Abstract
The contribution of the catalytic and noncatalytic domains of factor IXa to the interaction with its cofactor, factor VIIIa, was evaluated. Two proteolytic fragments of factor IXa, lacking some or all of the serine protease domain, failed to mimic the ability of factor IXa to enhance the reconstitution of factor VIIIa from isolated A1/A3-C1-C2 dimer and A2 subunit. Both fragments, however, inhibited this factor IXa-dependent activity. Selective thermal denaturation of the factor IXa serine protease domain eliminated its effect on factor VIIIa 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 indicate the importance of the serine protease domain as well as structures residing in the factor IXa light chain (gamma-carboxyglutamic acid and/or epidermal growth factor domains) for cofactor stabilizing activity. 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 (Fl-FFR-IXa). This effect was inhibited by a factor IXa fragment comprised of the gamma-carboxyglutamic acid and epidermal growth factor domains. The difference in Fl-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 contribution of the A2 subunit, A peptide corresponding to factor VIII A2 domain residues 558-565 decreased the factor VIIIa dependent-anisotropy of Fl-FFR-IXa to a value similar to that observed with the A1/A3-C1-C2 dimer. These results support a model of multiple interactive sites in the association of the enzyme-cofactor complex and localize sites for the A1/A3-C1-C2 dimer and the A2 subunit to the factor IXa light chain and serine protease domain, respectively.
Highlights
Factors VIII and IX are essential plasma glycoproteins, which when absent or defective, result in hemophilia A and hemophilia B, respectively
In this study we demonstrate that the serine protease domain of factor IXa is essential for stabilizing the structurally labile, factor VIIIa heterotrimer and likely contributes to this association by interaction with the A2 subunit
Role of the Serine Protease Domain in Factor IXa-dependent Enhancement of Factor VIIIa Reconstitution—The serine protease domain of factor IX is structurally labile and melts at a temperature of 42–55 °C depending on pH with an irreversible loss of esterolytic activity [30]
Summary
Factors VIII and IX are essential plasma glycoproteins, which when absent or defective, result in hemophilia A and hemophilia B, respectively. Factor IX circulates in blood as a single chain zymogen It is composed of an NH2 terminus Gla domain which is rich in ␥-carboxyglutamic acid, followed by two EGF-like domains, a 35-residue activation peptide and the serine protease domain (see Ref. 15 for review). While reconstitution of heterotrimer factor VIIIa from the isolated A2 subunit and A1/A3-C1-C2 dimer is enhanced severalfold in the presence of factor IXa and phospholipid [14], prolonged incubation of factor VIIIa with factor IXa results in a loss of factor VIIIa activity due to proteolytic cleavage within the A1 domain [19]. Peptides corresponding to the sequence surrounding the activated protein C cleavage site in the A2 subunit of factor VIIIa (Arg562) [8], inhibit the factor IXa-dependent enhancement of factor VIIIa reconstitution [25]. Interaction of factor IXa and factor VIIIa appears to involve multiple sites on each protein
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