A ferric chloride induced arterial injury model used as haemostatic effect model

Abstract

A number of experimental bleeding models have been applied to animal models of haemophilia in order to evaluate the acute haemostatic effect of procoagulant compounds. In contrast, in vivo thrombosis models (including the FeCl(3) induced injury model) have mainly been used to study antithrombotic pharmacological intervention. However, as there are limitations to existing bleeding models and as new recombinant FVIII, FIX, and FVIIa variants with increased and prolonged activity are generated there is an increasing need for new and optimized in vivo animal models for testing the efficacy of these haemostatic drug candidates. This led us to look at existing thrombosis models in a new perspective. We have studied the effect of a FeCl(3) induced arterial injury in both F8-KO and F9-KO mice using optimized conditions where exposure to FeCl(3) induces occlusion within 4.2 +/- 0.2 min in wild type mice with a normal coagulation system. In contrast, no occlusion was observed in haemophilic mice providing a therapeutic window in the model making it suitable for pharmacological testing of therapeutic intervention. We demonstrate that replacement therapy with a clinical relevant dose of rFVIII (Advate 20-80 U kg(-1)) and rFIX [(0.75 mg kg(-1) BeneFIX) approximately 50 IU kg(-1)] restored coagulation and normalized the time to occlusion following FeCl(3) induced injury in F8-KO mice and restored coagulation and nearly normalized the time to occlusion in F9-KO mice. In conclusion, we have demonstrated that under optimized conditions the FeCl(3) induced arterial injury model provides a therapeutic window that makes it an useful effect model for evaluation of the haemostatic potential of procoagulant drugs.

Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice

“…However, our data and published studies demonstrate human FVIII binds murine VWF, 17 has comparable cofactor activity as murine FVIII, 17 is stable in murine circulation, 28,29 is incorporated into murine clots, 17 is inactivated by murine-activated protein C (Steve Pipe, University of Michigan, personal communication, June 21, 2011), and supports clot formation in mice. 17,[24][25][26][27] Second, the FeCl 3 /saphenous vein injury model is not commonly used as a model of venous thrombosis; however, our findings are consistent with published data from other venous models and effectively demonstrate the pathologic activity of elevated human FVIII in thrombi formed in murine veins. [17][18][19] Finally, the FeCl 3 thrombosis model does not fully recapitulate the inflammatory process associated with atherosclerosis or plaque rupture in humans or mice, but rather, models acute thrombosis after vascular injury.…”

“…22 17 has comparable cofactor activity as murine FVIII, 17 and promotes coagulation after tail clipping and vessel injury in hemophilic mice. 17,[24][25][26][27] In addition, previous studies 28,29 and our findings (FVIII activity assay, see "FVIII activity assay") show FVIII has sufficient half-life in murine circulation for these experiments. The endogenous FVIII concentration in mice (1 U/mL, 100%) 30 was raised by infusing human FVIII to 285% (total murine plus human FVIII) of normal, consistent with levels associated with thrombosis in humans.…”

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

“…24 Injury was induced up to 5 days after administration of 0.75 mg/kg and the time to occlusion recorded. Except for the first time point at 5 minutes, the average occlusion time in mice receiving N9-GP was consistently shorter than in mice treated with rFIX ( Figure 6C).…”

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models

“…26 The carotid artery was exposed, and an ultrasound flow-probe (0.5 PSB Nanoprobe) placed around the artery. The injury was made 5 minutes, 24 hours, 48 hours, 60 hours, and 72 hours after dosing of 280 U/kg N8-GP, FVIII, or vehicle by applying a filter paper (2 3 5 mm) soaked in 10% FeCl 3 around the exposed artery for 3 minutes.…”