Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit

Abstract

Factor VIIIa can be reconstituted from A2 subunit and A1/A3-C1-C2 dimer in a reaction that is facilitated by slightly acidic pH. We recently demonstrated that a truncated A1 (A1 37–336) possessed markedly reduced affinity for A2 compared with intact A1, but retained 30% of native factor VIIIa activity in the presence of A3-C1-C2. We now identify A1-interactive regions for A2 using A1 fragments derived from a limited tryptic digest. Unfractionated trypsin-cleaved A1 inhibited reconstituted factor VIIIa activity. Two fragments, designated A1 37–121 and A1 221–336, markedly inhibited factor VIIIa reconstitution with either native A1 ( K i=340 and 194 nM, respectively) or with A1 37–336 ( K i=69 and 116 nM, respectively) at pH 6.0. A third fragment designated A1 122–206 did not possess inhibitory activity. At pH 7.2, the A1 221–336 partially inhibited reconstitution, whereas the A1 37–121 possessed little if any inhibitory activity. Both fragments inhibited factor VIIIa reconstitution as judged by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1 forms at pH 6.0. Furthermore, covalent cross-linking between A2 and A1 37–121 but not A1 221–336 was observed following reaction with a zero-length cross-linker. These findings demonstrate the presence of an extended, pH-dependent A2-interactive surface within regions 37–121 and 221–336 of A1. This interactive surface appears conformationally labile in the truncated A1 as judged by its apparent stabilization following association with A3-C1-C2.