Activation of blood 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 affinity tissue factor/factor VIIa complex which initiates blood coagulation. 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. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 x 0.12 x 0.6 mm and showed diffraction to a resolution of 3 A.
D003460:Crystallization; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000737:chemistry; D013381:Subtilisins / Q000494:pharmacology; D013925:Thromboplastin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D015843:Serpins; D013925:Thromboplastin; D013381:Subtilisins; D015942:Factor VIIa