ACTIVATION OF FLOOD COAGULATION-FACTOR VIIA WITH CLEAVED TISSUE FACTOR EXTRACELLULAR DOMAIN AND CRYSTALLIZATION OF THE ACTIVE COMPLEX

Exposure of blood to tissue factor leads to the formation of a high af finity tissue factor/factor VIIa complex which initiates blood coagula tion. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai ) and soluble tissue factor (sTF) was prepared for crystallization. Cr ystals were obtained, but only after long incubation times, Analysis b y SDS-PAGE and mass spectrometry indicated the presence of sTF fragmen ts similar to those formed by proteolytic digestion with subtilisin (K onigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost, 69: 1171, 1993). To test the hypothesis that limited proteolysis of sTF fa cilitated the crystallization of the complex, sTF fragments were gener ated by subtilisin digestion and purified. Analysis by tandem mass spe ctrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellula r domain. Enzymatic assays and binding studies demonstrated that an eq uimolar mixture of N- and C-terminal fragments bound to factor VIIa an d fully restored cofactor activity. A complex of F.VIIai and sTF fragm ents was prepared for crystallization, Crystals were obtained using mi croseeding techniques. The best crystals had maximum dimensions of 0.1 2 x 0.12 x 0.6 mm and showed diffraction to a resolution of 3 Angstrom . (C) 1995 Wiley-Liss, Inc.