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TMVA, a snake C-type lectin-like protein from Trimeresurus
mucrosquamatus venom, activates platelet via GPIb
Hong Taia,b,c,1, Qin Weia,1, Yang Jina, Min Suc, Jian-Xin Songc, Xin-Ding Zhoua,b,Hong-Mei Ouyangc, Wan-Yu Wanga, Yu-Liang Xionga,*, Yun Zhanga
aDepartment of Animal Toxinology, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming 650223, Yunnan, People’s Republic of ChinabGraduate School of the Chinese Academy of Sciences, People’s Republic of China
cThe First People’s Hospital of Yunnan Province, Kunming, People’s Republic of China
Received 5 May 2004; revised 28 July 2004; accepted 28 July 2004
Available online 28 September 2004
Abstract
TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In
the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib
monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The
aggregation was also inhibited by mAb P2 (an anti-GP IIb mAb). Flow cytometric analysis revealed that FITC-TMVA bound to
human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent
manner. Flow cytometric analysis showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV.
Moreover, the platelet aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a
snake venom protease that specifically cleaves human GPIb. These results suggest that TMVA is a strong platelet agonist via
GPIb and it might have multiple functional binding-sites on GPIb molecule or on other unknown receptor.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Platelet aggregation; TMVA; GPIb; Mocarhagin
Platelet membrane glycoprotein Ib (GPIb), one of the
major platelet membrane receptors, plays important roles in
platelet adhesion, aggregation and subsequent thrombus
formation in the vessels under high shear rates or with
damaged endothelium. Approximately 25,000 GPIb mol-
ecules are present on the platelet surface. GPIb consists of
two subunits, GPIba (143 kDa) and GPIbb (22 kDa). GPIbais disulphide-liked to GPIbb that is non-covalently associ-
ated with GPIX and GPV (Berndt et al., 2001). Virtually
0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2004.07.022
* Corresponding author. Tel.: C86-871-5192476; fax: C86-871-
5191823.
E-mail address: [email protected] (Y.-L. Xiong).1 Both authors contributed equally to this work.
the entire ligand-binding capacity of the GPIb-IX-V
complex is situated in the N-terminal globular region
(w45 kDa) of the GPIba chain, which can be removed from
the GPIba fragment glycocalicin through treatment with
trypsin or mocarhagin (Ward et al., 1996). The N-terminal
282 amino acids of the GPIba chain contains binding sites
for vWF and a-thrombin. The platelet GPIb-von Willebrand
factor (vWF) and GPIV-collagen interactions triggered
intracellular signals leading to degranulation, elevation of
cytosolic Ca2C, cytoskeletal rearrangements, and ‘inside-
out’ activation of the integrin, GPIIb-IIIa, that binds von
Willebrand factor (vWF) or GPVI-fibrinogen and mediates
platelet aggregation (Kroll et al., 1996; Weiss, 1995;
Watson and Gibbins, 1998). Monoclonal antibody HIP1
Toxicon 44 (2004) 649–656
www.elsevier.com/locate/toxicon
H. Tai et al. / Toxicon 44 (2004) 649–656650
against GPIb inhibits the ristocetin-dependent binding of
von Willebrand factor (vWF) to platelets (Matsubara et al.,
2002).
A lot of snake venom C-type lectin-like proteins have
been purified from different species. Snake venom C-type
lectin-like proteins, which affect platelets by binding to
specific receptors such as GPIb, GPIa-IIa and GPVI have
been characterized and widely used as tools to investigate
platelet function and regulation. The C-type lectin-like
proteins acting via GPIb have similar primary structure but
different function. Most of the GPIb-binding proteins inhibit
platelet activation by blocking the interation of GPIb with
von willebrand factor (vWF) or thrombin, such as agkicetin
(Chen and Tsai, 1995), CHH-A (Fujita et al., 1998), CHH-B
(Fujita et al., 1998), echicetin (Peng et al., 1993, 1994),
flavocetin-A, flavocetin-B (Taniuchi et al., 1995), jararaca
GPIb-BP (Fujimura et al., 1995; Kawasaki et al., 1996) and
tokaricetin (Kawasaki et al., 1995). Most of them are
heterodimeric with the two subunits (a and b subunits)
linked through a disulfide. Flavocetin-A, which does not
directly activate platelets, binds with high affinity to the
platelet GP-Iba and functions as a strong inhibitor of vWF-
dependent platelet aggregation (Fukuda et al., 2000).
However, alboaggregin B (Peng et al., 1992) mamushigin
(Sakurai et al., 1998), bitiscetin (Hamako et al., 1996) and
TSV-GPIb-BP (Lee and Zhang, 2003) have platelet
agglutination activity.
Recently, we have isolated and characterized a novel
platelet agonist, designed as TMVA, from Trimeresurus
mucrosquamatus venom (Wei et al., 2002). It is a C-type
lectin-like protein consisting of two subunits, a (15,536 Da)
and b (14,873 Da). The amino acid sequences of two
subunits show great similarity to other C-type lectin-like
venom proteins. Native TMVA exists as two convertible
multimers of (ab)2 and (ab)4 with molecular weights of
63,680 and 128,518 Da, respectively. Previous studies
showed that TMVA has potent platelet aggregation activat-
ing effect. However, its receptor(s) on platelet surface is not
determined. In this study, we present the evidences that
TMVA is a GPIb binding protein and directly activates
platelet via GPIb.
1. Materials and methods
1.1. Materials
Lyophilized T. mucrosquamatus venom (Hunan, China)
was from the stock of Kunming Institute of Zoology,
Chinese Academy of Sciences. TMVA was purified as
previously described (Wei et al., 2002).
Fluorescein isothiocyanate (FITC)-conjugated mono-
clonal antibodies (mAb) against human GPIb (HIP1),
GPIX (ALMA16) were purchased from BD Biosciences,
USA. The mAbs against human GPIIb (P2), CDIIIa (SZ21),
GPIIa (CK20), GPIa (AK7), GPIV (CB38), P-selectin
(CLB-Thromb/6), FITC-Immunoglobulin and FITC were
purchased from Immunotech Co., France.
Mocarhagin was a kind gift from Dr Yang Shen
(Department of Biochemistry and Molecular Biology,
Monash University, Australia). Ristocetin was purchased
from Sigma Co., USA. vWF (lyophilized human plasma
containing vWF at approximately 100% activity) was
purchased from Diagnostica stago Co., France. Other
chemicals were of analytic grade.
1.2. Platelet preparation
Human platelet-rich plasma (PRP) was prepared by
dilution of the concentrated platelets from 3 to 5 healthy
donors (provided by Kunming Blood Transfusion Service)
using platelet-poor plasma of the same batch to a final
platelet count of 3!108/ml.
Washed human platelets were prepared as described by
Polg?r J et al. (1997). Briefly, platelet pellet obtained after
centrifugation of PRP was resuspended in 113 mM NaCl,
4.3 mM K2HPO4, 24.4 mM NaH2PO4, 4.3 mM Na2HPO4,
5.5 mM glucose, pH6.5 (buffer A) and centrifuged at
250!g for 10 min, and the platelets were washed once
more with buffer A. Washed platelets were then resus-
pended in 20 mM Hepes, 140 mM NaCl, 4 mM KCl,
5.5 mM glucose, pH 7.4 (buffer B), and the platelet count
was adjusted to 3!108 platelets/ml by dilution with buffer
B. The samples were kept at room temperature until used
for aggregation studies. Before platelet aggregation anal-
ysis, 2 mM CaCl2 was added and the platelets were
incubated at 37 8C for 2 min.
Fixed platelets were prepared as described by Kirby
and Mills (1975). Washed platelets were incubated with
equal volume of 1% formaldehyde in TBS at room
temperature for 0.5 h. Then platelets were washed twice
with TBS by centrifuging. Finally, the platelets were
resuspended to a final concentration of 3.1!107 platelets/
ml in TBS containing 2 mg/ml BSA and were stored at
K70 8C.
1.3. Platelet aggregation
Human PRP or washed human platelets were activated
by the indicated concentrations of TMVA under continuous
stiring at 1000 rpm in a LBY-NJ aggregometer (Precie
Group, Beijing, China) at 37 8C by the turbidimetric method
(Born and Cross, 1963).
To determine the effect of platelet glycoprotein anti-
bodies on TMVA induced aggregation, washed human
platelets were incubated with various concentrations of
mAb HIP1 against GPIb or mAb P2 against GPIIb (final
concentrations of 0, 5, 10, 20 or 40 mg/ml) for 30 min at
37 8C, before TMVA (final concentration of 10 mg/ml) was
added to trigger platelet aggregation.
H. Tai et al. / Toxicon 44 (2004) 649–656 651
1.4. Effect of mocarhagin on TMVA-induced platelet
aggregation
Aliquots of 100 ml washed human platelets were
incubated at 37 8C for 6 min in the absence or presence of
10 mg /ml mocarhagin. A final concentration of 1.5 mg/ml
ristocetin or 10 mg/ml TMVA was added to the pretreated
platelets in the presence of vWF (final concentration of
50 mg/ml). Platelet aggregation was determined as men-
tioned above.
1.5. Flow cytometry analysis of the effect of TMVA
on human platelet glycoprotein expression
TMVA (10 mg/ml) was incubated with washed human
platelets without Ca2C for 30 min at 37 8C. Then 100 ml
treated platelet suspensions were added into 20 ml FITC-
conjugated mAb of GPIb, GPIX, GPIIb, GPIIIa, GPIa, GPII,
GPIV or P-selectin for 20 min in dark at room temperature,
respectively. Then 500 ml PBS was added. 20 ml of FITC-
immunoglobulin were added in washed platelets as homo-
typal control. At least 50,000 gated platelets of TMVA-
treated washed human platelets were analyzed by an argon-
exciting laser at 488 nm, and the platelet glycoprotein
emitting mean fluorescence intensity was evaluated at
520 nm by flow cytometry.
1.6. FITC conjugation of TMVA
TMVA and BSA were conjugated with FITC by a
previously described method (Liu et al., 1994). TMVA
dissolved in 0.1 M sodium bicarbonate was mixed continu-
ously with FITC solution for 1 h, and the reaction was
stopped by the addition of 1.5 M hydroxylamine. The
conjugated TMVA was separated from free FITC using a
Sephadex G-10 column at 25 8C. The concentration of
FITC-TMVA was determined by protein assay kit (Bio-Rad,
USA). FITC-TMVA retained platelet aggregation activity
equivalent to intact TMVA.
Fig. 1. Dose-dependent platelet aggregation induced by TMVA.
Human PRP (black column) and washed platelets (light column)
were incubated at 37 8C with various concentrations of TMVA
under constant stirring. Platelet aggregation was monitored for
5 min. Values are meanGSE of three experiments.
1.7. Binding assay of FITC-TMVA by flow cytometric
analysis
Hundred micro-litres of fixed platelets (2.24!107
platelets/ml) were incubated with the various concentrations
of FITC-TMVA and FITC-BSA (as a negative control) for
30 min at room temperature in dark. After washed twice
with TBS, the platelet pellets were resuspended in 500 ml of
TBS. The mean fluorescence intensity of platelets was
determined by flow cytometry.
To determine the inhibitory effect of anti-GPIb antibody,
100 ml of fixed platelets were incubated and gently shaken
with the mAb HIP1 (0, 10, 20 or 40 mg/ml) for 15 min at
room temperature. And then FITC-TMVA (40 nM) were
added and incubated for another 15 min at room
temperature. The platelets were washed twice with TBS
before flow cytometry analysis.
2. Results
2.1. TMVA induced platelet aggregation of PRP
and washed platelets
TMVA (2.5, 5.0, 10 and 20 mg/ml) induced platelet
aggregation in both human PRP and washed platelet
suspensions in a dose-dependent manner (Fig. 1). TMVA
has similar aggregation activities in both PRP and washed
platelet, with the maximum aggregation achieved at a
concentration of 20 mg/ml. But platelet aggregation rate of
PRP was slightly higher than that of washed platelets. The
results suggested that TMVA did not modulate vWF as
ristocetin or botrocetin did (Cohen et al., 1999; McGregor
et al., 1983).
2.2. TMVA activated platelets and increase a-granule
secretion
Pre-incubating the washed platelets with final concen-
trations of 0, 1.25, 2.50, 5.0, 7.5, 10, 20mg/ml and 40 mg/ml
TMVA for 30 min at 37 8C was analyzed by flow
cytometry. The results showed that P-selectin mean
fluorescence intensity of TMVA-treated washed platelets
significantly enhanced in a dose-dependent manner
(Fig. 2).
2.3. Anti-GPIb antibody and Anti-GPIIb antibody inhibited
TMVA-induced platelet aggregation
TMVA-induced platelet aggregation of washed plate-
lets was inhibited by mAb HIP1 against GPIb and mAb
Fig. 3. TMVA-induced aggregation was inhibited by mAb HIP1 or
P2. (A) Various concentrations of MoAb HIP1 against GPIb were
incubated with washed human platelets (3!108/ml) at 37 8C for
30 min, and then 10 mg/ml of TMVA was added in a total volume of
200 ml for platelet aggregation. The results are representative of
three experiments. (B) Various concentrations of mAb P2 against
GPIIb were incubated with washed human platelets (3!108/ml) at
37 8C for 30 min, and then 10 mg/ml of TMVA was added in a total
volume of 200 ml. The results are representative of three
experiments.
Fig. 2. P-selectin fluorescence intensity on TMVA-treated washed
platelets. Washed human platelets without Ca2C was incubated with
various concentration of TMVA at 37 8C for 30 min. 100 ml of
TMVA-treated washed platelets was respectively added into 20 ml
of FITC-conjugated mAb CLB-Thromb/6 against P-selectin at
room temperature in dark for 20 min, and then 500 ml of PBS was
added. 20 ml of FITC-immunoglobulin was added in washed
platelets as negtive control. At least 50,000 gated platelets of
TMVA-treated washed human platelets were analyzed by an argon-
exciting laser at 488 nm, and FITC-conjugated P-selectin on
platelets emitting mean fluorescence intensity was evaluated at
520 nm by flow cytometry. Values are meanGSE of three
experiments.
H. Tai et al. / Toxicon 44 (2004) 649–656652
P2 against GPIIb in a dose-dependent manner
(Fig. 3A and B).
2.4. Effect of FITC-TMVA binding to formalin-fixed
platelets
Using FITC-TMVA as a probe, we further explored the
molecular mechanism of TMVA. FITC-TMVA possessing
equivalent platelet aggregation activity as intact TMVA
migrated equally with unconjugated TMVA on SDS-PAGE
(data not shown). The analysis of flow cytometry showed
that FITC-TMVA specifically bound to human fixed
platelets in a dose-dependent manner and reached a
saturable binding at a concentration of 40 nM (Fig. 4A).
As shown in Fig. 4B, HIP1 inhibited FITC-TMVA binding
to fixed platelets in a dose-dependent manner.
2.5. Effect of TMVA on the mean fluorescence intensity
of platelet glycoprotein
Washed platelet pre-incubated with various concen-
trations (0, 5.0, 10, 20, 40 and 80 mg/ml) of TMVA for
30 min at 37 8C was analyzed by flow cytometry. The result
showed that GPIb mean fluorescence intensity of TMVA-
treated washed platelets significantly decreased (Fig. 5).
However, mean fluorescence intensities of GPIX, GPIIb,
GPIIIa, GPIa, GPIIa and GPIV of washed platelets treated
by TMVA were not changed (data not shown).
2.6. Effect of mocarhagin on TMVA- or ristocetin-vWF-
induced platelet aggregation
Mocarhagin was used to test the binding domain of
TMVA on the platelet GPIb. Ristocetin could induce
aggregation of washed platelets with vWF (Fig. 6: column
2), but did not induce aggregation of washed platelets due to
absence of vWF (Fig. 6: column 1). After 6 min treatment,
mocarhagin completely blocked ristocetin-induced aggre-
gation of washed platelets with vWF (Fig. 6: column 3). The
result suggested that GPIb molecules on platelet membrane
were cleaved by mocarhagin. However, the pretreated
platelets aggregated when adding TMVA, though the
maximum aggregation rate was decreased (Fig. 6 column
4–6).
3. Discussion
TMVA, a novel platelet aggregation agonist purified
from T. mucrosquamatus venom, is a high molecular weight
heteromeric C-type lectin-like protein composed of a and b
Fig. 4. HIP1 inhibited TMVA binding to platelet. (A) Binding assay
of FITC-TMVA to fixed platelets. Human fixed platelets (2.24!
107/ml) were incubated with various concentrations of FITC-
TMVA with (Non-specific binding) or without 50-fold excessive
TMVA (Total binding) for 30 min at room temperature in dark, and
then analyzed by flow cytometry. Specific binding was calculated
by subtracting the nonspecific binding from total binding. Results
are averages of three determinations. (B) Inhibition effect of HIP1
on FTIC-TMVA binding to fixed platelets. Various concentrations
mAb HIP1 against GPIb were incubated with fixed platelets for
15 min at room temperature, and then a final concentration of 40 nM
FITC-TMVA was added for 15 min at room temperature in dark and
analyzed by flow cytometry analysis. Results are averages of three
determinations.
Fig. 5. TMVA inhibited HIP1 binding to GPIb. Washed human
platelets was incubated with various concentration of TMVA at
37 8C for 30 min. 100 ml of the TMVA-treated washed platelets was
respectively added into 20 ml of FITC-conjugated mAb HIP1
against GPIb at room temperature in dark for 20 min, and then
500 ml of PBS was added. 20 ml of FITC-immunoglobulin was
added in washed platelets as negtive control. At least 50,000 gated
platelets were analyzed for GPIb expression by flow cytometry.
Results are averages of three determinations.
Fig. 6. Inhibitory effect of mocarhagin on ristocetin or TMVA-
induced platelet aggregation. Washed platelets (WP) in presence of
vWF (column 2) or washed platelets (column 1) were incubated at
37 8C with a final concentration of 1.5 mg/ml ristocetin under
constant stirring for triggering platelet aggregation. Washed
platelets in presence or absence of vWF (column 4, 5) were
incubated at 37 8C with a final concentration of 10 mg/ml TMVA
under constant stirring for triggering platelet aggregation. Washed
platelets were preincubated with a final concentration of 10 mg/ml
mocarhagin for 6 min at 37 8C. Then the mocarhagin treated washed
platelet (mWP) were added with ristocetin (1.5 mg/ml) and vWF
(column 3) or TMVA (10 mg/ml) (column 6) under constant stirring
at 37 8C to trigger platelet aggregation. Values are meanGSE of
three experiments.
H. Tai et al. / Toxicon 44 (2004) 649–656 653
subunits. The amino acid sequences of a- and b-subunits are
highly homologous to those of other snake venom GPIb-
binding proteins. The identity levels of a- and b-subunits
between TMVA and flavocetin-A, a GPIb protein from
Trimeresurus flavoviridis venom (Shin et al., 2000) are 96.3
and 81.6%, respectively. In this study, we found TMVA
strongly induced aggregation in both human PRP and
washed platelet suspensions in a dose-dependent manner
(Fig. 1A).
It is known that platelet plug formation is dependent on
vWF and adhesive compounds from the vascular matrix
(Sakariassen et al., 1979). However, similar aggregation
activities of TMVA in both PRP and washed platelet
suggested that TMVA did not modulate vWF.
P-selectin belongs to the selectin family of adhesion
molecules (Hogg, 1992). It is located in membranes of
a-granules in resting platelets and redistributed to the cell
surface upon platelet activation and acts as a receptor that
supports binding of leukocytes to activated platelets and
endothelium (Berman et al., 1986). In washed platelets,
H. Tai et al. / Toxicon 44 (2004) 649–656654
TMVA caused strong increasing of the mean fluorescence
intensity of P-selectin (Fig. 1B). The evidence presented
here shows that TMVA causes P-selectin in a-granules
redistributed to the cell surface on platelet activation. The
effect of strong platelet aggregation induced by TMVA
may be related to an increase of platelet a-granule
secretion. Moreover, mAb P2, an antibody against platelet
GPIIb, dose-dependently inhibited TMVA-induced platelet
aggregation, suggesting that aIIbb3 is activated in the later
stage of TMVA-induced platelet aggregation.
A number of snake venom C-type lectin-like proteins
activate platelet aggregation, but their targets on platelet are
different (Clemetson et al., 2001). Up to date, there are three
major platelet receptors, GPIb, GPVI and GPIa/IIa, involved
in platelet activation by venom C-type lectin-like proteins.
The majority, including alboaggregin A (Asazuma et al.,
2001), aggretin (Chung et al., 2001), alboluxin (Du et al.,
2002) and convulxin (Kanaji et al., 2003), exhibit dual
specificity for these receptors. TMVA was recently
characterized as a potent platelet aggregation activator,
but its target(s) on platelet was unknown.
To determine the possible receptor of TMVA, effect of
GPIb antibody on TMVA-induced platelet aggregation was
investigated. The results showed that TMVA-induced
platelet aggregation was specifically inhibited by a GPIb
antibody, HIP1, suggesting that TMVA activates platelet via
GPIb. 20 mg/ml HIP1 almost completely blocked the
aggregation. This observation indicated that GPIb might
be the major receptor of TMVA on platelet membrane. To
confirm the hypothesis, we employed FITC-conjugated
TMVA as a probe in combination with flow cytometric
analysis to explore the molecular mechanism of TMVA.
FITC-TMVA specifically bound to human fixed platelets in
a dose-dependent manner and reached a saturable binding at
a concentration of 40 nM. In addition, mAb HIP1 against
GPIb specifically inhibited FITC-TMVA binding to fixed
platelets in a dose-dependent manner. To obtain direct
evidence of a specific interaction between TMVA and
GPIba, we investigated mean fluorescence intensity of
GPIb, GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV of
TMVA-treated (10 mg/ml TMVA) washed platelets by flow
cytometric analysis. The result showed that mean fluor-
escence intensity of GPIb remarkably deceased. However,
mean fluorescence intensity of GPIX, GPIIb, GPIIIa, GPIa,
GPIIa and GPIV did not alter (data not shown), indicating
that TMVA does not bind to these platelet glycoproteins. In
summary, TMVA is a GPIb-binding C-type lectin-like
protein and the binding domain for TMVA on GPIb
overlapes with that of HIP1, whose specific binding site
on GPIb was determined at the second leucine-rich repeat
domain (Cauwenberghs et al., 2001).
Several snake venom C-type lectin-like proteins were
characterized as platelet agonist or inhibitor, through
targeting platelet membrane GPIb. Members of the C-type
lectin family binding to GPIb are inhibitory and block vWF
binding (Clemetson et al., 2001), whereas higher multimers
are frequently activating, by either cross-linking GPIb-IX–V,
or by virtue of combining subunits that promote multivalent
interactions involving GPIb and either GPVI or GPIa–IIa
(Wang and Huang, 2001). But how these structurally similar
GPIb-binding venom proteins exhibit distinct biological
functions through the same receptor? Recently investigations
showed that different GPIb-binding venom proteins interact
with different site on GPIb and some of these protein have
more than one binding sites on GPIb (Andrews et al., 2003).
For example, echicetin has at least two binding site on GPIba,
one locating at N-terminal first leucine-rich repeat domain
and another locating at C-terminal flank domain (Peng et al.,
1995).
Based upon the finding that TMVA activates platelet via
GPIb specifically, we proposed to determine binding
domain of TMVA on GPIb. In this assay, mocarhagin, a
cobra venom metalloproteinase, which specifically cleaves
GPIba between Glu282/Asp283 (Ward et al., 1996), was
employed. It is well known that ristocetin does not only bind
to A1 of vWF, but also interacts with GpIba. GPIba
contains several distinguishable domains of residues 1–35
(N-terminal flanking sequence), residues 36–200 (seven
leucine-rich repeats), residues 201–268 (double-loop or
C-terminal flanking sequence) and residues 269–282
(anionic region). GPIba is the only detectable substrate on
the platelet surface of mocarhagin. Mocarhagin cleaves
GPIba between Glu282/Asp283 within an anionic sequence
(269–282) containing three sulfated tyrosine residues (276,
278 and 279), which is predominately for vWF-ristocetin
binding (Andrews et al., 2003). We confirmed the biological
action of mocarhagin by that ristocetin-induced platelet
aggregation was completely blocked by mocarhagin.
Mocarhagin only partly blocked TMVA-induced aggrega-
tion of washed platelets (Fig. 6). In combination with the
inhibitory effect of HIP1, the results suggested that there is
functional binding site(s) for TMVA located within the
N-terminal 282 amino acids of GPIba, because removing
this fragment dramatically decreased the aggregation rate
induced by TMVA.
To our surprise, TMVA still could aggregate platelet
pretreated by mocarhagin even with reduced maximum
aggregation rate. This result suggested that the mocarhagin-
treated GPIba might have functional binding-site for
TMVA, because 1–282 of GPIba is only part of the
extracellular domain of GPIba. We think that this extra
binding site for TMVA, if exists, is masked when GPIba
molecular is intact, because HIP1 nearly completely
inhibited TMVA binding to GPIb. However, when the
1–282 fragment is removed by mocarhagin, the extra site is
available for TMVA binding and this might be the reason
that why TMVA could aggregate mocarhagin-treated
platelet. Our results indicated that TMVA might have
other binding site/or unknown receptor apart from high
affinity GPIba 1–282 fragment. Further investigations are
carried out to check this hypothesis.
H. Tai et al. / Toxicon 44 (2004) 649–656 655
Acknowledgements
We thank Dr Yang Shen and Dr Robert K. Andrews of
the Department of Biochemistry and Molecular Biology,
Monash University, Australia, for mocarhagin.
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