Familial Thrombosis Due to Antithrombin III Deficiency - Blood

Blood.Vol 43. No 2 (February). 1974 219

Familial Thrombosis Due to

Antithrombin III Deficiency

By Ewa Marciniak, Claude H. Farley, and Philip A. DeSimone

A large kindred from eastern Kentucky, withextensive history of recurrent venous throm-basis and pulmonary embolism, was studied.Low antithrombin III titers, ranging from 26%to 49% of normal values, were found in plasmaof nine members in three consecutive genera-

tions; another five members, four of whomwere not available for study, are suspected ofhaving the biochemical defect. There was a

good correlation between clinical symptoms

and antithrombin Ill deficiency, although three

of the younger members with the defect stillremained free of thrombosis. In serum of the

affected subjects antithrombin Ill was almost

completely utilized, which indicates that stoich-iometric binding to coagulation enzymes domi-

nates under biological conditions. Antithrombinand antifactor Xa activities residing in themacroglobulin region of plasma and serum re-

mained unchanged. The responsiveness to hepa-

rin in vitro and in vivo confirmed the evidencethat antithrombin Ill is the sole blood com-ponent through which heparin exerts its anti-coagulant effect. In five affected memberstherapy with oral anticoagulants increased verysignificantly the level of antithrombin Ill in

plasma and contributed to a remarkable in-

crease of residual antithrombin Ill in serum.This objective improvement after warfarin ther-apy may create significant difficulties in thelaboratory diagnosis of antithrombin III de-ficiency.

T THE PRESENT time antithrombin III is recognized as an a2 globulin capable of

inactivating both thrombin and factor Xa in blood.”2’3 This antiproteinase has

been also found identical with so-called heparin cofactor.4’2 Reports on hereditary

deficiency of antithrombin III have been restricted to a single Norwegian family

studied by Egeberg and Abildgaard.5’6” A second large kindred from the Appalachian

Mountains, unusually susceptible to thromboembolic disease in consequence of a bio-

chemical defect, is the subject of the present study. The defect, inherited as an auto-

somal dominant, was limited to a single protein fraction bearing the characteristics of

antithrombin III. In the affected subjects we found a reduced neutralization of throm-

bin and factor Xa both in the absence and in the presence of heparin. During the

therapy with oral anticoagulants the level of antithrombin III in members with the

defect has been significantly increased. These findings substantiate the outstanding

biological role of antithrombin III in the support of blood fluidity and indicate that a

decrease in vitamin-K-dependent coagulation factors abates the utilization of anti-

thrombin III in vitro and probably also in vivo.

MATERIALS AND METHODS

Plasma was obtained by collecting 9 volumes of a fasting blood sample into 1 volume of citricacid-sodium citrate (0.1 M) applying a two-syringe technique. Immediately after drawing, blood

From the Department of Medicine. Unii’ersitr of Kentucky Medical center, Lexington, Ky.

40506.

Submitted May 7, 1973: revised July 5, 1973: accepted Juli’ 16, 1973.

Supported in part by the A merican Heart Association with contribution from the Kentucky

Heart Association, and in part bt’ the University of Kentucky Research Foundation.

Ewa Marciniak, M.D.: Associate Professor of Medicine, University of Kentucky Medical center,

Lexington. Ky. 40506. Claude H. Farley, M.D.: Assistant Professor of Medicine, University of

Kentucky Medical center, Lexington, Kr. 40506. Philip A. DeSimone, M.D.: cluiical Fellow,

Division of Hematology, Department of Medicine, Unii’ersit’i’ of Kentucky Medical center, Lexiizg-

ton, Kr. 40506.

©1974 by Grune & Stratton, inc.

For personal use only.on April 3, 2019. by guest www.bloodjournal.orgFrom

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220 MARCINIAK. FARLEY. AND DESIMONE

was centrifuged in a refrigerated centrifuge at 1 200 g for 20 mm. Plastic syringes, centrifuge tubes,

and storage containers were used. Serum was obtained from blood clotted in a glass tube and incu-

bated for 2 hr at 37CC prior to centrifugation. Ether extract of bovine brain, suspended in saline at

a concentration of 0.03%, was used as partial thromboplastin. This was mixed with an equal volume

of 4% (w/v) kaolin suspension for activated partial thromboptastin time (PTTa) determination.

Sodium heparin (Upjohn) was used. Purified bovine factor Xa,8 purified bovine thrombin,9 andpurified bovine antithrombin 10 were obtained as described before.

Prothrombin time was performed by the method of Quick.” Thrombin activity was standardized

in Iowa units.12 Two-stage prothrombin assay was performed according to the method of Ware and

3 Factors X and Xa were determined as described previously8’3 ; the activity in 1 ml of

normal human plasma was accepted for 100 U. Factor V. VIII, and IX assays were carried out by

one-stage technique on a substrate plasma deficient in the respective coagulation factor. Thrombin

time determination was performed by mixing equal volumes of plasma and purified thrombin

solution of varying activity, indicated in the text. Antitrypsin determination was according to

Homer Ct al.14

Gel Filiratioiz

A sampleof plasma or serum, exactly 5 ml in volume,was applied to the Sephadex G-150 column(Pharmacia) 45 x 2.5 cm, conditioned and eluted with 0.1 M NaCl in 0.04 M Tris-FIC1 buffer, pH

7.8. An up-flow adaptor was used giving the elution rate of 20-25 mI/hr. The efluent was collected

in fractions, exactly 3 ml in volume.

AiitiJ#{224}etor Xa Assay

One volume of the test fraction and 1 volume of saline containing 2 U of purified factor Xa perml and 0.2% of bovine albumin were mixed and incubated at 37#{176}Cfor 30 mm, unless stated other-wise. After the incubation the residual procoagulant activity of factor Xa was recorded as a

clotting time on the substrate bovine plasma3 and compared with a control value, ranging from 17

to 18 sec, and obtained by substituting buffer for the test fraction. The difference between thesetwo evaluations, expressed in seconds, represented the activity of antifactor Xa in the test fraction.

Applying this method of procedure to variable low concentrations of purified antithrombin III, a

rectilinear relationship between prolongations in clotting times and antithrombin III concentrations

was recorded. This did not, however, apply to higher concentrations of antithrombin III, rapidly

inactivating factor Xa, in which case the incubation time had to be considerably shortened.

Antithrombiii Assay

Purified thrombin, 0.1 ml of a 20 U/mI solution, was mixed with 0.1 ml of test fraction andincubated for 10 mm at 37#{176}C.In fractions obtained from plasma after heparin injection this incu-

bation was shortened to 5 mm. At the end of incubation 0.2 ml of acacia-calcium reagent’2 wasadded, immediately followed by addition of 0.1 ml of fibrinogen. The clotting time was recordedand compared with the control time (saline instead of the test fraction, usually 6.0-6.5 sec).Antithrombin activity in the test fraction was expressed as a difference in seconds between these

two clotting tunes.

Heparin �‘onceii tration

This was measured as based on the ability of heparin to potentiate the activity of purified anti-

thrombin Ill.’� Factor Xa was used as the substrate for the inhibition. One volume of the test

fraction was supplemented with 0.5 volume of purified factor Xa of high activity (20 U/mI) and

with 0.5 volume of purified antithrombin III (about 10% of the activity present in normal plasma).After 5 mm of incubation at 37#{176}Cthe residual activity of factor Xa was recorded. it was inversely

proportional to the heparin concentration in the test fraction. A reference curve was obtained with

known heparin concentrations by plotting heparin concentration versus the residual factor Xa

activity.

CASE REPORT

The propositus (ill-i 6) is a 24-yr-old white male who was in excellent health until June 3, 1972,

when waking from an afternoon nap he suffered a sudden onset of chest pain, dyspnca, and hemop-




FAMILIAL THROMBOSIS 221

Fig. 1. Pedigree of family H.

tysis. After admission to the University of Kentucky Medical Center pulmonary embolism wasdiagnosed. No signs of peripheral thrombophlebitis were noted on admission, but venograms per-

formed on the 3rd hospital day revealed the presence of thrombi in the left iliac and the right

femoral veins. Intravenous infusion of heparin was started on the 1st hospital day and continued

for 18 days. On the 8th hospital day the patient developed obvious signs of thrombophlebitis in

the left leg. Chest pain and hemoptysis reoccurred and lasted for a few hours. While continuingheparin, therapy with warfarin was initiated. Symptoms of thrombophlebitis resolved slowly and

disappeared completely by the 17th hospital day. Twenty-four hours after discontinuing heparin,

antithrombin III in patient’s plasma was evaluated and found markedly decreased . He was dis-

charged, continuing the medication with warfarin, and has been controlled periodically over 9 mo,

but no thrombotic episodes reoccurred. During the entire period of hospitalization the patient

demonstrated an unusual, apprehensive attitude. He stated repeatedly that symptoms similar tothese observed in him were seen previously in several members of his family causing sudden deathin two of his siblings.

History of Family H

The family has been known to inhabit an Appalachian region of Eastern Kentucky for at least acentury, and most of the members still reside there. A few of them moved to Ohio, Indiana, andMichigan. A history of consanguinity was denied. The four generations that we have examined are

presented in Fig. 1. Plasma level of antithrombin Ill for each investigated subject is shown in

Table 1.

The matriarch of the family is, at the age of 83, in considerably good health with normal anti-thrombin III level. Her husband died at age 70, of stroke. For many years he suffered recurrent

pain, swelling, and ulcerations of legs suggestive of thrombosis.

Subject 11-3, the 62-yr-old uncle of the propositus, with markedly decreased antithrombin III,

has a history of long-lasting swelling and pain in both legs with secondary crural ulcerations. Hewas hospitalized at the age of 58 with thrombophlebitis and suspicion of pulmonary embolism, and

treated briefly with oral anticoagulants. Thereafter he had at least one additional incident of

thrombophlebitis.

Subject 11-4, the 59-yr-old father of the propositus, suffered recurrent leg thrombosis since the

age of 26, and had two incidents of pulmonary embolism. When we initially evaluated his anti-

thrombin III and found it abnormally low, he had been irregularly treated with small dose ofhedulin for few years.

Subject 111-1 is a 34-yr-old woman, with decreased antithrombin III, who after the delivery of her

third child, at the age of 25, developed severe thrombophlebitis in left leg, which recurred several

times over the following few months. Treatment with oral anticoagulants lasted for 3 yr. Since

then she has remained free of thrombotic episodes, although for 3 yr she took oral contraceptives.

I �10ii -

‘U

� � r�56 7 S 9 0 II I��j�I4 1 9f 2Of 21 22 23 24

ASYMPTONATIC: HISTORY OF THROMBOSIS:

o normal A-� Q normal A-�

rA-nz<50S IA-Ill <50%

f�J not investigated � not investigated

�PROPOSITUS

f DEAD




222 MARCINIAK. FARLEY. AND DE SIMONE

Table 1. Antithrombin Ill Levels4 in Plasma from Members of Family H

Subject A�Ill (%) Subject A-Ill (%)

I-f 79 111-9

111-10

Ill-li

39

47

78

11-2 77 111-12 31

11-3 31 111-15 49

II.4t 32 III-16� 48

11-5 116 111-17 90

11-6 118 111-21

111-22

85

105

Ill-i 26 111-23 100

lll-4� 83

111-6 87 lV-1 91

111-7 89 IV-2 82

111-8 89 lV-3 32

Normal range 80%-117%.

tOn hedulin.

�On warfarin for 5 yr.

§ Propositus, on warfarin for a few days.

Subject 111-4 is a 26-yr-old man who had his first thrombosis and pulmonary embolism at the

age of 15 , after fracturing the left femur. Since then he has had 10 episodes of recurrent throm-bophlebitis and at least three additional incidents of pulmonary embolism. Continuous therapy

with warfarin was instituted at the age of 20, when multiple pulmonary embolization occurredafter a plastic surgery of the leg for crural ulcers. Since the age of 21 he has remained asympto-

matic but has continued the therapy with warfarin. Despite his dramatic clinical history the plasma

level of antithrombin III was now found within the normal range. After observing in the other

affected members of his family that a prolonged treatment with warfarin may considerably in-crease the biological activity of antithrombin III, we presume that this subject has the congenital

biochemical defect.

Subject IlI-lO, age 38, is the oldest brother of the propositus, with both legs amputated below

the hip. He lost his left leg at the age of 23, a few months after developing pain and progressive

numbness in this extremity.At that time he was told that he had Beurger’s disease. The right leg

was amputated at the age of 29 in consequence of severe circulatory disturbances indicative of

venous obstruction with secondary pretibial ulcerations, which lasted for 4 yr. He has had delayed

healing of the stump with recurrent ulcerations. We found that his antithrombin Ill is significantly

decreased.Subject 111-12 is a 33-yr-old brother of the propositus, with decreased antithrombin III. He had

a single incident of thrombophlebitis following appendectomy, at the age of 25.

Subject Ill-i 3, another brother of the propositus, is said to have had symptoms suggestive of

thrombophlebitis. He died suddenly at the age of 27, when in the military service in Korea. Hiscollegues were alarmed when they heard him breathing with an extreme difficulty, but found him

dead upon entering his room. On the autopsy a marked vascular congestion of the lungs, pulmon-ary edema, and right heart dilatation were noticed. This implies that pulmonary embolism was

most likely the cause of a sudden death.Subject 111-20, a sister of the propositus, died suddenly at the age of 18, with obvious signs of

pulmonary embolism. Shortly before death she was released from the local hospital where she

received treatment for bilateral pneumonia.Two individuals in the third generation (111-9, age 13, and 111-15, age 26) and one in the fourth

(IV-3, age 9) have the biochemical defect but have not shown any thromboembolic symptoms.They are of considerably young age. Since all of the older members of the family with the defecthave had symptoms, it can be predicted with a fair degree of probability that these three will also

in the future develop the clinical disease.




7

6

5

4

3

2

60

40

20 -�

U

In

w0�

I-

(9

60 Z

I-0-J

40 0

‘1

20

6

5

4

3

2


FRACTION NUMBER

Routine Laboratory Evaluations

RESULTS

The propositus and subjects 11-3, 11-4, and 111-1 were evaluated for evidence of

hemostatic abnormalities. The platelet count, fibrinogen level, PTTa, euglobulin lysis

time, and thrombin time with thrombin solutions ranging from 2 to 20 U of activity

per ml gave normal results. In addition, subjects 11-3 and 111-1 before the therapy with

oral anticoagulants had a norni�il prothrombmn time and two-stage prothrombmn level;

determinations of factOrs V, VIII, IX, and X were also within normal range. The

thromboplastin generation test with 11-3 plasma and serum, utilizing platelet substi-

tute, was normal. Cellulose acetate electrophoresis of serum from the propositus and

subject 111-1 showed no abnormalities. Serum levels of antitrypsin in subjects

11-3, 11-4, and 111-I were increased, assuming, respectively, values of 1.72, 2.22, and

1 .69 mg trypsin inactivated per ml of serum (normal range 1 - 19 + 0. 1 8).

Evaluation of Antithrombin III

Since in human blood �lt�tivities directed against thrombin’6”7 and against factor

)(.a3”8 are not limited to antithrombin III, but have been found also in connection

with macroglobulins, it was desirable to fractionate each specimen before analysis. For

this purpose plasma or serum was filtered on Sephadex G-1 50 and antiproteinases

were analyzed in isolated fractions.

Figure 2 compares the result obtaihed by measuring antithrombin and antifactor

Xa in normal plasma and serum with representative results from one of the affected

subjects (111-1). Antithrombin III, which is eluted from Sephadex with the last proiein

peak, was of significantly lower activity in 111-1 plasma than in normal plasma. In

Fig. 2� Antithrombin (trian-gles) and antifactor Xa (solid cir-cles) #{246}valuationsin plasmas andaera after gel filtration on Sepha-dex G-150.

C

0

4

NORMAL PLASMA

n�

Ill-I PLASMA

u�

NORMAL SERUM

�

ill-I SERUM

�30 40 50 6030 40 50 6O�




224 MARCINIAK, FARLEY. AND DE SIMONE

normal serum a partial utilization of antithrombin III during blood coagulation was

apparent. A total utilization took place in case of Ill-I serum from which antithrom-

bin III was virtually absent. This finding is ofparticular importance since it reflects an

extensive functional involvement of antithrombin III during the process of blood

coagdlatibn. The capacity of antithrombin in the macromolecular fraction from 111-1

serum was slightly greater than in control. In the macroglobulin fraction from plasma

aritithrombin was not analyzed since a considerable adsorption of thrombin on fibrin

that forms in these fractions interferes with the assay. Antifactor Xa in the macro-molecular region revealed activity in normal plasma and serum similar to that in ab-

normal plasma and serum.

The procedure of gel filtration was standardized and applied to plasma samples fromseveral normal subjects and to plasma from members of family i-i. Occasionally serum

samples were also analyzed. We limited the activity studies in filtered fractions to anti-

factor Xa assay because it gave in our hands steady results of a more reproducible

character than results obtained in the evaluation of antithrombin. The capacity of

antithrombin III in a given plasma sample was expressed as the sum of prolongations in

clotting time (during 30-nun incubation with factor Xa) in all fractions located within

the last protein peak. This value in the analysis of 14 normal plasmas ranged from 240to 350 sec with a mean of 299.6 ± 30.4 sec. Accepting antithrombin III activity in

normal plasma for 100% we thus obtained a normal range of 80%-l 17% and a standard

deviation of 10.1%. A normal plasma in which antithrombin III concentration was

evaluated as 92% was diluted with the buffer used in filtration and samples containing

80%, 50%, 40%, and 25% of plasma were subsequently analy±ed. Antithrombin III

titers of 73%, 43%, 35%, and 25% respectively, were obtained confirming the useful-

ness of the method for quantitative analysis.Table 1 shows the values obtained in plasma of family H members. Nine out of 24

analyzed subjects gave values that were significantly lower than those in the normal

group, ranging from 26%-49% of normal activity. Six of the biochemically abnormal

members had history of thrombosis. The seventh member with extensive history of

thrombosis (111-4) revealed normal antithrombin III level, bdt, as mentioned before,

he has been on long-term medication with warfarin. The effect of this medication on

antithrombin III in other members of the family H is documented below. Also the

values originally obtained in the propositus and in his father (11-4), although distinctlybelow normal, might have been to some extent altered by the theEapy with oral anti-

coagulants.

Effect of Heparin In Vitro and In Vivo

In vitro studies were performed on fractions from plasma and serum isolated by gel

filtration. Factor Xa was used as a substrate for inactivation. After addition of heparin

the inactivation of factor Xa was greatly accelerated, but limited to fractions located

in the antithrombin III region (Fig. 3). Plasma fractions from subject 11-4 showed a

diminished responsiveness to heparin. Fractions from 11-4 serum which contained

practically no antithrombin III were almost totally resistant to heparin. These results

are in agreement with the assumption that antithrombin III is the substance throughwhich heparin exerts its anticoagulant effect in blood.

The responsiveness in vivo was evaluated after intravenous injection of 100 U of




FRACTION NUMBER

60

40

Fig. 3. Effect of heparin in vi-tro on antifactor Xa activity (solidcircles) in fractions after gel fil- 20tration. Heparin at a concentra-tion of 0.25 �g/ml of fraction was

added; each fraction was then in-cubated with factor Xa (10 U/mI) 0for 5 mm and the loss of coagu-lant activity evaluated.

heparin/kg. In subjects 11-3 and 11-4 the hypocoagulable state, when evaluated by

thrombin time assay, was of much shorter duration and lower intensity than in normal

volunteers (Fig. 4). This was, however, less pronounced when we monitored the effect

of heparin by PTTa assay (Fig. 5). Particularly in the case of patient 111-1, the inhibi-

tion in PTTa after heparin was better expressed than in some of the controls and did

not correlate with her low antithrombin III level. Unfortunately, she was not followed

>6OQL CONTROL #{149}

CONTROL 0400 13-3 #{149}

fl-4 U

201

100

Fig. 4. Changes in thrombin time after heparin

injection in two normal volunteers and two sub-jects with antithrombin Ill deficiency.

I 2 3

HOURS AFTER HEPARIN


Ua,

U)

Lii

I.-

z

0cr

F-

EC

0

c’J4




15 60 120

MINUTES AFTER HEPARIN

up by thrombin time evaluations. However, we could demonstrate the insufficiency of

antithrombin III in her plasma even during the high heparin concentration in blood. As

shown in Fig. 6, the analysis of plasma obtained 15 mm after heparin injection re-

vealed a higher activity of heparin in subject 111-1 than in the normal volunteer. At the

same time, however, antithrombin III fraction of the affected subject remained in-

significant in its antithrombin activity, which was in sharp contrast to the eminent

elevation in the control. The discrepancy between this result and the marked hypo-

coagulability of whole plasma in subject III-! was apparently due to the fact that be-

fore the fractionation heparin at high concentration remained in contact with anti-

thrombin III compensating for its low level. During the fractionation on Sephadex

nuost of the heparin had been separated in association with other protein structures,’5

eliciting the defect.

Ua,10

Lii

I-

(9zF-F-0-JU

‘I

20 30 40 50 20 30 40 50

FRACTION NUMBER


>10001 II � ft

GOOL

Ua,

(I)

0F-I-a.

o CONTROL

o CONTROL ()�o CONTROL Q#{225}�

D fl-3d’#{149}11-4 0’

0111-I �

Fig. 5. Changes in PTTa after heparin in-

jection in three volunteers and three subjectswith antithrombin III deficiency.

Fig. 6. Gel filtration ofplasma from blood taken 15mm after intravenous injection

of 100 U heparin/kg. For eval-uation of antithrombin ac-tivity (dotted line) each frac-tion was incubated withpurified thrombin (20 U/mI)for 5 mm. Heparin (solid cir-cles) filters presumably in as-sociation with beta lipo-proteins.1 �





‘Patient postaortic valve replacement, on warfarin for 5 yr.tThis patient was taking hedulin irregularly at the time of analysis.

Table 2. The Effect of Therapy With Warfarin on Antithrombin Ill Level in Deficient Subjects

SubjectDuration of

TherapyProthrombin

Time (sect)

A-Ill Level

in Plasma (%)

11-3 Before4 days

4mo

12.9

18.3

17.3

31

41

56

11-4 Before’

5 days

3wk

2 mo

12.0

23.0

23.0

20.0

32

48

52

79

Ill-i Before

4 days1 mo

11.6

20.0

20.1

26

68

68

111-15 Before

6mo

11.8

18.5

48

76

111-16 14 days

20 days

4mo

6’/2 mo

9 mo

22.0

18.5

18.2

19.8

22.6

48

51

58

78

61

‘On hedulin.

Results of Therapy With Oral Antocoagulants

The propositus and subsequently four additional members of his family have been

treated with warfarin in the conventional manner. In response to this therapy a sig-

nificant increase in antithrombin III activity in plasma has been observed (Table 2).

Even after a few days of treatment, subject 111-1 showed more than twice as much of

antithrombin III as she had before. In subject 111-4 almost normal values were recorded

after 2 mo of the therapy. The propositus also gave objective evidence of improvement

over several months of treatment. In his case, however, the assessment is not quite

accurate, since the prewarfarin level of antithrombin III remains unknown.

The difference between the residual antithrombin III in serum before and during

the therapy with warfarin was remarkable (Table 3). In subjects with the defect,

serum-antithrombin III increased from near-zero level to a range seen in normal sera.

Higher residual values, however, were observed in the serum of a patient treated with

warfarin, who was without an apparent defect in antithrombin III synthesis. In this

Table 3. The Effect of Warfarin Therapy on Res idual Antithrom bin I II in Serum

Plasma A-Ill (%)Prothrombin

Subject 2-stage (U) In Plasma In Serum

Normal control 300 103 47

Control on warfarin’ 48 107 92

lI.4� 227 32 <5ll-4onwarfarin 53 79 56

Ill-i 250 26 <5

Ill-i on warfarin 57 68 55





latter case we could not account for a significant increase of plasma antithrombin III

above normal level. This leaves us with the impression that stern stochiometric propor-

tions are implicated in the neutralization ofcoagulation enzymes in blood.

DISCUSSION

The biochemical defect in family H is apparently transmitted by an autosomal gene

and has a dominant character with incomplete penetrance. Nine members are heter-

ozygotes with antithrombin III ranging approximately from 25% to 50% of normal

level, as evaluated by the anticoagulant properties. An additional five members, three

of whom died before we started the investigation, are suspected of having the de-

ficiency. Although there is a good correspondence between the presence of the bio-

chemical defect and the occurence of thrombosis, the clinical symptoms appear rather

late in life, frequently after the age of 20. A similar hereditary abnormality was re-

ported originally by Egeberg 8 yr ago.5 No other evidence in the literature indicates

that additional families are involved. Taking into consideration the dominant character

of this trait, one has to assume that the frequency of the abnormal gene must be

extremely low. On the other hand, the evaluation of antithrombin III, at the present

time, belongs to seldomly used laboratory procedures, and available methods are far

from being satisfactory. Thus, a number of cases may escape the evidence of the in-

vestigator, despite a remarkable family history. In studies conducted on sporadic cases

with thrombosis Abildgaard et al.’9 observed frequently a marked decrease in plasma

levels of antithrombin III, but attributed this to an enhanced consumption. Hensen

and Loeliger2#{176} recorded normal values in all their patients with thrombosis.

Antithrombin III has the distinction of being a protein with well-defined properties,

known to interact stoichiometrically with thrombin2’ and with factor Xa,’#{176}on ac-

count of which it can be visualized as an insurance agent against acute thrombotic

episodes. Nevertheless, the question which remains to be answered is, to what extent

normal persons benefit from these properties of antithrombin III. The existence of

individuals with incomplete antithrombin III synthesis due to a selective genetic de-

fect, many of whom develop deep vein thrombosis and pulmonary embolism without

any apparent cause, not only buttresses the biological significance of antithrombin III

but indicates that the tendency for intravascular coagulation has been generally under-estimated. The ratio of antithrombin III capacity to the potential capacity of thrombin

undoubtedly plays a very important role in the prevention of thrombosis. The liver

appears to be the only organ in which antithrombin III is produced. In addition to the

inborn defect, low levels are commonly observed in liver cirrhosis.’9’2#{176} The explana-

tion for the observation that thrombosis does not appear in the cirrhotic patient to the

same extent as in congenital deficiency of antithrombin III lies in the differential re-

quirements of the two abnormalities for antithrombin III. In liver cirrhosis the synthe-

sis of prothrombin and factor X is impaired, which lowers the requirement for the

inactivator. For members of family H with normal levels of vitamin-K-dependent

coagulants, higher levels of antithrombin III are required in order to support ade-

quately the fluidity of their blood. On this assumption one would expect a significant

improvement of the biologic equilibrium in patients with hereditary deficiency of

antithrombin III by lowering their level of circulating procoagulantsl The relatively

short time during which a systematical therapy with warfarin has been administered in

members of family H does not allow us yet to assess properly their clinical responsive-





ness. Nevertheless, as a result of the therapy, not only the residual antithrombin III in

serum appeared in quantities indicating a generous surplus over the capacity required

for thrombin inactivation, but a significant increase in plasma levels has been recorded.

The latter effect might be attributed to either the possibility that a catabolic pathway

of antithrombin III is also induced by its stochiometric binding to activated coagula-

tion enzymes, or that coumadin stimulates the synthesis. Of interest is the fact that,

although an increase in normal plasma levels of antithrombin III in patients on oral

anticoagulants has been noticed before,20’4 it was usually of a very moderate degree.

Similar therapy in some of the subjects with the defect whom we observed raised the

level of antithrombin III more than twice above the original value. At the present time

we are unable to offer an explanation for this discrepancy. The fact that must be taken

under consideration, however, is that a long-lasting therapy with oral anticoagulants

giv#{231}san objective improvement in patients with the hereditary antithrombin III de-

ficiency, which worsens the conditions for a laboratory diagnosis of this abnormality.

The responsiveness to heparin in vivo in nuembers of family H reflects a direct stimu-

lation of antithrombin III, which as a sole blood component in concert with heparin

provides the anticoagulant effect. Only in the event of a complete absence of anti-

thrombin III will this effect be abolished. Such a condition, however, in a human being

is unknown and probably incompatible with life. In heterozygotes, like the menubers

of family H, it is a question of supporting an adequate amount of heparin in order to

compensate for low antithrombin III level. This amount may not differ significantly

from a conventionally applied dose and is probably highly individual, since the distri-

bution of heparin in blood depends on other plasma components than antithrombin

111.15 When an abnormal type of gamma globulin appears in the circulation, the

therapy with heparin may be completely futile despite a normal antithrombin III

level.22 Partial neutralization of heparin by blood cell constituents23’24 found in

platelets, e�ythrocytes, and neutrophils also may contribute to a variable responsive-

ness. Furthermore, the rate of heparin decay or elimination from blood, the niecha-

nism of which remains unknown, will influence the duration and extent of the anti-

coagulant effect. Thus, variations in the responsiveness to heparin among persons with

an inborn error of antithrombin III metabolism, as unrelated to the profoundness of

the defect, are not surprising.

In the view of a high frequency of thrombosis in the family H, it seems unquestion-

able that antithrombin III represents the principal biochemical safeguard against fibrin

deposition inside the vessels. As documented previously,’0 under biological conditions

thrombin constitutes the main target for this antiproteinase, factor Xa being protected

by a complex formation with phospholipid and factor V. Of some concern, however,

is the specificity of other natural proteinase inhibitors. Despite elevated antitrypsin

activity in antithrombin Ill-deficient sera, we could not account for any thrombin

inhibition that might have corresponded to the alpha-i antitrypsin fraction. This con-

tradicts some of previously published suppositions25 in regard to the role of antitryp-

sin in binding and disposition of thrombin. In the case of this antiproteinase, however,

the time of interaction26 and species specificity of the reacting components21 may

contribute to outstanding discrepancies in experimental results. It is also difficult to

determine critically the biological implications of macroglobulin fraction for throm-

bosis and hemostasis. Although a bimolecular binding of thrombin’6 and factor Xa’8

by components of this fraction has been documented, the inhibitory capacity of




230 MARCINIAK. FARLEY, AND DESIMONE

macroglobulins for coagulation enzymes seems to remain unchanged after blood

clotting, even in subjects with antithrombin III deficiency. This may imply that the

biological role of macroglobulins is rather negligible. On the other hand, experimental

procedures may alter significantly the physiological environment in which macro-

globulins function as inactivators of coagulation enzymes.3”8

ACKNOWLEDGMENT

We would like to thank Dr. Douglas Rees for serum antitrypsin estimations. The technical

assistance of Mrs. Sharon Calhoun and the secretarial service of Mrs. Pat Schrils are gratefully

acknowledged.

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1974 43: 219-231

Ewa Marciniak, Claude H. Farley and Philip A. DeSimone Familial Thrombosis Due to Antithrombin III Deficiency

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