Abnormal hemostasis in a knock‐in mouse carrying a variant of factor IX with impaired binding to collagen type IV

Abstract

Summary. Background: Factor IX binds to collagen type -IV, but this binding has no known consequence. Objectives: To determine the effect of reduced binding of FIX to collagen IV. Methods: We constructed and characterized Ôknock-inÕ mice containing the mutation lysine 5 to alanine (K5A) in the Gla domain of their FIX. The K5A mutation dramatically reduced the affinity of FIX for collagen type IV, but had no measurable effect on platelet binding, phospholipid binding, or in vitro clotting activity. However, K5AFIX mice had a mild bleeding tendency, despite their in vitro clotting activity being normal. Hemostatic protection from delayed rebleeding was intermediate between wild-type and hemophilia B mice (which had no detectable clotting activity); moreover, survival of K5A FIX mice after nascent clot removal was dramatically improved as compared with hemophilia B mice. Importantly, there was no detectable difference between K5AFIX and wild-type mice in either a laser-induced thrombosis model or the chromogenic FIX activity assay. In contrast, after ferric chloride injury, which exposes collagen IV as well as other basement membrane proteins, intravital microscopy revealed that vessel occlusion was significantly slower in K5AFIX mice than in wild-type mice. Conclusions: Our results indicate that the FIX molecule with decreased affinity for collagen IV has altered hemostatic properties in vivo and that the binding of FIX to collagen IV probably plays a significant functional role in hemostasis.

Prophylactic efficacy of BeneFIX vs Alprolix in hemophilia B mice

“…9,10 Although the hemophilia B mice show undetectable circulating levels of FIX 7 days after an infusion of BeneFIX, an injured saphenous vein will nevertheless form clots on day 7. If clotting at the site of an injury were due to residual, nondetectable levels of circulating FIX, then one would expect the limit of clot recurrence at that wound to represent either the consumption of the residual circulating FIX, or conversely, exhaustion BLOOD, 14 JULY 2016 x VOLUME 128, NUMBER 2 BeneFIX VS ALPROLIX 287…”

“…This mouse exhibits a bleeding diathesis even though its circulating level of FIX K5A is about 20% higher than normal. 9 The specific activity of this FIX K5A in vitro is indistinguishable from that of wild-type FIX (FIX WT ); this suggests an important hemostatic role for FIX bound to extravascular collagen IV.…”

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

“…A recent study suggested that lack of collagen binding was associated with a partial hemostatic defect in models involving exposure of vascular collagen including ferric chloride and tail transection models. 46 It is presently not clear whether the preserved efficacy of N9-GP in the same models is explained by adequate residual affinity of N9-GP for collagen or by the higher in vivo recovery which may compensate for a reduced accumulation at vascular sites.…”

Animal Models of Hemophilia and Related Bleeding Disorders

“…54 Another knock-in mouse has been created in which lysine at amino acid 5 is mutated to alanine, which impairs binding of the factor IX protein to type IV collagen. 55 Interestingly, in vitro factor IX activity of this variant is normal, but in vivo hemostasis is impaired, though not as much as in the factor IX knockout mouse. 55 These animals are useful in studies on the effect of factor IX mutations in residual circulating protein on immunogenicity of factor IX.…”