PB0163 - Differential binding of recombinant FVIII products to platelets, and impact of FVIII on VWF expression and platelet contractility in vitro

Background: Binding of factor VIII (FVIII) to procoagulant platelets ensures efficient localisation and availability of thrombin at injury sites. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signalling.

Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of FVIII on von Willebrand factor (VWF) expression and platelet contractility.

Methods: Platelet-FVIII binding: Platelets isolated from healthy donors and people with haemophilia A were activated with thrombin and cross-linked collagen-related peptide (CRP-XL), and incubated with simoctocog alfa (Nuwiq®), efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by FVIII immunostaining and flow cytometry, and values were normalised to values for simoctocog alfa. Furthermore, identification of FVIII binding receptors using TriCEPS-based ligand-receptor capture technology was conducted. VWF expression: Isolated platelets were seeded before activation with adenosine diphosphate (ADP), or thrombin and CRP-XL, in the presence/absence of simoctocog alfa. Levels and locations of VWF and FVIII were assessed by immunostaining and confocal microscopy. Platelet contractility: Isolated platelets were seeded before activation with ADP, or thrombin and CRP-XL, in the presence/absence of simoctocog alfa. Myosin phosphorylation was quantified after Western blotting as a measure of platelet contractility.

Results: Binding to activated platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol (p < 0.05). Addition of simoctocog alfa increased the levels of endogenous platelet VWF, with the VWF levels varying depending on the type of platelet activation. Simoctocog alfa and endogenous VWF colocalised in stimulated platelets. In non-stimulated platelets, addition of simoctocog alfa increased the levels of phospho-myosin expression.

Conclusion(s): Simoctocog alfa displays increased binding to procoagulant platelets compared with other rFVIII products in vitro.