THE LOCATION OF THE ACTIVE-SITE OF BLOOD-COAGULATION FACTOR VIIA ABOVE THE MEMBRANE-SURFACE AND ITS REORIENTATION UPON ASSOCIATION WITH TISSUE FACTOR - A FLUORESCENCE ENERGY-TRANSFER STUDY

The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of f VIIa via a tripeptide tether to yield uorescein-D-phenylalanyI-L-proly l-L-arginy-L-fVIIa (FI-FPR-fVIIa). The location of the active-site pro be relative to the membrane surface was determined, both in the presen ce and absence of tissue factor (TF), using fluorescence energy transf er between the fluorescein dye and octadecylrhodamine (OR) at the phos pholipid vesicle surface. When FI-FPR-fVIIa was titrated with phosphol ipid vesicles containing OR, the magnitude of OR-, calcium ion-, and p hosphatidylserine-dependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the a ctive site of fVIIa and OR at the vesicle surface is 82 Angstrom assum ing a random orientation of donor and acceptor dyes (kappa(2) = 2/3; t he orientational uncertainty totals similar to 10%). The active site o f fVIIa is therefore located far above the membrane surface, and the e longated fVIIa molecule must bind at one end to the membrane and proje ct approximately perpendicularly out of the membrane. When FI-FPR-fVII a was titrated with vesicles that contained TF, the efficiency of ener gy transfer was increased by a TF-dependent translational and/or rotat ional movement of the fVIIa protease domain relative to the membrane s urface. if this movement was solely translational, the height of the a ctive site of fVIIa was lowered by an average of 6 Angstrom after bind ing to TF. The association of fVIIa with TF on the membrane surface th erefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the a ctive-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.