TISSUE FACTOR POSITIONS AND MAINTAINS THE FACTOR VIIA ACTIVE-SITE FARABOVE THE MEMBRANE-SURFACE EVEN IN THE ABSENCE OF THE FACTOR VIIA GLADOMAIN - A FLUORESCENCE RESONANCE ENERGY-TRANSFER STUDY

Coagulation factor VIIa (fVIIa), a soluble serine protease, exhibits f ull proteolytic activity only when bound to its cofactor, tissue facto r (TF). Both proteins interact with membranes; TF is an integral membr ane protein, while fVIIa binds reversibly to phospholipid surfaces via its Gla domain. In this study, we examine the extent to which the loc ation of the fVIIa active site in the fVIIa . TF complex is determined by the fVIIa Gla domain. A fluorescein dye was covalently attached to the active site of fVIIa lacking the Gla domain (Gla domainless fVIIa , GD-fVIIa) via a tripeptide tether to yield fluorescein- D-Phe-Pro-Ar g-GD-fVIIa (Fl-FPR-GD-fVIIa). The location of the active site of GD-fV IIa relative to the membrane surface was determined using fluorescence resonance energy transfer between the fluorescein dye in the active s ite of GD-fVIIa and octadecylrhodamine (OR) at the surface of phosphol ipid vesicles. As expected, no energy transfer was observed between Fl -FPR-GD-fVIIa and OR in vesicles composed of phosphatidylcholine/phosp hatidylserine (PC/PS, 4:1) because the Gla domain is required for the binding of fVIIa to phospholipid. However, when Fl-FPR-GD-fVIIa was ti trated with PC or PC/PS vesicles into which purified TF had been recon stituted, energy transfer was observed. Based on the dependence of flu orescence resonance energy transfer on OR density, the average distanc e of closest approach between fluorescein in the active site of Fl-FPR -GD-fVIIa . TF and OR at the vesicle surface was determined to be 78 A ngstrom (kappa(2) = 2/3). Since this value is nearly the same as that obtained with intact Fl-FPR-fVIIa bound to TF, the presence or absence of the fVIIa Gla domain has only a small effect on the location of th e active site in the fVIIa . TF complex. The extracellular domain of t issue factor therefore must be fairly rigid and fixed relative to the surface to position and maintain the fVIIa active site far above the m embrane even in the absence of the fVIIa Gla domain.