Calcium- and prothrombin-induced lateral phase separation in membranes

Calcium and prothrombin changed the observed phase transition temperature (T_m) of appropriately constituted phospholipid vesicles. The results are consistent with clustering of the acidic phospholipid molecules to form a prothrombin binding site. Transition temperatures were obtained by monitoring the fluorescence intensity and fluorescence depolarization of the lipid-soluble probes 8-anilino-1-naphthalenesulfonic acid and diphenylhexatriene, respectively. In all cases, calcium and prothrombin caused the observed T_m to increase or decrease toward the value of the pure neutral phospholipid. For example, calcium caused a 2°C increase in the observed T_m of vesicles containing 20% bovine brain phosphatidylserine (PS) and 80% dipalmitoylphosphatidylcholine (DPPC). Prothrombin binding to these vesicles caused a further 2°C increase in the T_m. Conversely, calcium or calcium plus prothrombin caused 2 and 3.5°C decreases, respectively, in the observed T_m of membranes of 30% dipalmitoylphosphatidic acid and 70% dimyristoylphosphatidylcholine. The changes are due to clustering of the acidic phospholipid molecules, which enriches the bulk membrane in the neutral phospholipid. The observed T_m therefore increases or decreases, depending on whether the acidic phospholipid component had a T_m higher or lower than the neutral phospholipid. In agreement with this interpretation and with previous studies, manganese caused negligible changes in the T_m and inhibited the effect of calcium. In addition, the concentration dependence of the calcium-induced increase in the T_m (for PS-DPPC vesicles) indicated a dissociation constant of about 0.5 mM for calcium-phosphatidylserine binding, which agrees with direct calcium binding measurements. The inhibitory effect of manganese on the calcium-induced changes was overcome by prothrombin; in the presence of manganese and calcium, prothrombin still induced the full 4°C increase. Light-scattering studies demonstrated that, in both cases, prothrombin-induced changes in the T_m correlate closely with actual prothrombin binding to the phospholipid vesicles. Lateral phase separation appears to be an integral part of prothrombin-membrane binding.