Protein S binds to and inhibits factor Xa.

Abstract

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...

Generation and phenotypic analysis of protein S–deficient mice

“…[9][10][11] In addition, PS might directly limit thrombin generation by binding to and inhibiting factor Xa and FVa in the prothrombinase complex. 12,13 APC-cofactor activity of PS is consistent with the similar clinical manifestations associated with deficiencies in PS and protein C, but is in marked contrast with the weak potentiating effect of PS on APC proteolytic activity in vitro. Indeed, an only approximately 20-fold stimulation of APC-catalyzed cleavage of FVa by PS has been reported in purified systems.…”

Lack of Protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis

“…It functions as an essential cofactor for activated protein C (aPC) in the degradation of coagulation factors FVa and FVIIIa (1)(2)(3) and thus operates at a central node in the coagulation cascade. In in vitro assays, ProS also binds directly to FVa, FVIIIa, and FXa (4,5), although the extent to which it functions as an aPC-independent anticoagulant in vivo is debated.…”

Different effects of oral contraceptives containing different progestogens on protein S and tissue factor pathway inhibitor

“…Protein S also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation [14,15]. Recently, it was reported that protein S stimulates the inhibition of the extrinsic coagulation pathway by TFPI [16].…”