Differences in venous clot structures between hemophilic mice treated with emicizumab versus factor VIII or factor VIIIFc

Abstract

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab-prophylaxis led us employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We thus analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab compared to rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab compared to rFVIII/rFVIIIFc. Scanning-electron microscopy and immunofluorescent spinning-disk confocal-microscopy of in vivo generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFc- and emicizumab-induced clots. Contrary to the regular fibrin-mesh obtained with rFVIII/rFVIIIFc, fibrin-fibers appeared to be fused into large patches upon emicizumabtreatment. Moreover, fewer red blood cells were detected in regions where these fibrin patches were present. The presence of highly-dense fibrin-structures associated with a diffuse fiber-structure in emicizumab-induced clots was also observed when using superresolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.