Influence of heparin cofactor II (HCII) on the determination of antithrombin III (AT)

THROMBOSIS RESEARCH 40; 571-576, 1985 0049-3848/85 $3.00 t .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

BRIEF

COMMUNICATION

INFLUENCE OF HEPARIN COFACTOR II DETERMINATION OF ANTITHROMBIN

(HCII) ON THE III (AT)

Tri H. Tran and F. Duckert Coagulation and fibrinolysis Laboratory, Kantonsspital, 4031 Basel, Switzerland (Received 10.5.1985; Accepted in revised form 15.8.1985 by Editor E.A. Beck)

INTRODUCTION Plasma thrombin-antithrombin heparin cofactor is generally attributed to the activity of antithrombin III (AT) (1,2). Recently, a new plasma inhibitor, heparin cofactor II, has been purifiedand characterized (3,4). Because of the presence of both thrombin inhibitors in plasma, the assay of plasma AT by heparin cofactor activity includes the effect of HCII. Furthermore, Friberger et al (5) have reported that the second heparin cofactor inhibits more pronouncedly human thrombin than bovine thrombin while comparing different assays of human AT. A reinvestigation of the assay of plasma heparin cofactor requires plasma depleted of either AT or HCII. In this report, to show quantitatively the contribution of HCII, the heparin cofactor activity is determined in plasmabefore and after immunoadsorption by using human or bovine thrombin. MATERIALS AND METHODS Bovine thrombin was a gift of Dr. C.M. Jackson sent from St-Louis, Missouri. Human thrombin was prepared by incubation of partially purified prothrombin obtained from commercial factor IX complex with Echis carinatus venom (6). Thrombin was purified by chromatography on Sulfopropyl-Sephadex C-50 (7). Other reagents were the same as described previously (8). IgG fraction containing antibodies to HCII or AT (81, anti-HCII respectively anti-AT, was coupled to CNBr-activated Sepharose4B with 2.8 mg per ml gel. One milliliter of anti-HCII-Sepharose 4B was calculatedto remove specificallyall HCII in 1.7 ml of plasma. Normal human plasma and HCII-free plasma were made free

Key words: Heparin cofactors, Thrombin 571

inhibition

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of AT by immunoadsorption on minicolumns (0.7 x 1.3 cm) of antiAT - Sepharose 4B (8) . A titrated human plasma pool (NP) obtained from 43 normal male donors, frozen at - 170 C was used throughout and contained by definition 1 U/ml AT and HCII. Plasma heparin

cofactor

activity

The plasma heparin cofactor activity was determined by measuring the rate of thrombin inhibition in the presence of heparin. Plasthrombin, heparin and chromozym TH all were diluted in 0.02 bl ??ls-HCl and 0.15 M NaCl, pH 8.0 containing 1 g of polyethylene glycol 6000 per liter. Plasma dilhtion (0.2ml) was incubated with 0. i ml human thrombin (75 nbl) , or bovine thrombin (100 nM) , at 37 C in the presence of 0.05 ml of heparin (20 USP-U/ml). At several time intervals, the residual thrombin activity was assessed by addition of 0.6 ml 0.15 mM Chromozym TH and 0.05 g/l polybrene and the release of p-nitroaniline recorded at 405 nm. The initial rate of substrate hydrolysis is inversely proportional to the heparin cofactor activity in sample. The curve of the initial rate of substrate hydrolysis versus plasma volume for an incubation time is a regression line. The same assay was performed for AT-free plasma, however with 0.1 ml of 25 nM human thrombin or 44 nM bovine thrombin. RESULTS Inhibition

of

human thrombin

with

NP and HCII-free

NP

The inhibition of human thrombin with NP and HCII-free NP was investigated for incubation times of 30 set to 10 min. The initial rate of substrate hydrolysis is shown in Fig.lA as a function of plasma volume for 30 set and 10 min. The slope of regression line of NP with 10 min incubation is steeper than that with 30 set, indicating that more heparin cofactor activity is measured after 10 min than after 30 sec. On the other hand, the difference in slope of the regression line of NP and of HCII-free NP with 10 min incubation represents the heparin cofactor activity of HCII. Using the regression line of NP with 10 min incubation as standard, the heparin cofactor activity in per cent was calculated as a function of plasma volume for each incubation time (Table 1). On the assumption of 100% for NP and 10 min, a 30-set incubation exhibits a heparin cofactor activity of 87% for NP and 72% for HCII-free NP. Whereas the heparin cofactor activity of HCII-free NP slightly increases from 72% after 30 set to 76% cofactor activity of NP is practically after 10 min, the heparin 100% after 5 min. These data indicate that the determination of the heparin cofactor activity of AT requires solely a 30-set and a 5-min incubation is needed to assay the heparin incubation, in heparin cofactor accofactor activity of HCII. The deviation tivity of NP and HCII-free NP for each incubation time expresses the heparin cofactor activity of HCII, which is 15% after 30 set then increases to 21% after 2 min, respectively 22% after 5 min and 24% after 10 min. Inhibition

of

bovine

thrombin

with

NP and HCII-free

NP

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The previous experiment was repeated with bovine thrombin.The residual thrombin activity is shown in Fig.lB as a function of plasma volume for 30 set and 10 min incubation. The slope of the regression line of NP with 10 min incubation is steeper than that with 30 set, reflecting the previous finding as reported with human thrombin. The regression line of NP with 30 set incubation seems identical to that of HCII-free NP with 10 min. Table 2 shows the heparin cofactor activity in per cent for each incubation time assuming 100% for NP and 10 min. The heparin cofactor activity of HCII-free NP (74-769,) remains practically constant, independently of the incubation time. Thus, an incubation of 30 set is sufficient to assay the heparin cofactor activity of AT. The heparin cofactor activity of NP increases from 78% after 30 set to 88% after 5 min and 100% 2 min, respectively 96% after after 10 min. Thus, the heparin cofactor activity of HCII is 4% at 30 set, respectively 12% at 2 min, 21% at 5 min and 26% at 10 min. The contribution of HCII to about 25% of the whole plasma heparin cofactor activity implies that the molar concentration of HCII corresponds to one third of that of AT. Inhibition

of

thrombins

with

AT-free

NP

The thrombin inhibition was investigated as a function of AT-free NP with 30 set to 10 min incubation (Fig.2). The heparin cofactor activity for each incubation time and plasma concentration is shown in Table 3. After 30 set, 67% of HCII participate in the inhibition of human thrombin, 89% of HCII after 2 min, and 985 of HCII after 5 min. Under similar conditions, only 21% of human HCII are involved in the inhibition of bovine thrombin, 61% of HCII after 2 min, and 90% of HCII after 5 min. These data are very similar to the results calculated from the difference in heparin cofactor activity of NP and HCII-free NP (Table 3). A

.

8 0.6 _

\ J

I 0.5

I 1.0 PLASMA

1.5 pi

D 213

25

FIG. 1

I OS

1 1.0 PLASMA

I 1.5

I 2.0

\ I 25

JJI

Inhibition of thrombins versus plasma concentration in the presence of heparin. A) human thrombin; B) bovine thrombin. NP was incubated with thrombin for 30 set (o---o) or 10 min (a.--a); HCII-free NP (o--o) instead of NP with 10 min incubation.

574

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t

I

AT FREE

I

I

I

0.5

1.0

1.5

PLASMA

2.0

III

l-c

I

I

0.5

2.5

pl

Vol. 40, No. 4

I

1.0 AT FREE

FIG.2

I

I

1.5 PLASMA

2.0

I

2.5

~1

Inhibition of thrombins by AT-free NP in the presence of heparin. A) human thrombin; B) bovine thrombin. AT-free NP was incubated with thrombin for 30 set (u---o)or 2 min (o----o) and 10 min (A.--A). TAB.1 Inhibition of human thrombin versus plasma volume and incubation time in the presence of heparin. Values of heparin cofactor activity in per cent are calculated from the standard curve with 10 min incubation. Columns A : NP, columns B : HCII-free NP. Plasma 011)

30 set A B

0.5

2 min A B

5 min A B

10 min A B

1.0 1.5 2.0 2.5

93 91 85 80

76 75 70 70 71

99 94 94 95 -

79 72 72 73 75

91 97 98 98 -

71 75 76 75 75

98 101 102 99 -

76 76 73 77 78

mean + S.D.

87 5

72 3

95 3

74 3

96 3

74 2

100 2

76 2

TAB.2 Same conditions as in Table 1, however with bovine thrombin. Plasma 011)

30 set A B

2 min A B

5 min A B

10 min A B

1.0 1.5 2.0 2.5

79 79 77 74

79 74 73 70

88 89 88 88

76 75 78 75

97 96 98 94

73 78 77 73

96 101 101 100

67 77 78 75

mean + S.D.

77 2

74 4

88 11

76

96 2

75 3

100 2

74 5

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TAB.3 Inhibition of thrombins by AT-free NP in the presence of heparin. Values of heparin cofactor activity in per cent are calculated from the standard curve of AT-free NP with 10 min incubation. Columns A : human thrombin ; columns B : bovine thrombin. Last line displays the contribution of HCII calculated from the difference in heparin cofactor of NP and HCII-free NP. 30 set A B

2 min A B

1.0 1.5 2.0 2.5

73 72 62 61

20 24 21 20

87 92 90 86

64 61 62 56

99 100 99 94

100 90 87 84

100 102 102 99

103 95 103 100

mean + S.D. Difference NP - HCII-free

67 6

21 2

89 3

61 3

98 3

90 7

99 5

100 4

60

16

84

48

88

84

96

100

Plasma (yl)

5 min A B

10 min B A

0.5

90

NP

-

DISCUSSION In the presence of heparin, the contribution of HCII to the thrombin inhibition is evident. The inhibition rate depends on thrombin species. Human thrombin is more rapidly inhibited by human HCII than bovine thrombin. With a 30-set incubation, while 20% of HCII participate in the inhibition of bovine thrombin, 60% of HCII are involved in the inactivation of human thrombin (Table 3). This result is consistent with the observation of Friberger et al (5). In fact, if HCII is not removed, the apparent heparin cofactor activity of AT is described in a simplified manner by the equation: = (AT) + f, (HCII) (1) (AT)app (AT) is the true heparin cofactor activity of AT and ft(HCII) the fraction of heparin cofactor activity of HCII dependent on the incubation time and thrombin species. (AT) and (HCII) can be written as follows: (AT)

=

cl (AT),

(HCII)

=

c2 (HCII)n

;:;

where (AT), and (HCII), are the heparin cofactor activities of AT and HCII in normal human plasma pool and cl, c2 are constants. Replacing (2) and (3) in (1): CATlapp

=

Cl

(AT),

+ ftc2

(HCII),

The relative (HCII), corresponds approximately (AT), 9 the equation (4) appears as follows:

(ATI

app

=

(cl + l/3 ftc2)

(AT),

(4) to one third of (5)

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For normal plasma pool cl = c2 = 1 ; assuming ftd 0.6 for human thrombin and ftd0.2 for bovine thrombin with 30 set incubation (Table 3), the equation (5) becomes: (AT)app (*T)app

= =

1.2 (AT) with human thrombin n 1.067 (AT), with bovine thrombin

The apparent heparin cofactor activity of plasma AT is 20% higher than its true heparin cofactor activity when human thrombin is used, respectively 6.7% when bovine thrombin is used. At low AT activity, i.e. AT deficiency or consumption, the term of l/3 ftc2 in equation (5) is no more negligible. The more important the term of l/3 ftc2 , the greater is the deviation between the true and apparent heparin cofactor activities of AT. Thus , to assay the true heparin cofactor activity of AT, HCII should be removed by immunoadsorption. To minimize the contribution of HCII in the assay of AT, bovine thrombin can be used with 30 set incubation at 37OC, which appears to be sufficient for a complete reaction between AT and thrombin (Table 2). The assay of AT can be also overcome by measuring the inhibition of factor Xa since the latter is not inhibited by HCII (3,4). REFERENCES 1. ODEGARD, O.R. Evaluation of an amidolytic heparin method. Thromb. Res. 1, 351-360, 1975.

cofactor

2. HANDELAND, G.F., ABILDGAARD, U. and AASEN, A.O. Simplified assay for antithrombin III activity using chromogenic peptide substrate. Scand.J.Haematol. 31, 427-436, 1983. 3. TOLLEFSEN, D.M., MAJERUS, D.W. and BLANK, M.K. Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J.Biol.Chem. 257, 2162-2169, 1982. 4. WUNDERWALD, P., SCHRENK, W.J. and PORT, H. Antithrombin BM from human plasma: An antithrombin binding moderately to heparin. Thromb.Res. 25, 177-191, 1982. 5. FRIBERGER, P., EGBERG, N., HOLMER, E., HELLGREN M. and BLOMBKCK, M. Antithrombin assay - the use of human or bovine thrombin and the observation of a "second" heparin cofactor. Thromb.Res. 25, 433-436, 1982. 6. FRANZA, B.R., ARONSON, D.L. and FINLAYSON, J.S. Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity. J.Biol.Chem. 250, 7057-7068, 1975. 7. LUNDBLAD, R.L., KINGDON, H.S. and MANN, K.G. Thrombin. Meth.Enzymol. 45, 156-177, 1976. 8. TRAN, T.H. and DUCKERT, F. Heparin cofactor II determination. Levels in normals and patients with hereditary antithrombin III deficiency and disseminated intravascular coagulation. Thromb. Haemostas. -52, 112-116, 1984