OC 50.4 - Differences in bleeding profiles and venous clot structures between emicizumab and factor VIII-treated mice

Background: Emicizumab is a bispecific antibody used to treat hemophilia A with/without inhibitors. We previously reported that emicizumab partially corrects bleeding in factor VIII (FVIII)-deficient mice following tail-clip injury. Using a milder bleeding model of tail vein-transection (TVT), we further analyzed emicizumab’s hemostatic efficacy in mice.

Aims: To analyze the bleeding profile and clot stability/structure following FVIII or emicizumab treatment.

Methods: FVIII-deficient mice received low-dose FVIII (5 IU/kg) or emicizumab (5 mg/kg) intravenously before TVT, and bleeding profiles were evaluated with/without removing the occlusive clot (wound challenge). Alternatively, tail sections were collected 10 minutes post-injury and prepared for microscopic analysis by immunofluorescence (fibrin, platelets and red blood cells (RBCs)) or scanning electron microscopy (SEM).

Results: Without wound challenge, blood loss was similar in FVIII-treated mice (28±7 µL) or emicizumab-treated mice (32±15 µL; p=0.99; Figure 1). In FVIII-treated mice, each wound challenge was followed by rapid bleeding arrest and minor blood loss (135±33 µL after 2 challenges). No differences were observed between mice treated with FVIII or FVIII-Fc. In emicizumab-treated mice, wound challenges were associated with prolonged times to arrest, spontaneous rebleeding thereafter and abundant additional blood loss (533±165 µL; p< 0.0001 versus FVIII with challenges). Immunofluorescence analysis of occlusive clots revealed that FVIII-derived clots contain larger areas of co-localization between aggregated RBCs and platelets compared to emicizumab-derived clots (21±4% vs 4±1% of RBC-signal overlapping platelet-signal; p=0.0031). SEM-analysis further confirmed differences in clot-structure between both treatments (Figure 2). Compact RBC-structures were observed in clots from FVIII-treated mice, with individual RBCs being mostly indistinguishable. In contrast, the outer-surface of clots from emicizumab-treated mice consisted of a fractured structure harbouring individual RBCs and apparent ruptured fibrils.

Conclusion(s): Clots forming in the presence of FVIII or emicizumab show different structure and density, which correlate with wound challenge response and clot stability. Our data suggest mechanistic differences in clot formation.