Determinants of the formation and activity of factor V-phospholipid complexes. I. Influence of phospholipid structure

Previous studies showed that factor V consists of multiple oligomeric forms including a minor component (form L) which contained lipid and was eluted in the excluded volume of a Sepharose 4B column. In the present and following study the hypothesis that factor V is a lipid requiring protein rather than a lipoprotein has been tested. Modification of a previous purification procedure resulted in the separation of factor V from lipoprotein. The lipid extracted from this lipoprotein was qualitatively similar to that previously isolated from form L. Removal of this lipoprotein by sucrose density gradient ultracentrifugation resulted in the isolation of factor V of high specific activity which contained no detectable phospholipid and less than 0.2% cholesterol. No component was excluded from Sepharose 4B after gel filtration of this purified preparation. It is concluded that factor V is not a lipoprotein, but rather a protein which requires lipid for its coagulant properties. To define this requirement further the ability of factor V-phospholipid complexes to accelerate the conversion of prothrombin to thrombin by factor Xa in the presence of calcium was tested. The rate of thrombin formation was highly dependent on the phospholipid employed with each active phospholipid exhibiting a characteristic optimal concentration. At 250 muM, the order of activity was phosphatidyl ethanolamine greater than phosphatidyl inositol greater than cardiolipin greater than phosphatidyl choline. Phosphatidyl serine was inert at all concentrations employed. No single fatty acid was consistently present in active lipid preparations, nor was a specific fatty acid absent from those which had no clotting activity. In comparing different lipid classes, a direct relationship between the overall degree of unsaturation and activity was not observed. However, bovine phosphatidyl inositol, which is highly unsaturated, is active, while plant phosphatidyl inositol, which contains only trace amounts of unsaturated fatty acids, were inert. At lipid concentrations below 300 muM, saturated phosphatidyl ethanolamine produced by hydrogenation of bovine phosphatidyl ethanolamine was less active than an equal concentration of the native lipid. In mixtures containing both lipids, artificially saturated phosphatidyl ethanolamine potentiated the activity of the native compound.