Role of Protease Exosites for In Vivo Hemostatic Activity and Clearance of FIX

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.1166-1166
• Main Authors: Westmark, Pamela, Sheehan, John
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-120212

Introduction: FIX(a) binds rapidly and reversibly to vascular endothelium and extravascular matrix, in part mediated by the interaction of specific Gla domain residues with collagen IV. Both anticoagulant heparan sulphate and collagen IV localize predominantly to the basement membrane and supramolecular structure analysis suggests that heparan sulphate chains are integrated into collagen IV-containing networks. Previous work has demonstrated the contribution of the heparin and antithrombin (AT)-binding exosites in the protease domain to the regulation of human FIXa. Here we evaluate the contribution of these protease exosites to FIX hemostatic function and clearance in the hemophilia B (HB) mouse. Methods: Recombinant human FIX variants expressed in HEK293 cells with substitutions in the heparin- (K126A/K132A or R170A) or AT-binding (R150A) exosites (chymotrypsinogen #) were purified to homogeneity. In vivohemostasis was assessed in a saphenous vein bleeding model in HB mice. Anesthetized adult mice were injected with a weight-based dose predicted to achieve 30% FIX plasma concentrations and the exposed left saphenous vein was punctured with a 29 gauge needle. Clot formation at the injury site was repeatedly disrupted over 30 min to determine the average time to clot formation and total #clots/30 min. FIX pharmacokinetics (PK) in HB mice were determined with a weight-based dose injected into the tail vein. Serial retro-orbital blood samples were collected, plasma isolated and FIX content determined with a human FIX specific ELISA. FIX concentrations were plotted versus time and initially fit to the equation: [IX]t = [IX]max*(C1*e(-k1*t)+ C2*e(-k2*t)), where k1 and k2 are rate constants for the initial and terminal elimination phases, respectively. As the 4-parameter fit resulted in some large error estimates, the two phases were fit separately to estimate the respective parameters. Results: Previous characterization of APTT-based coagulant activities for these FIX variants (% WT ± SEM) were as follows: FIX WT 100 ± 1.4, R150A 73.6 ± 1.4, K126A/K132A 20.6 ± 9.2, and R170A 609.2 ± 37. In the saphenous vein bleeding assay, HB mice had no clot formation over 30 min, while wild type mice demonstrated 11 clots/30 min. When HB mice were injected with increasing concentrations of FIX WT (20%-100%), a dose dependent increase was observed from 4.3 to 16.5 clots/30 min (Table 1). When injected with sufficient FIX to achieve 30% plasma levels, the AT-binding exosite v