Vascular remodelling is a prominent feature of haemophilic arthropathy (HA) that may underlie re-bleeding, yet the nature of vascular changes and underlying mechanisms remain largely unknown. Here, we aimed to characterize synovial vascular remodelling and vessel integrity after haemarthrosis, as well as temporal changes in inflammatory and tissue-reparative pathways. Thirty acutely painful joints in patients with haemophilia (PWH) were imaged by musculoskeletal ultrasound with Power Doppler (MSKUS/PD) to detect vascular abnormalities and bloody effusions. Nineteen out of 30 painful joint episodes in PWH were associated with haemarthrosis, and abnormal vascular perfusion was unique to bleeding joints. A model of induced haemarthrosis in factor VIII (FVIII)-deficient mice was used for histological assessment of vascular remodelling (α-smooth muscle actin [αSMA] expression), and monitoring of in vivo vascular perfusion and permeability by MSKUS/PD and albumin extravasation, respectively. Inflammatory (M1) and reparative (M2) macrophage markers were quantified in murine synovium over a 10-week time course by real-time polymerase chain reaction. The abnormal vascular perfusion observed in PWH was recapitulated in FVIII-deficient mice after induced haemarthrosis. Neovascularization and increased vessel permeability were apparent 2 weeks post-bleed in FVIII-deficient mice, after a transient elevation of inflammatory macrophage M1 markers. These vascular changes subsided by week 4, while vascular remodelling, evidenced by architectural changes and pronounced αSMA expression, persisted alongside a reparative macrophage M2 response. In conclusion, haemarthrosis leads to transient inflammation coupled with neovascularization and associated vascular permeability, while subsequent tissue repair mechanisms coincide with vascular remodelling. Together, these vascular changes may promote re-bleeding and HA progression.
Key Points• Fibrin aC389-402 binds a cleft in the b-sandwich domain of FXIII-A 2 * exposed only after cleavage of the activation peptide.• Binding of fibrin aC389-402 to FXIII-A 2 * regulates fibrin cross-linking and thus clot stabilization and fibrinolysis.Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the aC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A 2 (FXIII-A 2 *); however, the functional role of this interaction and binding site(s) on FXIII-A 2 * remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquidchromatography-tandem mass spectrometry the interacting residues on FXIII-A 2 * following chemical cross-linking of fibrin(ogen) aC389-402 peptides to FXIII-A 2 *; and (3) carried out molecular modeling of the FXIII-A 2 */peptide complex to identify contact site (s) involved. Results demonstrated that inhibition of the FXIII-A 2 */aC interaction using aC389-402 peptide (Pep1) significantly decreased incorporation of biotinamidopentylamine and a2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the b-sandwich domain of FXIII-A 2 * localizing aC cross-linking Q366 to the FXIII-A 2 * active site. Our findings demonstrate that binding of fibrin aC389-402 to the APcleft is fundamental to clot stabilization and presents this region of FXIII-A 2 * as a potential site involved in glutamine-donor substrate recognition. (Blood. 2013;121(11):2117-2126
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.
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