Protein S (ProS) is a blood anticoagulant encoded by the Pros1 gene, and ProS deficiencies are associated with venous thrombosis, stroke, and autoimmunity. These associations notwithstanding, the relative risk that reduced ProS expression confers in different disease settings has been difficult to assess without an animal model. We have now described a mouse model of ProS deficiency and shown that all Pros1 -/-mice die in utero, from a fulminant coagulopathy and associated hemorrhages. Although ProS is known to act as a cofactor for activated Protein C (aPC), plasma from Pros1 +/-heterozygous mice exhibited accelerated thrombin generation independent of aPC, and Pros1 mutants displayed defects in vessel development and function not seen in mice lacking protein C. Similar vascular defects appeared in mice in which Pros1 was conditionally deleted in vascular smooth muscle cells. Mutants in which Pros1 was deleted specifically in hepatocytes, which are thought to be the major source of ProS in the blood, were viable as adults and displayed less-severe coagulopathy without vascular dysgenesis. Finally, analysis of mutants in which Pros1 was deleted in endothelial cells indicated that these cells make a substantial contribution to circulating ProS. These results demonstrate that ProS is a pleiotropic anticoagulant with aPC-independent activities and highlight new roles for ProS in vascular development and homeostasis.

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...

The high expression of LEDGF/p75 in prostate tumors and BPH could be induced by inflammation and oxidative stress. LEDGF/p75 cleavage fragments generated during prostate tumor cell death might trigger autoantibodies under inflammatory conditions in certain patients.

The binding of human coagulation factor XI to washed human platelets was studied in the presence of zinc ions, calcium ions, and high molecular weight kininogen. Significant factor XI binding occurred at physiological levels of these metal ions when high molecular weight kininogen was present. Binding required platelet stimulation and was specific, reversible, and saturable. Scatchard analysis of the binding yielded approximately 1500 binding sites per platelet with an apparent dissociation constant of approximately 10 nM. Since the concentration of factor XI in plasma is about 25 nM, this suggests that in plasma factor XI binding sites on stimulated platelets might be saturated. Calcium ions and high molecular weight kininogen acted synergistically to enhance the ability of low concentrations of zinc ions to promote factor XI binding. The similarity between the concentrations of metal ions optimal for factor XI binding and those optimal for high molecular weight kininogen binding, as well as the ability of high molecular weight kininogen to modulate these metal ion effects, implies that factor XI and high molecular weight kininogen may form a complex on the platelet surface as they do in solution and on artificial negatively charged surfaces.

No abstract