Exosite binding drives substrate affinity for the activation of coagulation factor X by the intrinsic Xase complex

Abstract

Factor X activation by the intrinsic Xase complex, composed of factor IXa bound to factor VIIIa on membranes, is essential for the amplified blood coagulation response. The biological significance of this step is evident from bleeding arising from deficiencies in factors VIIIa or IXa in hemophila. Here, we assess the mechanism(s) that enforce the distinctive specificity of intrinsic Xase for its biological substrate. Active site function of IXa was assessed with a tripeptidyl substrate (PF-3688). The reversible S1 site binder, 4-aminobenzamidine (pAB), acted as a classical competitive inhibitor of PF-3688 cleavage by Xase. In contrast, pAB acted as a noncompetitive inhibitor of factor X activation. This disconnect between peptidyl substrate and protein substrate cleavage indicates a major role for interactions between factor X and extended sites on Xase in determining substrate affinity. Accordingly, an uncleavable factor X variant, not predicted to engage the active site of IXa within Xase, acted as a classical competitive inhibitor of factor X activation. Fluorescence studies confirmed the binding of factor X to Xase assembled with IXa with a covalently-blocked active site. Our findings suggest that the recognition of factor X by the intrinsic Xase complex occurs through a multistep “dock-and-lock” pathway in which the initial interaction between factor X and intrinsic Xase occurs at exosites distant from the active site, followed by active site docking and bond cleavage.

Probing activation‐driven changes in coagulation factor IX by mass spectrometry

“…In our studies, we used the inhibitor EGRck to assess the effect of active site occupation in FIXa. Binding of the natural substrate FX, however, is more complex and probably also involves exosites distant from the active site 46 . Therefore, substrate‐driven changes in FIXa may be more extensive than we observed in active‐site inhibited FIXa.…”

“…Binding of the natural substrate FX, however, is more complex and probably also involves exosites distant from the active site. 46 Therefore, substrate-driven changes in FIXa may be more extensive than we observed in active-site inhibited FIXa. Further studies could dissect substrate-and cofactor-driven changes in more detail.…”

Site‐specific functional roles of the Factor X activation peptide in the intrinsic tenase‐mediated Factor X activation

“…The intrinsic tenase activation of FX is driven by exosite binding [7], and as previous studies have suggested that the FX‐AP contributes to activator specificity and prevents the promiscuous activation of FX [8,11,19], the FX‐AP may be an important exosite. This was demonstrated in a competitive inhibition study, where a chimeric FX construct containing human FX and the AP of venom‐derived Pseudonaja textilis was a significantly weaker competitor of the intrinsic tenase‐mediated activation of human FX, compared with a nonactivable FX construct containing a Gln substitution of the Arg residue at the P1 site [12].…”

Mapping the zymogen to protease transition in FIXa

“…Thus, the contribution of interdomain interactions to protease conformational changes upon activation remain incompletely described. Second, while incorporation of EGR‐ck recapitulates a portion of the substrate interaction with the active site, FX activation by the intrinsic tenase complex involves exosite‐mediated substrate recognition, which likely induces additional conformational changes that contribute to activation of FIXa catalysis 17 . Finally, limitations in the sensitivity of the HDX‐MS approach exist due to back‐titration of deuterium during peptide processing.…”

“…Reduced deuterium uptake was noted for three α-helixes located in the basic exosite II of FIXa (Figure 1 changes that contribute to activation of FIXa catalysis. 17 Finally, limitations in the sensitivity of the HDX-MS approach exist due to backtitration of deuterium during peptide processing.…”