PROTHROMBIN ACTIVATION ON DIOLEOYLPHOSPHATIDYLCHOLINE MEMBRANES

Factor-Xa-catalyzed prothrombin activation is greatly accelerated by n egatively charged phospholipids plus calcium ions. In 1990, we reporte d that neutral phosphatidylcholine membranes also stimulated prothromb in activation [Gerads, I., Govers-Riemslag, J. W. P., Tans, G., Zwaal, R. E A. and Rosing, J. (1990) Biochemistry 29, 7967-7974]. In the pre sent study, we have performed a detailed analysis of the prothrombin-c onverting activity of phosphatidylcholine membranes. Stimulation of pr othrombin activation by phosphatidylcholine vesicles was particularly observed (a) with phosphatidylcholine molecules that contained unsatur ated hydrocarbon side chains, (b) in the presence of factor Va, (c) at low ionic strength and (d) when Ca2+ were present in the reaction med ium. It is unlikely that the prothrombinase activity of phosphatidylch oline preparations was due to contaminating anionic phospholipids. Thi s is concluded from the fact that thin-layer chromatographic analysis showed that dioleoylphosphatidylcholine [(Ole)(2)GroPCho] contained le ss than 0.1 mol/ 100 mol anionic phospholipid, and that incorporation of such amounts of anionic lipids in (Ole)(2)GroPCho membranes hardly increased their prothrombin-converting activity. At low ionic strength and in the presence of factor Va and Ca2+ (Ole)(2)GroPCho membranes a ccelerated prothrombin activation about 100-fold. At ionic strength (I ) 0.06, prothrombin activation on 100 mu M (Ole)(2)GroPCho was charact erized by a K-m for prothrombin of 2 mu M, a V-max of 3020 IIa min(-1) Xa(-1) and a K-d for factor XaVa complex formation at the membrane su rface of 7.5 nM. Prothrombin activation on (Ole)(2)GroPCho membranes w as drastically reduced when the ionic strength was increased. The inhi bition at high ionic strength could be explained by an effect on the K -d for XaVa complex formation which increased from 7.5 nM at I = 0.06 to 100 nM at I = 0.22. Prothrombin activation on (Ole)(2)GroPCho requi red Ca2+ and was dependent on the presence of gamma-carboxyglutamic ac id domains in prothrombin and factor Xa. This indicates that similar i nteractions may account for the assembly of prothrombinase complexes o n phosphatidylcholine and on anionic lipid-containing membranes.