Zymogen-Like Factor Xa Variants Restore Thrombin Generation and Effectively Bypass the Intrinsic Pathway in Vitro

Several clotting factors circulate as zymogens and are converted to the active protease state following proteolysis at a highly conserved site (R15–I16). Liberation of the new N terminus (I16-VGG) drives a conformational change which leads to maturation of the catalytic domain and imparts function. We recently characterized two variants, FXaI16L and FXaV17A and found that they are zymogen-like, have poor catalytic function and are ineffectively targeted by active site probes (JBC2008; 283: 18627). Surprisingly however, active site function could be rescued with saturating concentrations of FVa. Once assembled in prothrombinase the FXa variants could function in a comparable way to the wild-type (wt) enzyme. Based on these differential functional states, these zymogen-like FXa variants may offer a unique alternative to bypass deficiencies in the intrinsic pathway. Hemophilia is characterized by deficiencies of FVIII (HA) or FIX (HB) with inadequate production of FXa and subsequently thrombin (IIa) formation. Replacement therapy with FVIII or FIX is very effective except when inhibitory antibodies are present. To circumvent this, bypass agents such as activated prothrombin complex concentrates and recombinant FVIIa, were developed. In principle, FXa should also be effective; however, it has serious limitations as FXa could cause excessive activation of coagulation and its very short half life (<1–2 min) in plasma limits its utility. We examined whether FXaI16L could circumvent these associated problems and restore IIa generation using systems of increasing complexity. Factor XaI16L was first characterized on platelets and we also evaluated its sensitivity to antithrombin III (ATIII), an inhibitor of FXa in vivo. Limiting concentrations of FXaI16L on IIa-activated platelets with exogenously added FVa yielded rates of IIa generation that were comparable to wt-FXa (<2-fold difference), indicating this variant can assemble and function on a physiological surface. Inhibition studies revealed that the second order rate constant for ATIII with FXaI16L was reduced 40-fold relative to wt-FXa, consistent with the model that its active site is not readily accessible by the inhibitor. To examine whether FXaI16L promotes IIa generation in hemophilic plasma, clotting assays were employed. In this system, wt-FXa (0.1 nM) normalized the prolonged aPTT of HA or HB plasma while FXaI16L (0.1 nM) was ~70% effective. Whereas wt- FXa exhibits a half life of <2 min in either HA or HB plasma, FXaI16L retained >60% activity after 2 hr, indicating that the zymogen-like conformation protects it in a plasma environment. To extend these findings, we employed a IIa generation assay. In this system, initiation of coagulation with CaCl2, phospholipid and tissue factor (2 pM) yields robust IIa generation (endogenous IIa potential (ETP) ~4000 nM min; peak IIa, 300 nM) over a 60 min period in hemostatically normal plasma while very little IIa is generated in HA or HB plasma. Addition of FXaI16L to HA or HB plasma increased IIa generation in a dose dependent manner and completely restored the ETP at ~30–100 pM. Comparisons with recombinant FVIIa indicated the FXaI16L was effective at ~500-fold lower molar concentration in this system at restoring IIa generation. Both proteins (rFVIIa and FXaI16L) significantly shortened the initiation phase (lag time) relative to normal plasma. Since FVa is required to rescue the activity of the variant, we examined how the protein C pathway modulates the activity of FXaI16L. Introduction of either soluble thrombomodulin (1–25 nM) or activated protein C (1–20 nM) to HA or HB plasma containing FXaI16L reduced total IIa generation to baseline values in a dose dependent fashion. Collectively our data show that zymogen-like FXa variants restore IIa generation in hemophilic plasma and have a prolonged half-life. The data also indicate that the FVa generated in these in vitro systems is sufficient to rescue FXaI16L and that elimination of the cofactor by the protein C pathway effectively regulates FXaI16L function. The efficacy and prolonged half-life suggest that these FXa variants might prove useful as therapeutic procoagulant bypass agents to treat deficiencies upstream of the common pathway.