title: Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer.
abstract: Heterotrimeric factor VIIIa was reconstituted from isolated A2 subunit and A1/A3-C1-C2 dimer of thrombin-activated human factor VIII in a reaction that was sensitive to pH. Maximal levels of reconstituted factor VIIIa at pH 6.0 were as much as 20-fold greater than were values observed at pH 7.5. The presence of factor IXa and phospholipid resulted in a marked increase in factor VIIIa reconstituted at physiologic pH. However, the resultant factor VIIIa was unstable due to slow proteolysis of the A1 subunit. Factor IXa modified by the active site-specific reagent dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone (DEGR-IXa) increased the level of factor VIIIa reconstituted from subunits to a similar extent as was observed for unmodified factor IXa and yielded stable factor VIIIa. This enhancement was saturated above a 1:1 molar ratio of DEGR-IXa to factor VIIIa subunits and could be blocked by an anti-factor IX antibody, suggesting that the DEGR-IXa-dependent increase in factor VIIIa reconstitution correlated with assembly of the factor X-ase complex. At a saturating amount of DEGR-IXa, the level of factor VIIIa reconstitution at pH 7.5 approached values obtained at pH 6.0. Fluorescence polarization measurements indicated that factor VIIIa altered binding of DEGR-IXa to phospholipid. However, neither the A2 subunit nor the A1/A3-C1-C2 dimer alone produced this effect. This result suggested that both A2 and A1/A3-C1-C2 were necessary for association of the cofactor with factor IXa. These results suggest a model in which assembly of the intrinsic factor X-ase complex stabilizes factor VIIIa through inhibition of subunit dissociation.
authors.affiliation: Department of Medicine, University of Rochester School of Medicine & Dentistry, New York 14642.
authors.forename: B J
authors.initials: BJ
authors.lastname: Lamphear
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015949:Factor IXa / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010743:Phospholipids / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D010743:Phospholipids; C055180:dansylglutamyl-glycyl-arginyl chloromethyl ester; D015944:Factor VIIIa; D015949:Factor IXa; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone; D002118:Calcium