rFVIIIa–platelet Binding Enhances Platelet Procoagulant Activity Independent of Thrombin Generation Open Access

• FVIII potentiates procoagulant platelet activity by thrombin independent signaling involving integrin αIIbβ3 and GPVI

• rFVIII products modified to extend the half-life show reduced binding to proaggregatory platelets

Platelets play a critical role in hemostasis. In addition to adhesion at the site of injury, phosphatidylserine (PS) exposing platelets (procoagulant) bind activated factor VIII (FVIIIa), facilitating coagulation. Effects of FVIIIa-platelet interactions on platelet activity are unclear. We explored how FVIIIa-platelet interactions affect their transition from a proaggregatory to procoagulant phenotype and how molecular modifications of recombinant (rFVIII) products might tune this phenotype shift. Platelets were collected from healthy donors and people with severe hemophilia A (HA) and activated with thrombin and collagen-related peptide. Integrin αIIbβ3 activity and PS exposure were measured as markers of proaggregatory and procoagulant activities, respectively. Platelet phenotypes, rFVIIIa binding and calcium influx were assessed by flow cytometry and confocal microscopy. rFVIIIa binding potentiated the procoagulant activity, while having no appreciable effect on percentage of platelets with activated integrin αIIbβ3. Potentiation of procoagulant activity was mediated by a thrombin-independent outside-in signaling cascade but involving integrin αIIbβ3. Similar trends were observed in platelets from healthy donors and HA patients. A potential role for glycoprotein VI in rFVIIIa-platelet interactions was identified. rFVIIIa products with different modifications to extend the circulation half-life of FVIII showed reduced binding to proaggregatory platelets compared to non-modified rFVIIIa. Binding to procoagulant platelets was comparable across rFVIII products, except for rFVIII with site-specific PEGylation, which showed reduced binding. In conclusion, these results offer insights into FVIIIa-platelet interactions and the potential impact of rFVIII modifications on platelet binding may inform clinical decision making in HA.