File S1 - Role of Siglec-7 in Apoptosis in Human Platelets

Supporting information. Table S1, Monoclonal antibodies used for flow cytometry to analyze the membrane expression of platelets. Figure S1, Representative maximum projection confocal microscopy z series images of Siglec-7 (red) and CD62P (green), demonstrating colocalization (z tack  = 0.6 µm) (five series for each platelet sample: n = 3). Figure S2, Expression of CD33r Siglecs on the membrane surface of unstimulated and TRAP-stimulated platelets analyzed by flow cytometry. A. Representative scattergram from platelet samples of 10 healthy donors. Data expressed as percentage of CD41+Siglec+ cells. B. Percentage of CD41+Siglec+ cells. Data expressed as mean ± SEM, (n = 10). *: Significant difference (t-test, p<0.05) between TRAP-stimulated platelets vs unstimulated platelets. Figure S3, Concentration of soluble CD62P (A) and soluble Siglec-7 (B) in supernatants (n = 10) of resting and TRAP-induced platelets activation over time. Data are shown as pg/2×108 platelets/ml and expressed as mean ± SEM. * Significant differences in sCD62P levels in supernatants of TRAP-stimulated platelets vs unstimulated platelets (t-test, p<0.05), ¥ and #: significant difference of sCD62P concentration in supernatants over time vs 0 min (ANOVA, p<0.05). Figure S4, Concentration of soluble MMP-1 (A), MMP-2 (B), and MMP-9 (C) in supernatants (n = 5) from resting and TRAP-induced platelet activation over time. Data were adjusted to pg/200,000 plot and expressed as mean ± SEM. *significant difference (ANOVA, p<0.05) between MMP-1 concentration over time vs 0 min. Figure S5, Flow cytometry analysis of CD3, CD14, CD15, CD19, and CD41 expression in platelet preparations. Peripheral blood was collected from healthy donors in endotoxin-free tubes with 3.2% sodium citrate. Platelet-rich plasma (PRP) was prepared by centrifuging the blood at 150 ×g for 12 min at 22°C. PRP residual mononuclear cells (A,B) were counted by flow cytometry and compared with peripheral blood (C,D). There was a marked reduction in contaminating cells (CD3-T cells, CD19-B cells, CD15-neutrophils or CD14-monocytes) in platelet preparations in PRP conditions compared with peripheral blood (data are expressed percentage expression (± SD; n = 10 experiments). One representative experiment is shown (A, B).
(DOCX)