The Mechanism of Protein S as An Anticoagulant Independent of Activated Protein C.

Abstract 3186

Poster Board III-123

Thrombosis is a serious problem in the United States. The overall estimated incidence (annual occurrence) of deep venous thrombosis is 1 episode for every 1000 persons. Protein S, a vitamin K-dependent protein, is one of the natural anticoagulants found in the blood. Deficiency of protein S is most common protein deficiencies associated with familial venous thrombosis There are studies that suggest an association between arterial thrombosis (stroke, heart attack) in patients with protein S deficiency. At this time, the exact role of protein S deficiency and its relative importance in arterial disease is still being explored by physicians and scientists.

Protein S is known as a non-enzymatic cofactor of activated Protein C in the inactivation of factors Va and VIIIa, as part of a negative feedback loop to regulate blood coagulation. Plasma coagulation assays in the absence of activated protein C suggest that Protein S may have other anticoagulant role(s). For example, it has been suggested that Protein S down-regulates thrombin generation by stimulating FXa inhibition by the tissue factor pathway inhibitor (Rosing, J., et al., Thromb Res, 2008. 122 Suppl 1: p. S60-3). It has also been proposed that protein S can directly inhibit the intrinsic Xase complex (Takeyama, M., et al.. Br J Haematol, 2008. 143(3): p. 409-20). But the exact mechanism of how Protein S exerts its anticoagulant effect on factor IXa/VIIIa complex is still unclear. In order to determine the role of Protein S as an anticoagulant in the intrinsic Xase Complex, we have used C6PS (a small six carbon chain synthetic Phosphatidylserine (PS) molecule) that does not occur in vivo, but has been used as a powerful tool in demonstrating the regulation of both factors Xa and Va by binding of molecular PS. Soluble lipid binding can offer invaluable insights into events that would be next to impossible to document on a membrane surface which is complicated as it has surface condensation effect and allosteric effects of different factors. We focus here on the conformation changes of the proteins by using C6PS as a tool.

We have determined the binding of Protein S with C6PS by using tryptophan fluorescence and observed a stoichiometric K_d of ∼180 μM.We checked for micelles formation under each experimental condition. We have also determined the direct binding of factor IXa with Protein S by using DEGR-IXa ((5-(dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginyl chloromethyl ketone) in the presence and absence of C6PS. Our results show that the affinity of binding of DEGR-IXa to Protein S in the presence of C6PS is ∼22 fold tighter (K_d ∼15 nM compared to 324 nM) than without C6PS. We also measured the rate of factor X activation by factor IXa with the addition of increasing concentration of C6PS in the presence and absence of Protein S. We observed that Protein S decreased factor IXa mediated factor X activation by 14 fold. We had previously shown that apparent K_d of factor IXa binding to C6PS during factor X activation was ∼125 μM. But addition of Protein S had an effect on the apparent K_d as it increased to 700 μM indicating the affinity of factor IXa towards C6PS was decreased with the addition of Protein S during factor X activation. From these data we can speculate that Protein S induces a conformational change in factor IXa in the presence of C6PS which may affect the interaction of factor IXa with factor VIIIa, thus affecting the intrinsic Xase complex. Using this useful tool (C6PS), we will characterize the anticoagulant role of Protein S in the intrinsic Xase complex which in turn will give us some insights into this important protein which is a crucial target for therapeutic drugs for venous thrombosis.