LOCALIZATION OF FACTOR IXA AND FACTOR VIIIA INTERACTIVE SITES

The contribution of the catalytic and noncatalytic domains of factor M a to the interaction with its cofactor, factor VIIIa, was evaluated, T wo proteolytic fragments of factor Ma, lacking some or all of the seri ne protease domain, failed to mimic the ability of factor Ma to enhanc e the reconstitution of factor VIIIa from isolated A1/A3-C1-C2 dimer a nd A2 subunit. Both fragments, however, inhibited this factor Ma-depen dent activity, Selective thermal denaturation of the factor Ma serine protease domain eliminated its effect on factor Villa reconstitution, Modification of factor IXa with dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR-IXa) stabilized this domain, and heat-treated DEGR-IXa retained its ability to enhance factor VIIIa reconstitution. These results ind icate the importance of the serine protease do main as well as structu res residing in the factor Ma light chain (gamma-carboxyglutamic acid and/or epidermal growth factor domains) for cofactor stabilizing activ ity, In the presence of phospholipid, the A1/A3-C1-C2 dimer produced a saturable increase in the fluorescence anisotropy of fluorescein-Phe- Phe-Arg chloromethyl ketone-modified factor IXa (F1-FFR-IXa), This eff ect was inhibited by a factor Ma fragment comprised of the gamma-carbo xyglutamic acid and epidermal growth factor domains, The difference in F1-FFR-IXa anisotropy in the presence of A1/A3-C1-C2 dimer (Delta r = 0.043) compared with factor VIIIa (Delta r = 0.069) represented the c ontribution of the A2 subunit, A peptide corresponding to factor VIII A2 domain residues 558-565 decreased the factor Villa dependent anisot ropy of F1-FFR-IXa to a value similar to that observed with the A1/A3- C1-C2 dimer, These results support a model of multiple interactive sit es in the association of the enzyme-cofactor complex and localize site s for the A1/A3-C1-C2 dimer and the A2 subunit to the factor Ma light chain and serine protease domain, respectively.