PB0384 - GPVI is a binding site for factor VIII on platelets

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

RS

Rohini Sekar, PhD student

INSERM UMRS 1138, Sorbonne University
Paris, Ile-de-France, France

Co-Author(s)

AM

Angelina Mimoun

PhD student
INSERM UMRS 1138
Paris, Ile-de-France, France
Melissa Bou Jaoudeh, PhD

Ph.D.
INSERM UMRS1138 - Sorbonne University
Paris, Ile-de-France, France
SL

Stéphane Loyau

IE
INSERM UMRS-1148
PARIS, Ile-de-France, France
SD

Sandrine Delignat

ITA/ITRF/BIATSS
Inserm
Paris, Ile-de-France, France
VD

Victoria Daventure

ITA/ITRF/BIATSS
Inserm
Paris, Ile-de-France, France
JR

Julie Rayes

Researcher
University of Birmingham
Birmingham, England, United Kingdom
YB

Yacine Boulaftali, PhD

Dr
Unité INSERM U1148, Université Paris Cité
Paris, Ile-de-France, France
MJ

Martine JANDROT-PERRUS, PhD

Emeritus Professor
INSERM
Paris, Ile-de-France, France
VP

Valérie Proulle

Doctor
Service Hematologie Biologique et UF Hemostase Clinique, Hopital Cochin AP-HP.Centre - Universite Paris Cite
Paris, Ile-de-France, France
Sebastien Lacroix-Desmazes, PhD

PI
Centre De Recherche Des Cordeliers, Paris, France
Paris, Ile-de-France, France

Background: Factor VIII (FVIII) is an essential actor to form the tenase complex at the surface of activated platelets. FVIII binding partners on platelets include phosphatidylserine (PS) and fibrin-bound-αIIbβ3. Screening for FVIII interactions with platelet-expressed glycoproteins (GP) indicated FVIII binding to GPVI, the cross-linking of which by collagen, activates platelets.

Aims: To characterize the FVIII/GPVI interaction and decipher its influence on platelet functions.

Methods: Binding of full-length FVIII (FL-FVIII), B domain-deleted FVIII (BDD-FVIII) and BDD-FVIII mutated in the C1 and C2 domains (C1R2090A/K2092A/F2093A, C2R2215A/R2220A) was investigated using ELISA on immobilized human monomeric recombinant GPVI (rGPVI) or Fc-fused dimeric GPVI (GPVI-Fc), and by flow cytometry. FVIII was pre-incubated alone or with varying amounts of von Willebrand factor (VWF), or human monovalent monoclonal IgG anti-A2, anti-C1, anti-C2 FVIII domains. The effect of FVIII on collagen-induced platelet aggregation and GPVI-dependent downstream phosphorylation was further studied on washed platelets.

Results: FL-FVIII and BDD-FVIII, but not plasma-derived FVIII, bound in a dose-dependent manner to GPVI-Fc and rGPVI. VWF inhibited FVIII binding to GPVI. Interestingly, C1- and C2-specific, but not A2-specific, monovalent IgG also inhibited the interaction. Accordingly, FVIII variants mutated in the C1 and C2 target epitopes lost binding capacity to GPVI. VWF-free FVIII binding to resting and thrombin-activated platelets was confirmed using flow cytometry, albeit at high concentration. In these conditions, FVIII inhibited by more than 50% collagen-induced aggregation, while FIX had no effect; and by 52±19% global GPVI-dependent downstream phosphorylation, as compared to a 72±7% inhibition induced by the anti-GPVI Fab 9O12.

Conclusion(s): Our results identify GPVI as a novel non-PS binding partner for FVIII on platelets, in adjunct to fibrin-bound- αIIbβ3. We also observed that elevated FVIII concentrations inhibit collagen-mediated platelet activation. Whether the FVIII-GPVI interaction plays a modulatory role at the surface of activated platelets during thrombus formation, remains to be determined.