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

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.

Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models.

The lack of innovation in von Willebrand disease (VWD) originates from many factors including the complexity and heterogeneity of the disease but also from a lack of recognition of the impact of the bleeding symptoms experienced by patients with VWD. Recently, a few research initiatives aiming to move past replacement therapies using plasma-derived or recombinant von Willebrand factor (VWF) concentrates have started to emerge. Here, we report an original approach using synthetic platelet (SP) nanoparticles for the treatment of VWD type 2B (VWD-2B) and severe VWD (type 3 VWD). SP are liposomal nanoparticles decorated with peptides enabling them to concomitantly bind to collagen, VWF, and activated platelets. In vitro, using various microfluidic assays, we show the efficacy of SPs to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from mice with VWD-2B and deficient VWF (VWF-KO, ie, type 3 VWD). In vivo, using a tail-clip assay, SP treatment reduced blood loss by 35% in mice with VWD-2B and 68% in mice with VWF-KO. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen-binding peptide, although not sufficient by itself, was crucial for SP efficacy in VWD-2B; whereas all 3 peptides appeared necessary for mice with VWF-KO. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of mice with VWF-KO led to a strong clot, similar to those obtained in wild-type mice. Altogether, our results show that SP could represent an attractive therapeutic alternative for VWD, especially considering their long half-life and stability.

Menin inhibition suppresses castration-resistant prostate cancer and enhances chemosensitivity.

Disease progression and therapeutic resistance of prostate cancer (PC) are linked to multiple molecular events that promote survival and plasticity. We previously showed that heat shock protein 27 (HSP27) acted as a driver of castration-resistant phenotype (CRPC) and developed an oligonucleotides antisense (ASO) against HSP27 with evidence of anti-cancer activity in men with CRPC. Here, we show that the tumor suppressor Menin (MEN1) is highly regulated by HSP27. Menin is overexpressed in high-grade PC and CRPC. High MEN1 mRNA expression is associated with decreased biochemical relapse-free and overall survival. Silencing Menin with ASO technology inhibits CRPC cell proliferation, tumor growth, and restores chemotherapeutic sensitivity. ChIP-seq analysis revealed differential DNA binding sites of Menin in various prostatic cells, suggesting a switch from tumor suppressor to oncogenic functions in CRPC. These data support the evaluation of ASO against Menin for CRPC.

A reactive center loop-based prediction platform to enhance the design of therapeutic SERPINs.

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.