Characterisation of Platelet Sensitivity in Immune Thrombocytopenia Identifies Distinct Subgroups with Either Higher or Lower Platelet Sensitivity

Background: Immune Thrombocytopenia (ITP) is an autoimmune bleeding disorder characterised by a low platelet count (≤100 × 10⁹/L). Anti-platelet autoantibodies, present in approximately 60% of ITP patients, are known to target platelet glycoproteins (GP) Ib-IX and IIb/IIIa (integrin αIIbβ3) on the platelet surface, contributing to platelet clearance and impaired platelet production. Current therapeutic strategies, such as corticosteroids, intravenous immunoglobulins (IVIg), rituximab, and thrombopoietin receptor agonists (TPO-RAs) aim to increase platelet number. More recently, inhibitors of the Spleen Tyrosine Kinase (SYK) and Bruton's Tyrosine Kinase (BTK) are either recently licensed (Fostamatinib) or in clinical trial (Rilzabrutinib) for the treatment of ITP. However, diagnosis and treatment remain a challenge, as it can be difficult to predict treatment response and disease prognosis. One feature complicating the management of ITP is the wide heterogeneity of the disease. This extends also to haemostatic risks. While many patients have minimal symptoms, some patients have increased bleeding tendencies. Paradoxically, others suffer from thrombosis, even at low platelet counts. Hence, there is a need to further characterise patient variability within ITP and elucidate personalised therapeutic strategies for patients at varying stages of disease.

The Platelet Phenomic Analysis (PPAnalysis) identifies platelet sensitivity and functional response as independent measures of platelet reactivity by flow cytometry. The assay measures fibrinogen binding as a marker of αIIbβ3 activation and the exposure of P-selectin as a marker of α-granule secretion. We hypothesised that this assay could be used to categorise patients into different subgroups: those who are more at risk of bleeding or more at risk of thrombosis.

Methods: Patients and controls were recruited from the ITP Centre at Imperial College Healthcare NHS Trust and consented to the Multi Centre Research Ethics Committee in Wales MREC Wales reference 07/MRE09/54. Venous blood samples collected in sodium citrate vacutainers were processed immediately to obtain platelet-rich plasma (PRP). PRP was labelled with fluorescein isothiocyanate (FITC)-conjugated anti-fibrinogen and PE-Cy5-conjugated P-Selectin antibodies, and stimulated with increasing concentrations of the platelet agonists, adenosine diphosphate (ADP; 0.03-30 µM), collagen-related peptide (CRP; 0.003-3 µg/mL), and thrombin receptor activator peptide 6 (TRAP-6; 0.05-15 µM). Changes in platelet sensitivity, measured as LogEC50, were analysed using R.

Results: 126 patients (providing 262 samples at multiple time points) and 59 healthy control samples were included in the study: 5 patients had refractory disease, 7 were in remission, and 12 had vaccine-associated ITP. Patients with ITP had more variable platelet sensitivities than controls, with outliers both above and below the 95% centile of controls: 11-19% of ITP patients exhibited higher platelet sensitivity (below the 95% centile for controls) and 2-10% had lower sensitivity (above the 95% centile for controls), (Figure 1). The variability in platelet sensitivity did not correlate with platelet count (Figure 2). We also found no correlations between hypo- or hypersensitive platelets in ITP post COVID-19 vaccine. Patients with either higher or lower sensitivities were more likely to vary over time, suggesting a more volatile group of patients.

Conclusions: Here we show that cohorts of patients with ITP display either higher platelet sensitivity or lower platelet sensitivity compared to controls, irrespective of their platelet count. This signifies a role for aberrant platelet reactivity in the pathogenesis of ITP and may contribute to the heterogeneity of the disease. We also highlight the usefulness of PPAnalysis in distinguishing different subgroups of patients, demonstrating an assessment of platelet function, regardless of platelet count, that could ultimately allow for more personalised treatment for patients. Larger cohorts of patients and assessment of responses to treatment could help to further define this disease.

Cooper:Sanofi, Principia, Novartis, Griffols, Sobi, Argenyx, UCB, Rigel: Consultancy, Honoraria, Research Funding.