High total and free protein S in patients with acute deep vein thrombosis

THROMBOSIS RESEARCH59; 213-217,199O 0049-3848/90$3.00+ .OOPrintedinthe USA. Copyright(c) 1990 Pergamon Pressplc.All rights reserved.

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HIGH TOTAL AND FREE PROTEIN S IN PATIENTS WITH ACUTE DEEP VEIN THROMBOSIS

P. TOUl_ON1,3, S. GANDRILLE', J.F. VITOUX*, J.N. FIESSINGER' Y. SULTAN3, M. AIACH' 1. Laboratoire d'Hemostase and 2. Chaire de Pathologie vasculaire, HBpital Broussais, and 3. Laboratoire d'Hematologie, h6pital Cochin, Paris (France). (Received30.10.1989; acceptedinrevised form 21.3.1990by EditorM.-C.Boffa) INTRODUCTION Protein S (PS) is a vitamin K dependent coagulation moderator (1) which circulates in the plasma both in free form and bound to C4b-binding protein (C4b-BP) (2). Only free PS acts as the cofactor for the anticoagulant and profibrinolytic activities of activated protein C (PC) (3). Since hereditary PS deficiency has been reported to be associated with deep vein thrombosis (DVT) (4), its measurement in plasma is one of the usual screening tests for the development of thrombosis risk factors such as antithrombin III or PC (5). Recently, D'Angelo et al. (6) reported normal total PS and transient free PS deficiency in 8 patients with acute DVT, and concluded that acute DVT is associated with an augmentation of the C4b-BP bound form of PS at the expense of the free form. A retrospective study revealed a significantly higher total PS level (p < 0.0001) in patients with acute DVT under heparin therapy than in patients who had presented with a thrombotic event more than three months previously, and whose anticoagulant therapy has been stopped. The aim of the present study was to investigate more extensively the PS-PC pathway in patients with acute DVT. Key words: Protein S, acute phase reactant, acute deep vein thrombosis. Reprint request: Dr Pierre TOULON Laboratoire d'hemostase H6pital Broussais 96, rue Didot 75674 PARIS Cedex 14 (France) 213

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Patients Group I: II4 patients with acute DVT under heparin therapy, comprising 48 males and 66 females (mean age: 44.2 years, range: 18-75) were studied during their stay in an intensive care unit. Group 2: 132 patients who had had DVT, comprising 65 males and 67 females (mean age: 44.3 years, range: 17-85) who had presented with a thrombotic event more than three months previously and whose anticoagulant therapy has been stopped, were studied during follow up visit. Group 3: 9 inpatients consisting of 6 males and 3 females (mean age 61.4 years, range: 30-87) were studied during hospitalization for phlebographically-proven acute DVT. Blood samples were drawn on admission before any anticoagulant therapy was given (sample I) and 2 to 4 days after the initiation of heparin therapy (sample 2). Collection of plasma Venous blood. was collected into evacuated tubes containing 0.129 M trisodium citrate I:9 (Vacutainer # 606608, Becton Dickinson, Meylan, France). Plasma was obtained by sample centrifugation at 2,300 x g for 15 minutes (t 3°C). Samples were immediately analyzed for prothrombin time and plasma heparin anti-Xa activity. The remaining plasma was frozen at - 70°C until further assay. Laboratory assays Free PS was prepared by PEG precipitation of the bound form to C4b-BP (7). The absence of any C4b-BP-PS complexes in the supernatant was assessed by performing crossimnunoelectrophoresis: a single peak pattern could be revealed in the supernatant instead of the two peaks observed in the native plasma. The antigen concentrations of total and free PS were measured by rocket electrophoresis (8) using a specific polyclonal antiserum from American Diagnostica (New York, NY). The C4b-BP antigen concentration was evaluated according to Laurel1 (8) using a polyclonal antiserum (Diagnostica Stago, Asnieres, France). The PC antigen concentration was evaluated using an ELISA (Assera Protein C, Diagnostica Stago). Protein concentrations (i.e. PS, PC and C4b-BP) were expressed in units per ml, I U/ml being the concentration of protein found in 1 ml of pooled normal human plasma. Here, 1 U/ml was the concentration of free PS found in 1 ml of PEG treated normal plasma. The normal values were 0.96 + 0.17 U/ml for PC, 1.03 f 0.15 U/ml for total PS, and 1.00 f 0.15 U/ml for free PS. Fibrinogen was measured by a functional assay according to Clauss (9), and plasma heparin anti-Xa activity using a chromogenic substrate based assay (IO). Statistical methods Results were expressed as means + standard deviation (SD). Data were compared using Student's t test. Wilcoxon's U test for paired samples was used to compare data for the 9 patients in Group 3. The correlation coeffi-

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cient was used to test the interdependence of parameters. p values smaller than 0.05 were considered significant. RESULTS AND COMMENTS Total PS was significantly higher in Group 1 patients with acute DVT treated with heparin than in Group 2 patients who had presented with a thrombotic event more than three months previously and whose anticoagulant therapy had been stopped. PC was within the normal range, and was similar in the two groups (Table 1). TABLE 1 Total protein S (PS), protein C (PC), fibrinogen and heparin anti-Xa plasma activity (heparin) in the two groups of patients (for details, see Patients, Materials and Methods).

Group 1

Group 2

Patients with acute DVT under heparin therapy

Former DVT patients no longer under anticoagulant therapy

114

132

Total PS (U/ml)

1.23 + 0.26

1.04 f 0.27

PC (U/ml)

1.03 * 0.20

1.04 f 0.29

NS

Fibrinogen (g/l)

5.2 + 1.7

3.7 +_ 1.0

p < 0.00001

Heparin (III/ml)

0.38 f 0.15

ND

n

Comparison

p < 0.0001

Data were compared using Student's t test; p < 0.05 was considered significant. ND = not determined, NS = not significant.

The higher PS in Group 1 than in Group 2 was not due to a change in the migration of PS in heparinized plasma, because no modification of the PS concentration was observed after in vitro addition to human plasma of up to 2 IU/ml heparin (data not shown). This led us to study more extensively the PC-PS pathway in Group 3 patients with acute DVT. As shown in Table 2, total and free PS were elevated in sample 1 and further increased while patients were under heparin therapy (mean anti-Xa activity: 0.35 IU/ml, range: 0.25-0.68, sample 2). The free-to-total PS ratio was normal on admission but rose significantly after the initiation of heparin therapy. As expected for acute phase reactants, C4b-BP and fibrinogen were elevated in sample 1, and rose even higher in sample 2. PC was within the normal range in both samples 1 and 2, and did not change significantly. The significant correlations found on admission bet-ween total PS and fibrinogen (r = 0.845, p < 0.05) and total PS and C4b-BP (r = 0.862, p ( 0.05) suggest that total PS, like C4b-BP and fibrinogen, acts as a semi-delayed acute phase

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reactant. We did not observe the decrease in the free form of PS previously reported in patients with acute DVT (6). On the contrary, the free range on admission, rose fraction, which was within the normal significantly during the first days of heparin therapy, leading, as stated above, to a significant increase in the free-to-total PS ratio. Nevertheless, these results have.to be confirmed in larger scale series. TABLE 2 Protein C, total and free protein S (PSI, free to total PS ratio (F/T ratio), and C4b-binding protein (C4b-BP) in 9 patients (Group 3) with acute deep vein thrombosis, before heparin therapy (Sample 1) and 2-4 days after its initiation (Sample 2). Sample 1

Sample 2

Comparison

Protein C (U/ml)

1.05 t 0.21

1.07 +-0.18

NS

Total PS (U/ml)

1.30 +_0.36

1.48 + 0.37

p < 0.05

Free PS (U/ml)

1.19 f 0.27

1.41 + 0.34

p ( 0.05

F/T ratio

0.92 f 0.22

1.01 f 0.33

p ( 0.05

C4b-BP (U/ml)

1.66 f 0.19

2.06 + 0.35

p < 0.05

Fibrinogen (g/l)

4.85 + 1.27

5.23 + 1.39

p < 0.05

Results are means + standard deviation. Values were compared using Wilcoxon's U test for paired data. NS = not significant. Our results raised the question of whether acute phase or heparin infusion was responsible for the increase in the free-to-total PS ratio; this problem is currently being investigated. From the clinical viewpoint, the present findings indicate that screening for risk factors for thrombophilia must be performed some time after an acute event rather than during that event, and that when borderline PS is measured under acute conditions (whether or not patients are heparinized) it would have to be confirmed by a repeat measurement more than three months after the acute event. REFERENCES 1. DISCIPIO, R.G., HERMODSSON, M.A., YATES, S.A., DAVIE, E.W. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S. Biochemistry 16, 698-706, 1977. 2. DAHLBACK, B., STENFLO, J. High molecular weight complex in human plasma between vitamin-K dependent protein S and complement protein C4bbinding protein. Proc. Natl. Acad. Sci. USA 78, 2515-2526, 1981. Regulation of activated protein C by a new protein. J. 3. WALKER, F.J. Biol. Chem. 255, 5521-5524, 1980.

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4. SCHWARTZ, H.P., FISHER, M., HOPMEIER, P., BATARD, M.A., GRIFFIN, J.H. Plasma protein S deficiency in familial thrombotic disease. Blood 64, 1297-1300, 1984. 5.

ROSENBERG, R.D., BAUER, K.A. Thrombosis in inherited deficiencies of antithrombin III, protein C and protein S. Hum. Pathol. 18, 253-262, 1987.

6.

D'ANGELO, A., VIGANO-D'ANGELO, S., ESMON, C.T., COMP, P.C. Acquired deficiencies of protein S. Protein S activity during oral anticoagulation, in liver disease, and in disseminated intravascular coagulation. J. Clin. Invest. 81, 1445-1454, 1988.

7;

COMP, P.C., DORAY, D., PATTON, D., ESMON, C.T. An abnormal plasma distribution of protein S occurs in functional protein S deficiency. Blood 67, 504-508, 1986.

8.

LAURELL, C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45-52, 1966.

9.

CLAUSS, A. Gerinnungsphysiologische Schnell-Methode zur Bestimmung des Fibrinogens. Acta Haematol. 17, 237-246, 1957.

IO. AIACH, M., MICHAUD, A., BALIAN, M., LEFEBVRE, M., WOLER, M., FOURTILLAN, J. A new molecular weight heparin derivative. In vitro and in vivo studies. Thromb. Res. 31, 611-621, 1983.