Antithrombin- and Heparin-Binding Exosites Regulate Extravascular Binding and Clearance of Factor IXa

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.2392-2392
• Main Authors: Westmark, Pamela, Sheehan, John P.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-131580

Introduction: Administration of increasing doses of FIX to hemophilia B (HB) mice reveals that the extravascular compartment is capable of binding several-fold more FIX than circulates in the plasma (Cooley et al., Blood 2019). FIX binds rapidly and reversibly to vascular endothelium and the extravascular matrix, in part mediated by interaction of the Gla domain with collagen IV (Col IV). Anticoagulant heparan sulphate and Col IV localize predominantly to the basement membrane and supramolecular structure analysis suggests that heparan sulphate chains are integrated into Col IV-containing networks. Previous work has demonstrated the contribution of the heparin and antithrombin (AT) binding exosites on the protease domain to the regulation of FIXa activity. We now compare the contribution of these protease exosites to in vivo recovery and clearance of the FIX(a) protease and zymogen in hemophilia A (HA) or HB mice. Methods: Recombinant human FIX variants expressed in HEK293 cells with substitutions in the AT- (R150A) and heparin-binding (K126A/K132A) exosites (chymotrypsinogen #) were purified to homogeneity and activated to FIXa with FXIa. FIX(a) pharmacokinetics (PK) in HA or HB mice were determined with injection of a weight-based dose into the tail vein. Serial retro-orbital blood samples were collected, plasma isolated and FIX(a) content determined with a human FIX specific ELISA. FIX(a) concentrations were plotted versus time and fit to the equation: [IX]t = [IX]max*(C1*e(-k1*t)+ C2*e(-k2*t)), where k1and k2are 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: PK for clearance of predicted 30% plasma levels of human FIXa (5 min to 3 hr) or FIX (5 min to 25 hr) were examined in HA (Fig 1) or HB mice (Fig 2), respectively. HA mice were employed for FIXa to avoid in vivoclot formation triggered by active protease. Notably, FIXa plasma clearance was relatively prolonged with ~50% of recovered antigen levels present at 25-30 min post-injection. Similar to FIX, the pattern of FIXa plasma clearance suggested a 2-compartment model with initial and terminal phases of elimination. FIXa WT demonstrated approximately ~23% recovery of predicted levels at 5 min in HA mice, similar to the zymogen (21%) in HB mice (Table I). FIXa R150A (reduced AT affinity) demonstrated markedly enhance recovery