Abstract Number: PB1195
Meeting: ISTH 2022 Congress
Theme: Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies » Acquired Thrombocytopenias
Background: The removal of sialic acid residues (SA) from platelet glycoproteins is among the most important markers for platelet clearance. Decreased amount of SA was observed in patients with immune thrombocytopenia (ITP), an acquired autoimmune bleeding disorder. However, the existing approaches to platelet desialylation assessment provide controversial results, those limit their applicability for routine ITP diagnostics.
Aims: Evaluation of different protocols for the characterization of platelet desialylation.
Methods: Whole blood of 10 healthy donors and 13 ITP patients was collected into sodium citrate-containing tubes in accordance with the declaration of Helsinki. Platelets were washed using three sequential centrifugations and resuspended in Tyrode’s buffer in the presence/absence of BSA and calcium. Desialylation was measured using lectins from Erythrina cristagalli and Ricinus communis in normal or in 2% PFA fixed samples. The desialylation level was scaled to the desialylation maximum, obtained upon platelet incubation with neuraminidase from Cl.perfringens. Alpha- and dense granule release was monitored by anti-CD62p and mepacrine, correspondingly. Samples were analyzed by flow cytometry. Mann-Whitney test was used for statistical analysis.
Results: Lectin binding to platelets achieved saturation after 20 minutes of incubation at 37oC. Platelet stimulation with ADP (10 µM), SFLLRN (4.5 µM) or AYGPKF (4.5 µM) resulted in a detectable increase in the desialylation level only in presence of calcium. Furthermore, platelet desialylation correlated with P-selectin exposure and dense granule release (r = 0.935 and r = 0.503). Finally, fixation with PFA caused platelet desialylation even without platelet activation. The developed protocol was applied to blood samples from ITP patients. Refractory to first-line therapy patients had significantly increased platelet desialylation (p = 0.0061).
Conclusion(s): We conclude that the measurement of platelet desialylation should be performed in the presence of calcium, while fixation should be avoided. The protocol allowed to distinguish ITP patients, refractory to first-line therapy.
Study was supported with RSF grant 21-45-00012.
To cite this abstract in AMA style:
An O, Martyanov A, Ignatova A, Pankrashkina M, Ptushkin V, Ataullakhanov F, Panteleev M, Sveshnikova A. Optimized Protocol for Measurement of Platelet Desialylation Level Allows to Determine Immune Thrombocytopenia Patients’ Responsiveness to First-line Therapy [abstract]. https://abstracts.isth.org/abstract/optimized-protocol-for-measurement-of-platelet-desialylation-level-allows-to-determine-immune-thrombocytopenia-patients-responsiveness-to-first-line-therapy/. Accessed September 24, 2023.« Back to ISTH 2022 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/optimized-protocol-for-measurement-of-platelet-desialylation-level-allows-to-determine-immune-thrombocytopenia-patients-responsiveness-to-first-line-therapy/