PB0772 - Secondary activation of platelets is responsible for increased desialylation of platelets of ITP patients

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Olga An, MD

PhD Candidate
University Medical Center Mainz, Center for Thrombosis and Hemostasis
Mainz, Rheinland-Pfalz, Germany

Co-Author(s)

AM

Alexey Martyanov

Researcher
Dmitry Rogachev National Medical Research Centеr of Pediatric Hematology, Oncology and Immunology
Chapel Hill, North Carolina, United States
VV

Varvara Vasilevskaya

Master's Student
M.V.Lomonosov Moscow State University, Faculty of Physics, Moscow, Russia
Moscow, Moskva, Russia
AT

Anastasia Tarakanova

PhD Candidate
Center for Theoretical Problems of Physico-Chemical Pharmacology RAS, Moscow, Russia
Moscow, Moskva, Russia
AI

Anastasia Ignatova

Research associate
Dmitry Rogachev National Medical Research Centеr of Pediatric Hematology, Oncology and Immunology
Moscow, Moskva, Russia
MP

Mikhail Panteleev

Director
Center for Theoretical Problems of Physico-Сhemical Pharmacology
Moscow, Moskva, Russia
Carsten Deppermann, PhD

Assistant Professor
Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Germany
AS

Anastasia N. Sveshnikova

Head of Laboratory
Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology, Moscow, Russia
Moscow, Moskva, Russia

Background: Platelet desialylation is crucial for the removal of aged platelets from the bloodstream. Increased desialylation is also observed in activated platelets and in platelets from patients with immune thrombocytopenia (ITP). While it is generally acknowledged that autoantibodies play a role, the exact mechanism leading to enhanced platelet desialylation in ITP is not fully understood.

Aims: To assess potential mechanisms of platelet desialylation in ITP patients.

Methods: Whole blood of healthy donors (HD) and ITP patients was collected into sodium citrate and platelets were isolated using sequential centrifugation. E.cristagalli lectin (ECL) and R.communis agglutinin (RCA) were used to evaluate N-acetylglucosamine and β-galactose exposure, respectively. As desialylation control, platelets were incubated with neuraminidase from C.perfringens. Alpha- and dense granule release was quantified using anti-CD62p and mepacrine staining, respectively. Aspirin and apyrase were used to inhibit secondary platelet stimulation. To model different platelet counts in healthy and ITP patients, platelet concentrations of 300,000/µl (high), and 10,000/µl (low) were used. Samples were analyzed using flow cytometry.

Results: At high platelet concentrations, maximal platelet desialylation was achieved upon strong stimulation (combination of PAR-1AP and PAR-4AP), whereas at low platelet concentrations, platelet desialylation was significantly lower. ECL and RCA binding correlated with both alpha- and dense granule secretion at both concentrations of platelets. Inhibiting mediators of platelet secondary activation did not have a significant impact on platelet desialylation at high concentrations, however, at low concentrations, platelet desialylation was reduced. Studies with platelets from ITP patients showed that platelet desialylation in ITP depends to a higher degree on alpha- and dense granule release compared to healthy donors.

Conclusion(s): Platelet desialylation is ‘self’-triggered and enhanced with secondary activation. We assume that inhibiting secondary activation of platelets in ITP patients might help to reduce desialylation and destruction of platelets.