other proteinases with a2-
macroglobulin. This cleavage by the C. atrox toxins inactivated the inhibitor: however
the data were not clear as to whether the inhibitor had covalently entrapped the enzyme
upon the clipping of a 180kd subunit.
In summary, the role of a2-macroglobulin as an effective ir t vivo inhibitor of
venom metalloproteinases a t the moment seems inconclusive. Further studies need to be
conducted in order to clarify the interaction of the inhibitor with the various venom
metalloproteinases and determine the efficacy of the inhibitor against this class of venom
proteinases.
Another family of inhibitors of proteinases found in human plasma is called the
Currently, this family includes a l - serpins, for serine proteinase inhibitors (1 16).
antiproteinase, antithrombin, a, -antichymotrypsin, mouse contrapsin, and C1-inhibitor.
In light of the fact that certain venom metalloproteases could escape inhibition
by endogenous plasma a2-macroglobulin, Kress and Paroski searched for potential
plasma substrates which the unchecked metalloproteinases could attack (120). They
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BJARNASON AND FOX 186
reported that snake venom from the crotalid, viperid, and colubid families were capable
of inactivating human al -antiproteinase, an important endogenous inhibitor of elastase.
This inactivating property of the venom was experimentally attributed to the
metalloproteinases present in the venom. These studies were continued with two
purified metalloproteinases; proteinase I1 from C. adaniarzfeus venom and a-proteinase
from C . afrox venom. Proteinase I1 was demonstrated to inactivate, by specific cleavage
of the inhibitors, cxl-antiproteinase, C1-inhibitor, and antithrombin 111 (121-124). C.
utrox cx-proteinase was shown to cleave al-antichymotrypsin (124). These studies
demonstrated that the metalloproteinases in crotalid venom could inactivate the
endogenous enzyme inhibitor defense system in plasma resulting in a potential situation
in which other serine proteinases from the venom or endogenous proteinases could
escape inhibition in the plasma.
This situation is potentially devastating in that certain of these venom serine
proteinases have been implicated in some of the pathologies associated with crotalid
envenomation, such as disruption of the blood coagulation system and kinin production
from precursors (125,126). Thus, in addition to the metalloproteases’ ability to give rise
to hemorrhage, they can potentially serve to decrease the effectiveness of the victim’s
endogenous protease-inhibitor system.
It has been known for many years that certain snakes and mammals are resistant
to the toxic effects resulting from envenomation by crotalid snakes (127-131). Of
particular relevance to this review are the factors present in the sera of specific snakes
and animals which are antihemorrhagic. The first report of such a factor was by
Omori-Satoh et al. (132) in 1972 of an antihemorrhagic factor from the serum of
Trinieresurus flavoviridis. The factor, which has a molecular weight of 70,000 daltons
was demonstrated to inhibit the hemorrhagic activity of the toxins HRI and HR2 (HR2a
and HR2b) from the same snake. No precipitin line was observed in
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 187
immunoelectrophoresis experiments between the factor and H R l o r the crude T.
/lavoviridis venom. This was interpreted by the authors as evidence that the factor was
not an immunoglobulin. The factor was also able to inhibit the hemorrhagic activity of
other snake venoms of the genera Agkisirodon, Bothrops, Crotalus, Bitis, and Vipera,
although to varying extents.
Omori-Satoh continued with the characterization of the antihemorrhagic factor
with regard to its ability to act as a proteinase inhibitor (133). The antihemorrhagic
factor was capable of forming a 1:l molar complex with H2-proteinases from the same
venom. Heat inactivated antihemorrhagic factor did not bind to H2-proteinase which
suggested to the author that the inhibition mechanism was related to the proteolytic
activity of the proteinases. When the H2-proteinase/antihemorrhagic factor complex was
electrophoresed in a reducing SDS PAGE system no evidence was observed to suggest
that nicking or formation of an acyl covalent complex had occurred. This is dissimilar
to the mechanism of inhibition of the serpin class of proteinases inhibitors. However,
with regard to the possibility of nicking, the author did suggest that had a cleavage
occurred near a terminal of the inhibitor the difference of molecular weights between
the control inhibitor and the complexed inhibitor perhaps was undetectable with their
system.
In 1978 Ovadia (134) reported on the purification and characterization of an
antihemorrhagic factor from the serum of Vipera palaestiriae. This factor has a
molecular weight of approximately 80,000 daltons. The antihemorrhagic activity of the
factor was demonstrated against the crude venom of V . palaestiriae as well as Echis
colorata and Cerasies cerasies. Similar to the T . Jlavoviridis factor, the V . palaestiriae
factor did not form a precipitin line with the crude venom, leading the authors to
speculate that the factor was not an immunoglobulin but perhaps an albumin or a-
globulin.
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188 BJARNASON AND FOX
In 1978 Perez and co-workers demonstrated the resistance of sera from certain
mammals against hemorrhagic snake venoms (135). This report was followed in 1979 by
Perez et 01. (139) on the resistance of hispid cotton rat (Sigmodon hispidus), gray
woodrat (Neotonza micropus), and opossum (Didelphis virgirzinrza) to C. atrox crude
venom. The antihemorrhage activity was shown to be associated with the sera of the
animals with the opossum serum having the highest antihemorrhagic activity.
An antihemorrhagic factor was later isolated from the serum of opossum (137).
The factor has a molecular weight of 68,000 daltons. No precipitin line was formed
when incubated with crude C. ufrox venom. Based upon the isoelectric point (4.1) and
the molecular weight the authors suggested that the factor is similar to albumin. At that
time the authors could not suggest a mechanism for inhibition other than that the factor
was not proteolytic and did not form an antibody/antigen type complex.
An antihemorrhagic factor was isolated from the serum of the hispid cotton rat
which neutralized the hemorrhagic activity of crude C. alrox venom (138). The
molecular weight of the factor was determined to be approximately 90,000 daltons with
an isoelectric point of 5.4. No precipitin line was observed during immunodiffusion
with the crude venom and no proteolytic activity was observed for the factor. This
factor, judging from its biochemical properties, is more similar to the antihemorrhagic
factors isolated from T. flulJolJiridis and V . palasf irm sera than the factor from opossum.
The authors suggested the factor may be similar to a a-globulin.
Garcia and Perez (139) reported on the purification and characterization of an
antihemorrhagic factor from the serum of the woodrat. This factor was capable of
neutralizing the hemorrhagic activity of C. u1ro.x venom, and like the other factors
discussed above, did not form a precipitin line with the venom. The factor has a
molecular weight of 54,000 daltons and an isoelectric point of 4.1. This factor appears
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 189
similar in biochemical properties to the 56,000 dalton factor isolated from the serum of
V . palaestitiae (1 34).
All of the above work, although interesting, suffers from the lack of critical
biochemical characterization data on the various factors. None of the investigations
reported amino acid compositions, amino terminal sequence data, or peptide mapping
data which could be used to determine biochemical similarities or differences among
those factors. Furthermore, none of those investigations attempted to determine the
mechanism of inhibition other than to demonstrate that the inhibitors were not
proteolytic or that they did not function as multivalent binding proteins such as
antibodies. The reports discussed above also determined the antihemorrhagic activity of
the factor against crude venom rather than purified hemorrhagic toxins. Finally, the
above studies did not assay for the inhibition of proteolytic activity by the inhibitors.
The notable exception to this is the factor from T. flavoviridis venom which was shown
to neutralize the hemorrhagic toxins H R l and HR2 (133).
In 1985, Kress and Catanese (140) reported on the isolation of an proteinase
inhibitor from the serum of the opossum. This protein was termed oprin and was
demonstrated to inhibit the proteolytic activity of C. atrox metalloproteinases a, p, 7.
The inhibitor reacted stoichiometrically with the proteinases to form an inactive enzyme-
inhibitor complex. It appears that the inhibitor is somewhat specific in that it did
inhibit both the proteolytic and hemorrhagic activities of C. atrox hemorrhagic toxin b
but was not as effective against hemorrhagic toxin a. Furthermore, the inhibitor did not
inhibit any of the serine proteinases assayed as well as the non-venom metalloproteinases
thermolysin, clostridiopeptidase A, dispase, and Pseudon2onas aerugitiosa protease.
Another interesting property of oprin, in contrast to other members of the serpin class
of proteinase inhibitors, was its resistant to inactivation by C. atrox a-proteinase and
only minor inactivation by C. adanzatzfcus proteinase I1 (141).
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190
Kress has isolated another hemorrhagic toxin inhibitor named HTI from opossum
serum with somewhat different inhibitory properties (141). This inhibitor was shown to
inhibit the hemorrhagic activity of HT-a from C. afrox and proteinase H from C .
adanianfeus. Kress suggested that the mechanism of inactivation is via an inhibitory-
enzyme complex formation. The biochemical characterization of this inhibitor has not
yet been reported.
Recent investigations include the identification of three macroglobulin proteinase
inhibitors in the plasma of the hedgehog (Erimceus europaeus) which gave resistance to
the hemorrhagic venom from the European viper (Vipera berus) (142). The
macroglobulins were identified as a2-macroglobulin, a,-p-macroglobulin, and p-
macroglobulin. This is the first report of macroglobulins being responsible for the
neutralization of hemorrhagic venom. It is important to note with regard to our earlier
sections that these macroglobulins are well known inhibitors of all four classes of
proteinases.
From the above summary of reports on antihemorrhagic factors found in the
serum of various snakes and mammals it appears that perhaps two classes of proteolytic
inhibitors are involved. The lower molecular weight class (54.000-90,000 daltons) are
possibly similar to the inhibitor oprin from the opossum. The mechanism of inhibition
has not been fully elucidated for this class of inhibitors, however the preliminary work
of Kress and Catanese indicates that an inhibitor-enzyme complex is involved. Whether
the mechanism is in fact identical to that of the serpins remains to be seen. As for the
large molecular weight inhibitors isolated from the plasma of the hedgehog, the data is
clear that these proteins are macroglobulins and it is quite likely that the mechanism of
inhibition is identical to that observed for az-macroglobulin. Obviously, much more
information is needed in order to understand the overall mechanism of certain
organisms’ resistance to crotalid and viperid venoms in general, and their hemorrhagic
proteinases in particular.
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 191
5.2 3
Small organic compounds which have been shown to inhibit various hemorrhagic
proteinases include: EDTA, EGTA, cysteine, and 1,lO-phenathroline, among others.
Unfortunately, these inhibitors are rather broadly specific for metalloproteinases and
have been demonstrated to inhibit other metalloproteinases in addition to hemorrhagic
proteinases. A significant amount of data is available on synthetic inhibitors of
metalloproteinases. The most effective inhibitors have functional groups which can
complex to the active site zinc. This zinc ligand is coupled to a peptide which is
specific for the substrate binding sites of the enzyme (143-145). Some of the zinc
liganding groups that have been utilized include carboxyl, hydroxamic acids, phosphoryl,
and thiols. Therefore, our approach to the design and synthesis of inhibitors of
hemorrhagic proteinases has been to first elucidate the substrate binding specificity of
the proteinase. In our work with C. nfrox hemorrhagic toxins c and d we used various
peptide and small protein substrates to determine peptide bond cleavage specificity (64).
This data allowed us to design amino acids and peptides which could bind to the active
site of the proteinase. We then coupled the appropriate peptide or amino acid to a
functional group such as hydroxamate or carboxymethyl which could ligand to the active
site zinc.
The hydroxamic acid amino acid inhibitors we developed proved to be fairly
good inhibitors of the hemorrhagic proteinases Ht-c and Ht-d, albeit with a rather low
degree of specificity (see Table 16). This is not unexpected since only a single amino
acid residue bound to the hydroxamate moiety would not be expected to be highly
specific since we had shown that these proteinases have extended substrate binding sites
(64). The L-Leu-NHOH appears to be a slightly better inhibitor than the other
hydroxamic acids. This is probably due to contributions to binding by the L-Leu
residue and therefore gives rise to a certain degree of specificity. The primary
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192 BJARNASON AND FOX
TABLE 16
Synthetic Inhibitors of Ht-c,d
Inhibitor K i x lo6
9 10 1 1
L-Ile-NHOH L-Leu-NHOH L-Val-NHOH L-Phe-NHOH
Z-L-Leu-Gly-NHOH 2-L-Val-Gly-NHOH
H02C-CW2-L-Leu-L-Leu
MeO-S~c-Gly -Leu-Phe-CH~Cl L-Leu-CH2CI NH2-Ah-Leu-Tyr-Leu
2-L-Val-L- Ah-NHOH
M 920 150 480 430
60 240 70 20
430 15 25
Substrate used was Abz-Ala-Gly-Leu-Ah-Nba
Reference for inhibitors 1-4 and 9-1 I , 64. Inhibitors 5 - 8 , unpublished data from authors' laboratory.
mechanism of inhibition is probably mediated by the binding of the hydroxamate
function to the active site zinc with some degree of specificity being conferred on the
inhibitors by the amino acid side-chain. This mode of action has previously been
suggested for this type of inhibitor with thermolysin. Nishino and Powers referred to
the Z-Gly-Gly-NHOH, having a K i value of 94 x 10-6M, as a non-specific hydroxamic
acid inhibitor of thermolysin (146). The L-lle-NHOH inhibitor with Ht-c similarly had
a K i value of 92 x 10-6M, also indicative of a lack of specificity contributed by the L-
Ile moiety of the inhibitor. This is in agreement with our observation that the L-Ile
containing acetylated peptide 13 was neither a substrate nor an inhibitor of the
hemorrhagic toxins (see Table 12).
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HEMORRHAGIC T O X I N S FROM S N A K E VENOMS 193
Dipeptide and tripeptide hydroxamic acid inhibitors have been shown to be more
effective against thermolysin than the amino acid hydroxaniates, with K i values in the
micromolar range (146). This suggested to us that perhaps we could synthesize more
potent inhibitors against the hemorrhagic proteinases by taking advantage of the
extended substrate binding site of the proteinases. We have recently designed and
synthesized three dipeptide hydroxnmate inhibitors which are effective inhibitors against
hemorrhagic toxins c and d. The inhibitor 2-L-Val-Gly-NHOH gave a K i value of 2.4
x 10-6M against Ht-d (see Table 16). 2-L-Val-L-Ala-NHOH and 2-L-Leu-Gly-
NHOH both gave K i values in the IOW7M range. All of these dipeptide inhibitors were
significantly more potent than the amino acid hydroxamate inhibitors.
We have also synthesized a peptide inhibitor taking advantage of the proteinase
specificity and another zinc liganding functional group, the carboxymethyl group. This
class of inhibitor has proven effective with certain other zinc metalloproteinases (144).
The inhibitor we synthesized, H02C-CH2-L-Leu-L-Leu, also gave a K i value with Ht-d
in the 10-7M range. Our laboratory is currently involved in the synthesis of additional
peptide analog inhibitors using different peptide sequences in conjunction with zinc
liganding functional groups. From work on the development of synthetic inhibitors
against collagenases (145) it is apparent that short peptides with zinc complexing moieties
do not serve as particularly good inhibitors. This is perhaps due to the more stringent
substrate requirements that this class of enzymes demonstrate. From our studies, it
appears that the venom hemorrhagic toxins are probably similar in this regard to the
collagenases. As more data is gained on the endogenous substrate(s) of the hemorrhagic
proteinases and the interaction of both synthetic and natural inhibitors with the
proteinases, more potent inhibitors will most likely be developed.
6.0 Summary
In this review we have discussed the results of biochemical studies on some 42
hemorrhagic toxins from 13 species of snakes. These toxins have been under
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194 BJARNASON AND FOX
investigation for almost 30 years. The development of this field was rather slow during
the first half of this period due to the complexity of snake venoms and the lack of
powerful purification techniques. Uncertainties related to the concept of proteolysis and
the methods of detecting proteolytic activity has also caused considerable confusion in
the field. The past decade has witnessed rapid progress in our understanding of these
toxins, especially due to the detailed and extensive research on the hemorrhagic toxins
from Crotalus atrox venom.
The important questions to address in the study and comparison of the
hemorrhagic toxins from the various families and genera of snakes is the magnitude and
nature of the hemorrhagic and proteolytic activities of the toxins on the one hand, and
the chemical composition and protein structure of the toxins on the other. In spite of
the wealth of information on the various toxins, it is currently not feasible, in many
instances, for sensible comparisons to be made. Admittedly in some cases this is simply
because of the lack of appropriate data, but also sometimes data on similar phenomena
from different laboratories for various reasons are not readily comparable. Regardless of
these inherent difficulties, an attempt should be made to draw conclusions and form a
generalized concensus of the hemorrhagic toxins from the information available. This
should and will be refined, modified and corrected as more detailed information
becomes available.
As previously suggested (40) i t is possible and probably preferable to assign the
hemorrhagic toxins into categories or classes based on their sizes as expressed by the
published molecular weights. The importance of this could naturally be questioned but
as the data indicate, it does not appear to be a futile exercise. The categories thus
formed appear to bear upon the hemorrhagic activities of the toxins and could possibly
serve to identify classes and subclasses of the hemorrhagic toxins. Unfortunately,
sequence data on the toxins is not available in the scientific literature, if the short
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 195
segment of sequence from Ht-c and Ht-d from C. alrox venom is discounted (40).
Ultimately, the best method of classification will be based upon sequence comparisons.
We have previously suggested the existence of two classes of toxins depending
upon molecular weights: the large and small molecular weight toxins (40). In light of
additional data, we are now proposing the classification of the toxins into three classes
depending upon molecular weight. Class I, the small toxins, having molecular weights of
20 to 30 kD; class 11, the medium size toxins, with molecular weights of 30 to 60 kD,
and class 111, the large and most potent hemorrhagic toxins, having molecular weights of
60 to 100 kD.
Of the 42 hemorrhagic toxins reviewed in this chapter, 19 appear to belong to
class I, the low molecular weight toxins, 13 fall into class 11, the medium molecular
weight toxins, and the remaining 10 are placed into class 111, the high molecular weight
toxins (Table 17). It should be recognized and clearly stated that these assignments are
in some cases questionable for various reasons such as the lack of information and the
difficulty of comparing data from different laboratories.
Many of the class 111 toxins are very potent hemorrhagic agents according to
their published minimum hemorrhagic doses. Indeed, all of the most potent hemorrhagic
toxins fall into class 111. These toxins include HR-I f rom A . halys blomho//ii venom,
proteinase H from C . udanzanteus venom, hemorrhagic toxin a from C. atrox venom and
HR-IA and HR-2A from T. flavoridis venom. From their amino acid compositions,
among other data, it seems plausible to conclude that proteinase H and hemorrhagic
toxin a are homologous enzymes which define a subclass, IIIa. Also, HR-I from A .
halys boloniho//ii and HR-2A from T. flavoviridis could be members of this toxin
subclass (IIIa). However, more information, especially o n their amino acid sequences, is
needed to corroborate this classification. Subclassification of other members of class 111
is even more uncertain due to the lack of appropriate data.
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Tabl
e 17
Cla
ssifi
catio
n of H
emor
rhag
ic T
oxin
s Acc
ordi
ng to
Mol
ecul
ar W
cirh
ts
Cla
ss J
Cla
ss 11
C
lass
111
Toxi
n M
W(k
D)
MH
D(u
g)
Toxi
n M
W(k
D)
MH
D(u
g)
Toxi
n M
W(W
MH
D(%
)
Ac
1 (A
.acu
tus)
Ac
2 (A
.acu
tus)
Ac
5 (A
.acu
tus)
Aa
H I(
A.a
cutu
s)
Aa
H I
I(A
.acu
tus)
Aa
H II
I(A
.acu
tus)
FP
(A.a
cutu
s)
MPA
(Bm
ooje
ni)
Prot
eina
se I(
C.a
dam
ante
us)
Prot
cina
se II
(C.a
dam
ante
us)
Ht-b
(C
.atro
x)
Ht-
c (C
.atro
x)
Ht-
d (C
.atro
x)
Ht-
e (C
atro
x)
HR
-2a
(T.fl
avov
iridi
s)
HR
-2b
(T.fl
avov
iridi
s)
HR
1 (T
.gra
min
eus)
HR
-a(T
.muc
rosq
uam
atus
)
HR
-b(T
.muc
rosq
uam
atus
)
24.5
25
24
22
22
22
24
23
25
24
24
24
24
25.1
23
24
24
15
27
0.22
0.43
0.37
0.4 1.5
10
3.8
3 8 11
1
0.07
0.07
1.7
1.3
Ac
3 (A
.acu
tus)
Ac
4 (A
.acu
lus)
HF2
(B.ja
rara
ca)
BP
(Bia
rara
ca)
NH
F-a(
B.n
euw
icdi
)
NH
F-b(
B.n
cuw
iedi
)
HP
IV(C
.h.h
orrid
us)
HR
-l(V
.pal
aest
inac
)
HR
-2(V
.pal
aest
inae
)
HR
-3(V
.pal
aest
inae
)
Ht-l
(V.a
.am
mod
ytes
)
Ht-2
(V.a
.am
mod
ytes
)
Ht-S
(V.a
.am
mod
ytes
)
57
0.95
33
0.31
50
48
46
58
57
4
GO
0.2
60
0.2
60
0.4
60
60
60
HR
-I(A
.h.b
lom
hofii
) 85
0.0
3
HR
-II(
A.h
.blo
mho
lfii)
95
42
Prot
cina
se H
(C.a
dam
ante
us)
86
0.02
Ht-
a (C
.atro
x)
68
0.04
Ht-f
(Cat
rox)
64
0.5
HR
- lA
(T.fl
avov
iridi
s)
60
402
HR
-lB(T
.flav
oviri
dis)
60
0.01
Ht-
g (C
atro
x)
60
l.4
HR
2 (T
.gra
min
eus)
82
Mur
A(T
.muc
rosq
uam
atus
) 94
2.3
W
rd
?- z
c,
CZJ 0
x
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 197
Some of the most active low molecular weight toxins of class I, with similar
amino acid compositions and with isoelectric points in the weakly acidic range, appear to
define another subclass of homologous enzymes which we term Ia. The most obvious
members of this subclass seem to be hemorrhagic toxin e from C. ntrox venom, which
would be the standard for this subclass, and the apparent homologs from A . acutus
venom, Acl , Ac2, AaHI, and FP (fibrinolytic protease). Other potential members of this
subclass are Ac5 and AaI-lII also from A . acuius venom, HR,, from T. gramirzeus venom
and HR-a from T. niucro~quuniatu~ venom, although more data on these toxins is
required for these assignments to be confirmed.
The basicity of some of the toxins in class I suggests the need for a subclass for
these toxins (Ib). This subclass would contain toxins of relatively strong hemorrhagic
activity such as HR2a and HR2b from T. flavoviridis venom and also those of relatively
low activity such as Ht-b from C. atrox venom and HR-b from T. mucrosquatatus
venom as well as possibly AaHllI from A. acutus venom.
Some of the toxins in class I are so weakly hemorrhagic that they are barely
classifiable as hemorrhagic toxins. We have assigned these toxins to subclass Ic. Indeed,
some of these toxins have been given different biological activities. Based upon their
molecular weights, amino acid compositions, and low hemorrhagic activities, the toxins
Ht-c and Ht-d from C . ulrox venom define this subclass. Proteinase I and proteinase 11,
from C. adanwileus venom as well as moojeni protease A (MPA) from B. nzoojerii
venom are also likely members of this subclass. It is possible that the weak hemorrhagic
toxin from A . uculus venom, termed AaHIII, also belongs to this subclass although it is a
basic protein.
At this point, the only published amino acid sequences of hemorrhagic toxins are
of Ht-c and Ht-d from C. ulrox venom which suggested that they are isoenzymes (40).
There is also unpublished sequence homology data that indicate that Ht-e and Ht-a share
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198 BJARNASQN AND FOX
some homology with Ht-d (149). It is our opinion that toxins which belong to the same
subclass would have almost 100% sequence homology if they are from the same venom.
Members of a subclass isolated from venom of different species would probably
demonstrate sequence homologies between 80- 100%. Also, toxins belonging to different
subclasses could have considerable sequence homology, such as that observed between
Ht-d and Ht-e, with sequence homologies in the range of 20 to 70% depending upon the
species similarities and toxin relationships.
Of the 42 hemorrhagic toxins described in the review, proteolytic activities have
been found associated with all but one of them. Hemorrhagic toxin HR-3 from Vipera
palastiriae had no detectable gelatinase and caseinolytic activity, which does not, in our
opinion, constitute proof that HR-3 is not proteolytic. This is particularly noteworthy in
the hemorrhagic toxins, HR-2a and Hr-2b from T. flavoviridis and HR-I from A. halys
blomhoffi, which had previously been considered non-proteolytic but have recently been
shown to contain proteolytic activities (48).
All the hemorrhagic toxins examined have shown metal dependency when assayed
with metal chealators. Of the 42 hemorrhagic toxins, 12 have been analyzed for their
metal content and all found to contain zinc. Ten of the 12 toxins contained
approximately one mole of zinc per mole of toxin. Both hemorrhagic and proteolytic
activities of most of these toxins are dependent on presence of the zinc. Also, calcium
ions appear to stabilize the hemorrhagic toxins in aqueous solutions. Unfortunately, the
metal composition of the 30 remaining hemorrhagic toxins have not yet been determined.
Some of the hemorrhagic toxins have been demonstrated to hydrolyze basement
membrane preparations (59,106). Recent results from the authors’ laboratory have shown
that Ht-a,b,c,d, and e from C. atrox venom hydrolyze type IV procollagen, nidogen, and
laminin; all of which are basement membrane components (147). It is also noteworthy
that these hemorrhagic toxins do not cleave type I or type 111 native collagens but
rapidly digest gelatin.
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HEMORRHAGIC TOXINS FROM SNAKE VENOMS 199
It is therefore apparent that venom induced hemorrhage is primarily caused by
metal dependent, proteolytic activity of the hemorrhagic toxins, probably acting on
connective tissue and basement membrane components. The disruption of the intact
capillary basement membrane structure results in the escape of capillary contents.
The results of many of the investigators suggest this mechanism of direct
proteolytic action of the hemorrhagic toxins on tissue substrates. However, it is
conceivable that some toxins may induce hemorrhage by a different mechanism, such as
activation of endogenous hemorrhagic factors which could themselves cause hemorrhage.
No data supporting this model has yet been presented.
At this stage, it appears that the purification of additional toxins is not the most
important course of research for the understanding of toxin induced hemorrhage. Of
much greater significance to our understanding of the toxins and venoms and the
mechanism by which they produce hemorrhage would be to obtain a large body of
primary structural data on the toxins already isolated. Accompanying this would be
further research on the endogenous substrates of the toxins and how the proteolysis of
those substrates alters the macromolecular organization of the tissues. Finally, the
information gained from the hemorrhagic toxins must be considered in light of the
presence of thrombin-like, fibrinogenolytic, and endogenous proteinase inhibitor
inactivator activities that some of the venoms contain. All of these factors impinge upon
the total affect of the venom to produce the dramatic hemorrhage observed in many of
the crotalid envenomation cases.
REFERENCES
1. Friederich, C. and Tu, A.T., Role of metals irt snake veiloms for henrorrhage, esterase and proteolytic activities, Biochem. Pharmacol., 20:1549, 197 1.
MacKay, N., Ferguson, J.C., and McNicol, G.P., E//ects of three mamba ve?zoni~ on the haeniostatic niechaiiisnr, Br. J. Haematol., 15549, 1968.
2.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
BJARNASON AND FOX 200
3.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
MacKay, N., Ferguson, J.C., and McNicol, G.P., Effects of three cobra vetionis oti blood coagulatioti, platelet aggregatiori atid Fibritiolysis. J. Clin. Pathol., 22304. 1969.
MacFarlane, R.C., and Barnett, B., Heniosfalic possibilities of snake vetiont, Lancet, 2:985, 1934.
Boffa, M.C., Delori, P., and Soulier, J.P., Atiticoagularit factors from Viperidae vetionis: Plaielei phospholipid iiihibitors, Thromb. Diath. Haemorrh., 28509,
Bonilla, C.A., Defibritiatioti etizyme /rani tiniber rattlestiake (Crolnlirs horridus horridu.7) vetioni: A poietitial agent for flie therapeutic defibririatioti. I . Purificatioti atid properties, Throm. Res., 6151, 1975.
Markland, F.S., Damus, P.S., Davidson, T.M., and Shanley, J.D., Thronibiti etizynie front Cro/alus odoniotrteu.7, Lancet 1:1398, 1970.
Ouyang, C., and Teng, C.M., Purificatioti arid propertics of the ntilicoagulutit priticiple of A~/ii.T/~OdoIt ncit/rr.x vetioni, Biochim. Biophys. Acta, 278: 155, 1972.
Mebs, D., Atialysis of I ,cf /oder ' rn n i l t l l l ~ vc~rtoni, Herpetologica, 24:338, 1968.
Tu, A.T., Vetionis: Chemistry atid Molecular Biology, John \Viley & Sons, New York, 1977.
Dimari, S.J., Lembach, K.J., and Chatman, V.B., The cyloiositis of cobra veilonis: Isolatioti aiid partial characterizaiioti, Biochim. Biophys. Acta, 393:320, 1975.
Roy, A.C., Leciiliiri atid w o n t heniolysis, Nature, 155:696, 1955.
DeVries, A., Kirschmann, C., Klibansky, C., Condrea, E., and Gitter, S., Heniolytic actiori oJ itidirect lytic stiake vetiom & h, Toxicon, 1:19, 1962.
Bjarnason, J.B., Barrish, A., Direnzo, G.S., Campbell, R., and Fox, J.W., Kalli/creiti-li/ce ctizynies front Croin1ii.F n1ro.x vetioni, J. Biol. Cheni., 258:12566, 1983.
Ferreira, S.H., A brudpkitriti-potetitiatiti~~ factor (BPF) presetif iti the verioni of Bolhrons iarnmcn, Br. J . Pharmacol., 24163, 1965.
Feldberg, W., and Kellaway, C.H., Liberation of histaniirie from the perfused lurig by srialte iwionis, J. Physiol., 90:257, 1937.
Rothschild, A.M., Mechatiisnz of histaniirie release by atiinzal vetionzs, Mem. Inst. Butantan, 33:467, 1966.
Ownby, C.L., Bjarnason, J., and Tu, A.T., Heniorrhagic tositi front rattlestiake (Crotdri .~ ntrox) verioni: Paihogeriesis of hemorrhage itiduced by three purified toxitis, Am. J. Path., 93:201, 1978.
Stringer, J.M., Kainer, R.A., and Tu, A.T., Myotiecrosis ittduced by rattlesriake verioni: An electroti niicroscopic study, Am. J. Pathol., 67:127, 1972.
Stringer, J.M., Kainer, R.A., and Tu, A.T., Ultrastructural studies of niyoriecrosis iriduced by cobra iwiom iti mice, Toxicol. Appl. Pharmacol., 18:442, 1971.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
HEMORRHAGIC T O X I N S FROM S N A K E VENOMS 201
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Mohamed, A.H., Saleh, A.M., Ahnied, S., and El-Maghraby, M., Histopathological arid histochemical chairges in skeletal muscles a f ter Biris gnhoriica eiivenoniatioti, Toxicon, 13:165, 1975.
Harris, J.B., Johnson, M.A., and Karlsson, E., Pathological responses of rat skeletal niuscle to a sitrgle subcutarreous injection o f a toxiri isolated froni the venom o f the Australiau tiger snake. Notechis scutntus .sciiinius, Clin. Exp. Pharmacol. Physiol., 2383, 1975.
Ownby, C.L., Cameron, D., and Tu, A.T., Isolatioit o f niyoioxic coniporietit froni rattlesnake ( ~ r o t n l u s viridis viridis) venoni: Electron microscopic analysis of niuscle damage, Am. J. Path., 85:149, 1976.
Chang, C.C., and Lee, C.Y., Isolatiort o f rieurotoxitis froni the veiioni of Butionr14.s nirrltici~rctrrs arid their niodes o f iieuroniuscular blockitig actioti, Arch. Int. Pharmacodyn., 344241, 1963.
Halpert, J., Eaker, D., and Larlsson, E., The role o f phospholipase activity iti the actioti o f a presyuaptic rreurotoxiti froni the vetioni o f Notechis .sci~~oir~s .sciitntii.s (Australiari tiger sriake), FEBS Lett., 61:72, 1975.
Tchorbanov, B., and Aleksiev, B.J., A simple procedure for the isolatiotr o f viproxiii, a iieurotosiii with weak phospholipase activity f rom the vetiont of the Bulgariaii viper, J . Appl. Biochem., 3559, 1981.
Slotta, K.H., and Fraenkel-Conrat, H.L., Two active proteins froni ratilesriake veiionis, Nature 142:213, 1938.
Cate, R.L., and Bieber, A.L., Purification arid characierizatioti of Mojave (Crotnlris scutir/n/rrs .scuii~lnius) tosiri atid its suburiits, Arch. Biochem. Biophys., 189:397, 1978.
Mori, N., Nikai, T., and Sugihara, H., Purificatiori o f a proteiriase (Acs- Proteinuse) arid characterizatiori of hemorrhagic toxi~is froni the veiioni of the hutidred-pace srioke ( Arkislrorlori ncritus), Toxicon 22:45 1, 1984.
Nikai, T., Sugihara, H., and Tanaka, T., Enzymocheniical studies oiz make vetionis - I I Purification o f lethal proteiti Ac,-Proteiriase in the veiioni o f Ackistrodo1i w, J. Pharm. SOC. Japan 91:501,1971.
Sugihara, H., Nikai, T., Kawaguchi, T., and Tanaka, T., E~izyniochen~ical studies 011 snake vetionis IV. Puri Jicatiori o f lethal protein AcZ- Proteiiiase iri the veiioni o f Aakistrodoti n c ~ . , J. Pharm. SOC. Japan 98; 1523, 1978.
Sugihara, H., Nikai, T., Umeda, H., and Tanaka, T., Erizyniochemical studies oti siiake vetionis - V. Purificatiori of lethal protein Ac -Proleitiuse iri the vetioni o f Azkistrodoti ncutus, J. Pharm. SOC. Japan 99; 1161, f979.
Sugihara,-H., Nikai, T., Kinoshita, G, and Tanaka T., Enzyniochemical studies oti snake venoms - V I I . Purification o f hemorrhagic pririciple Acq-Proteitiase in the vetioni of Aoki~irodoir ncutus, J. Pharm. SOC. Japan 100; 855, 1980.
Nikai, T., Ishisaki, H., Tu, A.T., and Sugihara, H., Presetice of zitic iti proteolyfic heniorrhagic rosirr isolated from Aokislrodori ncutu.7 vetioni, Comp. Biochem. Physiol. c., 72103, 1982.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
202 BJARNASQN AND FOX
35. Bjarnason, J. and Tu, A.T., Heniorrhagic tuxitis from IVeJterti dianiotzdback raltlestzalie (Crotnlus atrox) vetioni; Isolatiuti arid characterizatioti of jive toxi/i.s arid the role of zitzc itz heniorrhagic toxiti e, Biochemistry, 17:3395, 1978.
Ouyang, C., and Huang, T., Purificatiori aiid characterizatioti of the fibrirzolytic principle of A~ki.~trodori acutud veiioni, Biochim. Biophys. Acta, 439: 146, 1976.
Ouyang, C., and Huang, T., The properties of the purified fibritiolytic principle from Arltirtrodor~ acutus snake verzoni, Toxicon, 15:161,1977.
Xu, X., Wang, C. Liu, J., and Lu, Z., Purijication arid charncterizatiorz of hcniorrhagic conipotietits froni A ?kisfrodoii aczrtu.7 (Huridred pace stiake) ivtioni, Toxicon, 19:633, 1981.
Zhang, J., Chen, 2, He, Y , and Xu, X . , Effect of calciuni oti proleolytic activity atid cut1 fornialiotz of heniorrhagic toxiti I froni f ive pace siiake (Arkistrodoti w) veiioni, Toxicon, 22931, 1984.
Bjarnason, J.B., and Fox, J.W., Characterizaliori of two hemorrhagic zinc proteiriases. ~oxii i c a d toxitz d , froni eastertz dianioizdback raltlestzake (Crotnlus u) vetioni, Biochim. Biophys. Acta, 911:356, 1987.
Omori, T., Iwanaga, S. and Suzuki, T., The relatiotiship between the heniorrhngic atid lethal actiilities of Japaiiese Maniushi (Arkistrodoti hnlvs hlonihoffii) irctiuni, Toxicon 2:1, 1964.
36.
37,
38.
39.
40.
41.
42. Satake, M., Murata, Y., and Suzuki, T., Studies on snake venom XIII. Chromatographic separation and properties of three proteinases from Asltistrodori hnlvs hlonihoffii, J. Biochem 57~392, 1965.
Iwanga, S., Omori, T., Oshima, G., and Suzuki, T., Studies ori sriake i’etionis X V I . Denioristratioii of a Proteiiiase with Heniorrhagic activity irz fhe vetzont of A,okislrodoii halss hlonihoffii, J. Biochem 57:392, 1965.
43.
44. Oshima, G., Iwanaga, S. and Suzuki, T., Studies oti sriake vetionis. X V I I I . Atz improved niethod for purijicatioti of the proteinuse b /rani the vetioni of Arkistrodoti halvs hlonihoffii atid its physiocheniical propevfies. J. Biochem. 64:215, 1968.
Oshima, B., Matsuo, Y., Fwanaga, S. and Suzuki, T., Studies oti sriake veiionis. X I X . Purijicaiiori and some physiochemical properties of proteitiase a arid c froni the vetioni of Arkistrodoil h a h ~ s hlonihoflii, J. Biochem. 64227, 1968.
Oshima, G., Iwanga S., and Suzuki, T., Sonie properties of proteinuse b iti the veiioni of Ackistrodotz halvs hlonihoflii, Biochim. Biophys. Acta, 250416, 1971.
Oshima, G., Omori-Satoh, T., Iwanaga, S. and Suzuki, T., Studies on make veiioni heniorrhagic faclor I (HR- I ) iti the vetzoni of Ackistrodoti hnlvs hlonihof fii. Its purification aiid biological properties, J . Biochem. 721483, 1972.
Nikai, T., Oguri, E., Kishida, M., Sugihara, H., Mori, N., and Tu, A.T., Reevaluatioti of heniorrhagic toxiti, HR-I, froni Arld~trodotz hal1I.s hlonihof fii veizoni: Proof a/ proleolytic etizynie. Itit. J. Biochem., 18:103, 1986.
Mandelbaum, F.R., Reichl, A.P. and Assakura, M.T., Sonie physical atid biocheniical characieristics of HF2, otie of the hemorrhagic factors i ~ i the vetioni of Bothrons iarornca, iti Atiimal, Platzt atid Microbial Toxitis, Val. I (Ohsaka, A., Hayashi, K., and Sawai, Y., Eds.) p. 11 1, New York, Plenum Press, 1976.
45.
46.
47.
48.
49.
Tox
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Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
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es o
n 06
/30/
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rson
al u
se o
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HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 203
50.
51.
52.
53.
54.
5 5 .
56.
57.
58.
59.
60.
61.
62.
63.
64.
Mandelbaum, F.R., Reichl, A.P., and Assakura, Isolalioti arid characteri:atiori o f a proteol3~tic erirynie from the vetioni of the snake Bo1hron.r iararncn (jaraca), Toxicon, 30:955, 1982.
Queiroz, L.S., Neto, H.S., Assakura, M.T., Reichl, A.P., and Mandelbaum, F.R., Pathological chaiiges h i niiiscle caused by heniorrhagic atid proteolytic factors from Rolhrons inrnrocg stlake vetioni, Toxicon 23:;341, 1985.
Assakura, M.T., Reichl, A.P., Asperti, M.C.A., and Mandelbaum, F.R., Isolatiori of the niajor proteolytic erirynie from the vetioni o f fhe snake Bolhrons niooierii (caissaca), Toxicon, 23:691, 1985.
Mandelbaum, F.R., Assakura, M.T., and Reichl, A.P., Characterizatioti o f two hemorrhagic factors isolated from the vetioni of Bolhrons rieuiviedi (jararaca pititada), Toxicon 22:193, 1984.
Fox, J.W., and Bjarnason, J.B., New proteases from Crotnlirs atrox iwioni, J. Toxicol. Toxin Reviews, 2:161, 1983.
Kurecki, T., and Kress, L.F., Purification arid partial characterization o f llie heniorrhagic factor from the verioni of Crolnlup ndanicttrteus (easterti dianioridback rattlesriake), Toxicon, 23:657, 1985.
Kurecki, T., Laskowski, M.Sr., and Kress, L.F., Purification arid sonie p r o p rties o f two proteitiases from Crolalus adaniniiteics verioni that itiactivate huniati a proteiriase irihibitor, J. Biol. Chem., 253:8340, 1978.
Kurecki, T., and Laskowski, M. Sr., Purification of collagetiases f rom Crotalus adanintifeu.r wrioni, in Toxicon supplement No. 1, Toxins, Animal, plant and Microbial, Elmsford, N.Y., Pergaman Press, P. 31 1, 1978.
Civello, D.J., Duong, H.L., and Geren, C.R., Isolalioti arid characterizatiori of a heniorrhagic proteitiase f r o m timber rattlesnake verioni, Biochem., 22:749, 1983.
Civello, D.J., Moran, J.B., and Geren, C.R., Substrate specificity o f a hemorrhagic proteiriase f rom riniber rattlestiake vetioni, Biochem., 22:755, 1983.
Bjarnason, J.B., and Fox, J.W., Proteolylic specificity arid cobalt exchange of hemorrhagic toxin e. a ziric protease isolated from the vetioni o f the western dianiotidback rattlesrialie (Crotnlus atrox), Biochemistry, 223770, 1983.
E '
Nikai, T., Mori, N., Kishida, M., Sugihara, H., and Tu, A.T., Isolatiori arid biocheniical characlerizatioti of heniorrhagic loxiti f froni the vetioni o f Crotnlus
(westerti dianiotidback rattlesnake), Arch. Biochem. Biophys., 231:309, 1984.
Nikai, T., Mori, N., Kishida, M., Tsuboi, M., and Sugihara, H., Isolation arid characteriratiori of hemorrhagic toxiri g f rom the verioni o f Crotalus nlrox (westerti dianiotidback rattlesnake), Am. J. Trop. Med. Hyg., 34:1167, 1985.
Komori, Y., Hagihara, S., and Tu, A.T., Specificity of heniorrhagic proleirime fronr Crotalics ntrox (ivesterri dianiotidback rattlestiake) vetioni, Biochim. Biophys. Acta, 829:127, 1985.
Fox, J.W., Campbell, R., Beggerly, L., and Bjarnason, J.B., Substrate specificities atid itihibitiori of two heniorrhagic zinc proteases HI-c arid Ht-d froni Cr0tnlu.s a veiioni, Eur. J. Biochem., 156:65, 1986.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
BJARNASON AND FOX 2 04
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
Ohsaka, A., Ikezawa, H., Kondo, H. and Kondo, S., Two hemorrhagic prirtciples dcrived from Hahu sriake verioni arid their differertce iri zorie electrophoret ical niobility, Jap. J. Med. Sci. Biol. 13:73, 1960.
Ohsaka, A., Ikezawa, H., Kondo, H., Kondo, S. and Ushida, N., Hemorrhagic activitics o f Habu sriakr vcrioni atid their relatiotis lo lethal toxicity. proteolytic activities arid other pathological aclivities, Br. J . Exp. Path. 41:478, 1960.
Omori Satoh, T., Ohsaka, A, Kondo, S., and Kondo, H., A simple arid rapid niethod for separalirig 1 )YO hcniorrhagic principles in the verioni of Triniere.rurrrr flnvoviridis, Toxicon, 517, 1967.
Takahashi, T. and Ohsaka, A., Purification arid sonie properties o f two heniorrhagic pririciples (HR2a arid HR2b) iri the verioni o f Triniere.~u,us
flavoviridis: coniplete separatiori o f the priiiciples froni proteolj~tic activity, Biochim. Biophys. Acta, 207:65, 1970.
Omori-Satoh, T., and Ohsaka, A., Purification arid sonie properties of heniorrhagic pririciple 1 iri the vertoni of Trinieriwrrrrs flavoviridis, Biochim. Biophys, Acta, 207:432, 1970.
Ohsaka, A., Just, M., and Habermann, E., Actioit o f sriake verioni hemorrhagic pririciples ori isolated glonierular baseniertt niembrarie, Biochim. Biophys. Acta, 323:415, 1973.
Omori-Satoh, T. and Sadahiro, S., Resolutiori of the niajor hemorrhagic coniporiertt of Trimeresi~rirs Navoviridis verioni irtlo two parts, Biochim. Biophys, Acta, 580:392, 1979.
Huang, T.F., Chang, J.H., and Quyang, C., Characterization of heniorrhagic pririciples fronz Trinieresi~rrrr ~raniirteus srtake verioni, Toxicon, 2245, 1984.
Sugihara, H., Moriura, M., and Nikai, T., Purificatiori arid properties o f a lethal heniorrhagic proteiri "Mucrotoxin A", /rani the verioni of the chiriese habu sriake (Trinii~reswus nii~crosartaniatus), Toxicon, 21:247, 1983.
Kishida, M., Nikai, T., Mori, N., Kohmura, S., Sugihara, H., Characterization o f niucrotosirt A front verioni o f Trinteresirrus nirrcrosaunnintlts (the Chiriese Habu srtalte) Toxicon, 23:637, 1985.
Nikai, T., Mori, N., Kishida, M., Kato, Y., Takenaka, C., Murakami, T., Shigezane, S . , and Sugihara, H., Isolatiori arid characterizatiori o f hemorrhagic factors a arid b f rom the vettoni of the Chinese habu sriake (Trinieresurus ntucrosairaniaius) Biochim. Biophys. Acta, 838:122, 1984.
Ovadia, M., Isolatiori arid characterization o f three hcniorrhagic factors from the venoni o f Vinera nalaestiriac, Toxicon, 16:479, 1978.
Grotto, L., Moroz, C., deVries, A., and Goldblum, N., Isolatiori o f palesliriae heniorrhagiri arid distirictiori bet weeri its heniorrhagic arid proteoljdic activities, Biochim. Biophys. Acta, 133356, 1967.
Fox, J.W., Nikolov, B., and Shkenderov, S. Manuscript in preparation.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
HEMORRHAGIC TOXINS FROM SNAKE VENOMS 205
79.
80.
81.
82.
83.
84.
8 5 .
86.
87.
88.
89.
90.
91.
92.
93.
94.
Arnold, R.E., Treatnietit o f Rattlestiake Bites, in Ratilestiake Verionis: Their Acfioris atid Treatnietit, edited by A.T. Tu, p. 315, Marcel Dekker, New York, 1982.
Denson, K.W.E., Coagularit arid atiticoagularit action of stlake vetionis, Toxicon, 75, 1969.
Cheng, H.C.., and Ouyang, C., Isolatioti of coagulatit arid atiticoagularit priticiples from the vetioni o f Arkistrodoti aculiq, Toxicon 4:235, 1967.
McKay, D.G., Moroz, D., deVries, A., Csavossy, I., and Cruse, V., The Actioti of heniorrhagiti atid pliospholipase derived front Vinern naleslittae l'erioni oti the Microcirculation, Laboratory Investigation, 22387, 1970.
Tsuchiya, M., Ohshio, C., Ohashi, M., Ohsaka, A., Suzuki, K., and Fujishiro, Y., in Platelets, thronibosis. inhibitors, edited by Didisheim, P., Shimamoto, T., and Yamazaki, H., p. 439, Schattauer, Stuttgart, 1974.
Ownby, C.L., Kainer, R.A., and Tu, A.T., Pathogetiesis of heniorrhage itiduced by raftlestiake verioni, Am. J. of Pathology, 76:401, 1974.
Ownby, C.L., Bjarnason, J., and Tu, A.T., Heniorrhagic toxitis front rattlesrialie (Croralrrs atrox) verioni, Am. J. Pathol., 93:201, 1978.
Minton, S.A., Jr., in Vetionis, edited by Buckley, E.E., and Porges, N., p. 145, American Association for the Advancement of Science, Washington, D.C., 1956.
Mitsuhashi, S., Maeno, H., Kawakarni, M., Hashimoto, H., Sawai, Y., Miyazaki, S., Makino, M., Kobayashi, M., Okonogi, T., and Yamaguchi, K., Sfudies on Habu snake verioni. I. Coniparisoti of several biological activities of fresh arid dried Habu siiake vetioni, Japan J. Microbiol., 3:95, 1959.
Kondo, H., Kondo, S., Ikezawa, H., Murata, R., Ohsaka, A., Studies oti the quatititative method for the deterniiriatioti of hemorrhagic activity of Habu snake vctioni, Japan J. Med. Sci. Biol., 13:43, 1960.
Ohsaka, A., Omori-Satoh, T., Kondo, H., Kondo, S., Murata, R., Biocheniical arid pathological aspects of hemorrhagic pririciples iri strake vetionis with special refererice to Habu (Trinieresurus flavoviridi.~) vetioni, Mem. Inst. Butantan, Simp. Internat., 33:193, 1966.
Ownby, C.L., Colbeng, T.R., and Odell, G.V., A tiew method for quatifitaliori of heniorrhage iriduced by rattlestiake verionis: Ability of polyvalerir atitivetioni to tieutralize heniorrhagic activity, Toxicon, 22221, 1984.
Miles, A.A. and Wilhelm, D.L., Br. J. Expt. Pathol., 3671, 1955.
Just, D., Urbanitz, D., and Habermann, E., Pharniakologische charalcterisierurig der vasculri'reri schratiketi futiktini gegetiuber erythrocyfeti utid albuniiti, Arch. Pharrnakol., 267:399, 1970.
Bonta, I.L., Vargaftig, B.B., Bhargava, N., and Vos, C.J., de, Method for study of stlake vetioni itiduced heniorrhage, Toxicon, 8:3, 1970.
Deutsch, H.F., and Diniz, C.R., Sonie proteolytic activities of stiake vetionis, J. Biol. Chem. 933, 1955.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
206 BJARNASON AND FOX
95. Kocholaty, W.F., Toxicity arid some e~izymatic properties arid activities i t i the verioni of Crotalidae, Elapidae arid Viperidae, Toxicon 9 13 1, 197 1.
Jimenez-Porras, J.M., Biochcniistry of srialie venoms, Clin. Toxicol., 4:235, 1970.
Pandya, B.V., and Budzynski, A.A., Arilicoagulatil proteases from westerr1 diamondback ratllesriake (crotnlus alrox) verioni, Biochemistry, 23:460, 1984.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
Kunitz, M., Crystallitie soybeart trypsiri irihibitor. Ira. Getieral properties, J. Gen. Physiol., 30:291, 1947.
Lin, Y., Means, G.E., and Feeney, R.E., The action o f proleolylic etizynies on N.N-diniethyl proteins, J. Biol. Chem., 244:789, 1969.
Lacks, S.A., and Springhorn, S.S., Reriaturation of enzymes ajler polyacrjdamide gel electrophoresis iti rhe preserice of sodium dodecyl sulfate, J. Biol. Chem., 255:7467, 1980.
Markland, F.S., and Perdon, A., Comparisori of two methods for proteolytic erizynies detection iti srialic vcrioni, Toxicon, 24:385, 1986.
Meier, K., Stocker, Svendsen, L.G., and Brogli, M. , Chroniogeriic proteitiase substrates as possible tools iti the characterizaliori o f Crotalidae and Viperidae sriake venoms, Toxicon, 23:393, 1985.
Timpl, R. and Dziadek, M. (1986) Sfructure. developmetit arid molecular pathology of basemerit membranes, I t i t . Rev. of Exp. Pathol. 29, 1-1 12.
Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Star, V.L., Cannon, F.B., Laurie, G.W., and Martin, G.R. (1986) Basenieril membrarte conipleses with biological activity Biochemistry 25, 312-318.
Bjarnason, J.B., Hamilton, D., Fox, J.W. (1988) Studies OH the niechariism of hemorrhage produclioti by free proteolytic hemorrhagic tosiii from Crotnlus ntrox vetiom, Biol. Chem. Hoppe-Seyler 369 Suppl. 121 -129.
Laurie, G.W., Bing, J.T., Kleinmann, H.K., Hassell, J.R., Aumailley, M., Martin, G.R., and Feldman, R.J. (1986) Localizatioti of bitidirig sites for lamitiiti, heparart sulfate. proleoglycati arid fibroriectiri OII basemerit membrnrie (type I V ) collageti, J. Mol. Biol. 189, 205-216.
Paulsson, M., Aumailley, M., Deutzmann, R., Timpl, R., Beck, K., and Engel, J. (1987) Lattuniriitrogert complex. Extracliori with chelatirig agetils arid slruclural characterizatiori. Eur. J. Biochem. 116, 11-19.
Tu, A.T., in Vetionis: Chemistry mid Afolecular BiOlOgJ), p. 104- 131, John Wiley, New York, 1977.
Hawgood, B.J., in Raltlesriake Verioms (Tu, A.T. ed.), p. 121, Marcel Dekker, New York, 1982.
Cameron, D.L. and Tu, A.T., Chemical arid furictiorial homology of nipotositi from prairie rattlesriake veriom atid crotaniine front South American rattlestiake vetiom, Biochim. Biophys. Acta, 532147, 1978.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
HEMORRHAGIC T O X I N S FROM SNAKE VENOMS 207
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
Carlson, R.W., Schaeffer, R.C., Whigham, H., Michaels, S., Russell, F.E., and Weil, M.H., Rattlestiaka veiioni shock iti the rat: Developmetit o f a nieihod, Am. J. Physiol., 229:1668, 1975.
I-leimburger, N., in Proteases atid Biological Control (Reich, E., Rifkin, D.B. and Shaw, E., eds), 2:367, Cold Spring Harbor Laboratory, New York, 1975.
Imber, M.J. and Pizzo, S.V., Clearaiice arid biiidiiig of iivo electrophoretic fast" forms of hiiniati a2-macroglobulin, J. Biol. Chem., 256:8134, 1981.
Travis, J. and Salveson, G.S. Human Plasma Proteinase Inhibitors in Annu. Rev. Biochem, 52:655, 1983.
Starkey, P.M. and Barrett, A.J., in Proteiriiases in Mammalian Cells arid Tissues (Barrett, A.J., ed.), North-Holland, New York, 1977.
Carrel1 R. and Travis, J., a, -Aiititrypsin arid the serpiiis: variatiori arid counteri~ariatiori, Tibs, Jan:2O, 1985.
Werb, Z., Burleigh, M.C., Barrett,A.J., and Starkey, P.M., The ititeruction o f a2- niacroglobuliti with proleitiam, Biochem. J., 139139, 1974.
Kress. L.F. and Kurecki. T.. Studies 017 the coninlex betweeti huniari a?- I ,
niacroglobuliti atid Crolnlirs ndnnintileuc proteitiase 11, Biochim. BiophGs, Acta, 613:469, 1980.
Fox, J.W., Hamilton, D., and Bjarnason, J.B., unpublished results.
Kress, L.F. amd Paroski, E.A., Enzymatic inactivation o f huntari serum proteitiase inhibitors by snake venom proteirrases, Biochem. Biophys, Commun., 83549, 1978.
Kress, L.F. and Catanese, J.J., Identification of cleavage sites resulting from eriryniatic itiaclivatiotz of human atitithronibiri I11 by Crolnlus ~dnniariteus proleiiiase II in the presetice arid absence o f hepariti, Biochemistry, 20:7432, 1981.
Kress, L.F. Kurecki, T., Chan, S.K., and Laskowski, Sr. M., Characterizafioti of the iriactive fragnietit resultitig f rom limited proteolysis o f huniaii al -pt.oteiriase inhibitor by Croln1it.v ndnmniileus, J. Biol. Chem., 25453 17, 1979.
Kress, L.F., Iiiactivalioti of human plasma seriiie proteitiase irihibitors (serpitis) by limited proteolysis o f the reactive site loop with snake vetioni arid bacterial nietalloproleitiases, J. Cell. Biochem., 2451, 1986.
Kress, L.F. Catanese, J.J. and Hirayama T., Analysis o f the e f fec t s o f m a k e veiioni proteiitases on the activity o f huntari plasma CI-esterase inhibitor, a l - aritichj~niolr.vpsiri arid a2-atiliplasmiri, Biochim. Biophys. Acta, 7451 13, 1983.
Boffa, M.C. and Boffa, G.A., Correlations betweeit the eiizyniatic activities and the factors active oti blood coagulation arid platelet aggregatioti f rom the venom o f Viaern nsois, Biochim. Biophys. Acta, 354:275, 1974.
Markland, F.S., Damus, P.S., Davidson, T.M., and Shanley, J.D., Thrombiti enzynie from Crotaltis adnnianteu$, Lancet, 1: 1398, 1970.
Sraight, R., Glenn, J.L, and Synder, C.C., Aiitiverioni activity of rattlesriake blood plasma, Nature, 261:259, 1976.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
208 BJARNASON AND FOX
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
Noguchi, H., Natural ininiutiity of certaiti ariinials froni stiake vetioni, Snake Venoms, p.268, Washington, D.C., Carnegie Institute.
Werner, M.R., and Vick, A.J., Resistatice o f the opossuni (Didelahis iiirgiiiinria) to etivetioniatioti by stiakes of the Janiily Crotalidae, Toxicon, 1529, 1977.
Keegan, H.L. and Andrews, T.F., Effects o f Crotalid verioni or1 North Aniericati sriakes, Copeia, 4:251, 1942.
Philpot, V.B. and Smith R.G., Neutralization o f pit viper veiioni by kirigsriake seruni, Proc. SOC. Exp. Biol. Med., 74521, 1950.
Omori-Satoh, T., Sadahiro, S., Ohsaka, A., and Murata, R., Purificatioti arid charactcriratiori of ati aritiheniorrhagic factor iri the seruni o f Triniere.surii.7 f1nimVridi.s. a crotalid, Biochim. Biophys. Acta, 285:414, 1972.
Omori-Satoh, T., Aiitiheniorrhagic factor as a proleitiuse irihibitor f rom the seruni of Trir?7eresrirrrs f lmwvir idis, Biochim. Biophys. Acta, 495:93, 1977.
Ovadia, M., Purificatioti atid characterizatiori o J ati atitiheniorrhagic factor froni the serum o f thc stinke
Perez, J.C., Haws, W.C., Garcia, V.E., and Jennings 111, B.M., Resistatice of ivarni-blooded atiiniuls to stinlie vetionis, Toxicon, 16661, 1978.
Perez, J.C., Pichyangkul, S . , and Garcia, V.E., The resistaitce o f three species of ivarnz-blooded ariinials to western dianioiidback rattlesrialte (Ciotalus atrox) veiioni, Toxicon, 17:601, 1979.
Menchaca, J.M. and Perez, J.C., The purificatiori atid characterizatiori o f all atitiheniorrhagic factor iti opossuni (Didelahis vir.eiriiarin) seruni, Toxicon, 19623, 1981.
Pichyangkul, S. and Perez, J.C., Purificatiori atid characterizatioii o f a riaturally occurririg atitiheniorrhngic factor it1 the seruni of the hispid cottori rat (Sipniodori hi.rnidi/.y), Toxicon, 19:205, 1981.
Garcia, V.E. and Perez, J.C., The purificatiori arid characterizatioti of ati atitiheniorrhagic factor iti woodrat (Neolonia micronus), Toxicon, 22: 129, 1984.
Kress, L.F. and Catanese, J.J., Isolatiori o f ati inhibitor o f snake vetioni nietalloprofeiriases froni opossuni scruni, Fed. Proc., 44:143 I , 1985.
Kress, L.F., The aclioti of stlake verioni nietalloproleitiases oti plasnia proleitiuse irihibitors, iri Vetioms Q& Honiostasis, (Pirkle, H. and Markland, F.S., eds.), Marcel Dekker, New York, in press.
De Wit, C.A. and Westrom, B.R., Verioni resistarice iri the hedgehog. Eririnceus errroaneus: Puri ficatioti arid idetiti ficatiori o f niacroglobtrliti irihibitors as plasnia atitiheniorrhagic factors, Toxicon, 25:315, 1987.
Nishino, N. and Powers, J.C., Peptide hydroxaniic acids as irihibitors of therniolysiti, Biochemistry, 17:2846, 1978.
Chu, T.G. and Orlowski, M., Active site directed N-carboxynieihyl peptide irihibitors of a soluble metalloetidopeptidase f rom rat brain, Biochemistry, 23:3598. 1984.
pnlnstiiine, Toxicon, 16:661, 1978.
Tox
in R
evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
HEMORRHAGIC TOXINS FROM SNAKE VENOMS 209
145. Vencill, C.F., Rasnick, D., Crumley, K.V., Nishino, N., and Powers, J.C., Clostridiuni histol vticuni collageiiase: Development of riew thio ester, fluorogeriic. aiid depsipeplide substrates atid rieiv itihibilors, Biochemistry, 2 4 3 149, 1985.
Fox, J.W., Shannon, J.D., Bjarnoson, J.B., and Hite, L. Unpublished data.
Baramova, G.N., Shannon, J.D., Bjarnason, J.B., and Fox, J.W. Manuscript submitted.
146.
147.
Tox
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evie
ws
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Yor
k U
nive
rsity
Lib
rari
es o
n 06
/30/
14Fo
r pe
rson
al u
se o
nly.
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