Sites in the A2 subunit involved in the interfactor VIIIa interaction.
Factor VIIIa is a trimer of the A1, A2, and A3- C1- C2 subunits. Regions in the A2 subunit that interact with the A1/A3- C1- C2 dimer were localized using synthetic peptides derived from A2 sequences showing high probability of being surface exposed. Peptides were restricted to residues 373-562 of A2 based on the earlier observation that this region of A2 reacts with A1 using a zero length cross-linker. Peptides were assessed for their capacity to inhibit the reconstitution of factor VIIIa from the isolated A1/A3- C1- C2 dimer and A2 subunit. Reconstitution was monitored using both regeneration of factor VIIIa activity and fluorescence quenching of an acrylodan-labeled A2 (Ac-A2) by fluorescein-labeled A1/A3- C1- C2. The activity assay identified four peptides as inhibitors, residues 373-395 (IC(50) = 65 micrometer), 418-428 (IC(50) = 25 micrometer), 482-493 (IC(50) = 325 micrometer), and 518-533 (IC(50) = 585 micrometer). The 373-395 and 518-533 peptides eliminated the fluorescence quenching of Ac-A2, whereas the 418-428 peptide reduced but did not eliminate Ac-A2 quenching. Peptide 482-493 had no effect on the fluorescence quenching of Ac-A2 suggesting that the peptide did not directly affect reassociation of the factor VIIIa subunits. These results identify three regions in the A2 subunit (373-395, 418-428, and 518-533) that interact with the A1/A3- C1- C2 dimer. Furthermore, comparison of results obtained using the two assays distinguish inhibition of the intersubunit interactions from intermolecular interactions.[1]References
- Sites in the A2 subunit involved in the interfactor VIIIa interaction. Koszelak, M.E., Huggins, C.F., Fay, P.J. J. Biol. Chem. (2000) [Pubmed]
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