The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor

Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa-TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa-TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIXWT), FIXQ50P, FIXPCEGF1 (EGF1 domain replaced with that of protein C), FIXDeltaEGF1 (EGF1 domain deleted), FXWT, and FXPCEGF1. Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa-TF or FVIIa(.)sTF activated each mutant significantly more slowly than the FIXWT or FXWT. Importantly, in ligand blot assays, FIXWT and FXWT bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K-i of similar to60 mum. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FXWT and FXPCEGF1, normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.