Sites in the A2 subunit involved in the interfactor VIIIa interaction

Factor Villa is a trimer of the Al, A2, and A3-C1-C2 subunits, Regions in t he A2 subunit that interact with the A1/A3-C1-C2 dimer were localized using synthetic peptides derived from A2 sequences showing high probability of b eing surface exposed. Peptides were restricted to residues 373-562 of A2 ba sed on the earlier observation that this region of A2 reacts with Al using a zero length cross-linker, Peptides were assessed for their capacity to in hibit the reconstitution of factor Villa from the isolated A1/A3-C1-C2 dime r and A2 subunit. Reconstitution was monitored using both regeneration of f actor Villa activity and fluorescence quenching of an acrylodan-labeled A2 (Ac-A2) by fluorescein-labeled A1/A3-C1-C2. The activity assay identified f our peptides as inhibitors, residues 373-395 (IC50 = 65 mu M), 418-428 (IC5 0 = 25 mu M), 482-493 (IC50 = 325 mu M), and 518-533 (IC50 = 585 mu M). The 373-395 and 518-533 peptides eliminated the fluorescence quenching of Ac-A 2, whereas the 418-428 peptide reduced but did not eliminate Ac-A2 quenchin g, Peptide 482-493 had no effect on the fluorescence quenching of Ac-A2 sug gesting that the peptide did not directly affect reassociation of the facto r Villa subunits. These results identify three regions in the A2 subunit (3 73-395, 418-428, and 518-533) that interact with the A1/A3-C1-C2 dimer, Fur thermore, comparison of results obtained using the two assays distinguish i nhibition of the intersubunit interactions from intermolecular interactions .