Effect Of Combined Heparin- and Antithrombin-Binding Exosite Mutations On Human Factor IX(a) Coagulant Activity, Thrombin Generation, and Protease Half-Life In Human Plasma

Hemophilia B is an X-linked genetic disorder characterized by defective factor IX activity. Recombinant factor IX (rFIX) is employed as protein replacement for the treatment and prophylaxis of bleeding episodes. Antithrombin is the primary plasma inhibitor of activated factor IX (FIXa), and inhibition is enhanced by heparin/heparan sulfate. We hypothesize that selective disruption of protease interactions with heparin and antithrombin via mutations in the respective heparin- and antithrombin-binding exosites may enhance rFIX(a) efficacy by prolonging protease half-life in vivo.

To assess the effect of mutations in the FIX(a) heparin- and antithrombin-binding exosites on traditional coagulant activity, thrombin generation, and protease half-life in human plasma.

Human FIX cDNA constructs with alanine substitutions (chymotrypsinogen numbering) in the heparin exosite (K126A, K132A, K126A/K132A), antithrombin exosite (R150A), or both (K126A/R150A, K132A/R150A, K126A/K132A/R150A) were expressed in HEK293 cell lines. Recombinant zymogens were purified from conditioned media, and a portion activated to protease with human factor XIa. Zymogen and protease forms were characterized in APTT-based clotting assays, and tissue factor (TF) and FIXa-initiated thrombin generation (TG) assays in pooled human FIX-deficient plasma, respectively. Comparisons were made with human plasma-derived factor IX (pFIX) and recombinant FIX wild type (WT). Protease half-life in pooled, citrated human plasma was determined using a novel assay that detects FIXa activity by TG response.

Zymogen coagulant activities (% WT ± S.E) were: pFIX 105.2 ± 2.8, WT 100 ± 7.1, K132A/R150A 75.8 ± 3.4, K126A 63.3 ± 2.3, R150A 62.4 ± 4.0, K132A 30.9 ± 1.0, K126A/R150A 27.0 ± 2.1, K126A/K132A 20.6 ± 9.2, and K126A/K132A/R150A 7.3 ± 3.8. Similarly, protease coagulant activities were: WT 100 ± 6.1, pFIXa 98.4 ± 11.4, K132A 91.4 ± 1.6, K132A/R150A 84.9 ± 2.8, R150A 77.1 ± 5.8, K126A 39.5 ± 2.4, K126A/R150A 25.3 ± 2.8, K126A/K132A/R150A 10.9 ± 0.6, and K126A/K132A 9.3 ± 0.6. In contrast to their relative coagulant activities, FIX K126A (1.9-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased peak thrombin concentrations during TF-triggered TG; pFIX, FIX K132A and K126A/R150A were similar to WT; and FIX K126A/K132A/R150A (0.6-fold) and K126A/K132A (0.2-fold) demonstrated marked reductions in peak thrombin relative to WT. In the FIXa-initiated TG assay, FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased peak thrombin concentrations; pFIXa, FIXa K132A, R150A, and K126A (0.8-1.0 fold) were similar to WT; while FIXa K126A/K132A and K126A/K132A/R150A demonstrated markedly reduced (0.2-0.3 fold) and delayed peak thrombin concentrations. In pooled, citrated FIX-deficient plasma, FIXa WT (40.9 ± 1.4 min) and K126A/K132A (37.2 ± 0.7 min) demonstrated similar half-lives, while FIXa R150A, K126A/R150A, and K132A/R150A all had half-lives > 2 hr.

Single exosite mutations resulted in mild to moderate reductions in coagulant activity, while the double mutation in the heparin exosite (K126A/K132A) markedly reduced activity, likely due to a synergistic effect on cofactor binding. Traditional coagulant activity did not accurately represent the ability of the mutant proteins to support thrombin generation. Despite variable reductions in coagulant activity, FIX K126A, K132A, R150A, K126A/R150A and K132A/R150A supported levels of plasma thrombin generation that were equal to or greater than FIX WT. The plasma half-life of FIXa WT activity was remarkably lengthy, and while mutations in the heparin exosite had negligible effects, R150A in the antithrombin exosite substantially increased protease half-life, consistent with a primary role for antithrombin in the plasma inhibition of FIXa. Thus, single exosite mutations did not significantly disrupt the procoagulant function of human FIX(a), and combined exosite mutations (K126A/R150A and K132A/R150A) maintain or enhance plasma thrombin generation while disrupting exosite-mediated regulatory mechanisms. The combination of intact procoagulant function with disruption of antithrombin- and heparin-mediated regulation of FIX(a) will potentially enhance in vivo recovery, prolong plasma half-life, and enhance the efficacy of hemophilia B replacement therapy.

Sheehan:Novo Nordisk Access to Insight Basic Research Grant: Research Funding; Bayer Hemophilia Awards Program: Research Funding; Diagnostica Stago: reagents, reagents Other.