A study of hemostasis in ischemic cerebrovascular disease I. Abnormalities in factor VIII and antithrombin

THROMBOSIS RESEARCH 26; 183-192, 1982 0049-3838/82/030183-10$03.00/O Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.

A STUDY OF HEMOSTASIS I.

ABNORMALITIES

IN ISCHEMIC CEREBROVASCULAR IN FACTOR

DISEASE

VIII AND ANTITHROMBIN

K.L. Mettinger Departments of Neurology and Blood Coagulation Disorders, Karolinska Institute, Karohnska Hospital, S-104 01 Stockholm, Sweden

(Received

14.1.1982; in revised form 25.2.1982. Accepted by Editor H. Stormorken)

ABSTIUCT A sample of in all 119 young adults below the age of 55, with ischemic cerebrovascular disease (TIA and minor stroke), was investigated later than three months after acute disease. Factor VIII biological activity and antithrombm antigen were significantly (p ( 0.001) increased as compared to 80 healthy controls. In combination, these two variables correctly classified 85percent of patients and controls at a stepwisediscrirninant analysis. Factor VIII reiated antigen was increased (p ( 0.02) in patients with atherosclerotic signs at cerebral angiography and in postmenopausal female patients (p (0.00 1). It is suggested that high levels of factor VIII might predispose for thrombosis/atherosclerosis. Antithrombin biological activity was normal in spite of high antithrombin antigen levels, possibly indicating a relative insufficiency in the antithrombin defense line. It is concluded that young stroke patients provide good opportunities to look for early operating factors and predictors in human atherosclerosis and arterial thromboembolism.

INTRODUCTION After 30 years of clinical trials of antithrombotic therapy in ischemic cerebrovascular disease (ICD) a therapeutic dilemma still remains (l-2). Since ICD, as well as atherosclerosis, most likely consist of heterogenic disease entities, identification of pathogenic subgroups might be useful in future guidelines for a differentiated prevention and therapy. Earlier studies of blood coagulation in ischemic cerebrovascuhu disease (ICD) (3-10, Table I) have often the drawbacks of diagnostic insecurity as the clinical subclassification of stroke until recently has been invalid in more than 25% of the cases (11). Furthermore, most studies have been performed in the acute phase, when the results are biased by acute phase reactions. Thus, persistent hemostatic abnormalities, associated with genuine ICD, have been difficult to evaluate (Table I). Aim and Design of the Present Study The present study is part of an extensive case-control investigation of hemostasis, lipoprotein and glucose metabolism in patients with ICD occurring before the age of 55. Al1 biochemical tests have been performed after the acute stage of the illness, when acute phase reactions should have elapsed. We have focused our investigations on young adults, where the results are less likely to be biased by vascular and metabolic derangement than in the elderly.

Key Words: Ischemic cerebrovascular disease, factor VIII complex, antithrombin, discriminant analysis, predictors.

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TABLE I Authors

n

Diagnosis

VIII C(f)

AT

Fibrinogen

MisceBaneous

A. Acute Phase Gastonetal(1971)

14

“Infarction”

Todd etaI(l973)

33

“Infarction”

Ettinger

203

“Infarction”

Fletcher eta1 (1976)

182

Walsh et al (1976)*

22

B. Recovery Phase Pilgeram (1974)

406

“ICD”

TIA

/

N

11(f), V ( f 1,X N, P-I-T\\ P-l-T/ VII-X N, II N Hypercoagulability in one third (thromboelastograph) HMWFC / PTT/,VN

Turnover /

PTT 1, soluble

i’

/

CN

“ICD”

/

fibrin monomer /

Mettinger ef al (1979)

52

ICD

R:Ag N

/

Summary of Earlier Case-control Studies of Blood Coagulation in ICD. ICD = Ischemic cerebrovascular disease, ‘ICD”, ‘Tnfarctiori = diagnostic classification unreliable, AT = antithrombin, PTT = partial thromboplastin time, FDP = fibrinogen degradation products, HMWFC = high molecular weight fibrinogen complex, II, V, VII, VIII. X = blood coaguIation factors, VIIIC = clotting activity, VIIIRAg = related antigen, / = increased, \= decreased, N = normal, n = number of patients, * = time after onset not stated.

The aim of the study was to look for possible pathogenic factors and risk indicators (markers, predictors) in the early phase of arterial occlusive disease. Since the brain is our most sensitive structure, it is a useful indicator of early occlusive events. Young stroke patients thus provide excellent possibilities in the search of the early operating factors in human atherosclerosis and/or arterial thromboembolism. Later, the interrelation of possible pathogenic factors will be evaluated in a multifactorial analysis in order to look for pathogenic subgroups to get guidelines for a differentiated prevention and therapy. Recently, technical advances in the study of hemostasis have created new possibilities to search for etiological factors in thrombotic disease. Also, modem diagnostic tools, cranial computed tomography and cerebrospinal fluid spectrophotometry, enable a better separation of hemorrhagic and ischemic cerebral lesions (12), which provides an improved basis to determine the appropriate pathogenic factors for clear-cut subclasses of stroke in a clinical material. However, the high costs of advanced technology restricts the possibilities of screening large cohorts in prospective population studies and necessitates the performance of small well-designed case-control studies or prospective studies of well-defined high risk groups. This part of the study includes the following blood coagulation variables: a) Factor VIII related antigen (= VIII R:Ag). It is synthesized by the endothelium (13) and is essential for platelet adhesion at endothelial damage (14) b) Factor VIII procoagulant activity (= VIII:C). Its site of synthesis is still unknown (15). It is one of the regulatory proteins in the multienzyme system of blood coagulation and promotes blood clotting (16) c) VIII RAg/VIII.C ratio. This has been suggested to be a useful index of hypercoagulability (17) d) Antithrombin-antigen method (= ATAg). This measures free as well as complex-bound antithrombin (16) e) Antithrombin-substrate method (= AT:S). This measures only free antithrombin (16) f) AT:S / AT:Ag ratio Antithrombin, by tradition called antithrombin III, is thought to be synthesized by the liver (16) but binds e.g. to heparin localized at the endothelial surface (18). It is the main inhibitor of blood clotting ( 16).

I

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TABLE II n = 61 IMOrS

8

Uncertain CVD* Uncertain ICD** Drop outs: - presenile dementia - major strokes*** - alcoholism - drug addiction - others Malignant disease LED

15 15 1 14

1

* Transitory global amnesia (2), migraine accompagnde (3). psychogenic? (10). ** Admitted too late to allow diagnostic classification (15). *** Subtotal or total (hemi) paresis or aphasia at three months follow-up.

: 2 1

Exclusion Criteria.

MATERIALS AND METHODS Patient Material The present material includes 119 cases.from a district of Stockholm.with minor ischemic cerebral lesions occurring before the age of 55 years, admitted to the Department of Neurology, Karolinska Hospital, during a three year period, 1976-1978. Exclusion criteria are specified in Table II. Age, sex and blood group distribution of the final material are shown in Table III. Seventy-seven cases had cerebral infarction (minor strokes) and 42 cases transient ischemic attacks (TIA, meaning duration of focal neurological deficit was less than 24 hrs). Clinical characteristics are presented in Table IV. The diagnosis was based on the observation of acute focal neurological signs, quantitative cerebra-spinal-fluid (CSF) spectrophotometry and in the cases of cerebral infarction also computed tomography ( 12). Aortic arch angiography, in most cases combined with selective carotid angiography on both sides, was performed in 86 patients, 28 females and 58 males. Presence or absence of probable atherosclerotic signs at angiography was the basis for a preliminary subdivision of the material in angiopositive and angionegative groups. Blood sampling for hemostatic tests was performed later than three months (mean 10.0 _+ 7.7) after the onset of the disease or the latest cerebrovascular incident. Patients receiving anticoagulant therapy were generally not investigated earlier than at least 1-2 months after the treatment had been interrupted. Special instructions were given to avoid platelet inhibiting therapy 10 days before blood sampling. Oestrogen treatment and oral contraception (n = 9) was discontinued upon admission to the hospital. Control Material Reference values were obtained from a control group of 80 healthy and able males and females in Stockholm, of corresponding age. Age, sex and blood group distributions are presented in Table III. Detailed data on the control material has been published elsewhere ( 19). TABLE III Patients Subgroups

Sex Males Females Blood Group 0 A, B, AB

Age

45.6 f 9.4 (20-56) 42.8 C 11 .O (17-56)

Total n= 119

controls Group I n=52

Group II n=67

72

33

39

47

19

28

31/101* 70/101*

14 26

17 41

Age

41.1 * 5.4 (24-6 1) 39.1 +6.3 (21-61)

Patient and Control Populations. Distribution of Age, Sex and Blood Groups. Mean Age (Years) f SEM, and Range, is Shown. * As blood groups were introduced at a later stage of the study, some individuals could not be investigated.

n = 80

40 40

36/71* 36/71*

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TABLE IV Patients

Family History of Case History of ICD’ TLQ Infarction Vascular Disease’ Vascular Disease3

Hypertension4 Diabetes Smoking’ MeIIitus4

Patient Material, clinical Characteristics.

’ Vertebral arterial territory involved in 9 cases; amamosis fugax in 4 cases. * Coronary heart disease (31). stroke (13), claudication (5), venous thrombosis (12) before the age of 70. 3 Coronary heart disease (13), stroke ( 6), claudication (S), venous thrombosis (12). 4 History of medical treatment at the time of investigation. s Amount of tobacco equivalent to or exceeding 10 cigarettes/day. Statistical

Methods

fl statistical calculations were performed using the computer program ‘STATPAC’on the Nord IO-43 S III 80 A of the Karolinska Institute. The program for discrlminant analysis of multiple covarlates was used to analyze each test separately allowing correction of any possible imbalance between the groups of patients and controls due to clinical and biochemical variables. For classification of patients and controls stepwise discrbninant analyses were used (20). Since positively skewed distributions were observed in the results of factor VIII variables in controls, a logarithmical transformation was performed to create gaussian distributions of this variable necessary for the validity of the discriminant analysis. Correlation analyses were performed by the Spearman rank correlation test (21). Laboratory Methods Blood Sampling. All patients, as well as controls were fat fasting for 6-8 hours prior to investigation. Brachial vein puncture with a WR-needle was performed strictly between 7.30 and 10.30 a.m. Blood-sampling was always done at rest in supine position from a non-paralyzed arm. The first 5 ml of blood was discarded, then 9 ml venous blood was sampled into 1 ml trisodiumcitrate, 0.13 mol/l, pH 7.5 and the contents were mixed thoroughly. After centrlfuga tion of the blood, at room temperature, for 30 min. at 3,000 x g, the plasma was dispensed into plastic tubes and kept frozen at -7OOC until tested. Laboratory Tests. For technical reasons, the study was completed in two steps (Table V). The first step included 52 patients (Group I) admitted from January 1976 to June 1977 and the second step 67 patients (group II) from July 1977 to December 1978. It was considered important to include some new methods ln group 11 on the basis of preliminary findings in group I (10). Vlll~G and ATAG were assayed in the total material. VIIIE and AT:S were only assayed in Group 11 (AT:S also in part of Group I). In addition, erythrocyte sedimentation rate (= E-SR) was determined in the total material and fibrinogen levels in Group Il. All laboratory tests were performed without access to clinical data in the laboratory.

TABLE V Group I n=52 VIII R:Ag

AT:Ag

E-SR

(AT:Sy

Group II n=67 VIII R:Ag

AT:Ag

E-SR

AT:S

Vlll:C

Fibrinogen

Tests Performed in Respective Patient Group. VIIIC - Two-stage thrombin generation assay according to Savidge er al (22). VIII RAg - Quantitative electroimmunoassay according to Laurell (23). Antiserum, raised in rabbits against human factor VIII related antigen, adsorbed von Wilebrand plasma, was used. Electrophoresis, at pH 8.6, was interrupted after 23 hours. ATAg - Radial immunodiffusion according to Mancini er al. (24). lmmunodiffusion plates, containing specific antibodies against antithrombin, were used (M-Partigen, Antithrombin Ill, Behringwerke, Marburg, West Germany). ATS - A heparin co-factor activity assay was used according to Abildgaard er al (25). based on a synthetic chromogenic peptide substrate (S-2238, H-D-PhePip-Arg-p-nitroanilid (pNA). specific for thrombin, Kabi Diagnostica AB, Stockholm, Sweden). Fibrinogen - Determination of fibrin polymerization time (26). Erythrocyte Sedimentation Rate - According to the conventional technique.

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Units for Pammeters. All assays were calibrated against an internal plasma standard obtained from 20 healthy and able men (mean age 50 years, range 40-53 years). The results were expressed in percent per ml of the internal standard, which is considered fo contain 100 percent per ml.

RESULTS Basic Considerations

of Some Sources of Variability

in Controls and Patients, Respectively.

Acute Disease: The erythrocyte sedimentation rate and fibrinogen levels in patients were found to be normal (means and SEM 9.4f 1.02 mm/h, and 3.62 +1.6 g/l, respectively) and did not correlate to any of the coagulation variables studied. No correlation was found between time for investigation (months after the last incident) and the coagulation variables. Thus, the results were believed to represent a steady state. Sex: In controls, no significant differences between male and female subjects were found for any of the variables studied (19). In thepatien t group, females had significantly higher VIII R:Ag (p ( 0.00 1) and VIII:C (p ( 0.01) than males (Table VIII). Age: In controls (19) and patients no significant correlations were found between age and any of the laboratory variables. Blood Groups: Controls (19) and patients (Table VIII) with blood group A had significantly (p ( 0.001) higher VIII R:Ag than subjects with blood group 0. Patients Compared to Controls (Tables VI-W) Factor VIII:C was significantly higher (p ( 0.001) in patients as compared to controls. Factor VIII RAg was significantly increased (p ( 0.001) in female patients older than 40 years as compared to female controls of the same age group. The individual VIII RAg/VIIIE ratios, especially in young patients, were in most cases less than 1.O against more than 1.O in controls. In patients ATAg was significantly increased (p( 0.001). In contrast AT:S showed no difference between patients and controls. The individual AT:S/ATAg ratios in patients were generally less than 1.O, whereas more than 1.O in controls. Comparisons

Between Subgroups of Patients (Table VIII)

VIII R:Ag was significantly higher in angiopositive patients (p( 0.02) and tended to be increased in patients with a history of occlusive vascular diseases. Correlation

Between Tests

There was a higher correlation between VIII:C and VIII R:Ag in patients (r = 0.81) in comparison to the controls (r = 0.72). A discrepancy was also found for correlations between the two AT methods in patients (r = 0.65) as compared to controls (r = 0.84). No other correlations between coagulation variables were found. Stepwise Discriminant

Analysis

A stepwise discriminant analysis was performed within group II where all variables were included. Seventy-two percent of the patients and 85 percent of the controls could be correctly classified as patients and controls, respectively, by VIIIE. If ATAg was added, about 85 percent of the subjects in each group could be classified. No significant additional increase in classification rate was obtained by introducing more variables. The same rate was obtained by antithrombin ratio as by VIII:C. AT:Ag alone, or factor VIII ratio, were weak indicators, as the classification rate was less than 60 percent. DISCUSSION To establish a cause-and-effect relationship between a laboratory abnormality and thrombosis, not only must the abnormality precede the thrombotic event. Further support is needed from interventional studies, i.e. demonstration, that a therapeutic or prophylactic maneuver normalizes the result of the blood test, at the same time reducing the frequency of thrombosis. However, the first step would always be to demonstrate markers of disease after onset of symptoms, preferably in late recovery phase, to get a basis for selection of tests to be evaluated as predictors in a longitudinal study.

P-value

Patients

84f5.1 77k5.3

76Icr6.2 74zh7.4 92f7.8 82f7.6

40 40

20 20 20 20 (0.02 (0.001

( :soo 1

ns ns

7 13 32 15 39 28

110f14.0 121+11.1 115rt 5.5 132f 8.8 110* 5.0 126 + 7.0

8Of3.7 74+3.3

75C4.2 68+3.3 84+6.0 80-l-5.7

P-value (0.01 (0.001 (0.001 (0.001 (0.001 (0.001

n 20 20 20 20 40 40

1.OSfO. 1 1.04fO.l

0.95fO.O 0.94+0.1

39 28

Controls l.O1fO.l 1.09fO.l 1.10+0.7 1.03+-o. I

n

7 0.80fO.l 0.78-f-0.1 13 0.96-kO.O 32 1.02+0.I I5

Patients

llOk3.2 106k2.1

lOlk1.8 106k2.6

103k1.7 106k1.7

240 Males Females

Males Females

106&2.0 102+2.4

104*1.7 102+2.5

105k1.3 102*1.7

11 15

40 21

51 36

111+5.0

104k4.5 103+1.7 108f1.4

28

47 36 47

126k7.0

119f7.5 106k1.7 109f1.7

VIllE

VIII RAg ATS AT&

ns ns

ns ns

ns ns

P-value 10M 1.9 lCOk2.4

15 18 57 29 72 47

112k2.3 11N1.8 106+1.7 109+2.5 108-11.4 109k1.7 99*1.3 99k1.7

98f 1.9 97+2.4

Controls

n

Patients

(9

(0.001 (0.001 (0.02 (0.01 (0.001 (0.001

20 20

40 40

20 20

P-value

n

Antigen (ATAg)

0.96f0.01 0.97+0.02

0.96kO.03 0.96+0.02 0.96f0.01 0.98kO.02

Patients

72 51 72

39

n

n

31 58 42 58

Pos

122k6.4 llW6.2 lOS-I:1.9 llof1.7

P-vahle

(0.001 (0.001 ns ns

lll+!l.l 88&6.6 105f2.2 108+2.2

Neg

Angiography (cKf?r)

(;;sO2 ns

16 28 16 28

ns

P-value

n

131f7.1 119f7.6 106f2.2 109f2.1

A

n

51 36

20

n

0

9W6.3 92f5.7 lOl-k2.3 106+_1.6

n

24 46 35 46

Blood groups (a P)

1.05f0.01 1B4f0.01

1.06f0.02 1.05f0.01

17 31 22 31

n

40 40

20 20

1.03+0.01 20

1.07f0.01

Controls

Ratio ATS / ATAg

11 15 40 21

Antithrombin (AT). Mean k SEM is Shown and Significant Differences are Indicated. (Discriminant Analysis with Correction for Age.) TABLE VIII

40 40

20 20

20 20

n

Antithrombin

Blood Coagulation Parameters in Subgroups of Patients - Multivariate Analysis. Mean f SEM is Shown and Significant Differences are Indicated. (Discriminant Analysis with Correction for Age Q, Sex fl and Blood Groupsr).

Males

n

Females

Sex (0 Y)

Controls

n

(%)

Antithrombin Substrate (ATS)

TABLE VII

40 40

20 20 20 20

n

Ratio VIII RAg / VIllE

Factor VII1 Related Antigen (RAg) and Clotting Activity (C). Mean F SEM is Shown and Significant Differences are Indicated. (Discriminant Analysis with Correction for Age and Blood Croups.)

(40 Males Females

Parameters (W

15 18 57 29

4.5 72 7.5 47

Patients

104f 119f

Age(years) Sex

Group

Males Females

88+_ 6.6 94+_ 8.5 107+ 5.3 135flO.l

n

Controls

n

Controls

n

Age(years) Sex

Patients

VIIIC (“/4)

VIII RAg (%)

TABLE VI

Group

( 40 Males Females 1 40 Males Females

1

(ZO2

(0.001 (0.00 1

P-value

(0.001 (0.001

(0.001 (0.001 (0.001 (0.02

P-value

(0.02 11s

(iSO5 ( 0.02 ns

P-value

w

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The acute phase reaction after an acute cerebrovascular incident is likely to have elapsed within 4-6 weeks (3). Thus, in the present study, hemostatic tests were performed at a later stage. A steady state at the time of testing could be assumed, since normal levels of erythrocyte sedimentation rate and fibrinogen levels (both established acute phase indicators) were found, and since there was no correlation of results to the time interval (months) following onset. Thus, it is reasonable to assume that the results are essentially representative for the situation before the acute incident. Hemostatic changes in chronic vascular diseases could hypothetically reflect iterated endothelial lesions, i.e. iterated minor tissue injuries, causing acute phase reactions and low grade activation of hemostasis (27). However, as fibrinogen levels were normal in the present study, the presence of such acute phase reactions are unlikely. Factor VIII There is, normally, in plasma a state of equilibrium between factor VIII subunits, i.e. VIII:C and VIII R:Ag (21). The mean ratio of VIII RAg/VIII:C in the internal plasma standard of the laboratory is, by definition, 1.0. Increased levels of antigen and biological activity are regularly found (28) in the presence of acute disease or physiological stress. In situations with clot formation a high VIII RAg/VIIIC ratio has been demonstrated (17), which should be expected as the coagulation sequence includes thrombin formation, destroying VIII:C by a specific negative feed-back. Even in the absence of acute disease, increased levels of VIII subunits are sometimes found for unknown reasons, but VIII R:Ag/VIII:C ratio in these cases is rarely studied (29, 30). It has been suggested (lo), that increased levels of VIII R:Ag in such cases may be caused by continuous endothelial damage in atherosclerotic lesions releasing VIII R:Ag from its endothelial pool. This is, however, unlikely as the overwhelming portion of endothelium is found in the microcirculation and only l-2 percent in arteries (3 1). Furthermore, endothelial damage causes adsorption of VIII RAg from plasma (14), and the plasma levels, if affected at all, are more likely to decrease. Thus, it seems necessary to look for other explanations. In this study performed in the absence of acute disease, increased levels of VIII R:Ag as well as VIII:C were found in patients. A discrepancy was noted between the two subunits, indicated by the higher levels of VIII:C than VIII R:Ag, i.e. the low ratio VIII R:Ag/VIII:C, in patients. Both showed a slight tendency to increase with age (especially in patients) and were significantly higher in blood group A than in blood group 0 subjects, which is in agreement with earlier investigations (32). VIII R Ag and Atherosclerosis

The present study confirms our earlier observation of significantly higher VIII R:Ag in patients with atherosclerotic lesions at angiography (10). A challenging observation is the much more pronounced increase of VIII RAg in female patients older than 40 (Table VI) as compared to controls and the concomitant changes of VIII:C. This is the age when overt atherosclerosis becomes prevalent in females. For reasons mentioned above it is unlikely that the increase of VIII R:Ag levels is secondary to atherosclerosis. As VIII RAg is required for platelet adhesion in endothelial damage (14) and adhering platelets might release a growth factor capable of inducing extensive proliferation of smooth muscle cells (33), it is a reasonable postulation that high levels of VIII R:Ag might predispose for atherosclerosis. This hypothesis is further supported by the recent observations, that pigs with genetic von Willebrand’s disease, having decreased levels of VIII RAg as well as VIII:C, are resistant to development of atherosclerosis, e.g. when fed cholesterol- rich diet (34). Reasons for Persistently High Factor VIII Levels

Possible explanations for persistently high factor VIII levels should be looked for. These levels are most probably caused by increased synthesis. Possibly, a direct or indirect hormonal regulation of the rate of synthesis might be involved to some extent, indicated by higher levels after the menopause, particularly of VIII R:Ag in patients (Table VI). It is also possible that other circulatory factors, e.g. hyperlipidemia and hypertension could affect the physiology of e.g. endothelial cells and cause increased synthesis of factor VIII. However, several reasons point towards genetic mechanisms. VIII:C has been shown to be under X-chromosomal control, whereas VIII RAg appears to be under autosomal control (35). Thus, low levels of VIIIC are found in classical hemotilia A, and low levels of VIII RAg are found in von Willebrand’s disease, both recognized genetic diseases. Higher levels are found in blood group A than blood group 0 subjects, and it is thought that arterial occlusive disease, e.g. IHD, is more common in blood group A than in blood group 0 (16). Thus, it seems logical to postulate that high levels of VIII:C and VIII RAg in some cases might

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represent genetically determined disorders predisposing to thrombosis and atherosclerosis, and thus be reversed states of the hemorrhagic disorders mentioned above. To prove this hypothesis it would be necessary to perform family studies of patients as well as of controls. It should be noted that two reports describe families with increased levels of factor V or factor VIII and a predispositon to thrombosis (36, 37). Reasons for a Low VIII R.Agf VII.I:C Ratio So far, no explanations have been found for the low ratio VIII RAg/VIII:C in the present patient material. Exposure of collagen by perpetual endothelial damage might cause adsorption of VIII RAg from plasma with a concomitant rise of VIIIC, as shown experimentally with certain collagen preparations (38). Also, reduction-oxidation processes may be of importance for the integrity of the factor VIII complex, and possibly red-ox systems of platelets (e.g. lipoic acid or thioredoxin), adhering to the sites of endothelial damage, could cause dissociation of the complex and raised plasma levels of VIIIC (39). VI.II:C as a Predictor It thus seems logical to expect a low ratio in chronic arterial diseases with iterated minor endothelial lesions. VIII:C could be a most sensitive indicator of such diseases, as the already high levels would further increase, whereas the high VIII RAg levels would be counteracted by adsorption. As a matter of fact, VIII C was significantly increased in all age groups and in the stepwise discriminant analysis it correctly classified 72 percent of patients and 85 percent of controls. In conclusion, VIII:C might be a useful indicator of a high risk population. This is in agreement with preliminary results from a recently published extensive population study aimed to identify predictors of cardiovascular disease (40). A strong association between VIII:C and cardiovascular death was found. Antithrombin As antithrombin is the main inhibitor of blood coagulation, low levels might be associated with risk for thrombosis. Hereditary deficiency of antithrombin is a rare cause of venous thrombosis. More often decreased levels are caused by consumption in the clotting process, e.g. post operation, in acute myocardial infarction and in patients with angina pectoris associated with coronary atherosclerosis (29,30). Antithrombin in most patient materials(25), including ours, (Table VIII) shows a tendency to decrease with increasing age, which is consistent with increasing risk for thrombosis with aging. A high level of antithrombin would theoretically increase the risk of bleeding but has not been reported in disorders with such a tendency. Apparently raised levels have been reported in those, who are at risk of ischemic heart disease (41) or have already experienced a clinical event (41,42), and might indicate a protective response. It was a paradoxical observation in the present study, that antithrombin levels in all groups of patients were most significantly increased, as demonstrated by the antigen method, with a concomitant normal biological activity of antithrombin, as demonstrated by the substrate method. This suggests that antithrombin molecules could be functionally defect, and thus an increased antithrombin synthesis would be needed to maintain normal homeostasis. Furthermore, the immunologic method measures not only free antithrombin, but also antithrombin complex bound to thrombin and other proteases. Both explanations are possible. These could explain the ratio discrepancy between patients (AT:S/ATAg always less than one) and controls (the ratio always over one) as well as the weaker correlation between the two methods in patients (r = 0.65) than in controls (r = 0.84). Conclusions. Therapy and Prevention The present case-control study has shown that young stroke patients provide good opportunities to look for early operating factors in human atherosclerosis and arterial thromboembolism. Abnormalities in factor VIII and antithrombin might be useful markers (predictors) of cases with predisposition for thrombo-embolic disease. It should be noted that the profile of hemostatic variables in ICD seem to differ from that of coronary heart disease, as indicated by different trends in antithrombin antigen levels. The usefulness of these abnormalities as predictors for ICD should be evaluated in a multifactorial analysis of a longitudinal follow-up study. Their usefulness as markers of a thrombosis prone population should be evaluated in a randomized trial of long-term anticoagulant treatment.

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ACKNOWLEDGEMENTS Iwould

like to thank my teacher Associate Professor Margareta Blombkk for painstaking Interest and continuous advice in the present study. I am also grateful to the statisticians Ulf Brodin and Elisabeth Berg for invaluable assistance with the discriminant analyses. The study was supported by grants from the Swedish Medical Research Council (19X 520, M. Blomblck), the Hans and Loo Osterman Fund and the Karolinska Institute.

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