Heparin-PEAR1 Interaction Contributes to Platelet Activation By HIT Antibodies

Introduction

Heparin is a negatively charged, heavily sulfated polysaccharide that is used in medicine as a highly effective anticoagulant. In about 1% of patients who receive unfractionated heparin, heparin-induced thrombocytopenia (HIT) can occur. This is a disorder characterized by an intensely prothrombotic phenotype and thrombocytopenia that typically occurs 5-10 days after heparin exposure. In HIT, heparin binds to positively charged platelet factor 4 (PF4), exposing neoepitopes on PF4 to which heparin-dependent anti-PF4 antibodies can bind. This results in immune complexes which activate platelets through the low affinity Fc receptor, FcγRIIA. Recently, heparin was shown to activate platelets by binding to platelet endothelial aggregation receptor 1 (PEAR1). We therefore hypothesized that heparin itself may have a role in platelet activation in HIT in cooperation with activation through FcγRIIA.

Methods

We assessed activation of healthy donor washed platelets (2 x 10⁸/mL) to the HIT-like monoclonal antibody (mAb) 5B9 and HIT sera in the presence of heparin by light transmission aggregometry. Experiments were performed in the presence or absence of the PEAR1 nanobody, Nb138, which was raised against the heparin-binding domain of PEAR1.

Results

Heparin (0.5 IU/mL) but not the HIT-like mAb 5B9 (20 µg/mL) stimulated slow and sustained aggregation of washed platelets with a second, more rapid phase at ~20 min. The response to heparin was blocked by the PEAR1 nanobody, Nb138 (100 nM). In contrast, the combination of heparin and mAb 5B9 stimulated robust aggregation of washed platelets within 15 min (n=7). In 3/7 donors, the response was blocked by Nb138 and was delayed by over 30 seconds in two others. We then tested the effect of Nb138 on two HIT sera that also induce strong activation of platelets in the presence of heparin. Nb138 delayed aggregation to the first HIT serum in all 7 donors. Nb138 blocked aggregation to the second HIT serum in 4 donors and significantly delayed aggregation in 2 others.

Conclusions

The present results demonstrate that the platelet heparin receptor, PEAR1, potentiates the response to HIT-like mAb and HIT sera and that this is an important variable in diagnostic assays using HIT sera. Further studies are required to establish whether activation of PEAR1 contributes to the pathogenesis of HIT and whether this is due solely to binding of heparin to PEAR1 or to binding of heparin to PEAR1 when present in an immune complex.

Acknowledgements

RJB is supported by a British Heart Foundation Accelerator Award (AA/18/2/34218) and SJM by a British Heart Foundation Project grant (PG/23/11230). SPW holds a BHF Chair (CH03/003).

Buka:AstraZeneca: Research Funding; Viatris: Honoraria; Bayer: Honoraria; Sobi: Honoraria; Sanofi: Consultancy. Rollin:Stago: Honoraria, Research Funding. Gruel:Stago: Honoraria, Research Funding. Nicolson:Sobi: Honoraria; AstraZeneca: Honoraria, Research Funding.