Effects of Joint Blood Volume and Hemostasis Correction on Vascular Changes in the Joint after Induced Hemarthrosis in Hypocoagulable Mice

Abstract Introduction:Hemophilia causes recurrent bleeds into 'target joints' despite clotting factor replacement. Vascular remodeling associated with hemarthrosis is thought to contribute to vessel "leakiness" and perpetuated bleeding, but the underlying mechanisms remain unknown. Here, we studied the effects of joint blood volume and timing of hemostasis correction on vascular changes after induced hemarthrosis in FVIII-deficient mice, and in BALB/c mice treated temporarily with warfarin and anti-FVIII to create reversible hemostasis suppression. Methods: Mice were injured by sub-patellar puncture of the right knee to induce hemarthrosis. FVIII-deficient mice were treated with two doses of recombinant human FVIII (rhFVIII) or saline with an 8-12 hour interval. The first dose of rhFVIII was given i) 2 hours before injury (prophylaxis), ii) 2 hours after injury (early), iii) 8 hours after injury (intermediate), or iv) 16 hours after injury (late). BALB/c mice were administered 10 µg/ml warfarin in drinking water for 1 week plus 0.25 mg/kg anti-FVIII 2 hours prior to injury. This dose of warfarin yielded a ~4-fold increase in prothrombin time that was reversed with 100 IU/kg 4-Factor prothrombin complex concentrate on day 2 post-injury in one group of mice, and maintained in another. Clearance of anti-FVIII within 4 days was indicated by normalization of blood loss in tail clip experiments. On day 2 after injury, the hematocrit was determined as a measure of joint bleeding and incapacitance was monitored by left:right weight bearing. Vascularity was assessed at baseline and 1-2 weeks post-injury by high resolution musculoskeletal ultrasound with Power Doppler (MSKUS/PD) and histology with Safranin-O-Fast Green staining. Statistical analyses were conducted by Mann-Whitney test. Results: In FVIII-deficient mice, knee injury significantly reduced the mean hematocrit from 47 % to ~25 % for mice receiving late or no rhFVIII treatment (p Bleeding tendency and weight bearing deficits of the injured leg in BALB/c mice treated with warfarin and anti-FVIII were comparable to those observed in FVIII-deficient mice. Mean hematocrit decreased to 28 % (p Conclusions: FVIII prophylaxis or early treatment reduced blood loss into joints of FVIII-deficient mice and preserved joint function. However, neovascularization and vascular changes consistent with remodeling were not proportional to joint blood volume and were mostly unresponsive to short-term FVIII replacement, regardless of timing. Similarly, neovascularization in joints of hemostatically compromised BALB/c mice after induced hemarthrosis persisted despite normalization of hemostasis. Changes in vessel architecture, however, could be abrogated. This suggests that long-term hemostasis correction may protect against irreversible vascular remodeling in the joint, thought to underlie re-bleeding and development of hemophilic arthropathy. Further investigations are required to determine the effects of prolonged hemostasis correction on re-bleeding tendencies and joint health in hemophilia. Disclosures Mosnier: The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau. von Drygalski: Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau.