Abstract
Introduction: Protein S (PS) is a cofactor for two important endogenous anticoagulant proteins, tissue factor pathway inhibitor α (TFPIα) and activated protein C (aPC), and thus a critical negative regulator of thrombin generation. VGA039 is a fully human, IgG4 anti-PS monoclonal antibody in clinical development for the treatment of bleeding disorders. VGA039 binds PS and inhibits its cofactor activity for both TFPIα and aPC, thereby enhancing thrombin generation to restore both primary and secondary hemostasis. Here, we use an ex vivo vascularized hemostasis-on-a-chip bleeding test to assess how VGA039 promotes and restores primary and secondary hemostasis using samples from patients with von Willebrand disease (VWD), a disease characterized by deficits in both primary and secondary hemostasis. Our ex vivo hemostasis-on-a-chip is a fully endothelialized mechanical vascular injury model that monitors hemostasis under a physiologically relevant flow condition and enables measurement of an “ex vivo” bleeding time (Sakurai et al., Nat. Comm, 2018). We evaluated VGA039's potential for enhancing hemostasis by real-time monitoring of endothelial injury, platelet deposition, fibrin accumulation, bleeding, and hemostasis within an all-human cell-based environment.
Methods: Polydimethylsiloxane-based hemostasis-on-a-chip is constructed from an endothelialized vascular channel and a pneumatic valve which disrupts a part of the channel membrane to cause endothelial injury and creates an opening for “bleeding”. The research was conducted in accordance with Declaration of Helsinki, and blood from healthy donors and VWD patients was collected under our institutional review board approval with informed consent. Whole blood treated with VGA039 or IgG4 isotype control was perfused into our hemostasis-on-a-chip at 1500 s-1. Bleeding and platelets/fibrin accumulation were observed in real-time under a confocal microscope, and time-lapse images were obtained every 10 seconds. Ex vivo bleeding time was measured by visual confirmation of blood flow cessation at the injury area and was capped at 600 seconds if ex vivo bleeding did not stop.
Results: Blood from 13 VWD patients (type 1: n=11, type 2: n=2) showed significantly longer bleeding time (p=0.02, Mann-Whitney test) compared to blood from healthy donors (n=8). Treating the same VWD patient blood with VGA039 significantly reduced the ex vivo bleeding time compared to the IgG4 isotype control (p=0.002, Wilcoxon test). Median bleeding time of VWD blood treated with VGA039 (399 seconds) was not significantly different from healthy (273 seconds), demonstrating the ability of VGA039 to restore hemostasis in VWD patient blood. VGA039 also enhanced platelet adhesion and fibrin formation at the site of injury compared to the IgG4 isotype control. Significant increases in both mean platelet deposition and fibrin accumulation by VGA039 occurred as early as 150 seconds (p=0.038) and 190 seconds (p=0.043), respectively, peaking at 300 seconds (p=0.004) and 330 seconds (p=0.008) which aligned with the reduced ex vivo bleeding time by VGA039.Conclusion: In this study, we showed that VGA039, an anti-PS monoclonal antibody, promoted both platelet deposition and fibrin accumulation at sites of endothelial cell injury, thus enhancing both primary and secondary hemostasis that led to the restoration of hemostatic activity of VWD patient blood in our ex vivo vascularized hemostasis-on-a-chip bleeding tests. The data presented here support recent clinical trial results in which VGA039 administration led to significant reductions in annualized bleeding rates (ABR) in VWD patients with high historic ABR (Wheeler et al., HTRS 2025). Our assessment positions VGA039 as a promising prophylactic treatment option for patients with bleeding disorders, including VWD.
