rFVIIIa-platelet binding enhances platelet procoagulant activity independently of thrombin generation

TL;DR

This study shows that FVIIIa enhances platelet activity through a process not involving thrombin, and that modified FVIII products bind differently to platelets.

Contribution

The study reveals a thrombin-independent mechanism of FVIIIa-platelet interaction and how rFVIII modifications affect platelet binding.

Findings

FVIIIa enhances procoagulant platelet activity via integrin αIIbβ3 and glycoprotein VI without thrombin involvement.

Modified rFVIII products show reduced binding to proaggregatory platelets compared to nonmodified versions.

Site-specific PEGylation of rFVIII reduces binding to procoagulant platelets.

Abstract

•FVIII potentiates procoagulant platelet activity by thrombin-independent signaling involving integrin αIIbβ3 and glycoprotein VI.•rFVIII products modified to extend the half-life show reduced binding to proaggregatory platelets.

FVIII potentiates procoagulant platelet activity by thrombin-independent signaling involving integrin αIIbβ3 and glycoprotein VI.

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 FVIII (rFVIII) products might tune this phenotype shift. Platelets were collected from healthy donors and people with severe hemophilia A (HA) and were 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 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 involved integrin αIIbβ3. Similar trends were observed in platelets from healthy donors and patients with HA. A potential role of glycoprotein VI in rFVIIIa-platelet interactions was identified. rFVIIIa products with certain modifications to extend the circulation half-life of FVIII showed reduced binding to proaggregatory platelets compared to nonmodified 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.

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