FLUORESCENCE RESONANCE ENERGY-TRANSFER STUDY OF SHAPE CHANGES IN MEMBRANE-BOUND BOVINE PROTHROMBIN AND MEIZOTHROMBIN

Prothrombin activation to thrombin is a key control reaction. in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg(323)-Ile and Arg(274)-Thr) by factor X-a to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-cont aining membranes from platelets are believed to facilitate this two-st ep process, Using fluorescence energy transfer (FRET), we determined t he distances of closest approach between a specifically located C-term inal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C )-FM) or meizothrombin (M(S528A, G581C)FM) and phosphatidylethanolamin e-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed o f PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissoci ation constants and binding stoichiometries as global parameters and s aturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were est imated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 mu M with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothro mbin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the ac ceptor (PE-Rh) surface density. With the assumptions of kappa(2) = 2/3 and n = 1.4, the distances were 94 +/- 3 Angstrom for prothrombin and 114 +/- 2 Angstrom for meizothrombin. Since both prothrombin and meiz othrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 Angstrom, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essen tial role in the sequential alignment of the bond Arg(323)-Ile in prot hrombin and Arg(274)-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating proc ess.