Defective TAFI activation in hemophilia A mice is a major contributor to joint bleeding

Abstract

Joint bleeds are common in congenital hemophilia but rare in acquired hemophilia A (aHA) for reasons unknown. To identify key mechanisms responsible for joint-specific bleeding in congenital hemophilia, bleeding phenotypes after joint injury and tail transection were compared in aHA wild-type (WT) mice (receiving an anti-factor VIII [FVIII] antibody) and congenital HA (FVIII) mice. Both aHA and FVIII mice bled severely after tail transection, but consistent with clinical findings, joint bleeding was notably milder in aHA compared with FVIII mice. Focus was directed to thrombin-activatable fibrinolysis inhibitor (TAFI) to determine its potentially protective effect on joint bleeding in aHA. Joint bleeding in TAFI mice with anti-FVIII antibody was increased, compared with WT aHA mice, and became indistinguishable from joint bleeding in FVIII mice. Measurements of circulating TAFI zymogen consumption after joint injury indicated severely defective TAFI activation in FVIII mice in vivo, consistent with previous in vitro analyses in FVIII-deficient plasma. In contrast, notable TAFI activation was observed in aHA mice, suggesting that TAFI protected aHA joints against bleeding. Pharmacological inhibitors of fibrinolysis revealed that urokinase-type plasminogen activator (uPA)-induced fibrinolysis drove joint bleeding, whereas tissue-type plasminogen activator-mediated fibrinolysis contributed to tail bleeding. These data identify TAFI as an important modifier of hemophilic joint bleeding in aHA by inhibiting uPA-mediated fibrinolysis. Moreover, our data suggest that bleed protection by TAFI was absent in congenital FVIII mice because of severely defective TAFI activation, underscoring the importance of clot protection in addition to clot formation when considering prohemostatic strategies for hemophilic joint bleeding.

Mechanisms of vascular permeability and remodeling associated with hemarthrosis in factor VIII‐deficient mice

“…20 The effect of this antibody on tail bleeding in FVIIIdeficient mice has been shown previously. 20 After knee injury, Hypo BALB/c mice were divided into three groups and either continued on 10 μg/mL warfarin for 2-4 weeks or discontinued from warfarin and injected with 100 IU/kg 4F-PCC on day 2 +/− twice weekly doses of 0.25 mg/kg anti-FVIII for 2 to 4 weeks. A summary of the treatment strategy for the Hypo BALB/c hemarthrosis model is depicted in Figure 1.…”

“…For induction of hemarthrosis, mice were fed 10 μg/mL warfarin dissolved in drinking water for 7 days and injected i.v. with 0.25 mg/kg anti‐FVIII (GMA‐8015) 2 h prior to knee injury ( Hypo BALB/c model) since warfarin alone does not elicit notable hemarthrosis (internal observation), nor does anti‐FVIII treatment . The effect of this antibody on tail bleeding in FVIII‐deficient mice has been shown previously .…”

“…This effect was somewhat recapitulated in Hypo BALB/c mice with acquired hemophilia A, whereby a milder and delayed response may reflect residual FVIII activity. Indeed, it has previously been shown that, in contrast to FVIII‐deficient mice, thrombin generation in mice with acquired hemophilia A is sufficient to activate thrombin activatable fibrinolyisis inhibitor, resulting in less joint bleeding via suppression of urokinase‐type plasminogen activator‐mediated fibrinolysis . Thus, residual FVIII activity may dampen other potential FVIII‐mediated processes, such as vascular remodeling.…”

“…Indeed, it has previously been shown that, in contrast to FVIII-deficient mice, thrombin generation in mice with acquired hemophilia A is sufficient to activate thrombin activatable fibrinolyisis inhibitor, resulting in less joint bleeding via suppression of urokinase-type plasminogen activator-mediated fibrinolysis. 20 Thus, residual FVIII activity may dampen other potential FVIII-mediated processes, such as vascular remodeling. Interestingly, compromise in vascular permeability after joint bleeding was also worse with congenital FVIII deficiency and only partially corrected with short-term rhFVIII treatment.…”

“…with 0.25 mg/kg anti‐FVIII (GMA‐8015) 2 h prior to knee injury ( Hypo BALB/c model) since warfarin alone does not elicit notable hemarthrosis (internal observation), nor does anti‐FVIII treatment . The effect of this antibody on tail bleeding in FVIII‐deficient mice has been shown previously . After knee injury, Hypo BALB/c mice were divided into three groups and either continued on 10 μg/mL warfarin for 2‐4 weeks or discontinued from warfarin and injected with 100 IU/kg 4F‐PCC on day 2 +/− twice weekly doses of 0.25 mg/kg anti‐FVIII for 2 to 4 weeks.…”