Selective disruption of heparin and antithrombin-mediated regulation of human factor IX

Abstract

To cite this article: Westmark PR, Tanratana P, Sheehan JP. Selective disruption of heparin and antithrombin-mediated regulation of human factor IX. J Thromb Haemost 2015; 13: 1053-63.Summary. Background: Interaction with antithrombin and heparin regulates distribution, activity, and clearance of factor IXa (FIXa). Hemophilia B prophylaxis targets plasma FIX levels > 1% but neglects extravascular FIX, which colocalizes with antithrombin-heparan sulfate. Objective: Combined mutagenesis of FIX was undertaken to selectively disrupt heparin-and antithrombin-mediated regulation of the protease. Methods: Human FIX alanine substitutions in the heparin (K126A and K132A) and antithrombin (R150A) exosites were characterized with regard to coagulant activity, plasma thrombin generation, antithrombin inhibition, and plasma half-life. Results: Single or combined (K126A/R150A or K132A/R150A) exosite mutations variably reduced coagulant activity relative to wild-type (WT) for FIX (27-91%) and FIXa (25-91%). Double mutation in the heparin exosite (K126A/K132A and K126A/K132A/R150A) markedly reduced coagulant activity (7-21%) and plasma TG. In contrast to coagulant activity, FIX K126A (1.8-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased tissue factor-initiated plasma TG, while FIX K132A and K126A/ R150A were similar to WT. FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased FIXa-initiated TG, while FIXa K132A, R150A, and K126A (0.8-0.9-fold) were similar to WT. Dual mutations in the heparin exosite or combined mutations in both exosites synergistically reduced the inhibition rate for antithrombin-heparin. The half-life of FIXa WT in FIX-deficient plasma was remarkably lengthy (40.9 AE 1.4 min) and further prolonged for FIXa R150A, K126A/ R150A, and K132A/R150A (> 2 h). Conclusion: Selective disruption of exosite-mediated regulation by heparin and antithrombin can be achieved with preserved or enhanced thrombin generation capacity. These proteins should demonstrate enhanced therapeutic efficacy for hemophilia B.

Elevated Plasma Factor IXa Activity in Premenopausal Women on Hormonal Contraception

“…While this clinical association is well established, the mechanism(s) underlying this induced hypercoagulability remain incompletely understood. As FIXa plays a critical role in both hemostatic and thrombotic responses in vivo 32 , the highly sensitive eTGA was developed to detect plasma FIXa activity 29 . The major findings from these studies are: 1) a subset of donors on hormonal contraception demonstrate marked elevations in plasma FIXa activity, 2) elevated plasma FIXa activity correlates with reduced levels of TFPIα and PS, 3) TFPIα and PS directly impact the extent of TF-induced FIX activation in human plasma, and 4) increased FIXa levels induce APC resistance in the TGA.…”

“…The specificity of this assay was confirmed using inhibitory antibodies to block FXIa-dependent and TF-dependent activation of FIX within the assay and confirm the FIX(a) dependence of the thrombin generation signal. The eTGA assay has previously been employed to determine contaminating FIXa levels in recombinant FIX zymogen preparations and FIXa half-life in citrated plasma 29 . The prolonged plasma half-life (~41 min) of FIXa makes these measurements possible on clinical samples.…”

“…The poor reactivity of FIXa with both its substrate and inhibitor reflects the partially occluded active site of the isolated protease, which is dramatically relieved by incorporation into the FIXa-FVIIIa complex 28 . As a consequence of these structural constraints, FIXa has a markedly prolonged plasma half-life (~41 min) 29 , when compared to other coagulation proteases such as FXa and thrombin (<1 min) 30, 31 . Further, FIXa demonstrates enhanced potency for triggering in vivo thrombosis relative to other coagulation proteases in a rabbit jugular vein stasis assay 32 .…”

“…The combination of poor protease reactivity and high in vivo potency poses a challenge for accurate FIXa activity measurements. Here, a novel method to determine plasma FIXa activity was developed employing a modified TGA with FIX-deficient plasma to enhance sensitivity and a panel of inhibitory antibodies to confirm specificity 29 . This enhanced TGA (eTGA) was used to measure plasma FIXa activity in citrated plasma samples from volunteer blood donors.…”

New developments in the management of moderate-to-severe hemophilia B

“…These approaches include incorporation of the FIX Padua mutation or combined mutations to enhance coagulant activity, 79 introduction of additional N -glycosylation sites to enhance pharmacokinetics and subcutaneous (SC) absorption, 80 and disruption of antithrombin- and heparin-mediated regulation to enhance in vivo protease activity. 81 Although the overall risk of inhibitors is lower in hemophilia B (see “Inhibitor formation and anaphylaxis” section), variant FIX sequences will require careful analysis for potential inhibitor formation. 82 …”