Heparin-PEAR-1 interaction contributes to platelet activation by HIT antibodies.

Abstract

Background: Heparin is a negatively charged, polysaccharide used as an anticoagulant. In 0.5-1% of patients receiving unfractionated heparin, heparin-induced thrombocytopenia (HIT) occurs 5-10 days after exposure. In HIT, heparin binds to platelet factor 4 (PF4), exposing PF4 neoepitopes, allowing binding of heparin-dependent anti-PF4 antibodies, forming immune complexes that activate platelets through FcγRIIA. Recently, we showed that heparin mediates weak/slow aggregation of platelets via platelet endothelial aggregation receptor-1 (PEAR1).

Objectives: To investigate the role of the heparin-PEAR1 interaction in platelet activation in HIT.

Methods: Activation of washed platelets from multiple responsive donors to 5B9 (HIT-like mAb) and eight HIT sera in the presence of heparin, and in the presence or absence of the inhibitory PEAR1 nanobody, Nb138, was assessed. Light transmission aggregometry over 30 minutes was used to measure the kinetics of platelet activation for three sera, whilst flow cytometry for P-selectin expression gave an end read-out after 60 minutes for all eight sera.

Results: Aggregation and activation to heparin (0.5 IU/mL) was blocked by Nb138 (100 nM). 5B9 and HIT patient sera in the presence of heparin induced full platelet aggregation within 15 minutes, which was blocked or delayed by Nb138 in most donors. P-selectin expression was increased with 5B9 and HIT sera in the presence of heparin and was reduced by Nb138 in all donors.

Conclusions: Our results demonstrate that PEAR1 blockade reduces/blocks platelet activation to a HIT-like monoclonal antibody and HIT sera in the presence of heparin suggesting the heparin-PEAR1 axis may contribute to the pathophysiology in HIT.