Activated Factor IX As a Candidate for the Initial Protease in the Coagulation Cascade

As currently understood, activated factor VII is the initiating protease of the coagulation process. The identity of the protease(s) that cleaves factor VII to yield its circulating activated form (~1% of the total factor VII protein in normal individuals) remains uncertain. Although, measurement of plasma concentrations of factor VIIa in normal human subjects and those with specific factor deficiencies suggests that factor IX has a factor VIII-independent role in generating circulating factor VIIa (Blood80:25-28, 1992).

Previous work using purified plasma proteins yielded evidence that factor IX cleaved only at residue R226 (factor IXaα) was a competent protease, yet did not efficiently bind factor VIIIa (JBC23:14484-14490, 1995). We investigated whether this form of factor IX might activate factor VII, as it seems to have the appropriate characteristics (proteolytic activity that is VIIIa independent). We created and purified recombinant mutant forms of factor IX and factor VII that enabled exploration of the functions of these separate cleavages without the complication of unanticipated proteolysis that is inherent when using plasma derived proteins.

Wild-type factor IX and R191H factor IX (the Hemophilia B _{Chapel Hill} variant) were activated to yield factor IXaβ and IXaα, respectively, as demonstrated by SDS-PAGE under reducing and non-reducing conditions, and incorporation of fluoresceinated EGR-chloromethyl ketone into the active sites, confirming that activity correlated with the appropriate molecular mass subunit. As predicted, factor IXaβ and R191H IXaα demonstrated identical catalytic efficiency in the amidolytic cleavage of a small molecular weight substrate, Pefachrome IXa. In contrast, in the presence of factor VIIIa-phospholipid, compared to factor IXaβ, R191H IXaα exhibited greatly reduced ability to cleave the physiologic macromolecular substrate, factor X. Consistent with previous findings, R191H factor IXaα had appreciably lower affinity for factor VIIIa than did factor IXaβ, and this likely explains the difference in factor X activating activity. This difference in activity was also seen in a plasma-based system, where R191H factor IXaα demonstrated only 1.6% of the coagulant activity of factor IXaβ in an aPTT assay in factor IX immunodepleted plasma.

There was a distinct difference with a second physiologic macromolecular substrate. Factor R191H factor IXaα and factor IXaβ activated factor VII to VIIa with identical kinetic parameters in a purified system. Intriguingly, the addition of factor VIIIa did not change the kinetic parameters of factor VII activation by either form of factor IXa. This observation is unlikely to be due to factor VIIa contaminating the factor VII preparation, as catalytically incompetent factor VII (S344A) was cleaved at indistinguishable rates by factors R191H IXaα and IXaβ.

Experiments with control proteins that also contained mutations at the active site, showed that factor IX R191Q;S411A demonstrated the appropriate sized bands on SDS-PAGE for factor IXaα, but did not incorporate fluoresceinated EGR-chloromethyl ketone, and had no detectable amidolytic, proteolytic, or coagulant activity, consistent with the active site serine mutation. Factor IX R191Q;R226Q was not cleaved by factor VIIa-tissue factor, and factor IX R226Q was cleaved to subunits consistent with factor IXα. Neither of these proteins incorporated fluoresceinated EGR-chloromethyl ketone, and neither demonstrated detectable amidolytic activity.

These experiments with recombinant forms of factor IX support the hypothesis that factor IXaα exhibits active site enzymatic competency equal to factor IXaβ, and that cleavage at R191 is required to generate fully competent factor VIIIa binding determinants. Moreover, factor IXaα and factor IXaβ are fully competent to activate factor VII, without influence by factor VIIIa. Given this mechanistic demonstration and the previously noted clinical observations (Blood 80:25-28, 1992), we propose that activated forms of factor IX are responsible for generating circulating plasma factor VIIa, which is available for the initiation phase of coagulation. These data suggest that activated factor IX is as plausible a candidate for the initiating protease of the coagulation cascade as is activated factor VII_.