The Regulation of Factor IX_a by Phosphatidylserine.

A number of clinical studies have demonstrated correlation between elevated levels of factor IX and the risk of coronary heart disease. Studies by different groups have shown that blocking procoagulant activity of factor IX_a could reduce the risk of thrombosis. We propose to study the regulation of factor IX_a by phosphatidylserine (PS) which further could open up possibilities to design drugs that might inhibit the up regulation of the procoagulant protein factor IX_a. The activated form of factor IX, IX_a, forms the Xase complex together with factor VIII_a and Ca²⁺ on an activated platelet membrane surface. We hypothesize that PS regulates factor IX_a by binding to specific sites on factor IX_a and thereby enhancing its catalytic activity. We used a soluble form of phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS) as a tool in order to address the role of PS in regulating factor IX_a. Intrinsic fluorescence measurements demonstrate that C6PS binds tightly (K_d ~1.3 μM) to and induces changes in conformation of factor IX_a. We also monitored the amidolytic activity of 300 nM factor IX_a in the presence of C6PS using synthetic substrate Leu-PHG-Arg-pNA. There is a reduction in the amidolytic activity of factor IX_a with increasing addition of C6PS and the hyperbolic fit gives an apparent K_d of 130 μM. Recent results show that calcium is required for both amidolytic and proteolytic activity of factor IX_a in the presence of C6PS. Our data shows that amidolytic activity of factor IX_a in the presence of 600 mM C6PS is saturated at a concentration of calcium near 3 mM. We have also demonstrated that both C6PS and calcium enhance proteolytic activation of factor X by factor IX_a. The data shows that as the concentration of Ca²⁺ increases, the proteolytic activation of factor X by factor IX_a increases as well. Enhancement of factor X activation appears to saturate at a calcium concentration of 3 mM. Based on these results, we conclude, 1) C6PS induces a conformational change in factor IX_a, 2) C6PS regulates the amidolytic and proteolytic activity of factor IX_a, 3) Ca²⁺ is needed for PS mediated regulation of factor IX_a.