protein C (blood clotting/factor V/clotting inhibitors)

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1 Proc. Nati Acad. Sci. USA Vol. 80, pp , March 1983 Biochemistry Human coagulation factor Va is a cofactor for the activation of protein C (blood clotting/factor V/clotting inhibitors) HATEM H. SALEM, GEORGE J. BROZE, JOSEPH P. MILETICH, AND PHILIP W. MAJERUS* Divisions of Hematology-Oncology and Laboratory Medicine, Departments of Internal Medicine, Biological Chemistry, and Pathology, Washington University School of Medicine, St. Louis, Missouri Communicated by Stuart Kornfeld, December 28, 1982 ABSTRACT During blood clotting in vitro, protein C is converted in part to protein Ca. Protein Ca, in turn, inactivates factor V.. This is evidenced by the rapid inactivation of factor Va coagulant activity after clot formation which is associated with the cleavage of the Mr 110,000 peptide of factor Va. When exogenous factor Va is added to serum, it is inactivated only after a lag of min. Using purified coagulation factors in the presence of EDTA, we demonstrated that factor Va enhances the rate of protein C activation by thrombin by 50-fold. The Km for factor Va in the reaction is 14 nm, 100 times higher than its Km for accelerating platelet surface prothrombin activation by factor X.. By this mechanism, factor Va can act as a procoagulant as well as limit dissemination of the coagulation process through the activation of protein C and the subsequent inactivation of both factor V. and factor VIlla. Factor V is a single-chain polypeptide, Mr 330,000, that is relatively inactive until cleaved by thrombin to form factor Va (1). Factor Va serves as a cofactor for prothrombin activation by factor Xa (1). Human platelets possess approximately 300 high-affinity binding sites for factors Xa and Va, and bound factor Xa catalyzes prothrombin activation 300,000-fold faster than free factor Xa in solution (2-5). Protein C, a vitamin K-dependent glycoprotein, is also activated by thrombin with the release of a small peptide (Mr, 1,000) from the amino terminus of the heavy chain (6-9). Activated protein C (Ca) functions as an anticoagulant through the selective proteolysis of factor Va (10, 11) and factor VIIIa (12), the cofactors for thrombin and factor Xa generation, respectively. The physiological importance of protein Ca has been questioned because of its relatively slow activation by thrombin, particularly in the presence of Ca2+. Evidence that protein C is physiologically important includes the finding that patients with combined factor V and VIII deficiencies lack an inhibitor of protein C (13) and the finding of low levels of protein C in a family with multiple thromboses (14). More recently, Esmon and co-workers (15-17) proposed and identified an endothelial cell cofactor they termed "thrombomodulin" which enhances the rate of protein C activation by thrombin. In this paper we present evidence for the activation of protein C during blood clotting in vitro. We further demonstrate, using purified proteins, that factor Va, a substrate for protein Ca, acts as a cofactor in the activation of protein C by thrombin. MATERIALS AND METHODS Except where indicated, all chemicals were purchased from Sigma. Factors II and X were purified and activated as described (18). Human factor V was isolated (19) and had an activity of 130 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact units/mg of protein. It was radiolabeled by using Nal251 (Amersham) and IODO-GEN (Pierce Chemical). lodinated factor V had the same coagulation activity as the starting material. Factor V coagulant activity was measured by a one-stage assay utilizing purified coagulation factors as described (3). Thrombin was used to activate factor V to Va. Full (20- to 50-fold) enhancement in factor V coagulant activity was consistently obtained within 10 min at 370C at all thrombin concentrations used, ranging from 0.03 to 12 nm. Human protein C was purified as described by Suzuki et al (20) except that DEAE-Sepharose 6B-CL (Pharmacia) was used instead of DEAE-Sephacel. It was fully activated by thrombin, at a substrate-to-enzyme weight ratio of 85:1, at 370C for 3 hr. Thrombin was neutralized with hirudin and full activation of protein C was documented by NaDodSO4/polyacrylamide gel electrophoresis. That thrombin fully activated protein C was further documented by showing that the shift in migration of the protein C heavy chain on NaDodSO4 gel electrophoresis under reducing conditions was accompanied by the ability to incorporate [3H]diisopropyl fluorophosphate into the cleaved heavy chain. The effect of factor V on human protein C activation in the presence of thrombin was tested by incubating protein C with 0.6 nm thrombin and various concentrations of factor V or Va in a buffer containing 10 mm Tris Hepes (ph 7.8), 0.15 M NaCl, 0.1% polyethylene glycol, and 6 mm EDTA. After 60 min at 370C, the thrombin was neutralized with hirudin (20 units/ml) and the amount of protein Ca formed was determined by measurement of hydrolysis of benzoylarginine ethyl ester (BAEE) (17). For this, 5.2 mm BAEE was incubated with protein Ca in a final volume of 25 Al in the same Tris Hepes buffer described above for 30 min at 370C. Ethanol release was measured by using alcohol dehydrogenase: 12.5-,ul samples were added to 310,ul of 70 mm sodium pyrophosphate/20 mm glycine/75 mm semicarbazide, ph 8.8, containing 2 mg of,b-nad and 60 units of alcohol dehydrogenase per ml; absorbance changes at 340 nm were recorded over a 10-min interval at room temperature. Hydrolysis of BAEE was linear with respect to time and protein Ca concentration under the conditions used. Human protein Ca cleaved BAEE at &,at values of 7-28 sec-l in different preparations. IgG from control and anti-factor V (21) serum was purified by using protein A-Sepharose (Pharmacia). NaDodSO4/polyacrylamide gel electrophoresis was performed according to Laemmli with a 4% stacking gel (22). The acrylamide concentration was 7.5% for factor V and 10% for protein C. Protein concentrations were determined spectrophotometrically from absorbance at 280 nm with the following values used for El%:280 thrombin, 16.2; factor V, 8.9; protein C, 14.5; and IgG, Abbreviation: BAEE, benzoylarginine ethyl ester. * To whom reprint requests should be addressed.

2 RESULTS Formation and Inactivation of Factor Va During Blood Clotting. Factor V is a relatively inactive procofactor which, when converted to factor Va, increases in activity by 20- to 50-fold as measured by our coagulation assay. Using purified components, we developed a highly sensitive assay for factor V(Va) (3) that allows its measurement in whole blood. This is possible because plasma samples are diluted 1:104 or greater prior to assay, thereby preventing interference from thrombin or other factors generated in clotting of whole blood. Thus, the assay is insensitive to small changes in factor Xa concentration (z10 ng/ml, even if all of factor X was activated during blood clotting). Concentrations of thrombin up to 10 units/ml did not affect the assay when diluted 1: 104 to 1: 10. Using this assay, we followed the time course of change in factor V activity during and after blood clotting (Fig. 1). Factor V activity increased 2- to 3-fold, from 1 unit/ml, by the time of clot formation and peaked at units/ml several minutes later, suggesting conversion of factor V to Va. Thereafter, factor Va activity decreased progressively and after 1 hr, less than 1% of the activity remained. Because purified factor Va is relatively stable, the loss of activity in blood clotting suggested that it was destroyed by a factor in serum. We repeated the experiment in Fig. 1 but added "2I-labeled factor V to the blood (0.75 pug/ml) at the time of collection in order to follow the pattern of factor Va formation and inactivation (Fig. 2). By 2 min the high molecular weight factor V disappeared and a pattern of proteolysis characteristic of thrombin action was apparent with major bands at M, 150,000, 110,000, and 78,000 in addition to a higher molecular weight intermediate. By 5 min, factor Va formation was complete, and thereafter the peptide of M, 110,000 disappeared as factor Va activity decreased. The cleavage of the M, 110,000 component of factor Va is characteristic of the inactivation of factor Va by protein Ca (10, 11, 20). This result suggests that protein C is converted in part to Ca during blood coagulation. In three other experiments we collected blood in Microfuge tubes without anticoagulant and rapidly prepared platelet-poor plasma (within 10 sec). Factor V activation is faster in the presence of platelets (23). However, in glass tubes, factor Va formation was complete by 5 min in both whole blood and in samples centrifuged to remove platelets, leukocytes, and erythrocytes. Thereafter, there was inactivation of factor Va at the same initial rate of 2-4 units/ml per min in both types of samples. 30 -> l0 o 20 Biochemistry: Salem et al. Time, min FIG. 1. Activation and inactivation of factor V during blood clotting. Fresh blood (2 ml) was collected in a glass tube and incubated at 370. At the times indicated, 5-,ul samples were removed, diluted 1: 104 to 1:105 in 10 mm Tris/0.15 M NaCl, ph 7.4, containing 10 mg of albumin per ml, and immediately assayed for factor V coagulant activity. 40 Proc. Natl. Acad. Sci. USA 80 (1983) * _ Time Activity FIG. 2. Autoradiography of tracer 125I-labeled factor V during blood clotting. Fresh blood (2 ml) was mixed with 1.5,ug of 125I-labeled factor V (3.8 x 104 cpm/ng) and incubated at 370C. At the times (in minutes) indicated, 5-Al samples were removed, diluted in 10 mm Tris/0.15 M NaCl, ph 7.4, containing albumin (10 mg/ml), and immediately assayed for factor V coagulant activity (results shown as "Activity" are factor V activity in units/ml). At the same time, 50-,ul samples were centrifuged for 30 sec at 12,000 rpm in a Brinkman 3200 centrifuge. The supernatant plasma was added to an equal volume of 6% Na- DodSO4, boiled for 5 min, and analyzed by NaDodSO4/polyacrylamide gel electrophoresis. The other components at the top of the geljust under factor V (lane for 0 time) represent partial degradation of factor V (5). Mrs are shown x 10'. Inactivation was >90% by 60 min, indicating that protein C activation does not require platelets or other blood cells. Effect of Factor Va on Protein C Activation. In an attempt to identify the protein C activator in serum we added factor Va to serum samples in which the endogenous factor Va was already inactivated. In these experiments we observed that factor Va was inactivated only after a lag period of min and that the lag period was shortened by increasing concentration of factor Va. These results suggested that factor Va might participate in protein C activation. We therefore studied the effect of factor Va on thrombin-catalyzed protein C activation by using purified protein C. Factor Va was stable in 0.6 nm thrombin (Fig. 3, curve A). When factor Va was incubatedwith thrombin, protein C, and rabbit brain cephalin there was progressive inactivation of factor Va, as measured by coagulation assay, after a lag of min (Fig. 3, curve B). Prior incubation of protein C with thrombin for 1 hr in the absence of factor V shortened the lag slightly (Fig. 3, curve C). When factor Va was incubated with protein C and thrombin for 1 hr in the absence of rabbit brain cephalin, inactivation of factor Va was only 20%. Upon addition of rabbit brain cephalin, rapid inactivation of factor Va was followed (t1,2 = 2 min) without any lag period (Fig. 3, curve D). These experiments suggest that factor Va accelerates thrombin-induced protein C activation and that the inactivation of factor Va by Ca depends, in part, on abbit brain cephalin as has been reported (11). The effect of increasing factor Va concentration on protein C activation by0.6nm thrombin is shown in Table 1. In these studies protein C was used at the physiological level (0.1,tM). The rate of protein C activation increased with increasing factor Va concentrations and appeared to reach saturation at 0.12,uM factor Va. In these experiments the rate of factor Va inactivation is presumed to be proportional to protein Ca concentration. The actual amount of protein Ca formed was determined by other methods (see below).

3 1586 Biochemistry: Salem et al Proc. Natd Acad. Sci. USA 80 (1983) 150 bio a -4 0) :4; Cd 10 FB 0 0 ' ~4Z u D Time, min FIG. 3. Inactivation of factor Va by protein C in the presence of thrombin. Reaction mixtures were as follows: curve A, factor Va, thrombin, and rabbit brain cephalin; curve B, factor Va, protein C, rabbit brain cephalin, and thrombin; curve C, protein C, rabbit brain cephalin, and thrombin preincubated for 60 min at 3700 followed by addition of factor Va; curve D, factor Va, protein C, and thrombin preincubated for 60 min at 370C followed by addition of rabbit brain cephalin. Final concentrations were: protein C, 0.5 pm; factor Va, 0.1 1LM; rabbit brain cephalin, 80 Ag/ml; thrombin, 0.6 nm. Incubations were in 30 1d of 10 mm Tris/0.15 M NaCl/2.5 mm CaCl2, ph 7.4, containing albumin at 10 mg/ml. At the points indicated, 2-,ul samples were removed, diluted in 10 mm Tris/0.15 M NaCl, ph 7.4, containing albumin (10 mg/ml), and assayed for factor V coagulant activity. Protein C Activation Measured by Esterolytic Activity. We measured protein Ca formation by assaying hydrolysis of BAEE. BAEE hydrolysis was linear with time and protein C. concentration under the conditions used (data not shown). In the absence of factor V., 0.6 nm thrombin caused no detectable formation of protein Ca after 1 hr at 37 C. When factor Va was added at 0. l,um, in addition to 0.6 nm thrombin, protein Cawas formed linearly with increasing concentrations of protein C (Fig. 4). No protein Ca was formed when factor Va was incubated with pro- Table 1. Stimulation of protein C activation by factor Va as measured by coagulation assay Factor Va Protein C. formed, added, nm units/ml per min Protein C (0.1,uM) was incubated at 37 C with the indicated concentrations of factor Va along with 0.6 nm thrombin in 10 mm Tris, ph 7.4/4 mm CaCl2 containing 5 mg of bovine serum albumin per ml. After 60 min the samples were adjusted to contain 150 nm factor V. and assayed for factor V. activity. Rabbit brain cephalin was then added to a final concentration of 80,ug/ml. The decay in factor V. activity was measured 2 and 5 min later. Protein C, formed is expressed as the rate of factor V. inactivation. 2 Protein C, FIG. 4. Protein C-dependent generation of esterolytic activity. Protein C at the indicated concentrations, factor V8 at 0.1 pm, and thrombin at 0.6 nm were incubated at 37 C for 1 hr. Protein C activation was stopped by the addition of hirudin (20 units/ml) and samples were assayed for protein C8. tein C or protein C8 plus protein C in the absence of thrombin. We were unable to achieve a saturating concentration of protein C; however, the concentrations used greatly exceeded those found in human plasma ( ,iM) (20). Furthermore, we observed factor V.-dependent protein C activation at physiological concentrations (Table 1). The effect of varying thrombin concentration on protein C activation is shown in Fig. 5. In this experiment, factor V was used rather than factor V,. At all thrombin concentrations used, V8 formation was complete within 10 min and therefore the thrombin was required primarily for protein C activation. The effect of varying factor V8 concentration on protein C8 formation is shown in Fig. 6. The half-maximal rate of protein C8 formation occurred at 14 nm factor V8 in this experiment oa0)/ LM 0.25 Thrombin, nm FIG. 5. Thrombin dependence of protein C activation in the presence of factor V8. Factor V at 0.1,uM and protein C at 1.9 AM, were incubated at 37 C for 1 hr with thrombin at the concentrations indicated. Hirudin (20 units/ml) was used to inhibit the thrombin at the end of this period, and the amount of protein C8 generated was measured. 4

4 200 Biochemistry: Salem et al 0~~~~~~~~~~.4 o /~~~~~~~/ [Factor V] Factor V, WIM FIG. 6. Effect of factor Va on activation of protein C by thrombin. Protein C at 3.9 ym was incubated with 0.6 nm thrombin and factor V at the concentrations indicated for 60 min at 370C. Hirudin (20 units/ ml) was used to inhibit the thrombin at the end of this period and the esterolytic activity of the sample was measured. (Inset) Double reciprocal plot for the same experiment. The formation of protein Ca in the presence of factor Va and thrombin did not require Ca2O (Table 2). Factor Va increased the activation of 1,uM protein C by thrombin by approximately 50-fold, as also shown in Table 1. The ability of factor Va to accelerate protein C activation does not depend on the same sites as those required for accelerated prothrombin activation. We incubated 50,ug of factor Va in 6 mm EDTA for 60 min, during which time the activity decreased from 84 units to 1 unit as measured by coagulation assay. In contrast, this preparation was still able to accelerate protein C (2,uM) activation by 0.6 nm thrombin. Protein Ca was formed at 112 pmol/ml per hr with EDTAinactivated factor Va compared to 126 pmol/ml per hr with native factor Va. We used a described (21) monoclonal antibody against factor V to establish that the effects observed were due to factor Va perse. In this experiment we incubated factor Va (0.7,uM) with anti-factor V IgG or control IgG (1.2,uM) for 60 min at 37 C. Samples were assayed for factor V coagulant activity and for the ability to generate protein Ca in the presence of0. 6 nm thrombin and 2,uM protein C. Factor Va incubated with control IgG had factor Va activity of750 units/ml and protein Ca was formed at 97 pmol/ml per hr. Factor Va incubated with anti-factor V IgG had factor Va activity of 3.5 units/ml and no protein Ca was formed. DISCUSSION Suzuki et al (23) recently showed that factor V is fully activated to Va as determined by NaDodS04/polyacrylamide gel electrophoresis when human blood clots in vitro. However, it has long been known that factor V activity is absent from human serum (24). We have shown that factor V is fully activated during in Table 2. Activation of protein C by factor V and thrombin in the presence and absence of Ca2" Protein Ca formed. pmol/ mol/mol Incubation mixture ml/hr thrombin/hr Protein. C, 1 ym; factor V, 0.5 utm; thrombin, 0.6 nm; Ca2+, 2.5 mm Protein C, 1 M;factor V, 0.5 AM; thrombin, 0.6 nm; EDTA, 6 mm Protein C, 1 pm; thrombin, 60 nm; Ca mm Proc. NatL Acad. Sci. USA 80 (1983) 1587 vitro blood coagulation as measured by both coagulation assay and NaDodSO4 gel electrophoresis and that the factor Va is subsequently inactivated by the action of protein Ca. Full activation of factor V to Va does not take place until 3-5 min after clot formation, by which time a 20- to 30-fold increase in activity is observed. Previous investigators have shown that thrombin, the only known circulating activator of protein C, forms protein Ca at a very slow rate at levels of thrombin that are achieved physiologically. Esmon and co-workers (15-17) discovered that a protein present on endothelial cells, designated "thrombomodulin," could accelerate thrombin-catalyzed protein Ca formation. The current work indicates that factor Va is also able to serve this function. The relationship, if any, between factor Va and thrombomodulin remains to be elucidated. Factor Va is at least as potent as thrombomodulin. Owen and Esmon (16) found that 1.1 nm thrombin incubated with 2 AM protein C plus thrombomodulin generates approximately 1 pg of protein Ca per ml in 1 hr. Under similar conditions with 2 AM protein C, 0.1 um factor Va, and 0.6 nm thrombin, we found 5-10 pg of protein Ca formed per ml in 1 hr. The amount ofprotein C activated during in vitro blood clotting is probably very small. Kisiel et al (8) found that protein C in plasma was indistinguishable from that in serum. We do not know the steady-state levels of protein Ca in serum (because of concomitant activation and inactivation by inhibitors), but our results indicate that only small.amounts of protein Ca are required to account for factor Va inactivation. In the experiment reported as curve D of Fig. 3, we found that approximately 50 units offactor Va was inactivated in 2 min by the protein Ca formed after incubation of 0.5,M protein C with 0.6 nm thrombin and factor Va for 1 hr. We estimate from Fig. 4 that approximately 20 pmol of protein Ca was present during this incubation. Thus, the initial loss of Va activity in serum, 1.8 units/ml per min (Fig. 1), could be accounted for by 1-2 pmol of protein Caper ml. This would require only 1-2% activation of protein C. The concentration of factor Va required to stimulate protein C activation is greater than that required for platelet surface prothrombin activation. Half-maximal thrombin generation on platelets occurs' at factor Va concentrations of nm (5) which is approximately 1% of that required for protein C activation. When plasma factor V is fully activated to Va, its concentration is approximately 30 nm which is sufficient to accelerate protein C activation markedly. In this way, factor Va may serve to localize prothrombin activation to the platelet surface. As coagulation is initiated, low levels of factor Va accelerate thrombin formation which, in turn, activates larger amounts of factor V, ultimately generating factor Va in amounts that convert thrombin to a protein C activator. With the formation of protein Ca, factors Va and VIIIa are inactivated and dissemination of the coagulation process is prevented. The effect of factor Va on protein C activation that we have described can explain the inactivation of factor Va during in vitro blood clotting. However, other factors must be involved in vivo if factor Va indeed serves to localize coagulation by activating protein C. Thus, the time required to inactivate factor Va (Fig. 1) is too slow to prevent dissemination of coagulation. Cellular factors such as thrombomodulin may enhance the efficiency of this sytem. We thank Dr. Craig Jackson for helpful discussions and for supplying a sample of bovine protein Ca. This research was supported by Grants HLBI (Specialized Center in Thrombosis) and HL from the National Institutes of Health and by a grant from Monsanto Chemical Corporation. 1. Jackson, C. M. & Nemerson, Y. (1980) Annu. Rev. Biochem. 49,

5 1588 Biochemistry: Salem et al 2. Miletich, J. P., Jackson, C. M. & Majerus, P. W. (1977) Proc. Nati Acad. Sci. USA 74, , 3. Miletich, J. P., Majerus, D. W. & Majerus, P. W. (1978)J. Clin. Invest 62, Miletich, J. P., Kane, W. H., Hofimann, S. L., Stanford, N.. & Majerus, P. W. (1979) Blood 54, Kane, W. H. & Majerus, P. W. (1982) J. Biol Chem. 257, Stenflo, J. (1976) J. Biol Chem. 251, Esmon, C. T., Stenflo, J., Suttie, J. W. & Jackson, C. M. (1976) J. Biol Chem. 251, Kisiel, W., Ericsson, L. H. & Davie, E. W. (1976) Biochemistry 15, Kisiel, W. (1979) J. Clin. Invest. 64, Canfield, W, Nesheim, M., Kisiel, W. & Mann, K. G. (1978) Circulation 58, Suppl. 2, 210 (abstr.). 11. Walker, F. J., Sexton, P. W. & Esmon, C. T. (1979) Biochim. Biophys. Acta 571, Vehar, G. A. & Davie, E. W. (1980) Biochemistry 19, Marlar, R. A. & Griffin, J. H. (1980)J. Clin. Invest. 66, Griffin, J. H., Evatt, B., Zimmerman, T. S., Kleiss, A. J. & Wideman, C. (1981) J. Clin. Invest. 68, Proc. Natl. Acad. Sci. USA 80 (1983) 15. Esmon, C. T. & Owen, W. G. (1981) Proc. Nad. Acad. Sci. USA 78, Owen, W. G. & Esmon, C. T. (1981) J. Biol Chem. 256, Esmon, N. L., Owen, W. G. & Esmon, C. T. (1982) J. Biod Chem. 257, Miletich, J. P., Broze, G. J. & Majerus, P. W (1980) Anal Biochem. 105, Kane, W. H. & Majerus, P. W. (1981)J. Biod Chem. 256, Suzuki, K., Stenflo, J., Difhlback, B. & Teodorsson, B. (1983)J. Biod Chem. 258, Coots, M. C., Muhleman, A. F. & Glueck, H. Y. (1978) Am. J. HematoL 4, Laemmli, U. K. (1970) Nature (London) 227, Suzuki, K., Ddhlback, B. & Stenflo, J. (1982)J. Biol Chem. 267, Murphy, R. C. & Seegers, W. H. (1948) Am. J. PhysioL 154,

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