Interaction between Collagen IV and Heparin-Binding Sites in the Activity, Distribution and Clearance of Factor IX(a)
Authors: Annis Douglas S. , Westmark Pamela R. , Amend Christina E. , Torres Brianna , Sheehan John P. 
Published: December 2024

Introduction: Factor IX (FIX) is unique among vitamin K-dependent coagulation factors in that a substantial portion is bound to extravascular sites. The extravascular compartment of the hemophilia B (HemB) mouse can bind ~3-fold more FIX than is present in plasma and contributes to in vivo hemostasis. FIX Gla domain variants with decreased (K5A) or increased (K5R) collagen IV binding were evaluated in combination with the protease domain variant K126A/K132A, which disrupts heparin binding, to assess effects on extravascular binding of FIX(a).

Methods: Human FIX wild type (WT), K5A, K5R, K126A/K132A, K5A/K126A/K132A and K5R/K126A/K132A were expressed in VKOR-293 cells and purified to homogeneity. FXIa activation of zymogens, enzymatic characterization, APTT-based coagulant activity and tissue factor (TF)-triggered thrombin generation were previously described (Westmark et al. J Thromb Haemost 2015;13(6):1053-1063). Pharmacokinetics (PK) of FIX and FIXa variants were characterized in HemB and HemA mice, respectively, following tail vein injection (N=5-7 mice/variant), retro-orbital blood sampling and determination of FIX(a) concentration by human FIX(a)-specific ELISA. FIXa variants were injected into HemA mice to avoid triggering coagulation. PK of FIX(a) variants were fit to a 2-compartment model and murine plasma volume assumed to be 40 ml/kg body weight. Legacy (linear) numbering was used in the Gla domain and chymotrypsinogen numbering in the protease domain. Correlation of numbering systems (chymo/legacy/HGVS) for variants is as follows: K5 = –/5/51; K126 = 126/293/339; K132 = 132/301/347.

Results: All FIXa variants had similar catalytic efficiency (kcat/Km) for peptide substrate. FIXa-FVIIIa affinity determined in a functional binding assay ranked FIXa variants as follows: K5R &gt; WT &gt; K5R/K126A/K132A &gt; K5A &gt; K126A/K132A &gt; K5A/K126A/K132A; with Kd(app) ranging from 0.9 to 13.3 nM and similar Bmax for all variants (25-27 nM/min). FX activation showed similar Km (14-24nM) and kcat (2.9-5.3 sec-1) for all FIXa variants. APTT-based coagulant activities for FIX/FIXa were: WT 100/100%, K5A 44/48%, K5R 87/201%, K126A/K132A 16/14%, K5A/K126A/K132A 5.2/6.3% and K5R/K126A/K132A 24/39%. TF-triggered (0.2 pM) peak thrombin values for 90 nM FIX variants in FIX deficient plasma were: WT 72 nM, K5A 71 nM, K5R 182 nM, K126A/K132A 23 nM, K5A/K126A/K132A 22 nM and K5R/K126A/K132A 60 nM.

FIX (HemB mice) and FIXa (HemA mice) variants all demonstrated a biphasic clearance pattern with poor plasma recovery at 2 min for wild type proteins. FIX WT demonstrated ~15% recovery of the total dose in the plasma compartment. FIX K5A enhanced (39%) and FIX K5R reduced (8%) plasma recovery of zymogen. The FIX K126A/K132A heparin-binding variant increased (37%) plasma recovery, FIX K5A/K126A/K132A showed an additive increase (60%), while FIX K5R/K126A/K132A reduced (25%) recovery relative to FIX K126A/K132A. In HemA mice, FIXa WT recovery (16%) at 2 min was similar to zymogen above. In contrast, FIXa K5A dramatically (77%) and FIXa K5R moderately (29%) increased protease recovery. The FIXa K126A/K132A heparin-binding variant showed a similar effect (40%) on protease recovery. FIXa K5A/K126A/K132A (43%) had minimal additional effect, while FIXa K5R/K126A/K132A demonstrated additive (55%) effects on plasma recovery.

Conclusions: K5A/R substitutions in the collagen IV binding site located within the Gla domain had opposing effects on cofactor affinity, coagulant activity and thrombin generation, either alone or in combination with the K126A/K132A protease domain variant. PK of FIX(a) variants demonstrated biphasic clearance and poor recovery of WT proteins, consistent with extravascular binding of both zymogen and protease. Disruption of collagen and heparin binding sites had additive effects on zymogen redistribution to plasma, while K5R reduced plasma recovery. The collagen site had a dominant effect on protease distribution, while the heparin site had similar effects on both zymogen and protease. Disruption of the heparin site modulated the impact of the K5A variant in the protease, and along with the contradictory effects of K5R, suggest potential interaction between these binding sites in the protease.