Measurement of procoagulant platelets provides mechanistic insight and diagnostic potential in heparin-induced thrombocytopenia

Abstract

Background: Heparin-induced thrombocytopenia (HIT) is a prothrombotic, immune-mediated adverse drug reaction associated with high rates of thrombosis-related morbidity and mortality caused by FcγRIIa-activating pathogenic antibodies to PF4-heparin. Procoagulant platelets are a platelet subset that promote thrombin generation, are clinically relevant in prothrombotic diseases, and are formed when platelet G-protein-coupled receptor (GPCR) and ITAM-linked receptors are co-stimulated.

Objectives: We examined the procoagulant platelet response of healthy donors to platelet agonists in the presence of HIT plasma and determined the contribution of FcγRIIa.

Patients/methods: Our previously established flow cytometry-based procoagulant platelet assay was modified to incorporate plasma samples, performed using FcγRIIa-responsive donor platelets. Plasma samples were serotonin-release assay-confirmed HIT (HIT+), or negative on HIT screening.

Results: In response to GPCR stimulation, only HIT+ plasma produced a heparin-dependent sensitization that required active FcγRIIa. As a potential diagnostic tool, the procoagulant platelet assay achieved 98% accuracy in identifying clinically verified HIT when performed blinded to the diagnoses of a validation cohort. Samples inducing a higher procoagulant platelet response were more likely from patients with thrombotic complications. Thrombin stimulation markedly increased the procoagulant platelet response with HIT+ plasma that was heparin independent and only partially reversed by FcγRIIa blockade, possibly reflecting ongoing thrombotic risk after heparin cessation.

Conclusions: We demonstrate that HIT plasma together with platelet agonists increased the procoagulant platelet proportions, which may contribute to thrombotic risk in HIT. Targeting procoagulant platelet activation may represent a novel treatment strategy. This assay may be a rapid, clinically relevant functional assay for accurately detecting pathological HIT antibodies.

Keywords: FcγRIIa; flow cytometry; heparin-induced thrombocytopenia; procoagulant platelets; thrombosis.