Recent studies from our laboratory showed that clotting factor VIIa (FVIIa), whose primary function is to initiate the coagulation cascade following its binding to procoagulant cofactor tissue factor (TF), also binds anticoagulant cofactor, endothelial cell protein C receptor (EPCR). EPCR plays a key role in the activated protein C (APC)-mediated anticoagulant pathway by promoting the activation of protein C bound to it. Pharmacological concentrations of rFVIIa could compete with protein C for the EPCR and downregulate APC generation by preventing protein C binding to the EPCR. Our recent studies showed that FVIIa binding to EPCR also induces barrier-protective and anti-inflammatory effects both in vitro and in vivo model systems. Bio-distribution of rFVIIa in mice revealed that a small fraction of rFVIIa administed to mice i.v. accumulates in knee joints and retained there for three to seven days. The present study was conducted to investigate the relevance of FVIIa binding to EPCR in pharmacological FVIIa treatment of joint disease in hemophilia. For this, we first generated hemophilia A mice lacking EPCR or overexpressing EPCR. To generate EPCR deficient FVIII-/- mice, Procrflox/flox and Procr+/floxMeox2+/cre mice were first backcrossed with FVIII-/- mice to generate Procrflox/flox/FVIII-/- and Meox2+/cre/FVIII-/- mice. EPCR deficient FVIII-/- mice (Procr-/-/FVIII-/-) were generated by breeding female Procrflox/flox/FVIII-/- with male Procr+/floxMeox2+/cre /FVIII-/- mice. EPCR- overexpressing FVIII-/- were generated by crossing Tie2-EPCR mice with FVIII-/- mice. We induced joint bleeding in FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice by needle puncture injury. Right knee joint intra articular space was pierced with 30G needle to induce joint bleeding; left knee joint served as an uninjured control. Injured mice were treated with a single dose of rFVIIa (1 mg/kg) at 20 min following the injury or three doses of rFVIIa (1 mg/Kg) at 20 min, 24 h and 72 h following the injury. In controls, saline was administered in place of rFVIIa. The knee injury was evaluated by measuring knee joint diameter and visual bleeding score. At the end of 14 days post injury, mice were euthanized, and the knee joints were excised and fixed for immunohistochemical analysis. Hemophilic arthropathy was evaluated by scoring a set of pathological parameters (e.g., synovial hyperplasia, neoangiogenesis, presence of blood, iron score, villus formation, and cartilage degeneration). A subset of mice was used to collect synovial fluid at 7 days post injury. Assessment of joint bleeding at 5 h following the needle injury, by extracting and measuring hemoglobin levels in joint tissues and monitoring hematocrit, showed an equal amount of bleeding in all three genotypes of mice (FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice). Administration of three doses of FVIIa was effective in preventing hemophilic arthropathy in FVIII-/- and EPCR-overexpressing FVIII-/- mice, whereas a single dose of rFVIIa had a minimal effect in reducing hemophilic arthropathy. Interestingly, a single dose of FVIIa fully corrected the needle injury-induced hemophilic arthropathy in EPCR-deficient FVIII-/- mice. More importantly, EPCR-deficient FVIII-/- mice failed to develop full-blown hemophiic arthropathy even in the absence of any treatment. Measurement of inflammatory cytokines in the synovial fluid showed a robust increase in IL-6 levels in FVIII-/- mice and a single dose of FVIIa substantially reduced IL-6 levels. Interestingly, IL-6 levels in the synovial fluid of injured EPCR-overexpressing FVIII-/- mice were markedly higher compared to injured FVIII-/- mice, three doses of rFVIIa was required to attain a significant reduction in IL-6 levels in these mice. In contrast to these data, we found very little IL-6 levels in the synovial fluids of injured EPCR-deficient FVIII-/- mice. Overall, these data indicate that EPCR levels profoundly influence hemophilic arthorpathy. EPCR-deficiency protects from development hemophilic arthropathy because down-regulation of APC generation in EPCR deficiency could allow sufficient thrombin generation in hemophilia to prevent joint bleeding and bleeding-associated inflammation.


Rao:Takeda: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.