In vitro characterization of K5A and K5R variants of Factor IX

Objective Recent studies indicate that binding of coagulation factor IX (FIX) to extracellular matrix (ECM) contributes to its extravascular (EV) storage and its hemostatic efficacy. It is postulated that the binding affinity of FIX to ECM, more specifically to Collagen IV (Col-IV), increases when Lysine at position 5 is mutated to Arginine (K5R) and decreases when Lysine is mutated to Alanine (K5A). To understand the physiological role of FIX in EV space we aimed to characterize recombinantly generated K5 variants in comparison to wildtype rFIX molecules. We used albumin fused (FP) rFIX variants (rFIX-FP, K5A-FP, K5R-FP) for this purpose.

Material and Methods A functional characterization of the K5 variants was conducted in a FIX one stage clotting assay and in a modified FIX chromogenic activity assay, mimicking the tenase complex in vitro. In addition, enzymatic activity of rFIX variants was characterized using a FIXa-specific chromogenic substrate. Both characterizations were analyzed on a Michaelis-Menten kinetic based model, and the kinetic parameter Km was used as key comparator. Binding of rFIX molecules to Col-IV was measured by SPR.

Results All K5 variants decreased the clot formation time substantially from 76 to 37-38 seconds (≈1IU/mL FIX clotting activity). In the tenase complex both mutants K5A-FP and K5R-FP showed an identical Km (0.20 nM). These results are consistent with the observation that the specific activity (FIX clotting activity per total protein amount) of K5R-FP and K5A-FP displayed similar specific activities, 60 IU/mg and 59 IU/mg, respectively. For the FIXa specific substrate, K5 variants showed comparable rates of enzymatic activity relative to wildtype. Finally, no detectable interaction between immobilized Col-IV and rFIX or rFIX-FP or K5 variants at 0.2 µM was observed.

Conclusion Our experiments showed the structural integrity of the K5 variants and their comparable enzymatic characteristics to wildtype. In-house data on binding properties of these rFIX to Col-IV contradicts published results that posit Col-IV as the binding partner of FIX in the EVS.

Publication History

Article published online:
18 June 2021