OC 27.5 - Impact of differential binding of recombinant factor VIII concentrates to platelets on platelet clotting functionality

Background: Haemophilia A (HA) is rare genetic bleeding disorder caused by a deficiency of functional coagulation factor VIII (FVIII). Upon injury, FVIII physically interacts with platelets in order to ensure efficient blood clot formation. One possibility to compensate for the lack of functional FVIII in HA patients relies on the regular infusion of recombinant FVIII (rFVIII). However, the impact of various modifications to extend the half-life of rFVIII products on FVIII-platelet interaction, platelet phenotype and intracellular signalling remains unclear, thus limiting the treatment efficacy.

Aims: To evaluate the binding of different rFVIII products to platelets in vitro and their impact on platelet phenotype and the intracellular signalling involved in blood clot formation.

Methods: Activated platelets from healthy donors and HA patients were incubated with rFVIII molecules presenting different modifications. rFVIII-platelet binding, platelet phenotype (proaggregatory vs procoagulant) and platelet signalling were evaluated with flow cytometry through immunostaining, while rFVIII-platelet physical interaction was characterised by fluorescence microscopy. The role of integrin αIIbβ3 in rFVIII-platelet interaction was assessed with an inhibitory approach.

Results: rFVIII (simoctocog alfa) exhibited significantly higher binding to platelets than other rFVIII molecules. Through a time-course analysis, we found that rFVIII interaction occurs already in proaggregatory platelets. This early interaction upregulates the intracellular signalling cascade leading to an increased shift from proaggregatory to procoagulant phenotype. Fluorescent microscopy revealed co-localisation of rFVIII with integrin αIIbβ3 on platelets. Inhibition of integrin αIIbβ3 decreased binding of rFVIII to platelets and the associated phenotype-shift.

Conclusion(s): rFVIII-platelet binding triggers a signalling cascade leading to an increased phenotype-shift towards procoagulant platelets, essential for blood clotting. The associated signalling was blocked by inhibition of integrin αIIbβ3, suggesting a crucial role for integrin αIIbβ3 signalling upon FVIII-platelet binding. Our results indicate that differences in platelet binding may affect the efficacy of rFVIII products in the treatment of HA.