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CLIN. CHEM. 35/8, 1644-1648 (1989) Functional Activity of Protein S Determined with Use of Protein C Activated by Venom Activator Iaao Kobayashl,’ Norlhlko Amemlya,2Takeehl Endo,2 KohJlOkuyama,2 KohjI Tamura,1 and ShojI Kume2 A new screening procedure, an easy and specific assay for determining functional Protein S activity, has been developed, with use of Protein C activated by venom activator (Protac). Purified Protein C (100% amidolytic activity) was activated by venom activator (6 units/mL). To a mixture of 100 L of Protein S-deficient plasma, 20 L of sample plasma, 100 L of cephalin (Actin), and 20 L of activated Protein C we added 100 L of 25 mmol/L CaCI2 solution and measured the clotting time with a KC 10 coagulometer. The functional Protein S activity correlated well with the concentrations of Protein S antigen measured by enzyme immunoassay and the Laurell rocket technique (r = 0.810 and 0.850, respectively) in normal subjects, patients with myocardial infarction undergoing warfarin therapy, and patients with liver cirrhosis. Addftlonal Keyphrasea: screening . anticoagulant therapy liver cirrhosis . enzyme immunoassay and electroimmunoassay compared coagulation assays myocardial infarction The naturally occurring anticoagulant proteins antithrombin Ill, Protein C, and Protein S function to inhibit blood clotting. Several hereditary deficiencies and abnormalities among these proteins have been linked topredisposition to recurrent thrombosis (1). Protein S, a vitamin K-dependent plasma protein, functions to inhibit the procoagulant cofactors Va and Villa by serving as a cofactor for another plasma protein, activated Protein C (2-4). Patients deficient in Protein C are subject to severe, recurrent venous thrombosis (5-7) because of their inability to regulate intravascular clot formation. Because Protein S is required for the expression of the anticoagulant activity of activated Protein C, a hereditary deficiency of it has also been associated with recurrent thrombosis (8-10). Several specific methods for determining Protein S antigen have already been established (11-14). Two types of Protein S deficiency are recognized. In type I, the patient has little or no free Protein S but has significant quantities of Protein S associated with C4b-binding protein. In type H, the patient has no free Protein S and very little Protein S associated with C4b-binding protein (11). In some cases of Protein S deficiency the presence of an abnormal Protein S molecule with decreased functional activity has been recognized (8, 15). Therefore, a functional activity assay method for Protein S that could be performed in a clinical laboratory would be useful. Several assay methods specific for protein S functional activity have been reported (15-17) but are unsuitable for use as a screening assay. For example, they require specific purified coagulation factors or are subject to nonspecific ‘2nd Department of Internal Medicine and 2Depent of Hematology of the Central Clinical Laboratory, Yamanashi Medical College, Shimokato 1110,Tainahocho, Nakakoxnagun, Yamanashi 409-38, Japan. Received February 3, 1989; accepted May 8, 1989. 1644 CLINICAL CHEMISTRY, Vol. 35, No. 8, 1989 activation of plasma by venom activator, as we have already reported (18). Now we have developed a new, simple, specific assay for determining Protein S functional activity by using Protein C activated by venom activator (Protac), without interference from the nonspecific activation of plasma. MaterIals and Methods Materials Purified venom activator (“Protac,” one vial contains 3 goat IgG against human Protein S, and Protein C were obtained from American Diagnostica, Greenwich, CT. One unit of Protac activates all the Protein C in 1 mL of normal plasma. Protein S-deficient human plasma was obtained from Diagnostica Stago, Asnieres, France. The concentration of Protein S antigen in Protein S-deficient plasma was less than 2% of the normal concentration in plasma as determined by enzyme-linked immunosorbent assay (EusA). Cephalin (Actin) and Owren’s barbital buffer (50 mmolJL, pH 7.35) were purchased from American Dade, Miami, FL. Blood was collected into 38 g/L trisodium citrate solution, in a 9:1 ratio of blood to anticoagulant. Platelet-poor plasma (ppp) was prepared by centrifugation at 4#{176}C (2000 x g for 20 mm), then stored at -70 #{176}C until use. Normal plasma pooled from 26 ostensibly healthy normal subjects who had not been taking any drugs was used as standard plasma. Because there is yet no generally purified reference standard for Protein 5, the activity in normal plasma is usually used in defining the unit of biological and immunological activity of ProteinS, i.e., the amount of activity in 1 mL of average normal plasma (usually a pooled specimen of 20 normal plasmas) (19). The advantage of this system of units is that the severity of Protein S deficiency in a patient can be easily related to normal on a percentage scale. The Protein S activity in normal pooled plasma is stable for a month at -70 #{176}C, and the coefficient of variation (CV) for Protein S activity under these conditions is <5%. We also used citrated plasma samples from patients with myocardial infarction who were being treated with warfarin and from patients with liver cirrhosis. Substances used for interference experiments were as follows. Biirubin (161 mgldL) was an aqueous standard solution (Nippon Shoji Co., Ltd., Tokyo, Japan), hemoglobin was a hemolysate of normal erythrocytes washed with isotonic saline solution and then lysed with distilled water, lipid was a fat emulsion used for intravenous administration (Intrafat; Takeda Co., Ltd., Tokyo, Japan). Vitamin C (L-ascorbic acid), heparin (heparin sodium salt, 198.3 units/ mg), and dipotassium ethylenediaminetetraacetic acid (EDTA) were from Wako Pure Chemical Industries Co., Ltd., Tokyo, Japan. units of activity), purified human Methods Activated Protein C was prepared by mixing 100 L of Protein C (diluted with Owren’s barbital buffer, pH 7.35, to 100% amidolytic activity) with 20 zL of venom activator (diluted with distilled water to 5 unita/mL), then incubating at 37#{176}C for 15 mm. We used an automated KC 10 coagulometer (Heinrich Amelung GmbH, Lieme, F.R.G.) to measure the clotting time. To prepare the standard curve, we assayed standards having 0%, 6.25%, 12.5%, 25%, 50%, 100%, and 200% of the amount of Protein S in pooled normal plasma. The pooled normal plasma and the sample plasma were diluted with Owren’s barbital buffer, pH 7.35, or Protein S-deficient plasma. When sample plasma was diluted twofold, pooled normal plasma without dilution thus contained twice the concentration of Protein S activity, comparatively speaking. The assay was performed as follows. To the well of the KC 10 coagulometer add 20 pL of the standard or sample plasma, and 100 zL of Protein S-deficient plasma. After a 1-min incubation at 37#{176}C, add 100 p.L of cephalin. After 1 mm, add 10 zL of activated Protein C (69.7 g/mL). Exactly 2 mm later, add 100 1zL of 25 mmol/L CaC12 solution and measure the clotting time. We also assayed total Protein S antigen with an ELISA method (Asserachrom ProteinS; Diagnostica Stago, Francoville, France) and the Laurell rocket technique (11), using sample plasma. Protein S complexed to C4b-binding protein was precipitated with 3.75% polyethylene glycol 8000 according to the report of Comp et al. (11) and free Protein S antigen in the supernate was also assayed by the ELISA method and the Laurell rocket technique. Protein C amidolytic activity was measured by our method (18). Interference experiments were done as follows: To one volume of test substance add nine volumes of normal plasma and measure Protein S functional activity of the mixture after 30 mm of incubation at room temperature. We computed the linear-regression analysis as the principal standardized component, using the method proposed by Feldmann et al. (20). Results Figure 1 shows the influence of nonspecific activity of venom activator on coagulation time in the assay system. The clotting time of normal plasma was markedly prolonged in proportion to the concentration of venom activator present. In Protein S-deficient plasma the clotting time was also slightly prolonged at high concentrations of venom activator (2-4 units/mL). There is ordinarily some #{149} Normal o 80 Protaim plasma S depleted nonspecific activation of Protein S-deficient plasma by venom activator. In our assay system the final concentration of venom activator was <1 unit/mL. Therefore, there may have been no influence of nonspecific activation of Protein S-deficient plasma by venom activator in this assay system. After mixing 10 pL of venom activator (6 units/mL) with 100 ,uL of Protein C (100% amidolytic activity), we studied the activation of Protein C kinetically at 37#{176}C (Figure 2). The amidolytic activity of Protein C increased quickly, reaching a maximum after 15 miii, and after 45 mm it gradually decreased. Therefore, we measured Protein S activity from 15 to 45 miii after the activation of Protein C by venom activator. To determine the effect of incubation time, after adding activated Protein C, on clotting time, we assayed normal plasma, using incubation periods from 1 to 5 mm. Clotting time was longest after a 2-mm incubation. Therefore, we incubated the assay mixtures for 2 miii after adding activated Protein C. The standard curve from 6.25% to 200% is shown in Figure 3. Pooled normal plasma was diluted with Owren’s barbital buffer or with Protein S-deficient plasma to obtain the various Protein S concentrations.Both standard curves are linear on a semilogarithm scale, but the slope is steeper for the standard curve incorporating data for standard diluted with Protein S-deficient plasma. Therefore, we usually used the latter standard curve but measured sample plasma without dilution. We measured total Protein S antigen, using the standard curve for pooled normal plasma, and the value was the (LI Afmjn) 0 30 60 90 120 (mm) Fig.2. Kinetics forvenom activator actionon ProteinC activity After 100 pL ofProtein c (100% amidolytlc activity) was mixedwIth10 p1.of venom activator (6 unfts/mL), theamidolytic activity of Protein C was measuredserially by a Idnetic assay,at 405 nm plasma #{149} Diluted with Buffer o with Protein (S) 300 Diluted depleted S plasma #{149}2O0 ‘I, 01 20 100 Q U 0 1 2 3 L Protac (unlts/mL) FIg.1. Effect of venom activator (Protac)on coagulationtime of normal plasma (#{149}) and ProteinS-depletedplasma(0) To themixture of 100 1L of normal plasmaorProtein S-deficient plasma,100 uL of cephalin, and 20 pL of venom activator in variousconcentrationswe addediOOpL of 25mmol/L.CaCI2and measured theclotting time(In seconds) ..-/25 6.25 12.5 50 100 Protein S activity 200(0/0) Fig. 3. Standardcurvefor ProteinS activity from 6.25% to 200% of normal (S) = seconds CLINICAL CHEMISTRY, Vol. 35, No. 8, 1989 1645 Table 1. PrecisIon of the Assay Mean SD Sampi. Within-run 23.5 95.2 2.49 154.6 5.48 7.83 2.67 3.54 19.2 104.7 164.3 3.11 16.25 5.46 6.22 5.21 Low Normal High Between-run Low Normal High 1.84 3.79 n = 20 each. percentage of normal for total ProteinS antigen in pooled normal plasma. Free Protein S antigen was measured by using the standard curve for free Protein S of poolednormal plasma, and again the value was in terms of the percentage of free Protein S antigen in pooled normal plasma. Table 1 shows the precision, within-run and betweenrun. Resultsof interference experiments are shown in Table 2. A biirubin final concentration of 26 mg/dL, >0.1 unit of heparin per milliliter, and EDTA all interfered with the assay of Protein S functionalactivity. Figure 4 summarizes assay results for plasma samples from 31 normal subjects, 25 patients with liver cirrhosis, and 15 warfarun-treated patients with myocardial infarction. Free Protein S antigen as measured by the Laurell rocket technique or by ELISA exhibited good correlation with the Protein S functionalactivity (r = 0.850 and 0.810, respectively). Values for Protein S functional activity and antigen (total and free) and results for two casesof Protein S deficiency are shown in Table 3. DIscussIon The naturally occurring anticoagulantproteins-Protein C, ProteinS, and antithrombin Ill-inhibit blood clotting, and several hereditary deficiencies and abnormalities among these proteins have been linked to predisposition to recurrent thrombosis (5, 8,21). The frequencies of heterozygous Protein C and Protein S deficiencies detected by total antigen concentrations in plasma of young patients with venous thrombosis are 4% for type I ProteinC deficiency and 5% for type I Protein S deficiency (22). Many previously reported Protein S deficiencies were detected because of the decreased concentration of antigen. Protein in plasma is present both in the free form, which is the active Protein C cofactor, and in a functionally inactive form that is complexed to complement compound C4bbinding protein (23). These two forms of Protein S complicate diagnosis of Protein S deficiencies and abnormalities, because ordinary immunoassays do not distinguish between the free and bound forms (11). Most homozygous Protein S-deficient patients lack the free Protein S form but may have significant quantities of the bound inactive form (11). But most heterozygotes have concentrations of total Protein S antigen that are below the lower limit of the normal range (24). Therefore, methods for assaying functional Protein S activity are needed, but existing ones are not yet feasible in most laboratories. Our assay system of functional Protein S activity is simple and specific, because measurement of Protein C activity activated by venom activator (Protac) is easy, the standard curve is practicably steep, and in addition the functional activity is not influenced by the nonspecific activity of venom activator and shows good correlation with Protein S antigen as measured by ELISA and the Laurell rocket technique. Therefore, this assay method is useful in clinical laboratories for screening assay of functional Protein S activity. In normal subjects, functional Protein S activity was 102% (SD 21%) and values for total and free Protein S antigen were 106% (SD 25%) and 106% (SD 20%), respectively. The ratio of Protein S functional activity to the Table 2. Interference ExperImentsa Measured Rscovsry Substances Bilirubin,mg/dL Hemoglobin, mg/dL Lipid, % Added 2 5 7 10 26 (%) rate (%) 78 105 72 97 101 98 37 10 50 100 300 72 72 72 72 500 75 75 73 28 0.5 74 1.0 77 1.5 75 2.0 2.5 74 78 97 Materials VIt C, mg/dL Heparin,unltlmL 2 5 7 Measured (%) 63 75 76 Recovery rate (%) 85 101 102 10 30 75 74 101 Added 100 97 0.01 0.03 70 75 94 101 97 97 101 0.05 0.07 0.1 75 77 100 101 104 135 10 20 39 49 52 50 70 100 50 53 40 67 71 54 100 104 101 100 105 EDTA, mg/dL 66 100 Control 0 74 100 0 74 Control Theunit of each substance Is the final concentrationof the mixture. Isotonic saline solution was added to the mixture insteadofsubstance Incontrol. 1646 CLINICAL CHEMISTRY, Vol. 35, No. 8, 1989 Table 3. Plasma Content of Protein S (PS) Functional ActIvity and Antigen In VarIous Subjects Total PS PS activity n 31 Normal antigen Free PS antigen 102 ± 21 Free PSI total PS S 106 ± 25 Warfarintherapy 15 31 ± 16 59 ± 13 Livercirrhosis 25 52 ± 22 66 ± 29 106 ± 20 (E) 110±18(L) 51 ± 12(E) 48±17(L) 64 ± 18 (E) 1.00 0.85 0.97 63 ± 25 (L) Protein S deficiency a 2 37 15 ± 78 ± 2 61 ± 52± 8(E) 4(L) 0.78 PSactivity! free PS antigen 0.96 0.93 0.61 0.65 0.81 0.83 0.60 0.72 E, ELISA; L Laurell’s rockettechnique. Activity #{149} Liver 150 0 o Normal A Wararin therapy disease 0 0 0 0 0 100 % c% #{149} a0 000. ‘S ‘ n-li r-0 :9 #{149}I. I I #{149} #{149}#{149} 50 n-li A .850 #{149}r-0.8i0 Pathol Lab Med 1988;112:28-36. 2. Walker FJ. Regulation of activated Protein C by a new protein. J Biol Chem 1980;255:5521-4. 3. Walker FJ. Regulation of activated Protein C by Protein S. the role of phospholipid in factor Va inactivation. J Biol Chem 1981;256:11128-31. 4. Suzuki K, Nishioka J, Hashimoto S. Regulation of activated Pi-otein C by thrombin modified Protein S. J Biochem (Tokyo) 1983;94:699-705. 5. Griffin JH, Evatt B, Zimmerman TS, Wideman C. Deficiencyof Protein C in congenital thrombotic disease. J Clin Invest 1981;68:1370-3. 6. Comp PC, Nixon ER, Eamon CT. Determination of functional levels of Protein C, an antithrombotic protein, using thrombin 150 100 50 100 150 0 (() Laurell 50 thrombomodulin complex.Blood 1984;63:15-21. ELISA (‘Ia) 7. Broekmans AW, Veltkamp JJ, Bertina EM. Congenital Protein Fig. 4. Correlationbetween ProteinS functional activity and Protein C deficiency and venous thromboembolism: study of three Dutch S antigen (free form) measured by Laurell rocket techniqueand families. N Engl J Med 1983;309:340-4. ELISA in normal subjects (0), patIents with myocardlal Infarction undergoingwarfarintherapy(Lx),and patientswithlivercirrhosis(#{149})8. Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with a partial deficiencyof Protein S. N Engi J Med 1984;311:1525-8. 9. Broekmans AW, Bertina RW, Reinalda-Poot J, et al. Herediconcentrations of total or free forms of Protein S assayed by taly ProteinS deficiencyand venousthromboembolism.A studyin ELISA in normal subjects is nearly 1.0. In our patients with three Dutch families. Thromb Haemost 1985;53:273-7. myocardial infarction undergoing therapy with warfarin or 10. Kamiya T, Sugthara J, Ogata K, et al. Inherited deficiencyof in those with liver cirrhosis, the decreases in Protein S Protein Sin a Japanese family with recurrentvenous thrombosis: functional activity and antigen concentration (total and study of three generations. Blood 1986;67:406-10. 11. Comp PC, Doray D, Patton D, Esmon CT. An abnormal free forms) are prominent. And the ratio of functional plasma distribution of Protein S occurs in functional Protein S activity to the free form of Protein S as assayed by ELISA deficiency. Blood 1986;67:504-8. decreased more prominently in patients undergoing ther12. BertinaEM, Von Wijngaarden A, Reinalden-PoolJ, Poort SR, apy with warfarin than in patients with liver cirrhosis. But BornVJJ. Determination of plasma Protein S, the proteincofactor the ratio of the concentrations of free Protein S antigen to of activated Protein C. Thromb Haemost 1985;53:268-72. total Protein S antigen as assayed by ELISA is also nearly 13. Mannucci PM, Tripodi A, Bertina EM. ProteinS deficiency associated with juvenile arterial and venousthrombosis.Thromb 1.0. Therefore, in patients being treated with warfarin Haemost 1986;55:440. there would be decreased synthesis of the total and free 14. Bennhagen MJ, Holmberg R, DahlbOck B. Plasma concentraforms of Protein S antigen accompanying the production of tionsof C4b-binding protein and vitamin K-dependentProtein Sin incompletely y-carboxylatedand dysfunctional free form of term and preterm infants: low levels of protein S-C4b-binding Protein S. These discrepancies between Protein S funcprotein complexes.Br J Haematol 1988;68:445-9. tional activity and Protein S antigen were also recognized 15. CompPC, Nixon RB, Cooper MB, EsrnonCT. Familial Protein S deficiency is associatedwith recurrent thrombosis. J ChinInvest in other reports (15, 17). 1984;74:2082-8. During a screening examination of healthy subjects, we 16. Van de Waart P, PreissnerKT, Bechtold JR, Muller Berghans found two cases of Protein S deficiency. Both cases were G. A functional test for Protein S activity in plasma. Thromb Res type ll, as based on a report by Comp et al. (11), and the 1987;48:427-37. patients themselves had no thrombotic episodes nor did 17. Suzuki K, Nishioka J. Plasma Protein S activitymeasured their families. using Protac,a snake venom derived activator of Protein C. Thromb Res 1988;49:241-51. Evaluation of Protein S functional activity by a simple 18. Kobayashi I, Amemiya N, Endo T, et al. Amidolytic kinetic and specific screening assay method, both in healthy subassayof Protein C by selectivespectrophotometryin a centrifugal jects and patients with thrombosis, is quite useful and is analyzer. Clin Chem 1988;34:2260-3. urgently needed to treat such patients and to analyze the 19. Barrowcliffe TW. Standards and controls in assay of blood mechanism and pathophysiology of thrombosis. coagulation factors. In: Biggs R, Rizza CR, eds. Human blood coagulation, haemost.asis and thrombosis, 3rd ed. Oxford-LondonReferences Edinburgh-Boston-Palo Alto-Melbourne: Blackwell Scientific 1. High KA. Antithrornbin ifi, Protein C, and Protein S. Arch 1984:563-82. p<0.001 p(0 I .001 AS CLINICAL CHEMISTRY, Vol. 35, No. 8, 1989 1647 20. Feldmann U, Schneider B, KlinkersH, HaeckelR.A multivariate approach for the biometric comparison of analytical methods in clinical chemistry. J Chin Chem Clin Biochem1981;19:121-30. 21. Thaler E, Lechner K. Antithrombin ifi deficiencyand thromboembolisni.Clin Hematol 1981:369-90. 22. Gladson CL, Scharrer I, Hach V, Beck K!!, Griffin JH. The frequency oftype I heterozygousProtein Sand ProteinC deficiency in 141 unrelated young patients with venous thrombosis. Thromb 1648 CLINICAL CHEMISTRY, Vol. 35, No. 8, 1989 Haemost 1988;59:18-22. 23. DahlbOck B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependentProtein S and complement component C4b-binding protein. Proc Natl Acad Sci USA 1981;78:2512-6. 24. Engesser L, Broekmans AW, Briet E, Brommer EJP, Bertina EM. HereditaryProtein S deficiency:clinical manifestations. Ann Intern Med 1987;106:677-82.