Inherited Antithrombin III Deficiency and Cerebral Thrombosis in a Child

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PEDIATRICS Vol. 65 No. 1 January 1980 125

Inherited

Antithrombin

Ill Deficiency

and

Cerebral

Thrombosis

in a Child

Daniel R. Ambruso, MD, Linda J. Jacobson, and William E.

Hathaway, MD

From the Department of Pediatrics, University of Colorado Medical Center, Denver

ABSTRACT. Identification of a family affected by anti-thrombin Ill-heparin cofactor (AT-Ill) deficiency was made after diagnosis of the index case, a 15-year-old boy

who suffered cerebral thrombosis. The proband had a

two-year history of recurrent thrombosis involving the lower extremities. His mother and sister were also af-fected. Studies showed a decreased biological activity (AT-IlL) and antigen (AT-III5) by the Laurell technique in the proband (AT-III = 0.32, ATIIIag 46%), his sister

(AT-III = 0.29, AT4IIag 47%), and his mother (AT-III, = 0.41, AT4II8g 56%). Crossed immunoelectropho-resis (CIE) of the affected individuals’ plasma in

agarose-containing heparin demonstrated a normal pattern of

migration. Treatment with warfarin sodium (Coumadin) resulted in an increase in activity in two of three affected family members, and in antigen in all three. Anticoagu-lant therapy did not affect the pattern of AT-Ill on CIE. This family represents a quantitative deficiency in anti-thrombin III. A review of the reported cases of antithrom-bin III deficiency indicates that individuals with this disorder may have thromboembolic disease in childhood. Pediatrics 65: 125-131, 1980; antithrombin III, anti-thrombin III deficiency, cerebral thrombosis.

Antithrombin III deficiency is a rare disorder

inherited as an autosomal dominant trait. It is

manifested by recurrent thrombotic episodes as a

result of decreased levels of antithrombin Ill-hep-arm cofactor (AT-Ill). Although complications of

this disease usually occur during the early adult

years, several instances of thrombosis associated with AT-Ill deficiency have been reported in the pediatric age range. ‘ The index case of the present

Received for publication Jan 30, 1978; accepted Aug 4, 1978. Publication of this article was unavoidably delayed by adminis-trative problems.

Reprint requests to (D.R.A.) Department of Pediatrics, Box C220, University of Colorado Medical Center, 4200 E Ninth Aye,

Denver, CO 80262.

report had confirmed thrombotic episodes begin-ning in early adolescence. Evaluation ofthe biologic activity, antigenic levels, and pattern of the protein on crossed immunoelectrophoresis (CIE) indicated a quantitative deficiency of AT-Ill in three family members.

METHODS

Coagulation studies and AT-Ill biological activity were performed on plasma drawn from the subjects by the two-syringe technique; nine volumes of blood were drawn into one volume of anticoagulant (three parts 0.1 M sodium citrate and two parts 0.1 M citric acid). Plasma was separated by centrifugation at 1,400 g for 30 minutes at 4 C. Platelet count, prothrombin time (PT), kaolin-activated partial thromboplastin time (KPTT), thrombin time (TT), fibrinogen levels, and euglobulin lysis time were determined by standard methods. Fibrin-fibrinogen split products (FSP) were measured by the FDP latex suspension test,8 and fibrin monomer was measured by a protamine precipitation test.9 The AT-Ill clotting activity was determined in defibri-nated plasma by a previously described method.’#{176} The results are expressed as an index based on normal subject pooled plasma tested simultane-ously. The normal range was obtained by

determin-ing AT-Ill activity in 24 normal adults.

Serum was prepared by allowing a clot to form in

whole blood in glass tubes for two hours at 37 C.

The sample was then centrifuged at room temper-ature at 1,400 g. The AT-Ill antigen levels were determined by the immunoelectrophoresis tech-nique of Laurell,” using an antihuman AT-Ill an-tibody, and compared to serial dilutions of a normal

serum pool. The normal range was obtained by

determining AT-Ill antigen levels in 57 normal adults.

Crossed immunoelectrophoresis analysis of

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ATllIag

ATlllc

.29

ATlIlag

477.

ATlllag

93

ATlllc

.32

ATlIlag

46%

/

Fig I . AT-Ill activity and serum antigen levels in family members. AT-IlL indicates AT-III clotting activity index (normal, 0.66 to 1.38); AT-IlL5, quantification by immunologic method (normal, 80% to 116%); shaded symbols, affected family members.

III was performed according to the technique of Sas et 12Glass plates, 75 x 50 mm, were covered with

6 ml of 1% agarose containing 16.6 units/mI of

heparin. A 2.7-mm well was cut on the cathode end of the slide, and the plasma sample of 5 .tl was placed in the well. A second well was cut 2 mm below the first, and dye-tagged albumin marker was introduced here. An electrophoretic field of 10 mamp was applied to the slide to produce migration

of 3.5 cm of the marker. The upper portion of

agarose on the plate was removed and replaced with fresh agarose containing 2% AT-Ill antibody. An electrophoretic field of 10 mamp was again applied to migrate the albumin marker 3.5 cm

ver-tically. The area under the peaks was estimated by

the formula A = H x W, where H represents peak

height and W represents width at one-half height.’3 The ratio of the area of the first peak (the more

slowly migrating peak, which has been described as

AT-Ill bound to activated procoagulant molecules and thrombin) to that of the second (the more rapidly migrating peak, which has been described

as free AT-Ill) was calculated for plasma in 17

normal individuals of various ages.’4 The mean and standard deviation were computed for the control group. The ratio of areas of the peaks were calcu-lated for the affected individuals in the family in the same manner.

CASE REPORT

A 15’/2-year-old white boy was seen by his private physician with a one-week history of left eye pain and confusion. He was admitted to a hospital near his home with aphasia and proprioceptive agnosia. Laboratory studies included a lumbar puncture with normal opening

pressure, spinal fluid with RBCs and xanthochromia, a

normal complete blood cell count, and an EEG that

showed abnormal spike and wave pattern over the left

midtemporal area. Because of increased somnolence over the next few days, the patient was transferred to Colorado General Hospital, Denver, for further evaluation.

Physi-cal examination at this time showed normal vital signs,

blurred disk margins, nuchal rigidity, receptive aphasia, and proprioceptive and visual agnosia. He weighed 55 kg.

The patient had a superficial venous thrombosis re-lated to minor trauma in his right calf two years earlier. This diagnosis was made on clinical grounds. At 14 years of age, he was hospitalized for viral pneumonia, and deep vein thrombosis (confirmed by venography) developed in the left groin. This episode was treated with heparmn, acutely followed by therapy with warfarin sodium (Cou-madin). However, the warfarin treatment was terminated three months prior to his most recent illness. In the past, the patient’s sister had suffered from venous thrombosis in the lower extremities. Venography at the time of diagnosis indicated bilateral iiofemoral thrombosis that was more extensive on the left. The patient’s mother also had a history of several episodes of superficial throm-boses.

An arteriogram and computerized tomographic scan of the cranium demonstrated a large, avascular, left tem-poral mass. Craniotomy and biopsy disclosed a hemor-rhagic infarct of the left temporal lobe. Examination of the biopsy material revealed multiple areas of venous

thrombosis without involvement of the arterial system. The patient’s postoperative course was complicated by inappropriate antidiuretic hormone secretion, nuchal n-gidity, meningitis, hypersensitivity reaction to phenytoin (Dilantin), and several episodes of superficial thrombosis associated with the use of intravenous catheters. The pediatric hematology service was consulted on the sixth postoperative day.

Coagulation studies included the following values:

platelets, 260,000/cu mm (normal, 200,000 to 450,000); bleeding time, normal; KPTT, <30 seconds (normal, 37

to 50); PT, 16.7 seconds (normal, 12.0 to 14.5); TT, 12.4 seconds (normal, 8 to 10); fibrinogen, 140 mg/100 ml (normal, 200 to 450); FSP, <200 but >100 tg/ml (normal, <10); fibrin monomer, positive (normal, negative); AT-Ill activity index, 0.25 (normal, 0.66 to 1.38); AT-Ill antigen,

60% (normal, 80% to 1 16%). Heparin was started on the 15th hospital day; although the patient received as much as 130 units/kg every four hours, his KPTT remained below 37 seconds. On the 25th hospital day, therapy was switched to warfarin sodium, 8 mg/day orally. By the

time of discharge, the FSP decreased to 20 jig/ml; fibrin

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ARTICLES 127

Peak

1 Normal

Adult

Fig 2. Plasma AT-Ill by immunoelectrophoresis. Top left, Normal adult; top right, patient after warfarin

ther-TABLE 1. AT-Ill Activity Index and Serum Antigen Levels of Affected Family Members Before and After Treatment with Warfarin

Before Treatment

After Treatment

Activity Serum Activity Serum

Index Antigen (%) Index Antigen (%)

Proband 0.32 46 0.45 65

Sister 0.29 47 0.28 62

Mother 0.41 56 0.79 65

* Normal ranges: activity index, 0.66 to 1.38; serum

anti-gen, 80% to 116%.

KPTT, 37 seconds; PT, 19.2 seconds; AT-Ill activity index, 0.32; and AT-Ill antigen, 46%. He continued to receive warfarin at the time of discharge from the

hospi-RESULTS

As indicated in the family history, three family

members had significant problems with thrombosis.

apy; crossed bottom left, patient’s sister before warfarin therapy; and bottom right, patient’s sister after therapy.

TABLE 2. Ratio of Area of Peak 1 (Bound) to Peak 2 (Unbound) Plasma AT-Ill on Crossed Immunoelectro-phoresis

Ratio

Before After

Treatment Treatment

Proband 0.18 0.23

Sister 0.23 0.22

Mother 0.24 0.18

Control (N = 17) 0.24 ± 0.06*

* Mean ± 2 SD.

When screened for levels of AT-Ill, these three individuals-the proband, his mother, and his sis-ter-were affected with the deficiency (Fig 1). All three had a decrease in clotting activity levels of AT-Ill as well as in serum (Fig 1) or plasma (Fig 2) antigenic levels. Coagulation studies (PT, KPTT, TT, fibrinogen, euglobulin lysis time, factor VIII level, platelet count, and bleeding time) in the pro-band’s sibling and mother all gave normal findings.

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TABLE 3. Clinical and Laboratory Findings in Hereditary AT-Ill Deficiency

Source No. of

Affected Family Members

Age at 1st Thrombosis

Type of Thrombosis

Range of AT-Ill Levels Comments

(% of Normal)

Coagulation Immunologic Method Method

Egeberg’ 9/27 40-60 ...

Mean AT-Ill

time, 16.6 sec; con-trols, 40.2 sec

van der Meer3

Shapiro et al#{176}6

20/45 19-44 yr

>30 yr 50

Normal in proband

Sas et al52’ 9/33 18 yr-mid-20s Lower extremity

thrombophlebi-tis; postpartum

thrombosis

Egeberg2 4 24-29 yr

von Kualla and von Kualla2 Marciniak et al4 Mid-20s; 2 children, 10

and 11 yr

12/26 (3 Mid-20s-early

families) 30s; one

pa-tient, 16 yr

9/24 15-58 yr; most in mid-20s Lower extremity venous throm-bosis; puhno-nary embolus; cerebrovascu-lar thrombosis Lower extremity thrombophlebi-tin; pulmonary embolus; Budd-Chiari syndrome; postpartum thrombosis

Lower extremity 54-74

thrombophlebi-tin; pulmonary

embolus;

post-partum throm-bosis

Lower extremity 50

thrombophlebi-tis; pulmonary embolus

Lower extremity 26-49

thrombophlebi-tis; 2 sudden deaths due to pulmonary em-bolus

Lower extremity 50

thrombophlebi-tis; pulmonary embolus

5-30 in 7 af-fected members

Plasma, 25-57 Serum, 10-28

Decreased “hepa-rin cofactor” ac-tivity

3 children <12 yr affected, but not sympto-matic; fibrino-gen and pro-thrombin me-tabolism not

in-creased

. . . In vivo heparin resistance in 3 affected individ-uals; warfarin therapy related with a signifi-cant increase in AT-Ill activity in 5 affected in-clividuals

Proband had de-creased inacti-vation of Xa and thrombin5;

immunoelectro-phoresis of AT-HI suggestive of abnormal

pro-tein2’

. .. Warfarin therapy

increased

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ARTICLES 129 TABLE 3.-Co ntinued

Source No. of Age at 1st Type of Range of AT-Ill Levels Comments

Affected Family Members

Thrombosis Thrombosis (% of Normal)

Coagulation Immunologic

Method Method

Gruenberg et 1 35 yr Mesenteric in- .. . 25-30 ...

al23 farction related

to venous thrombosis

#{216}deg#{226}rdand 1 51 yr Mesentenc ye- 49 50 ...

Fagerhol’TM nous

thrombo-sis; lower ex-tremrty

throm-bophlebitis

Carvalho and Ellman6

5/6 16-38 yr Lower extremity

thrombophlebi-tis; pulmonary embolism

58-75 40-58 One family

mem-ber died at 18 yr with pulmo-nary embolism; amount of thrombin neces-sary to induce aggregation in gel-filtered platelets de-creased

Gomperts et 3 siblings Early 20s .. . .. . 25-35 Immunologic

al24 levels of AT-Ill

increased to 42%-50% during warfarin ther-apy

Filip et al7 10/34 Proband, 1 1

yr; sibling, 16 yr

Lower extremity thrombophlebi-tis; pulmonary embolism

28-67 37-56 ...

Stanthakis et 5/12 20-36 yr Lower extremity 50-58 38-55 All affected

mdi-al25

thrombophlebi-tis; pulmonary

embolism; 1

death due to mesenteric thrombosis

viduals have de-creased in vitro heparin toler-ance tests in plasma-throm-bin system

After the initial studies, the affected family mem-bers were treated with warfarmn. The antigen levels of all three patients increased, as did activity levels in two of the three, while they received the drug

(Table 1). Fig 2 illustrates CIE patterns for normal plasma, for the patient while receiving warfarin, and for his sister before and after therapy with warfarin. The patterns appear identical except for a decrease in absolute size of the peaks in the patients with AT-Ill deficiency. The patient’s sister showed an increase in the size of the peaks during therapy. The patterns of the proband’s mother demonstrated the same abnormality. Table 2 sum-marizes the results of the ratio of the area under the peaks in plasma. The ratios calculated for the family members with AT-Ill deficiency are within the range of values obtained from normal

individ-uals, and there are no differences in the ratios

during therapy.

DISCUSSION

Antithrombi III-heparmn cofactor is an a2-glob-ulin that is a major physiologic inhibitor of acti-vated coagulation serine proteases.’5 Heparin af-fects this interaction by altering the configuration of the AT-Ill molecule and allowing it to bind the activated procoagulant molecule more quickly.’6 AT-Ill can be measured in several ways. Its biolog-ical activity is estimated by measuring the macti-vation of thrombin or activated factor X in a clot-ting assay. Heparin cofactor assay, which is also a test of AT-Ill activity, measures the inactivation of

thrombin in the presence of heparin. The amount of protein in plasma is detected by either single radial immunodiffusion or immunoelectrophoresis

techniques using an antibody to AT-Ill. Recent

reports indicate that both AT-Ill activity and

an-tigen levels are low at birth (45% and 65% of normal,

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respectively) and that AT-Ill reaches adult levels by 6 to 12 months of age.’7 At least in men, the levels of AT-Ill gradually decrease again after 30 years of age.’8

Until 1975, information accumulated on patients with AT-Ill deficiency, including measurements of activity and antigen levels, suggested that the con-dition was a true deficiency state with a low level of biologically active protein; the possibility that the protein molecule itself was functionally abnormal was not excluded. In 1975, Sas and others,’2”9 using CIE in agarose-containing heparin, demonstrated that two or three peaks were formed instead of a single peak. Analysis of the peaks using a

gel-filtra-tion technique with Sephadex chromotography

in-dicated that the slower-moving peak (peak 1)

con-tamed AT-Ill with antigenic properties but no clot-ting activity, while the faster-moving peak (peak 2) contained both antigen and activity for AT-Ill.’4 Therefore, peak 1 is considered to represent AT-Ill bound to thrombin or other activated clotting fac-tors and migrates more slowly toward the anode in

an electrophoretic field in the presence of heparin than the antithrombin in peak 2 or unbound AT-III. Modifying this technique by using albumin as a marker, the amount of bound and unbound AT-III in plasma can be reproducibly quantitated in our studies.

Over the past decade, as the assays for measuring AT-Ill have been developed, the clinical syndrome of AT-Ill deficiency has emerged. Egeberg’ de-scribed the first family afflicted with this disorder in 1965. In his study, the deficiency followed an

autosomal dominant mode of inheritance, with the

heterozygous state demonstrating an increased tendency to severe thromboembolic disease. Since this report, other families with AT-Ill deficiency have been described27”8’#{176} (Table 3). Thrombosis can occur spontaneously in these individuals, but it can also occur as a result of trauma, infections,

burns, and childbirth. Indeed, many affected

women may have their first episodes of thrombo-phlebitis after childbirth. Although the thrombotic episodes usually involve superficial or deep venous systems in the lower extremities, pulmonary emboli, cerebrovascular accidents, and arterial occlusions have all been reported. The recurrent nature of thrombotic episodes in an individual is an impor-tant clue in the diagnosis of this disease. During treatment of these episodes, the patient may show an intolerance to heparmn, and large doses are needed to achieve an adequate anticoagulant effect. The age at onset of thrombotic events varies; most cases are first diagnosed in the third and fourth decades. However, thrombosis may occur at as young as 10 years of age and on into the adolescent age range.’7

The family described here conforms to the cmi-cal reports previously described for AT-Ill defi-ciency. The lack of verification beyond two gener-ations may indicate the occurrence of a spontaneous

mutation. Both secondary thrombosis (trauma,

in-fection) and spontaneous thrombosis (cerebrovas-cular accident) occurred in our patient. The recur-rent nature of the disorder as well as a resistance to

heparmn is demonstrated in the proband. The

aS-fected members of this family have a comparable decrease in both AT-Ill clotting activity and anti-gen whether measured in serum or plasma. The immunologic studies (CIE) in our family suggest a quantitative deficiency of AT-Ill rather than a functionally abnormal molecule.

To date, the therapy for patients with AT-Ill deficiency has consisted of treatment of the acute thrombotic episode with heparin. With resolution of the acute phase, warfarin derivatives may be substituted for heparin. Using this regimen, several groups have found a decrease in the number of thrombotic episodes as well as an increase in both AT-Ill activity and antigen.4’22’24 The mechanism for this increase is unknown, and it is not clear whether warfarin decreases consumption of AT-Ill by decreasing ongoing, low-grade activation of clot-ting factors, or whether it stimulates increased pro-duction of the molecule.

The incidence of thrombosis in the pediatric age range is low. In adults, a deficiency of AT-Ill may account for 2% of venous thrombosis. This report and others have ifiustrated that thrombosis related to AT-Ill deficiency can occur in the pediatric and adolescent age groups. Indeed, spontaneous deep vein thromboses may occur more often than prey-ously realized in adolescents.27 Children with recur-rent thrombotic episodes, a positive family history

of thromboembolic disease, or spontaneous throm-bosis without associated cause should have mea-surements of AT-Ill performed.

ACKNOWLEDGMENT

We wish to thank Dr. Wolf Kirsch for referring the index case for evaluation.

REFERENCES

1. Egeberg 0: Inherited antithrombin deficiency causing thrombophiia. Thromb Diath Haemorrh 13:516, 1965 2. von Kualla E, von Kualla KN: Deficiency of antithrombin

III activity associated with hereditary thrombosis tendency.

J Med 3:349, 1972

3. van der Meer J, Stoepman-van Dalen EA, Jansen JMS: Antithrombin III deficiency in a Dutch family. J Clin Pathol 26:532, 1973

4. Marciniak E, Farley CH, de Simone PA: Familial thrombo-sis- due to antithrombin III deficiency. Blood 43:219, 1974

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ARTICLES 131

III (AT-Ill ‘Budapest’) as a cause for familial thrombophilia.

Thromb Diath Haemorrh 32:105, 1974

6. Carvalho A, Ellman L: Hereditary antithrombin III defi-ciency: Effect of antithrombin III deficiency on platelet function. Am JMed 61:179, 1976

7. Fiip DJ, Eckstein JD, Veltkamp JJ: Hereditary antithrom-bin III deficiency and thromboembolic disease. Am J He-matol 1:343, 1976

8. Thrombo Wellcotest Latex Suspension, bulletin HAO8.

Beckenham, England, Weilcome Research Laboratories, 1975

9. Kidder WR, Laurence JL, Rapaport SI, et al: The plasma protamine paracoagulation test: Clinical and laboratory

eval-uation. Am J Clin Pathol 58:675, 1972

10. von Kualla E, von Kualla KN: Antithrombin III and diseases. Am J Clin Pathol 48:69, 1967

11. Laurell CB: Electroimmunoassay. Scand J Clin Lab Invest 29:21, 1972

12. Sas G, Pepper DS, Cash JD: Plasma and serum antithrombin III: Differentiation by crossed immunoelectrophoresis. Thromb Res 6:87, 1975

13. Weekes B: Crossed immunoelectrophoresis. ScaM J Im-munol 2(suppl 1):47, 1973

14. Sas G, Pepper DS, Cash JD: Investigations on antithrombin

III in normal plasma and serum. Br JHaematol 30:265, 1975 15. Rosenberg RD: Chemistry ofthe hemostatic mechanism and its relationship to the action of heparin. Fed Proc 36:10, 1977 16. Villaneuva GB, Danishefsky I: Evidence for heparin-induced

conformational change on antithrombin III. Biochem Bio-phys Res Commun 74:803, 1977

17. Teger-Nilsson A: Antithrombin in infancy and childhood. Acta Paediatr ScaM 64:624, 1975

18. #{248}deg#{224}rdOR, Fagerhol MK: Heparin co-factor activity and antithrombin III concentration in plasma related to age and

sex. Scand J Haematol 17:258, 1976

19. Sas G, Pepper DS: Detection of thrombin-antithrombin III complex by crossed immunoelectrophoresis. Thromb Res 9: 95, 1976

20. Shapiro SS, Prager D, Martinez J: Inherited antithrombin

III deficiency associated with multiple thromboembolic

phe-nomena. Blood 42:1001, 1973

21. Sas G, Pepper DS, Cash JD: Further investigations on antithrombin III in the plasma of patients with the abnor-mality of ‘antithrombin III Budapest.’ Thromb Diath Hae-morrh 33:564, 1975

22. Egeberg 0: Inherited antithrombin-HI deficiency and throm-boembolism. Thromb Diath Haemorrh 34:366, 1975 23. Gruenberg JC, Smallridge RC, Rosenberg RD: Inherited

antithrombin III deficiency causing mesenteric venous

in-farction. Ann Surg 181:791, 1975

24. Gomperts ED, Feesey M, van der Walt JD: Two dimensional immunoelectrophoretic studies in antithrombin III defi-ciency. Thromb Res 8:713, 1976

25. Stanthakis NE, Papayannis AG, Antonopoulos M, et al:

Familial thrombosis due to antithrombin III deficiency in a Greek family. Acta Haematol 57:47, 1977

26. Rosenberg R: Actions and interactions of antithrombin and heparin. N Engl JMed 292:146, 1975

27. Hartwitz J, Shenker IR: Spontaneous deep vein thrombosis

in adolescence. Clin Pediatr 16:787, 1977

GOSSYPOL, A NEW CONTRACEPTIVE FOR MEN

A new approach to contraception is being tested in China. Gossypol is a yellowish phenolic compound which has been isolated from the seeds, stem, and root of the cotton plant. Its effects were first noticed in surveys, because cooking with crude cottonseed oil led to infertility, especially in men, and gossypol was found to be the effective agent. It has been extracted, purified, and derivatives have been prepared which are being tested in clinical studies.

Gossypol was first used clinically in 1972. By now, more than 4,000 healthy men have used the treatment for more than six months, and more than half of them have been observed clinically for two years. Some men have used the treatment for four years continuously.

It is reliable, relatively safe, if the dosage is kept at the level used to treat infertility. There is an abundant supply of the compound; it is economical to produce; and it appears to be effective and safe. More work is needed to reduce some if its side-effects, to elucidate the mechanism of its infertility action, and to clarify its effects on potassium metabolism. The drug appears to be a potent addition to contraceptive armory in the coming years.

From the National Co-Ordinating Group On Male Infertility Agents, 1978. Chinese Medical Journa4 4, New Series, 417.

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1980;65;125

Pediatrics

Daniel R. Ambruso, Linda J. Jacobson and William E. Hathaway