| Abstract
| - The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissuefactor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interactionthrough its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminalTF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF)mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activationwas measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showedthat factor IX and factor X interacted with the same TF region located proximal to the putative phospholipidsurface. According to the degree of activity loss of the sTF mutants, this TF region can be divided intoa main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 Å2 and an extended region which comprises an additional 7−8 residues, includingthe distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158and those within the loop Lys159−Lys165, are near the factor VIIa γ-carboxyglutamic acid (Gla) domain,suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, thesurface identified as important for substrate interaction carries a net positive charge, suggesting that chargeinteractions may significantly contribute to TF−substrate binding. The calculated surface-exposed areaof this substrate interaction region is about 1100 Å2, which is approximately half the size of the TF areathat is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 Å2) is engagedin protein−protein interactions during substrate catalysis.
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