Although human protein S binds to human factor Va and inhibits prothrombinase activity, this inhibition is not totally dependent on factor Va. Hence, we investipted possible interaction of protein S with human factor Xa. Factor Xa, dilsopropyiphospho-factor Xa and their biotin derivatives ligand blotted specifically to protein S and protein S ligand blotted specifically to factor X and factor Xa. Biotinylated factors X and Xa bound to immobilized protein S and, reciprocally, protein S bound to immobilized factor Xa with a Kd of -19 nM. In fluid phase, protein S bound to factor Xa with a Kd of 18 nM. Protein S at 33 nM reversibly inhibited 50% of factor Xa amidolytic activity. Protein S inhibition ofprothrombin conversion to thrombin by factor Xa was phospholipidindependent and was 1.6 times stimulated by Ca+ ions. Inhibition of prothrombinase activity by protein S was 2.3-fold more potent in the presence of factor Va, with 50% inhibition at _4 tM protein S. Protein S prolonged the factor Xa one-stage clotting time of protein S-depleted plasma in a dose-dependent manner. These data demonstrate mecanims of antr gulant action for protein S that are independent of activated protein C and that involve direct binding to factors Xa and Va and direct inhibition of factor Xa.Protein S is a vitamin K-dependent plasma protein that can act as a cofactor for the anticoagulant functions of activated protein C (1-3). Deficiency of protein S is associated with venous thrombosis (4-6) or arterial thrombosis (7,8). Among young adult patients with venous thrombosis, 4-12% have hereditary protein S deficiency (9). Mechanisms of action of protein S as an antithrombotic factor are not fully understood, although it exerts negative feedback on blood coagulation pathways (10). Protein S increases the affinity of activated protein C for phospholipid vesicles (11), platelets (12), and endothelial cells (13). In purified systems using platelets, the cofactor activity of protein S is modest, increasing the effect of activated protein C by a factor of 2 (14,15 IN) and enzymes were active-site-titrated (21). FVII was from Celsus Laboratories (Cincinnati). Proteins were >95% pure by SDS/PAGE and were stored in aliquots at -700C. Proteins were biotinylated as described (22). Diisopropylphospho (DIP)-FXa (99%6 inactivated) was prepared by incubation of FXa at 1 mg/ml with 2 mM diisopropyl fluorophosphate (Sigma) on ice for 2 hr and dialysis against Tris-buffered saline (TBS: 50 mM NaCl/100 mM Tris HCl, pH 7.4). DEGRFXa was prepared by incubation of FXa with a 1.5-molar excess of 1,5-dansyl-Glu-Gly-Arg (DEGR) chloromethyl ketone (Calbiochem) until >99% inactivated and dialysis against TBS. Goat antibody to protein S was immunoaffinitypurified (17). Monoclonal antibody (mAb) S7 to protein S was prepared (23) and purified (24) as described. p-Amidinophenylmethylsulfonyl fluoride and biotin N-hydroxysuccinimide were from Clontech. Chromatography gels were from Pharmacia.Purifiation of Protein S. For most experiments below, protein S was puri...

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x lo5 M -l s-'. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 pM phospholipid vesicles composed of 20 moly" dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of OlezGroPSer some inhibition of APC-catalyzed factor Va inactivation was observed.Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine < oleic acid < phosphatidic acid < phosphatidylglycerol < phosphatidylmethanol < phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the K , for factor Va and the V,,, of factor Va inactivation were a function of the phospholipid concentration. The K , increased from 1 nM at 2.5 pM phospholipid (Ole2GroPSer/Ole2GroPCho 20: 80, mol/mol) to 65 nM at 250 pM phospholipid. The V,,, increased from 20 mol factor Va inactivated * min-' . mol APC-' at 2.5 pM phospholipid to 62 mol factor Va inactivated . min-' . mol APC-' at 10 pM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation, Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.

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