Complexes of Prothrombin with Calcium Ions and Phospholipids

The ability of phospholipids to bind prothrombin in the presence of Ca2+ ions has been studied as a function of lipid composition. Two distinct but complementary methods have been devised to evaluate binding. The first method involves gel filtration on Sephadex G-200 at pH 9.0 to separate the lipid-bound and free prothrombin; the second involves precipitation of the complexes at pH 6.5. Phosphatidylcholine (PC) or a mixture of this lipid with phosphatidylethanolamine (PE) bound very little prothrombin, whereas mixtures of phosphatidylserine (PS) with PC were very effective in binding and the complexes that formed were highly active in the one-stage prothrombin assay. PS alone, phosphatidic acid (PA) alone, PA-PS mixtures, and PA-PC mixtures containing high proportions of PA, bound prothrombin very effectively, but the complexes formed were much less active than when PS-PC was used. Bound protein could be much more readily removed from PS-PC than from PS-PA by treatment of the complexes with EDTA. It was suggested that an optimal surface condition is required to bind reversibly prothrombin prior to conversion to thrombin. Anticoagulant effects ascribed to acidic phospholipids may be due to irreversible binding of the clotting factors to lipid surfaces.