Patients with immune thrombocytopenia and antiplatelet antibodies show increased platelet activation and desialylation

Abstract

Abstract Background Antiplatelet autoantibodies contribute to ITP pathogenesis by enhancing platelet clearance through multiple mechanisms. However, not all patients with ITP have detectable autoantibodies, and the haemostatic differences between patients with and without antibodies remain unclear. Objective We aimed to compare the haemostatic profiles of patients with ITP, stratified by the presence or absence of detectable antiplatelet antibodies, to determine if this distinction had potential clinical relevance. Methods This prospective study was approved by the Ethics Committee of Hospital Universitario La Paz. We included 41 adult ITP patients with detectable antiplatelet antibodies (ITP_Ab+, platelet count: 91 (31-214)x109/L, 34% female) and 68 without (ITP_Ab-, platelet count: 122 (64-208)x109/L, 33% female). Patients with thrombotic history, bleeding disorder other than ITP, or on treatment with antiplatelet drugs were excluded. Antiplatelet antibodies (anti-GPIIb/IIIa, anti-GPV, and anti-GPIb/IX) were detected in serum using MAIPA assay. We evaluated platelet activation markers (FITC-PAC1 binding and FITC-P-selectin exposure at baseline and after stimulation with 100 μM TRAP) and caspase-3,-7, -8 and -9 activities in platelet rich plasma (PRP). FITC-Ricinus Communis Agglutinin (RCA) binding studies to indirectly assess sialic acid content by targeting exposed galactose residues were done on washed platelets. Neuraminidase 1 (NEU1) exposure on quiescent platelet was detected using an anti-NEU1-Alexa Fluor546 antibody. All samples were analysed by flow cytometry. Rotational thromboelastometry (ROTEM) was performed on fresh PRP adjusted to 25×109 platelets/L with the patient's own platelet-free plasma. Parameters recorded included clotting time (CT), alpha angle, maximum clot firmness (MCF), and lysis at 60 minutes. Plasma levels of B-cell activating factor (BAFF) and plasminogen activator inhibitor-1 (PAI-1) were quantified using ELISAs kits and cell-free DNA (cfDNA) by the Quant-iT PicoGreen dsDNA assay. Statistical analysis was performed with GraphPad Prism software. Results are expressed as median (25%-75% percentile) and a p-value<0.05 was considered statistically significant. Results ITP_Ab+ showed antibodies targeted to the following platelet glycoproteins: 7.3% anti-GPIIb/IIIa; 9.7% anti-GPV; 14.6% anti-GPIb/IX; 2.4% anti-GPIIb/IIIa+anti-GPV; 14.6% anti-GPIIb/IIIa+anti-GPIb/IX; 22.0% anti-GPV+anti-GPIb/IX; and 29.3% anti-GPIIb/IIIa+anti-GPV+ anti-GPIb/IX. ITP_Ab+ exhibited increased basal platelets activation, with significantly higher PAC1 binding [0.58 (0.245–1.445) vs. 0.35 (0.13–0.76), p=0.019] and P-selectin expression [6.60 (0.91–10.26) vs. 3.01 (0.89–5.40), p=0.012] compared to ITP_Ab-. However, no significant differences were observed between groups in response to agonist stimulation. Platelets from ITP_Ab+ exhibited increased RCA binding [504.8 (253.3–809.1) vs. 301.3 (122.2–629.1), p=0.0123] compared to platelets from ITP_Ab-, indicating decreased surface sialic acid content. This finding is consistent with the elevated levels of membrane-associated NEU1 detected in platelets from this cohort (614.0 (47.72–2497) vs. 51.77 (7.107–282.1), p=0.0079). Caspase activities were similar in both groups. Our findings showed that there were no significant differences in cfDNA and PAI-1 plasma levels, nor in MCF and Ly60 parameters of ROTEM. This suggests that there is no major systemic prothrombotic or fibrinolytic imbalance. It is noteworthy that the CT parameter of ROTEM was shorter in ITP_Ab+ [871.5 (685.3–962.0) vs. 916.5 (843.0–1155), p=0.0328], which may be attributable to the observed increased basal platelet reactivity. Elevated plasma levels of BAFF in ITP_Ab+ [778.6 (598.2–1414) vs. 550.8 (426.4–677.1), p=0.0193] support immune activation of antibody-producing B cells. Conclusions ITP patients with detectable antiplatelet antibodies exhibit increased baseline platelet activation and desialylation, related to their elevated NEU1 levels. These findings are consistent with previous studies (Marini et al., PMID: 31857361; Zheng et al., PMID: 35199503; Amini et al., PMID: 35901281). The results underscore the importance of conducting mechanistic studies to further explore how antiplatelet antibodies influence haemostasis in ITP patients. Project “PI22/01489”, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union.