Impact of Variable Recombinant Factor VIII Binding on Platelet Functions

Introduction: Hemophilia A (HA) is a rare genetic bleeding disorder caused by a deficiency of functional coagulation factor VIII (FVIII). Platelets have important functions throughout the various stages of hemostasis. Upon injury, platelets become activated in a pro-aggregatory state, and a subpopulation subsequently undergoes a phenotype shift to a pro-coagulant state which ensures efficient FVIII localization and bleeding protection through fibrin clot formation at the injury site. One treatment option to compensate for the lack of functional FVIII in patients with HA is the regular use of recombinant FVIII (rFVIII). However, the impact of structural modifications (chemical modification or protein fusion) to rFVIII products on FVIII-platelet interactions and subsequent platelet function remains unclear.

Aim: To examine the binding of different rFVIII products to platelets in vitro and their impact on platelet phenotype shift, including the intracellular signaling pathways involved.

Methods:Platelet activation: Platelets isolated from healthy donors and patients with HA were activated with thrombin and cross-linked collagen-related peptides. rFVIII-platelet binding: Activated platelets were incubated with simoctocog alfa (Nuwiq®), efmoroctocog alfa (Elocta®), rurioctocog alfa pegol (Adynovate®) or damoctocog alfa pegol (Jivi®). rFVIII-platelet binding was quantified by flow cytometry either by indirect immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or by direct measurement of rFVIII labeled with AF647. Phenotype shift: Phosphatidylserine (PS) becomes exposed on the membranes of platelets during the shift to a pro-coagulant state. To assess PS exposure, activated platelets were incubated with Annexin V-BV421. Intracellular signaling: Activated pro-aggregatory platelets exhibit “supramaximal” intracellular Ca²⁺ signaling committing them to shift to pro-coagulant. Ca²⁺ levels in platelets were quantified by flow cytometry with Fluo-5N AF488. Thrombin inhibition: PS exposure on platelets was investigated after thrombin inhibition with hirudin. Platelets were activated with TRAP-6 instead of thrombin. Inhibition of integrin αIIbβ3 signaling: To assess the role of integrin αIIbβ3 in rFVIII-platelet binding, activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5.

Results: Binding of rFVIII to platelets was associated with increased PS exposure over time in the platelets of healthy donors and patients with HA. Time-course analysis indicated an early interaction between rFVIII and pro-aggregatory platelets which upregulated intracellular Ca²⁺ signaling, leading to an increased shift to the pro-coagulant phenotype. Early interactions of rFVIII with pro-aggregatory platelets correlated positively with PS exposure, with simoctocog alfa demonstrating the highest binding compared to the other rFVIII concentrates (p < 0.05). Following thrombin inhibition, increased PS exposure was still present on the platelets of healthy donors and patients with HA following rFVIII addition. Inhibition of integrin decreased binding of rFVIII to platelets and the associated phenotype-shift.Simoctocog alfa exhibited significantly higher binding to platelets when compared to the other rFVIII concentrates in the platelets of healthy donors and patients with HA (p < 0.05).

Conclusion: Simoctocog alfa demonstrated early interactions with platelets, which resulted in an increased shift from the pro-aggregatory to the pro-coagulant phenotype in both healthy and HA-derived isolated platelets, indicating upregulated hemostasis in the presence of rFVIII. Increased PS exposure on platelets occurs in the absence of thrombin generation, indicating that rFVIII-platelet interactions may trigger outside-in signaling and downstream effects for the phenotype shift independent from the tenase complex formation. The associated signaling was blocked by inhibition of integrin αIIbβ3, suggesting a crucial role of αIIbβ3 signaling in FVIII-platelet binding. The rFVIII products explored in this study bound to platelets with varying strength, with simoctocog alfa demonstrating the highest amount of platelet binding, as well as the highest interaction with pro-aggregatory platelets. These findings indicate that variations in platelet binding may influence the efficacy of rFVIII products in the treatment of HA.

Strebel:Octapharma: Other: Travel grant. Pennacchio:Octapharma: Other: Travel grant. Lickert:Octapharma: Other: Travel grant. Klamroth:Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; BioMarin: Consultancy, Honoraria, Speakers Bureau; Biotest: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Consultancy, Honoraria, Speakers Bureau; Grifols: Consultancy, Honoraria, Speakers Bureau; Octapharma: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Roche / Chugai: Consultancy, Honoraria, Speakers Bureau; Sobi / Sanofi: Consultancy, Honoraria, Speakers Bureau; LFB: Consultancy, Honoraria, Speakers Bureau. Vogel:Octapharma: Other: Travel grant, Research Funding.