Binding to Platelets Differs Among Recombinant Factor VIII Products in Vitro

Abstract

Introduction: Platelets are activated in response to injury and play a key role in blood clotting. In primary hemostasis, platelets adhere to exposed subendothelial matrix proteins (e.g., collagen) and subsequent downstream signaling pathways induce granule secretion and platelet aggregation. In secondary hemostasis, procoagulant platelets in the periphery activate clotting factors, leading to a more stable fibrin clot. Binding of factor VIII (FVIII) to procoagulant platelets ensures efficient production and localization of thrombin at the site of injury. However, it is less clear how platelet-FVIII interactions spatiotemporally control the initial thrombus formation and pave the way for successful tissue repair. In patients with hemophilia A, replacement FVIII therapy is used to treat and prevent bleeds. A number of recombinant FVIII (rFVIII) products are indicated for use in patients with hemophilia A. Simoctocog alfa (Nuwiq®) is a chemically unmodified rFVIII with no fusion protein produced in a human cell line. Damoctocog alfa pegol (Jivi®) and rurioctocog alfa pegol (Adynovate®) are pegylated rFVIII products, while efmoroctocog alfa (Eloctate®) consists of FVIII fused with a Fc protein. It is not clear whether such modifications impact the platelet binding potential of FVIII. This study aims to examine platelet binding of different rFVIII products in vitro. Methods: Platelets were isolated from healthy donors, activated using thrombin and collagen-related peptide (CRP-XL) and incubated with individual rFVIII concentrates. After 30 min, the reaction was stopped by dilution. Binding was quantified by flow cytometry using one of two approaches: rFVIII immunostaining with a fluorescent (Alexa Fluor 647; AF647) anti-FVIII antibody; or using rFVIII molecules that had been pre-labeled with AF647. For comparison among products, experiments were performed at equimolar concentrations (17.6 nM) or equal activities of rFVIII concentrates. Values were normalized to simoctocog alfa. Statistical analyses were performed using one-way ANOVA with Tukey post-hoc testing. Results: Platelets were obtained from 10 healthy donors. At equimolar concentrations, efmoroctocog alfa and damoctocog alfa pegol bound significantly (p < 0.001) less than simoctocog alfa to stimulated platelets, as visualized via immunostaining (n = 10). Binding to platelets was significantly lower with damoctocog alfa pegol than efmoroctocog alfa (p < 0.001). Similar results were obtained from experiments with pre-labelled rFVIII (n = 6) or when using equal activities of rFVIII concentrates. In experiments with platelets from 2 healthy donors, rurioctocog alfa pegol exhibited lower platelet binding compared with simoctocog alfa, when tested using the immunostaining method and equimolar concentrations of rFVIII. Conclusions: Simoctocog alfa showed increased binding to procoagulant platelets compared with other rFVIII concentrates tested. Further studies, including additional functional platelet assays, will be performed using samples from a larger number of healthy donors and from patients with hemophilia A. The results of this study may provide new insights into the function and efficacy of different rFVIII products in patients with hemophilia A.