Glycoprotein VI is a receptor for the PF4/anti-PF4 IgG complex and contributes to platelet activation in HIT.

Abstract

Background: Platelet factor 4 (PF4) plays a central role in heparin-induced thrombocytopenia (HIT). PF4 is a positively charged tetramer that forms immune complexes with anti-PF4 IgG, activating platelets via FcγRIIA. Despite extensive study, a platelet receptor for PF4 within these complexes has not been clearly defined.

Objective: We hypothesized that glycoprotein VI (GPVI), a collagen receptor known to interact with multiple ligands, binds PF4 and contributes to platelet activation in HIT.

Methods: We used platelet aggregation assays, surface plasmon resonance (SPR), and AlphaFold 3 modeling to investigate interactions between PF4, the HIT-mimicking monoclonal antibody KKO, and GPVI.

Results: Platelet aggregation triggered by PF4/KKO or HIT patient sera was reduced by anti-GPVI antibodies or the anti-GPVI Fab fragment ACT017. SPR showed that PF4 binds GPVI with a dissociation constant of Kd ≈ 1 μM, but PF4/KKO complexes exhibited enhanced binding, with a 5-17-fold reduction in dissociation rate and Kd ≈ 30-100 nM. This interaction was blocked by heparin. In contrast, the non-pathogenic anti-PF4 antibody RTO had no effect. AlphaFold modeling suggested electrostatic interactions between anionic GPVI surfaces and the PF4/KKO complex, with PF4 tetramers contacting both the GPVI D2 domain and KKO Fab.

Conclusion: GPVI functions as a receptor for PF4/anti-PF4 IgG immune complexes, engaging FcγRIIA and contributing to platelet activation in HIT.