Protein S and Thrombosis*

Transcription

1 ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 19, No. 3 Copyright 1989, Institute for Clinical Science, Inc. Protein S and Thrombosis* ROBIN L. GRAVES-HOAGLAND, P h.d. and FR ED ER IC K J. WALKER, P h.d. American Red Cross Blood Services, Connecticut Region, Farmington CT ABSTRACT Thrombosis is a serious problem in the U nited States. Activated protein C is a vitamin K -dependent serine protease that seems to be an im portant regulator of the hemostatic process. Protein S is another vitamin K -dependen t protein that is essential for the expression of th e anticoagulant activity of activated protein C. Evidence suggests that defects in either of these proteins may be related to the developm ent of thrombosis. Therefore, the accurate m easurem ent of these proteins is im portant in the study and in the treatm ent of thrombosis. Sixty percent of the protein S found in hum an plasma is bound to the com plem ent com ponent C4b-binding protein, and when in this complex, protein S is inactive. The remaining 40 percent is the free, functional form of protein S. This paper describes the m easurem ent of both free and total protein S by two types of assays. The first assay involves immunoelectrophoresis, also known as the Laurell Rocket m ethod. The second assay is a capture enzyme linked immunosorbent assay (ELISA). The two assays are compared as to relative ease and cost. Introduction The problem of thrombosis cannot be understated. Throm bosis and diseases related to throm bosis rank first among the causes of death in the United States. In addition, venous throm bosis afflicts approxim ately five m illion individuals per year. O ne aspect of research into the causes of throm boem bolic disease has been the search for the regulatory processes of the blood coagulation cascade. * Acknowledgements: This work was supported by grants from th e A m erican Red Cross and the National Institutes of Health (HL38388). Approximately 10 years ago, a vitamin K -dependent protein named protein C was discovered. This protein is of in te r est because, in its active form as a serine protease, it is able to inhibit blood coagulation and stim ulate fibrinolysis. These in vitro findings suggest that protein C may be an im portant regulator in the hem ostatic process. M uch work has been carried out in recent years to ascertain the mechanisms by which the activity of protein C is regulated. T hese m echanism s are often referred to as the protein C pathw ay (review ed in reference 9). In this pathway, protein C is first converted into a /89/ $01.20 Institute for Clinical Science, Inc.

2 PROTEIN S AND THROMBOSIS 209 serine protease which can express either anticoagulant or proiibrinolytic activity. In addition, a m echanism exists for its inactivation and clearance from th e circulation. It has been known for some tim e that throm bin can convert protein C into a serine protease. 15 However, protein C is a poor substrate for throm bin. It was observed that if throm bin and protein C w ere incubated together in the presence of e ith e r cu ltu red endothelial cells or vein segments, that the rate of protein C activation could be enhanced. 11 This led to the discovery and isolation of throm b o m o d u lin. T h ro m b o m o d u lin is an intrinsic m em brane protein found exclusively in endothelial cells. It has a high affinity for throm bin and, w hen throm bin is bound to throm bomodulin to form a throm bin-throm bom odulin complex, 13 the substrate specificity of throm bin is altered such that it will no longer clot fib rin o g e n 10 o r a c tiv a te p la te le ts. 12 Instead, it becomes a potent activator of protein C. Infusion of throm bin into dogs will produce a dram atic anticoagulant effect. This is thought to be due to the production of activated p rotein C. 4 Purified activated protein C is a potent inhibitor of blood coagulation in vitro as well. 17 It has been observed that activated protein C can proteolytically inactivate both coagulation factors V26 and V III. 20,25 Facto r V and factor V III are cofactor prote in s in v o lv e d in th e activ atio n of prothrom bin and factor X, respectively. Like other reactions that involve vitamin K -dependent proteins, the proteolysis of these two proteins is dependent upon the presence of calcium and phospholipids. 26 It is thought that these proteolytic events occur on a m em brane surface. The observation that factor V could be cleaved m ore rapidly in plasma than with p u rifie d co m p o n en ts su g g ested th at plasma m ight contain a cofactor for activated protein C. Purification of the factor in plasm a that could stim ulate proteolysis of factor V by activated protein C led to the discovery that protein S, another vitam in K -d e p en d en t p ro tein, was a cofactor for activated pro tein C. 22 P rotein S has been observed to form a complex on m em b ran e surfaces w ith activ a te d p ro te in C. 21,24 T his com plex appears to be essential for th e rapid inactivation of factors V and VIII. Infusion of p rotein C into dogs has been observed to lead to a shortening of the euglobulin clot lysis tim e. 19 It has also been observed to cause a rise in the plasma levels of plasminogen activator. 4 In a n u m b er of in vitro system s, activated protein C has been observed to have a shortening effect on clot lysis. Though not all of the studies are entirely clear, it appears that at least one effect of activated protein C is to inactivate a plasm a protein known as plasm inogen activator inhibitor. 714 A ctivated protein C is inhibited by se v e ra l p lasm a p ro te a s e in h ib ito rs. These include proteins known as protein C inhibitor and plasm inogen activator inhibitor. These proteins form a covalent complex with protein C which is cleared by the hepatic system. The protein C pathway provides for the activation, expression of activity, and inhibition of protein C, and provides a basis for looking for clinical defects that might lead to thrombosis. A num ber of patients have been found with defects in the protein C pathway. The inheritance of p ro te in C deficiency is autosom al dom inant. Individuals who are hom ozygotes for th e deficiency always have some form of thromboembolic disorder from b irth. 2 Throm bosis in heterozygotes is more complex and may involve other factors. 18 One im portant elem ent in the expression of the anticoagulant activity of activated protein C is protein S. Like prot e i n C, p r o t e i n S is a v i ta m i n K-dependent protein. However, among

3 210 GRAVES-HOAGLAND AND WALKER this fam ily of p ro te in s, p ro te in S is unique because it is not a zymogen of a serine protease. Like the other vitamin K -dependent proteins, protein S contains 1 0 gamma-carboxy glutam ic acid residues and an epiderm al growth factorlike dom ain. 16 It does not, how ever, have the trypsin domain. Instead, it has a region that has some homology with ste ro id b in d in g p ro te in s. 1 A second major difference betw een protein S and other factors involved in blood coagulation is its m echanism of regulation. Protein S does not circulate as an inactive p ro te in th at req u ires proteolysis for activity. The single chain protein is fully active. No proteolysis has been observed to enhance its activity. Protein S is, however, susceptible to proteolysis. It has b een observed to be cleaved by throm bin, resulting in an inactive two-chain protein.23 Cleavage by throm bin results in a protein that can no longer interact with membranes. In addition to the free protein, protein S has been observed to circulate in complex w ith the com plem ent com ponent C4b-binding protein. 8 W hen in complex with this protein, protein S is unable to act as a cofactor for activated protein C. Approximately 60 percent of the total protein S found in plasm a is found in association w ith C 4b-binding protein. Protein S deficiency has been found to b e of several types. The first type consists of low antigenic levels of the protein. This type can be detected by several different immunoassays. The second type consists of normal levels of protein S b u t w ith an abnorm al d istrib u tio n betw een the free and bound forms. The third type consists of norm al antigenic levels b u t no functional activity of the protein S. As large scale studies have not been carried out, it is not possible to d e te rm in e th e relativ e abundance of each type of deficiency or to estim ate th eir prevalence in the general population. Several kits are available to evaluate protein S levels and to determ ine free and bound levels of protein. The first to be discussed is an electroim m unodiffusion (EID ) assay called Rellplate S.* The second kit to be discussed is an enzy m e-lin k ed im m unosorbent assay (ELISA) called Asserachrom Protein S.f Protein S Assay by E ID P r in c ip l e In the determ ination of plasma protein S by E ID, also known as the L aurell Rocket technique, protein S in plasm a m igrates through an agarose gel under the influence of an electric field. P recipitation of protein S occurs owing to the presence of specific antibodies to protein S in the gel, and the height of the peak ( rocket ) is proportional to the concentration of total protein S originally in the plasm a sample. R e a g e n t s a n d S t o c k S o l u t io n s Two E ID plates containing goat antiserum to hum an protein S are provided in the kit. They are one percent agarose with 1 2 wells each and contain sodium azide as a preservative. The electrophoresis buffer is provided as two vials of dry salt, each enough for one liter of 0.08M Tris, 0.024M Tricine, and 3.5 mm ethylenediam ine tetraacetic acid (EDTA) at ph 8.6. The Coom assie Blue staining solution is provided as a two-fold concentrate of Coomassie Brilliant Blue in m ethanol and acetic acid. F ilter paper wicks and polyethylene glycol (PEG) solution (25 percent) are also provided in the kit. Pooled normal plasma (PNP) for standards or a com m ercial p ro tein S standard is needed and is not contained in the kit. A washing solution of 0.9 percent saline needs to be made by dissolv- * American Diagnostica, New York, NY. t Diagnostica Stago, France.

4 PROTEIN S AND THROMBOSIS 211 ing nine g of NaCl in one liter of distilled water. M ethanol and glacial acetic acid are n eeded to prep are th e destaining solution. They are mixed as follows: 4.5 parts methanol, 4.5 parts purified water, and 1.0 part glacial acetic acid. S p e c ia l A ppa r a t u s An electro p h o resis cham ber and a c o n sta n t c u rre n t p o w e r su p p ly a re needed for this assay. A microcentrifuge and m icrocentrifuge tu b es w ould be helpful although not necessary. Plastic tu b e s (12 X 75 m m ) m ay b e u se d instead. P r o c e d u r e S tandards for th e m easu rem ent of total protein S are prepared by diluting PN P with the electrophoresis buffer at d ilu tio n s o f 1:2, 1:4, 1:8 and 1:16 (defined as 100, 50, 25 and 12.5 percent, respectively). Unknown plasma samples are diluted 1:2. The m easurem ent of free protein S is made by first precipitating bound protein S with 15 fjd of 25 percent PE G added to 85 xl of either PN P or unknow n plasm a. A fter vortexing and incubating on ice for 30 min, the samples are centrifuged for two min at room tem p e ra tu re in a m icro cen trifu g e. The supernatant is used to m easure free protein S. The PN P su pernatant is used undiluted (defined as percent) and diluted at 1:2, 1:4 and 1:8 (50, 25 and 12.5 percent, respectively) for the standards. Unknown samples are used undiluted. Exactly 10 xl of the standards or samples are loaded into each w ell, and, using th e electro p h o resis buffer and wicks provided, the gel is electrophoresed for three hrs at 16 ma per gel. The plates are positioned in the apparatus with the agarose side down. Following electrophoresis, the plates are rinsed overnight in 0.9 percent saline. Plates are pressed, dried, and stained for 15 to 20 m in w ith the staining solution supplied in the kit. They are then destained w ith the destaining solution until relatively free of background. C a l c u l a t io n The distances from the sample wells to the peaks of the rockets are m easured for standards and samples. The standards for total and free protein S are plotted as log p e rc e n t p ro te in S v erses log ro ck et height or on log-log graph paper, and total and free p ro te in S in unknow n plasma samples are determ ined by interpolation from th e a p p ro p riate curve. Values are expressed as relative p ercen t ages of the PNP. Typical standard curves for total and free protein S are shown in figure 1. Protein S Assays by ELISA P r in c ip l e The determ ination of plasma protein S utilizing an ELISA involves th e sandw ich principle. M icrotiter plates, p re coated with antibodies to protein S, are allowed to react w ith plasma, and pro log % Protein S F i g u r e 1. Typical standard curves for total (circles) and free protein S (squares) using the EID assay kit by American Diagnostica (New York, NY).

5 212 GRAVES-HOAGLAND AND WALKER tein S in the plasma binds to the plate. Next, a rabbit anti-protein S antibody coupled to peroxidase is added, which binds to p rotein S at a free antigenic d eterm in an t and forms a sandw ich. The presence of the enzyme peroxidase is rev ealed by adding th e su b strate, o rth o -p h enylenediam in e (OPD ). The color change is proportional to the concentration of p rotein S in the plasm a sample. R e a g e n t s a n d S t a n d a r d S o l u t io n s A 96-well m icrotiter plate, pre-coated with rabbit antiserum to hum an protein S, is supplied in the kit. The plate may be broken into six groups of two columns each, w ith e ig h t w ells p e r colum n. Therefore, six groups of 16 wells each can be run successively. Dilution buffer is prepared by diluting a concentrated phosphate buffer (provided in the kit) containing bovine album in and Tween- 2 0, at a rate of 1 :1 0 with distilled water. The anti-protein S conjugated to peroxidase is also provided, lyophilized for reconstitution to 2 0 ml w ith dilution buffer. The washing solution is supplied as a 2 0 tim es concentrated solution of phosphate, sodium chloride and Tween- 20. It is prepared by diluting the 50 ml of concentrate with distilled water to a total volume of one liter. A vial containing the O P D su b stra te, ly o p h ilized w ith its buffer, is reconstituted just prior to use with 20 ml of distilled water. E ither 10 xl of 30 percent or 50 (xl of seven percent hydrogen peroxide are added to com plete the O P D solution. A 25 p ercent solution of PE G is needed if the m e a s u r e m e n t o f fre e p r o te in S is desired, and 3 M sulfuric acid or 1 M HC1 is needed to stop the developm ent reaction. S p e c ia l A p pa r a t u s An adjustable m ultichannel pipette is needed to dispense xl, xl, and 50 xl. M ultichannel washing equipm ent for m icrotiter plates is also quite helpful. A plate reader set at 490 to 492 nm is necessary. P r o c e d u r e E ith e r P N P or p ro te in S c o n tro l plasm a is d iluted 1 : w ith dilution buffer for the m easurem ent of total protein S. By definition, this dilution represents 100 p ercent total protein S. The standards are further dilutions of this at the rate of 1:2 (50 percent), 1:4 (25 p ercent), 1:10 (10 percent), 1:20 (5 percent), and a blank of dilution buffer alone (0 percent). Sam ple plasm as are diluted with dilution buffer at 1:100 and 1:200. If protein S concentration is expected to be low, a dilution of 1:50 or 1:20 is preferable. For the m easurem ent of free protein S, 300 jl1 plasma are precipitated with 50 xl of 25 percent PEG, shaken well, incubated for 30 min on ice, and centrifuged at 3,000 rpm for 10 min. The standard and sample plasmas are both treated in this way, and the supernatants are used to m easure free protein S. A 1:100 dilution of the PNP supernatant is defined as 100 percent free protein S, and further dilutions are made as for total protein S described previously. D iluted standard and sample plasmas ( jl1) are applied in duplicate to precoated wells, and are incubated for two hrs at room tem perature. The wells are then washed five times with the washing solution, follow ed by the im m ediate addition of 200 xl of the anti-protein S conjugate. After a two hr incubation at room tem perature, the wells are again washed five times with washing solution. The O PD substrate (200 jxl) is added, allow ed to react for th re e m in, after which 50 xl of 3 M sulfuric acid or 100 jjli of 1 M HC1 are added to stop the reaction. After allowing the plate to stand for 10 min, the optical density (O.D.) is read at 490 to 492 nm.

6 PROTEIN S AND THROMBOSIS 213 C a l c u l a t io n The standard curves for total and free p rotein S are plotted as the log of p e r cent protein S verses log O.D., or on log-log paper, with percent protein S on the X-axis and the corresponding O.D. values on the Y-axis. If sample plasmas w ere diluted at 1 :1 0 0, the percent protein S may be read directly off the calibration curve. If diluted at 1:200, the percent protein S m ust be m ultiplied by 2, and if diluted at 1:50 or 1:20, they m ust be divided by 2 or 5, respectively. Typical standard curves for total and free protein S are shown in figure 2. D is c u s s io n Both the EID * and the ELISA t kits for m easuring protein S are relatively simple to use. The instructions provided by the manufacturers are clear, and all b u t a few com m on reagents are p ro v id e d. S om e s p e c ia l a p p a ra tu s is required for both but that required for the E ID assay (electrophoresis cham ber and current supply) is probably a smaller investm ent than that required for the ELISA (m icrotiter plate reader). The choice of an assay procedure is certainly d e p e n d e n t on w hat e q u ip m e n t m ay already be available in a laboratory. A lth o u g h th e to ta l a m o u n t of w ork involved with the E ID m ethod is probably less, th e p ro ced u re takes longer because of the three hrs to run the gel, the overnight saline wash, and the time to dry the gel before staining. The entire procedure takes almost two days. The ELISA can be finished in five hrs, so if results are needed quickly these factors should be considered. The E ID kit provides two agarose gels w ith 12 wells each. A set of four standards is needed for total protein S, four standards are n eed ed for free protein S * American Diagnostica, New York, NY. t Diagnostica Stago, France. log % Protein S F i g u r e 2. Typical standard curves for total (circles) and free protein S (squares) using the ELISA kit by Diagnostica Stago (France). a n d tw o w ells a re n e e d e d for each unknown (one for total and one for free). Thus, eight unknowns can be m easured with each kit. At a cost of $ per kit, this is $18.13 p e r sam ple. T he ELISA kit comes with a m icrotiter plate containing 96 wells. The manufacturers suggest all standards and samples be run in duplicate, so there is a potential for 48 different wells. Six standards are needed for total protein S and six standards are n eeded for free protein S, leaving 36 wells for unknowns. W ith th e m easurem ent of total and free protein S for each unknown, it is possible to m easure 18 unknown samples p er kit. At a co st'o f $ per kit, this is $13.11 per samp le. T h e refo re, alth o u g h th e in itial investm ent to m easure protein S with th e E L ISA k it m ay be g re a te r, th e amount of tim e and cost p er sample are less than that for th e E ID assay examined. At the present tim e, the Asserachrom kit* is the only ELISA available, whereas E ID kits are available from two c o m p a n i e s. O n e E ID kit$ was not exam ined here. t American Diagnostica, New York, NY. t A ssera P late P rotein S, D iagnostica Stago, France.

7 214 GRAVES-HOAGLAND AND WALKER Both companies also have available a rabbit antiserum to hum an protein S w ith w hich crossed or bi-dim ensional im m u n o ele c tro p h o re sis can b e p e r form ed. In this instance, only the antiserum and instructions are provided. Sources of Error The m anner of sample storage is a possible source of erro r for e ith er assay. Both manufacturers recom m end a maximum storage tim e of one m onth when stored at 20 C. Some evidence in our laboratory suggests th at m ethod and tim e of storage may be a critical factor. It is possible th at sam ples may be p re served b etter at 70 C. T h e E ID a ssay is b a s e d on th e assum ption that rocket height is proportional to protein S concentration in a fixed am ount o f sam ple. The m anufacturers suggest this fixed am ount be 1 0 xl, which seem s to be appropriate for the size of the wells. C are should be taken to see that these small volumes are accurately m easured because m inor variations in sam ple volum e could cause variations in rocket height that are unrelated to protein S concentration. In co m p lete w ashing of m ic ro tite r plate wells in th e ELISA could cause high nonspecific binding of subsequent reagents. To aid in com plete washing, wells should be filled to capacity and em ptied com pletely each tim e. These suggestions are m ade by the m anufacturer, as well as having b een independently observed to be im portant in our laboratory. Normal Ranges Normal ranges for total protein S have been reported 1 as 78 to 103 percent, 70 to 122 percent, and 58 to 106 percent for m en, w om en, and w om en taking oral c o n tra cep tiv e s, resp ectiv ely. C o rre sponding free protein S values have been reported as 69 to 149 percent, 50 to 130 p ercent, and 52 to 120 p ercent, respectively, for th e same groups of people. Resume of Clinical Interpretations A decrease in plasma protein S level has been reported with throm bosis, 6 oral anticoagulant treatm ent, 5 and in women being treated with oral contraceptives. 27 Congenital protein S deficiency of two types has been rep o rted.5 O ne type is characterized by a decrease in anticoagulant activity (associated with free protein S) b ut little loss in antigenic protein S level. The other is associated with a large decrease in both protein S activity and protein S antigen level. In principle, a functional assay for protein S would be most useful in the detection of deficiencies. Functional activity is m easured by the enhancem ent of the anticoagulant activity of activated p ro tein C in a clotting assay. For such an assay a stable preparation of activated protein C and protein S deficient plasma are needed. Developm ent of the latter reagent has proven to be a technical problem. This is because protein S activity is a function of two variables, protein S levels and C4b-binding protein levels. Preparation of a test plasm a th at has levels of C4b-binding protein that are m atched to patient samples may prove to be difficult. T herefore, th e antigenic assays rem a in th e b e s t m e th o d for detecting deficiencies. References 1. B a k er, M. E., F r e n c h, F. S., and J o s e p h, D. R.: Vitamin K-dependent protein S is similar to rat androgen binding protein. Biochem. J. 243: , B er t in a, R. M., Br o ek m a n s, A. W., Va n d e r L in d e n, I. K., and M e r t e n s, K.: Protein C deficiency in a D utch family w ith thrombotic disease. Thromb. Haemost. 48:1 5, Bo e r g e r, L. M., M o r r is, P. C., T h u r n e a u, G. R., E sm o n, C. T., and Comp, P. C.: Oral contraceptives and gen d er affect p ro tein S status. Blood 69: , 1987.

8 PROTEIN S AND THROMBOSIS COMP, P. C. and E s m o n, C. T.: Generation of fibrinolytic activity by infusion of activated protein C into dogs. J. Clin. Invest. 8: , C o m p, P. C., D o r a y, D., P a t t o n, D., and E s m o n, C. T. : An abnormal distribution of protein S occurs in functional protein S deficiency. Blood 67: , C o m p, P. C., N i x o n, R. R., C o o p e r, M. R., and E s m o n, C. T.: Familial protein S deficiency is associated with recurrent thrombosis. J. Clin. Invest. 74: , D A n g e l o, A., L o c k h a r t, M. S., D A n g e l o, S. V., and T a y l o r, F. B.: Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation released from platelets. Blood 69: , D a h l b a c k, B. and S t e n f l o, J.: High molecular weight complex in human plasma between vitamin K -dependent protein S and com plem ent com ponent C4b-binding protein. Proc. Natl. Acad. Sci. USA 78: , E s m o n, C. T.: The regulation of natural anticoagulant pathw ays. Science 235: , E s m o n, C. T., E s m o n, N. L., and H a r r is, K. W.: Complex formation betw een thrombin and thrombomodulin inhibits both thrombincatalyzed fibrin formation and factor V activation. J. Biol. Chem : , E s m o n, C. T. and O w e n, W. G. : Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc. Natl. Acad. Sci. U.S.A. 78: , E s m o n, N. L., C a r r o l, R. C., and E s m o n, C. T.: Thrombomodulin blocks the ability of thrombin to activate platelets. J. Biol. Chem. 258: , E s m o n N. L., O w e n W. G., and E s m o n C. T.: Isolation of m em brane-bound cofactor for thrombin catalyzed activation of protein C. J. Biol. Chem. 257: , H e e b, M. J., E s p a ñ a, F., G e i g e r, M., C o l l e n, D., S t u m p, D. K., and G r i f f i n, J. H.: Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3. J. Biol. Chem. 262: , Kis ie l, W., C a n f ie l d, W. M., E r ic sso n, L. H., and D a v ie, E. W.: Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry i 6 : , L u n d w a l l, A., D a c k o w s k i, W., C o h e n, E., S h a f f e r, M., M a h r, A., D a h l b a c k, B., S t e n f l o, J., a n d W y d r o, R.: I s o l a t i o n a n d t h e s e q u e n c e o f t h e c D N A f o r h u m a n p r o t e i n S, a r e g u l a t o r o f b l o o d c o a g u l a t i o n. P r o c. N a t l. A c a d. S c i. 8 3 : , M a r l a r, R. A., K l e i s s, A. J., and G r i f f i n, J. H. : Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 5 9 : , M i l e t i c h J., S h e r m a n, L., and B r o z e G.: Absence of thrombosis in subjects with heterozygous protein C deficiency. New Engl. J. Med. 317: , Se e g e r s, W., M c C oy, L. E., G r o b e n, H. D., Sakuragaw a, N., and Ag r a w a l, B. B. L.: Purification and properties of autoprothrombin Ha: A n anticoagulant perhaps also related to fibrinolysis. Thrombosis. Res. 1: , V e h a r, G. A. and D a v i e, E. W.: Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1 9 : , W a l k e r, F. J.: Protein S and the regulation of activated protein C. Semin. Thromb. Hemost. 1 0 : , W a l k e r, F. J.: The regulation of activated protein C by a new protein: A possible function for bovine protein S. J. Biol. Chem. 255: , W a l k e r, F. J. : Regulation of vitamin K-dependent protein S : Inactivation by thrombin. J. Biol. Chem. 259: , W a l k e r, F. J.: Regulation of activated protein C by protein S: The role of phospholipid in factor Va inactivation. J. Biol. Chem : , W a l k e r, F. J., C h a v i n, S. I., and F a y, P. J.: Inactivation of factor VIII by activated protein C and p ro te in S. A rch. B iochem. B iophys. 252: , W a l k e r, F. J., S e x t o n, P. W., and E s m o n, C. T.: The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim. Biophys. Acta : , W e i s s, P., S o f f, G. A., H a l k i n, H., and S e l i g - S O H N, U.: Decline of proteins C and S and factors II, VII, IX, and X during the initiation of warfarin therapy. Thromb. Res. 45: , 1987.