The tissue factor region that interacts with substrates factor IX and factor X

The enzymatic activity of coagulation factor VIIa is controlled by its cell ular cofactor tissue factor (TF). TF binds factor VIIa with high affinity a nd, in addition, participates in substrate interaction through its C-termin al fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, a nd their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divid ed into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys 166, Tyr185) forming a solvent-exposed patch of 488 Angstrom(2) and an exte nded region which comprises an additional 7-8 residues, including the dista lly positioned Asn199, Arg200, and Asp204. Some of the identified TF residu es, such as Trp158 and those within the loop Lys159-Lys165, are near the fa ctor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the fact or VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a ne t positive charge, suggesting that charge interactions may significantly co ntribute to TF-substrate binding. The calculated surface-exposed area of th is substrate interaction region is about 1100 Angstrom(2), which is approxi mately half the size of the TF area that is in contact with factor VIIa. Th erefore, a substantial portion of the TF surface (3000 Angstrom(2)) is enga ged in protein-protein interactions during substrate catalysis.