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Proteins C and S in patients with advanced prostatic cancer
THROMBOSIS RESEARCH 63; 195-200,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
BRIEF
---- COMMUNICATION ---___---
PROTEINS C AND S IN PATIENTS WITH ADVANCED PROSTATIC CANCER P. Dinis Oliveira, M. GraGa Oliveira, F. Pina, J. Aguiar Andrade, J.M. Pina-Cabral Centro de Fisiologiada Hemcktase (INK), Dpt. of Physiology, Oporto Medical School,Dpt- of Urology,Hosp. S. J&o, Porto 4200 Porto, PORlWAL (Received
7.1.1991;
accepted
in revised form 15.4.1991
by Editor U. Abildgaard)
INTRODUCTION inpatientswithprostatic Asin otherneoplasia,haemostaticalterationsarefrequentlyobserved cancer (1,2,3,4). These changes may occur due to the cancer itself or also as a result of therapy, particularly with oestrogens(6,7,8,). Protein C (PC) is avit Kdependent zymogen of a serine protease (9) which has an anticoagulant (10,11,12) andprofibrinolytic effect (13,14), after activation by thrombin on the endothelial surface Protein S (PS), another vit. K dependent protein, binds activated protein C (APC) to the endothelial surface (15,16) thus serving as a co-factor for APC, in the proteolytic cleavage of activated coagulation factors V and VIII. Although deficiencies in either PC or PS may be associated with thrombotic events (17,18,19), references concerning a possible alteration of these proteins in prostatic cancer are scarce(20). In this work the authors determined plasmatic levels of Proteins C and S in patients with prostatic cancer before and after total androgenic blockade with buserelin plus flutamide or orchidectomy plusflutamide, a therapy that is increasingly substitutingoesuogens in the management of this disease, due to a lesser incidence of cardiovascular effects.
MATERIALS AND METHODS Patient material: Thirty patients with either cytologically or histologically proven prostatic cancer ( stages C or D of the Whitmore classification) were selected. All patients had samples drawn twice, at least seven weeks apart (7), and in thirteen of them before initiating treatment. In all cases, second blood collections were done in patients under total androgen blockade, either with orchidectomy plus flutamide (250 mg tid) or buserilin (0.1 mg six times daily by nasal inhalation) plus flutamide. The control group was composed of fourteen patients with benign prostatic hypertrophy (20). Key Words: Protein C, Protein S, Prostatic Cancer 195
196
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
Laboratorv Methods: Blood Samples for PC and PS determination were collected in one tenth volume of 3.8% trisodium citrate and platelet poor plasma (PPP) was prepared by centrifugation for 15 mn at 2500 G at 4* C. Plasma was stored at -7OYJ. until used. Blood was also drawn for routine coagulation assays ( Pits, II VII X, FI, F XIII Quick and APTI’) as well as for liver and renal function indicators. Protein C Antigen was determined by E.IA. . Electrophoresis was carried out using the same buffer as described below, at 25 V/ 4cm . Plates were coated with 1% high EEO agarose in electrophoresis buffer containing .0.45% of antiserum from Diagnostica Stago (Assera Protein C) After electrophoresis over night plates were washed and stained with Coomassie blue. Functional Protein C levels were determined using a coagulometric test based on the APTT prolongation after Protein C activation by a snake venom (Acticlot C - American Diagnostica Inc ). Total and free nrotein S antigen were measured by electroimunoassay (EIA) Determination of Total Protein S (TPS) was carried out according to Laurel1 (21) using a buffer composed of 1.62 g/ L sodium veronal, 7.05 g/L glycine; 5.65g/L Tris, 1.80 g/L Na2 EDTA, pH 8.8 at 15 V/4cm. Plates were prepared usi.ng 1% high EEO agarose in electrophoresis buffer and 0.6% antiserum from Diagnostica Stago (Assera Protein S). After overnight electrophoresis, plates were washed and stained with Coomassie blue. Under these conditions only one protein S rocket was observed. Free Protein S (FPS) was measured after separation from Bound Protein S, acording to Comp et al(22): A 18.75% W/V solution of PEG 8000 (from Sigma Chemical Company) was prepared in water and stored at 4QC.200 pL plasma samples were warmed at 378 C , for five mn and 0.05 ml of pre-warmed PEG solution added to each sample. Tubes containing the samples were mixed twice for five seconds and left for 30 mn in melting ice. After centrifugation for 3 mn at 8000 rpm the supematant was removed. This was used immediately or stored at -70°C Normal pooled plasma was treated as described above to act as a free protein S standard. E.I.A. was carried out using the same electrophoresis buffer as for TPS. Plates were coated with 1% high EEO agarosis in electrophoresis buffer containing 0.1% P.E.G. 8000 (23) and 0.3% of antiserum . Electrophoresis conditions, washing and staining were done as for Total Protein S. Each Laurel1 plate was calibrated with four dilutions ( 1: I,1 :2,1:4,1:8 ) of the normal plasmapool, obtained from ten donors (mean age: 53; range: 18-65). Antigenic levels were calculated relative to the normal pool level that was defined as 100%.
Statistical Methods: Mann Whimey U test was used to analyse differences between groups and a p value inferior to 0.05 was considered as statistically significant.
197
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
RESULTS Four patients from the carcinoma group died during the course of the study (one before initiating treatment) and seven others (two from the before treatment group and five already under androgenic blockade) failed to be present at the follow-up. No serious clinical complications were observed in the remaining patients. All patients showed liver and renal function tests within normal levels. Routine coagulation studies revealed consistently elevated levels of fibrinogen with no other alterations.
Protein C and S results Antigen levels as well as functional activity of PC showed no significant differences either between the control and the total patient groups , between the control and after treatment or before treatment groups or between the two latter groups (Figure n”l and n”2). Significant higher levels of TPS were found in the disease groups when compared with the control group (Figure ngl), but no such differences were seen, when values of before treatment and after treatment patient groups were compared (Figure 11~2).FPS levels failed to yield any significant differences between group
I?; an_d__pCJn prostatlc
PSUree)
PXtotal)
PC runt. C
controls
PC ag.
carcinoma
PXlree)
P - patiti;
PSRotal) ??
- p
(n-14)
Figure n91
Figure np 2
c0.05
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
DISCUSSION Protein C may have its function altered either by a variance in its total antigen level or an alteration of its molecular structure, hence the interest in determining both functional and antigenic levels (11). Higher antigenic and functional PC levels have been found in patients with prostatic cancer submitted to treatment with oestrogens (20).In our patient groups no such alterations were found. Plasma Protein S, ace-factor for PCA anticoagulant activity, can be found in two forms: a free fraction which is the active one, and an inactive fraction bound to Complement component C4bbinding protein, C4b-BP, (16,24). PS is synthesized in the liver, endothelium, and in megakariocytes, being also found stored in platelets (15, 16). Immunoelectrophoresis, which was used in our study does not permit the assay of TPS, isolated from C4b-BP. This protein has recently been identified as an acute phase reactant, and may vary in its degree of binding to PS (25,26,27). For these reasons possible implication of C4b-BP in PS levels encountered must be considered. We found no references to Total Protein S determinations in prostatic neoplasia. The fact that there were no significant differences for Free Protein S among the groups studied supports the idea that the high Total Protein S levels, found in the carcinoma patient groups, are dependent on the bound fraction. No significant differences in TPS levels were found when before and after treatment groups were compared. This excludes the possibility of the observed increase in TPS being dependent on therapy. Further studies are needed in order to clarify the role of C4b-BP in our findings and to conclude whether the observed increase in TPS levels is a situation specific of prostatic carcinoma or an acute phase reaction as is the case with the increase of other coagulation factors, namely fibrinogen.
REFERENCES 1. AL-MONDHIRY, H. , DRAY, J., BARTHOLOMEW, M. J. The fibrinolitic system in experimental prostate tumor. Thromb. Haemostas. ,56 (2), 133-136, 1986 2. KOLLER, A., KIRCHEIMER, J., PFLUGER, H.,BINDER, B. R. Tissue plasminogen activator activity in prostatic cancer. Eur. Urol., 10 (6), 389-394, 1984. 3. KARPOV, A.B., UDUT, V.V., TSISIK, R.M., SOLOKHINA, E. A. Disorders of the haemostatic system in patients with disseminated stomach cancer. Vour. Onkol. 35(4) 460-465, 1989. 4. MILROY, R., DOUGLAS, ST., CAMPBELL, J., CANTER, R., LOWE, G. D., BANHAM, S.W. Abnormal haemostasis in smal cell lung cancer. Torax, 43 (12) 978-981, 5. RAPAPORT,S.I., CHAPMAN, C. G. Coexistent hypercoaguability and acute hypofibrinogenemia in a patient with prostatic cancer. Am. J. Med., 27, 144-153,1959.
Vol. 63, No. 1
PROTEINS C & S: PROSTATIC CANCER
199
6. AL-MONDHIRY, H., MANNI, A., OWEN, J., GORDON, R. Haemostatic effects of hormonal stimulation in patients with metastatic prostate cancer. Am. J. Hematol. ,28 (3), 141-145, 1988 7. BLOMBACK, M., HEDLUND, P.O., SAWE, U. Changes in blood coagulation and fibrinolysis in patients on different treatment regimens for prostatic cancer. Predictors for cardiovascular complications? Thromb. Res. 49 (1) 11-121, 1988. 8. HENRIKSSON,P.,BLOMBACK,M., BRATT,G.,EDHAG, O.,ERIKSSON, A.. Activators and inhibitors of coagulation and fibrinolysis in patients with prostatic cancer treated with oestrogens or orchidectomy. Thromb. Res. 44 (6), 783-79 1, 1986.
9. STENFLO, J. A new vitamin k-dependent protein; purification from bovine plasma and preliminary characterization. J. Biol. Chem. 251: 355, 1976 10. KIESEL, W., CANFIELD, W., ERICSSON, L., DAVIE, E., Anti-coagulant properties of bovine plasma protein C following activation by thrombin. Biochemistrv 16: 5824; 1977 11. MARLAR, R., KLEISS, A., GRIFFIN, J. Mechanisms of action of human activated protein C a thrombin-dependent anticoagulant enzyme. Blood 59: 1067, 1982. 12. VEHAR, G. , DAVIE, E., Preparation and properties of bovine factor VIII. Biochemistry 19,401, 1980. 13.ZOLTON,R., SEEGERS, W. Autoprothrombin II-A: thrombin removal and mechanisms of induction of fibrinolysis. Thromb. Res. 3:23, 1973 14. COMP, P.C., ESMON, C.T., Generation of fibrinolytic activity by infusion of activated protein C in dogs. J. Clin. Invest., 68, 1221, 1981. 15. WALKER, F.J., Regulation of activated protein C by protein S. The role of phospholipids in factor Va inactivation. J. Biol. Chem. 256, 11128-l 1131, 1981 16.GLADSON,C.L., SCHARRER, I.,HACH, V., BECK,K.H., GRIFFIN, J.H. The frequency of type I heterozygous Protein S and Protein C Deficiency in 141, unrelated young patients with venous thrombosis Thromb Haemostas. 59 (l), 18-22, 1988 17. COMP, P.C.V., NIXON, R.R., COOPER, M.R., ESMON, C.T. Familial protein S deficiency is associated with recurrent thrombosis. J. Clin. Invest. , 74 2082-2088, 1984 18. GRIFFIN, J.H., EKATT, B., ZIMMMERMAN,T.S., KLEISS, A.J., WIDEMAN,C. Deficiencies of protein C in congenital thrombotic disease. J. Clin. Invest. 68 1370-1373, 1982 19. SCHWARZ, H.P. FISCHER, M. HOPMEIR, P. BATARD, M.A., GRIFFIN J.H. Plasma protein S deficiency in familial thrombotic disease. Blood 64 1297-1300, 1984
200
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
20. ESPARAZ, R., GREEN,D., A study of proteins C and S in patients with prostatic disease Thromb. Haemostas. 60 (1) 122 ,1988
21. LAURELL, C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containig antibodies. Analvt. B&hem., 1545-47, 1966. 22. COMP,P.C., DORAY, D. PATTON D., ESMON, C.T. An abnormal plasma distribution of protein S occurs in functional protein S difficiency. Blood 67, 504-508, 1986 23. WOODHAMS, B.J. Simultaneous measurement of total and free protein S by Elisa. Thromb. Res. 50; 213-220, 1988. 24. DAHLBACK, B., STENFLO, J. High molecular weight complex in human plasma between Vitamin K-dependent Protein S and Complement component C4B-Binding Protein. Proc. Natl. Acad. Sci.,78,2512-2516,1981. 25. SACKI, T., HIROSE, S., NAKATSUKA, M., KUSUNOKI, Y., NAGASAWA, S.. Evidence that C4b binding protein is an acute phase protein. Biochem. Biophvs. Res. Commun.,l64: 1446-14451, 1989. 26. COMP. P. C., VIGANO, S., DiANGELO, A., THURNAU, G., KAUFMAN, C., ESMON, C.T.. AcquiredProtein S deficiency occurs in pregnancy, the nephrotic syndrome and acute sysremic lupus eritematosus. Blood, 65: 348 (Abstr), 1985. 27. COMP, P. C., THURNAU G. R., WELSH, J., ESMON, C.,T., Functional and immmunologic protein s levels are decreased during pregnancy. Blood, 68: 881-885, 1986.
BRIEF
---- COMMUNICATION ---___---
PROTEINS C AND S IN PATIENTS WITH ADVANCED PROSTATIC CANCER P. Dinis Oliveira, M. GraGa Oliveira, F. Pina, J. Aguiar Andrade, J.M. Pina-Cabral Centro de Fisiologiada Hemcktase (INK), Dpt. of Physiology, Oporto Medical School,Dpt- of Urology,Hosp. S. J&o, Porto 4200 Porto, PORlWAL (Received
7.1.1991;
accepted
in revised form 15.4.1991
by Editor U. Abildgaard)
INTRODUCTION inpatientswithprostatic Asin otherneoplasia,haemostaticalterationsarefrequentlyobserved cancer (1,2,3,4). These changes may occur due to the cancer itself or also as a result of therapy, particularly with oestrogens(6,7,8,). Protein C (PC) is avit Kdependent zymogen of a serine protease (9) which has an anticoagulant (10,11,12) andprofibrinolytic effect (13,14), after activation by thrombin on the endothelial surface Protein S (PS), another vit. K dependent protein, binds activated protein C (APC) to the endothelial surface (15,16) thus serving as a co-factor for APC, in the proteolytic cleavage of activated coagulation factors V and VIII. Although deficiencies in either PC or PS may be associated with thrombotic events (17,18,19), references concerning a possible alteration of these proteins in prostatic cancer are scarce(20). In this work the authors determined plasmatic levels of Proteins C and S in patients with prostatic cancer before and after total androgenic blockade with buserelin plus flutamide or orchidectomy plusflutamide, a therapy that is increasingly substitutingoesuogens in the management of this disease, due to a lesser incidence of cardiovascular effects.
MATERIALS AND METHODS Patient material: Thirty patients with either cytologically or histologically proven prostatic cancer ( stages C or D of the Whitmore classification) were selected. All patients had samples drawn twice, at least seven weeks apart (7), and in thirteen of them before initiating treatment. In all cases, second blood collections were done in patients under total androgen blockade, either with orchidectomy plus flutamide (250 mg tid) or buserilin (0.1 mg six times daily by nasal inhalation) plus flutamide. The control group was composed of fourteen patients with benign prostatic hypertrophy (20). Key Words: Protein C, Protein S, Prostatic Cancer 195
196
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
Laboratorv Methods: Blood Samples for PC and PS determination were collected in one tenth volume of 3.8% trisodium citrate and platelet poor plasma (PPP) was prepared by centrifugation for 15 mn at 2500 G at 4* C. Plasma was stored at -7OYJ. until used. Blood was also drawn for routine coagulation assays ( Pits, II VII X, FI, F XIII Quick and APTI’) as well as for liver and renal function indicators. Protein C Antigen was determined by E.IA. . Electrophoresis was carried out using the same buffer as described below, at 25 V/ 4cm . Plates were coated with 1% high EEO agarose in electrophoresis buffer containing .0.45% of antiserum from Diagnostica Stago (Assera Protein C) After electrophoresis over night plates were washed and stained with Coomassie blue. Functional Protein C levels were determined using a coagulometric test based on the APTT prolongation after Protein C activation by a snake venom (Acticlot C - American Diagnostica Inc ). Total and free nrotein S antigen were measured by electroimunoassay (EIA) Determination of Total Protein S (TPS) was carried out according to Laurel1 (21) using a buffer composed of 1.62 g/ L sodium veronal, 7.05 g/L glycine; 5.65g/L Tris, 1.80 g/L Na2 EDTA, pH 8.8 at 15 V/4cm. Plates were prepared usi.ng 1% high EEO agarose in electrophoresis buffer and 0.6% antiserum from Diagnostica Stago (Assera Protein S). After overnight electrophoresis, plates were washed and stained with Coomassie blue. Under these conditions only one protein S rocket was observed. Free Protein S (FPS) was measured after separation from Bound Protein S, acording to Comp et al(22): A 18.75% W/V solution of PEG 8000 (from Sigma Chemical Company) was prepared in water and stored at 4QC.200 pL plasma samples were warmed at 378 C , for five mn and 0.05 ml of pre-warmed PEG solution added to each sample. Tubes containing the samples were mixed twice for five seconds and left for 30 mn in melting ice. After centrifugation for 3 mn at 8000 rpm the supematant was removed. This was used immediately or stored at -70°C Normal pooled plasma was treated as described above to act as a free protein S standard. E.I.A. was carried out using the same electrophoresis buffer as for TPS. Plates were coated with 1% high EEO agarosis in electrophoresis buffer containing 0.1% P.E.G. 8000 (23) and 0.3% of antiserum . Electrophoresis conditions, washing and staining were done as for Total Protein S. Each Laurel1 plate was calibrated with four dilutions ( 1: I,1 :2,1:4,1:8 ) of the normal plasmapool, obtained from ten donors (mean age: 53; range: 18-65). Antigenic levels were calculated relative to the normal pool level that was defined as 100%.
Statistical Methods: Mann Whimey U test was used to analyse differences between groups and a p value inferior to 0.05 was considered as statistically significant.
197
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
RESULTS Four patients from the carcinoma group died during the course of the study (one before initiating treatment) and seven others (two from the before treatment group and five already under androgenic blockade) failed to be present at the follow-up. No serious clinical complications were observed in the remaining patients. All patients showed liver and renal function tests within normal levels. Routine coagulation studies revealed consistently elevated levels of fibrinogen with no other alterations.
Protein C and S results Antigen levels as well as functional activity of PC showed no significant differences either between the control and the total patient groups , between the control and after treatment or before treatment groups or between the two latter groups (Figure n”l and n”2). Significant higher levels of TPS were found in the disease groups when compared with the control group (Figure ngl), but no such differences were seen, when values of before treatment and after treatment patient groups were compared (Figure 11~2).FPS levels failed to yield any significant differences between group
I?; an_d__pCJn prostatlc
PSUree)
PXtotal)
PC runt. C
controls
PC ag.
carcinoma
PXlree)
P - patiti;
PSRotal) ??
- p
(n-14)
Figure n91
Figure np 2
c0.05
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
DISCUSSION Protein C may have its function altered either by a variance in its total antigen level or an alteration of its molecular structure, hence the interest in determining both functional and antigenic levels (11). Higher antigenic and functional PC levels have been found in patients with prostatic cancer submitted to treatment with oestrogens (20).In our patient groups no such alterations were found. Plasma Protein S, ace-factor for PCA anticoagulant activity, can be found in two forms: a free fraction which is the active one, and an inactive fraction bound to Complement component C4bbinding protein, C4b-BP, (16,24). PS is synthesized in the liver, endothelium, and in megakariocytes, being also found stored in platelets (15, 16). Immunoelectrophoresis, which was used in our study does not permit the assay of TPS, isolated from C4b-BP. This protein has recently been identified as an acute phase reactant, and may vary in its degree of binding to PS (25,26,27). For these reasons possible implication of C4b-BP in PS levels encountered must be considered. We found no references to Total Protein S determinations in prostatic neoplasia. The fact that there were no significant differences for Free Protein S among the groups studied supports the idea that the high Total Protein S levels, found in the carcinoma patient groups, are dependent on the bound fraction. No significant differences in TPS levels were found when before and after treatment groups were compared. This excludes the possibility of the observed increase in TPS being dependent on therapy. Further studies are needed in order to clarify the role of C4b-BP in our findings and to conclude whether the observed increase in TPS levels is a situation specific of prostatic carcinoma or an acute phase reaction as is the case with the increase of other coagulation factors, namely fibrinogen.
REFERENCES 1. AL-MONDHIRY, H. , DRAY, J., BARTHOLOMEW, M. J. The fibrinolitic system in experimental prostate tumor. Thromb. Haemostas. ,56 (2), 133-136, 1986 2. KOLLER, A., KIRCHEIMER, J., PFLUGER, H.,BINDER, B. R. Tissue plasminogen activator activity in prostatic cancer. Eur. Urol., 10 (6), 389-394, 1984. 3. KARPOV, A.B., UDUT, V.V., TSISIK, R.M., SOLOKHINA, E. A. Disorders of the haemostatic system in patients with disseminated stomach cancer. Vour. Onkol. 35(4) 460-465, 1989. 4. MILROY, R., DOUGLAS, ST., CAMPBELL, J., CANTER, R., LOWE, G. D., BANHAM, S.W. Abnormal haemostasis in smal cell lung cancer. Torax, 43 (12) 978-981, 5. RAPAPORT,S.I., CHAPMAN, C. G. Coexistent hypercoaguability and acute hypofibrinogenemia in a patient with prostatic cancer. Am. J. Med., 27, 144-153,1959.
Vol. 63, No. 1
PROTEINS C & S: PROSTATIC CANCER
199
6. AL-MONDHIRY, H., MANNI, A., OWEN, J., GORDON, R. Haemostatic effects of hormonal stimulation in patients with metastatic prostate cancer. Am. J. Hematol. ,28 (3), 141-145, 1988 7. BLOMBACK, M., HEDLUND, P.O., SAWE, U. Changes in blood coagulation and fibrinolysis in patients on different treatment regimens for prostatic cancer. Predictors for cardiovascular complications? Thromb. Res. 49 (1) 11-121, 1988. 8. HENRIKSSON,P.,BLOMBACK,M., BRATT,G.,EDHAG, O.,ERIKSSON, A.. Activators and inhibitors of coagulation and fibrinolysis in patients with prostatic cancer treated with oestrogens or orchidectomy. Thromb. Res. 44 (6), 783-79 1, 1986.
9. STENFLO, J. A new vitamin k-dependent protein; purification from bovine plasma and preliminary characterization. J. Biol. Chem. 251: 355, 1976 10. KIESEL, W., CANFIELD, W., ERICSSON, L., DAVIE, E., Anti-coagulant properties of bovine plasma protein C following activation by thrombin. Biochemistrv 16: 5824; 1977 11. MARLAR, R., KLEISS, A., GRIFFIN, J. Mechanisms of action of human activated protein C a thrombin-dependent anticoagulant enzyme. Blood 59: 1067, 1982. 12. VEHAR, G. , DAVIE, E., Preparation and properties of bovine factor VIII. Biochemistry 19,401, 1980. 13.ZOLTON,R., SEEGERS, W. Autoprothrombin II-A: thrombin removal and mechanisms of induction of fibrinolysis. Thromb. Res. 3:23, 1973 14. COMP, P.C., ESMON, C.T., Generation of fibrinolytic activity by infusion of activated protein C in dogs. J. Clin. Invest., 68, 1221, 1981. 15. WALKER, F.J., Regulation of activated protein C by protein S. The role of phospholipids in factor Va inactivation. J. Biol. Chem. 256, 11128-l 1131, 1981 16.GLADSON,C.L., SCHARRER, I.,HACH, V., BECK,K.H., GRIFFIN, J.H. The frequency of type I heterozygous Protein S and Protein C Deficiency in 141, unrelated young patients with venous thrombosis Thromb Haemostas. 59 (l), 18-22, 1988 17. COMP, P.C.V., NIXON, R.R., COOPER, M.R., ESMON, C.T. Familial protein S deficiency is associated with recurrent thrombosis. J. Clin. Invest. , 74 2082-2088, 1984 18. GRIFFIN, J.H., EKATT, B., ZIMMMERMAN,T.S., KLEISS, A.J., WIDEMAN,C. Deficiencies of protein C in congenital thrombotic disease. J. Clin. Invest. 68 1370-1373, 1982 19. SCHWARZ, H.P. FISCHER, M. HOPMEIR, P. BATARD, M.A., GRIFFIN J.H. Plasma protein S deficiency in familial thrombotic disease. Blood 64 1297-1300, 1984
200
PROTEINS C & S: PROSTATIC CANCER
Vol. 63, No. 1
20. ESPARAZ, R., GREEN,D., A study of proteins C and S in patients with prostatic disease Thromb. Haemostas. 60 (1) 122 ,1988
21. LAURELL, C.B. Quantitative estimation of proteins by electrophoresis in agarose gel containig antibodies. Analvt. B&hem., 1545-47, 1966. 22. COMP,P.C., DORAY, D. PATTON D., ESMON, C.T. An abnormal plasma distribution of protein S occurs in functional protein S difficiency. Blood 67, 504-508, 1986 23. WOODHAMS, B.J. Simultaneous measurement of total and free protein S by Elisa. Thromb. Res. 50; 213-220, 1988. 24. DAHLBACK, B., STENFLO, J. High molecular weight complex in human plasma between Vitamin K-dependent Protein S and Complement component C4B-Binding Protein. Proc. Natl. Acad. Sci.,78,2512-2516,1981. 25. SACKI, T., HIROSE, S., NAKATSUKA, M., KUSUNOKI, Y., NAGASAWA, S.. Evidence that C4b binding protein is an acute phase protein. Biochem. Biophvs. Res. Commun.,l64: 1446-14451, 1989. 26. COMP. P. C., VIGANO, S., DiANGELO, A., THURNAU, G., KAUFMAN, C., ESMON, C.T.. AcquiredProtein S deficiency occurs in pregnancy, the nephrotic syndrome and acute sysremic lupus eritematosus. Blood, 65: 348 (Abstr), 1985. 27. COMP, P. C., THURNAU G. R., WELSH, J., ESMON, C.,T., Functional and immmunologic protein s levels are decreased during pregnancy. Blood, 68: 881-885, 1986.