Differential binding of recombinant factor VIII concentrates to platelets may impact platelet functionality

Introduction After vascular injury, platelets become activated to a pro-aggregatory state, with a subpopulation undergoing a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind and localize factor VIII (FVIII) at injury sites. It is unclear whether modifications in recombinant (r)FVIII products impact FVIII-platelet binding and platelet signalling. Here we examined the binding of different rFVIII products to platelets in vitro, and the impact of rFVIII binding on the platelet phenotype and intracellular signalling.

Method Platelets isolated from healthy donors and people with severe haemophilia A were activated with thrombin and cross-linked collagen-related peptide.Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding of rFVIII to platelets was quantified by flow cytometry using immunostaining or by direct measurement of labelled rFVIII concentrates. The extent of phosphatidylserine (PS) exposure on the platelet surface was used to measure the shift to a pro-coagulant state. Annexin V-BV421 was used as a molecular probe to assess PS exposure levels. To assess the role of integrin αIIbβ3 signalling in the response of platelets to FVIII binding, platelets were concurrently activated and incubated with the integrin αIIbβ3 inhibitory antibody 10E5.

Results Binding to platelets of healthy donors was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of the detection method ([Fig. 1]). The rFVIII-platelet interactions observed in the static assay were validated for simoctocog alfa and efmoroctocog alfa using a dynamic platelet-binding assay ([Fig. 1]). Incubation of activated platelets with rFVIII resulted in increased PS exposure, with a greater effect observed with simoctocog alfa than with efmoroctocog alfa. Immunostaining revealed co-localization of simoctocog alfa and integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 decreased the binding of simoctocog alfa to platelets in a dose-dependent manner ([Fig. 2]). Similar trends were observed for the analysis of both platelet binding and phenotype shift in platelets from people with severe haemophilia A.

Fig. 1  Simoctocog alfa shows increased binding to activated platelets vs other rFVIII products

Fig. 2  Simoctocog alfa increases PS exposure, which is reduced by integrin αIIbβ3 inhibition

Conclusion Simoctocog alfa demonstrated higher binding to activated platelets from healthy donors and people with haemophilia A in vitro compared with other rFVIII products. The increased binding was associated with a phenotypic shift in platelets and was disrupted with inhibition of integrin αIIbβ3, suggesting a role of integrin αIIbβ3 signalling following binding of FVIII to platelets. Variations in platelet binding and signalling between different rFVIIIs might impact their therapeutic efficacy for the prevention of bleeds in people with haemophilia A.

Publication History

Article published online:
26 February 2024