Membrane lateral phase separation induced by proteins of the prothrombinase complex

Blood coagulation factors X and V, as well as prothrombin fragment 1 caused changes in the observed transition temperature (T_m) of appropriately constituted phospholipid vesicles upon binding to the membrane surface. Factor X- and prothrombin fragment 1-induced T_m shifts were calcium-dependent, while factor V changed the T_m in a calcium-independent manner. The results were consistent with clustering of the acidic phospholipid molecules due to protein binding. In all cases, protein binding to acidic phospholipid-containing vesicles caused the observed T_m to approach that for the neutral phospholipid. This resulted in a T_m increase for phospholipid mixtures containing bovine brain phosphatidylserine (PS) plus dipalmitoylphosphatidylcholine (DPPC) and a T_m decrease for mixtures of dipalmitoylphosphatidic acid (DPPA) and dimyristoylphosphatidylcholine (DMPC). Maximum T_m shifts induced in PS-DPPC (10:90) vesicles were very similar for all the prothrombinase proteins and the extent of the change was proportional to the actual amount of membrane-bound protein as determined by light-scattering techniques. For the vitamin K-dependent proteins, T_m changes were greater in the presence of protein plus calcium than in the presence of calcium alone, indicating that lateral phase separation occurs subsequent to initial protein-membrane contact. Lateral phase separation of acidic phospholipids appears to be an important process in the formation of the prothrombinase complex.