Fluorescence Resonance Energy Transfer Study of Shape Changes in Membrane-Bound Bovine Prothrombin and Meizothrombin

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Abstract
Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323−Ile and Arg274−Thr) by factor Xa to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0−8.7 mol %) contained in membranes composed of 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 dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 ± 0.10 and 0.28 ± 0.12 μM with stoichiometries of 42 ± 12 and 44 ± 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of κ2 = 2/3 and n = 1.4, the distances were 94 ± 3 Å for prothrombin and 114 ± 2 Å for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 Å, 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 essential role in the sequential alignment of the bond Arg323−Ile in prothrombin and Arg274−Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.

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