Cholesterol and Phosphatidylethanolamine Enhance the Binding of Factor Va and Factor Xa to Phospholipid Vesicles and Promote Assembly of the Prothrombinase Complex on Membranes Containing Low Concentrations of Phosphatidylserine.

Platelet membranes are composed of cholesterol (CH) and phospholipids including phosphatidylethanolamine (PE), sphingomeylin (SM), phosphatidylcholine (PC) and phosphatidylserine (PS). Phospholipid vesicles containing 20–25% PS have been used extensively to characterize assembly and regulation of the prothrombinase complex. However, the concentration of PS on the surface of activated platelets has been determined to be only 4–13%. Smeets et al (Thromb. Res. 81:419, 1996) demonstrated that CH and PE in the presence of 0–10% PS have stimulating effects on prothrombinase activity, while SM has an inhibitory one. To further investigate the roles of CH, PE and SM in the binding of factor Va and Xa to phospholipid vesicles and assembly of the prothrombinase complex, vesicles consisting of 1–25% DOPS plus 30% CH, 30% DOPE and/or 20% SM, were prepared for prothrombinase assays as a function of DOPS concentration. Compared to individual effect of DOPS alone or DOPS plus CH, PE or SM on prothrombinase activity, a synergistic effect was observed on vesicles containing CH, DOPE and SM, which was significant in the range of 1–15% DOPS. The PS concentration required for half-maximal rates of thrombin generation was 5.3% for vesicles containing CH, DOPE and SM, compared to 10.6, 11.2, 8.3 and 12.6% for vesicles containing DOPS alone, plus CH, plus DOPE or plus SM, respectively. This demonstrates that the requirement for PS is substantially decreased in the presence of CH, PE and SM. In order to further define the mechanisms for this effect, direct binding of factor Va to vesicles containing 30% CH, 30% PE and 20% SM was investigated using a fluorescence resonance energy transfer assay. These binding experiments demonstrated that factor Va bound to vesicles containing 1, 5, 10, and 20% DOPS with Kd values of 2.88±0.43 nM, 1.15±0.17 nM, 1.16±0.13 nM, and 1.18±0.19 nM, respectively. At low concentrations of PS (≤ 5%), CH, DOPE and SM increased the binding affinity of factor Va 2 to 6-fold compared to vesicles containing only DOPS. When prothrombinase activity was measured as a function of factor Va concentration on vesicles containing 5, 10, and 20% DOPS, K_1/2Va values of 0.63±0.08nM, 0.38±0.05nM, and 0.37±0.07nM were observed which were comparable to the Kd’s determined in direct factor Va binding experiments. These results indicate that factor Va binds tightly to phospholipid vesicles mimicking the phospholipid composition of activated platelets even at very low concentrations of PS. When direct binding of factor Xa to vesicles containing 5, 10, and 20% DOPS plus CH, DOPE and SM was examined, the Kd values were 70.5±6.8 nM, 62.6±6.6 nM, and 49.6±4.0 nM, respectively. When the interaction of factor Xa with membranes was investigated using prothrombinase assays, K_1/2Xa values of 0.12±0.03nM, 0.04±0.01nM, and 0.01±0.01nM were observed. In conclusion, the presence of CH and PE increases the affinity of factor Va and factor Xa binding to phospholipid vesicles containing physiologic concentrations of PS. The binding of factor Va to vesicles containing 1% PS is not sufficient for assembly and function of the prothrombinase complex, suggesting that higher concentrations of PS are needed to support the binding of factor Xa and prothrombin.