Molecular mechanisms that control platelet receptors in people with thrombocytopenia

Abstract

Platelet receptors are fundamental to the haemostatic properties of platelets. GPIb alpha binds VWF, and GPVI binds collagen, subsequently activating intracellular signalling cascades that culminate in platelet aggregation and clot formation. Pathological shedding of platelet receptors GPVI and GPIb alpha may contribute to platelet dysfunction and increased bleeding risk. Such changes are demonstrated in case reports of patients with acquired autoimmune diseases such as immune thrombocytopenia (ITP) where antibodies target platelets. A single-center, observational analysis was conducted among patients presenting with primary ITP as outpatients (platelet count < 50 x 109/L) or as hospital in-patients with platelet counts less than 120 x 109/L (TCP). ITP diagnosis was determined by clinical assessment. All cohorts were compared to contemporaneously collected healthy controls. Samples were collected in anticoagulants and processed and analysed within 3 h of collection. Parameters measured included platelet count, and platelet receptor quantitation by flow cytometry, in a whole blood assay designed to maximize platelet evaluation. Samples from 17 primary ITP patients, or 19 TCP patients with non-autoimmune thrombocytopenia, and 23 healthy donors were obtained. Surface GPIb alpha, alpha IIb integrin and GPVI levels were notably lower in patients with low platelet count compared to healthy controls analysed on the same day. Levels of tetraspanin CD9 and ADAM10 remained within normal ranges for both ITP and TCP patient samples. Regression analysis showed no relationship between platelet GPIb alpha or GPVI and platelet count in healthy donors but correlation between GPIb alpha or CD9 and platelet count in ITP samples, and between GPVI and platelet count in TCP samples. This difference may signal differences in the mechanism of thrombocytopenia between groups. Using our in-house ELISA, sGPVI levels in plasma from HD, ITP and TCP patients show no significant differences. GPVI and GPIb alpha were both significantly reduced (p<0.01 or smaller) in patients with TCP or with ITP, relative to healthy controls. There was some preliminary evidence of dysregulated glycosylation of the platelets in samples from people with ITP. These data imply that platelets generated by patients with ITP or TCP have reduced levels of the key adhesion-signalling receptors that initiate platelet activation and haemostatic responses. This molecular 7 deficiency may underpin bleeding that is otherwise not explained by the platelet count. These sorts of measurements may have future utility to examine mechanisms of cell receptor shedding, the nature of the anti-platelet autoantibody and may predict bleeding in ITP patients. Platelet protein surface levels may stratify ITP patients and may help in the evaluation of bleeding risk. Reduction in platelet surface receptors may indicate platelet activation and signal the presence of antiplatelet autoantibodies in ITP. The whole blood assay is rapid and easy to perform, with minimal handling of the samples and may be suitable for future standardisation in a clinical haematology facility. The relative contributions of platelet consumption and platelet production to the clinical observation of thrombocytopenia are an ongoing clinical problem. These studies have developed a new molecular analysis of the GPIb alpha receptor and established new clinical tools for monitoring platelet receptors relevant to platelet function. Show more