Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa.

The interaction of circulating factor VII/VIIa with tissue factor presented by cells in extravascular tissues represents the initial event in the extrinsic pathway of blood coagulation. To determine the tissue factor binding domains in human factor VIIa, we have subjected recombinant human factor VIIa to tryptic digestion and isolated two proteolytic fragments (molecular mass = 32 and 20 kDa) by a combination of immunoaffinity chromatography and reversed phase high performance liquid chromatography (HPLC) which strongly inhibits the tissue factor-enhanced amidolytic activity of factor VIIa and inhibits the activation of factor X by factor VIIa in the presence of tissue factor. The 32-kDa factor VIIa fragment consisted of residues 1-137/143 from the light chain of factor VIIa connected by a disulfide bond to residues 153-277 from the heavy chain. The 20-kDa factor VIIa fragment consisted of residues 1-137 of the light chain of factor VIIa in disulfide linkage with residues 248-266 of the heavy chain. The 32- and 20-kDa factor VIIa fragments inhibited the tissue factor apoprotein-enhanced factor VIIa amidolytic activity with Ki values of 35 and 65 nM, respectively. The Ki values for the inhibition of relipidated tissue factor apoprotein-enhanced factor VIIa amidolytic activity by the 32- and 20-kDa factor VIIa fragments were 70 and 610 nM, respectively. Factor X activation by factor VIIa-relipidated tissue factor was inhibited half-maximally by the 32- and 20-kDa factor VIIa fragments at 65 and 680 nM concentrations, respectively. Equilibrium binding studies indicated that the 32- and 20-kDa factor VIIa fragments interacted with cell surface tissue factor expressed on J82 cells in a specific and saturable manner with Kd values of 30 and 64 nM, respectively. In addition, a peptide consisting of residues 1-109 from the light chain of factor VIIa obtained by reduction and HPLC of the 20-kDa factor VIIa fragment retained inhibitory activity, but the selective removal of the gamma-carboxyglutamic acid domain from the 20-kDa factor VIIa fragment by cathepsin G cleavage resulted in the complete loss of inhibitory activity in this fragment. Our data strongly suggest that the epidermal growth factor-like domains covalently linked to the gamma-carboxyglutamic acid domain in factor VIIa constitute the high affinity tissue factor binding domain in this molecule.