Fig. 5: Desialylation directly induces apoptosis in platelets ex vivo and can be inhibited by DANA treatment.

A Flow cytometry analysis of freshly isolated human platelets after ex vivo treatment with 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) or neuraminidase for 24 h. Mean ± SEM is shown for n = 3 biological replicates, as indicated by individual data points. B RCA-I staining to detect exposed galactose in freshly isolated human platelets after ex vivo treatment with DANA, neuraminidase, ABT-263 or a combination of agents for up to 120 min. Mean ± StDev is shown for n = 2 biological replicates. Annexin V staining to detect apoptosis induced by C single agent treatments or D, E combination treatments with indicated agents for 24 h. Mean ± SEM is shown for n = 3 biological replicates in (C), mean ± StDev is shown for n = 2–4 biological replicates in (D) and mean is shown in (E) for n = 1–2 biological replicates. Flow cytometry analysis of platelets collected from WT mice treated with DANA or saline intraperitoneally every 12 h for 48 h total using F platelet counts, G RCA-I to detect exposed galactose, H anti-Neu1 antibody to detect surface expression of Neu1, and I Annexin V to detect apoptosis. Mean ± SEM is shown for n = 2–5 biological replicates, as indicated by individual data points. Platelet count (J) or platelet Annexin V positivity (K) from WT mice 4 or 12 h after a single intraperitoneal injection of ABT-263 (100 mg/kg) with or without DANA pretreatment. Data collected from each mouse as well as mean is shown for n = 2 (ABT-263) or 5 (DANA + ABT-263) mice/group.