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

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.