Page 243 - Haematologica April 2020

The role of neuraminidases in platelet function
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Figure 2. NEU1 and NEU2 membrane expression is mediated by von Willebrand factor-binding to glycoprotein Ibα. Platelets in platelet rich plasma (PRP) (200 x 106/mL) were pre-incubated with RGDS peptide (Arg-Gly-Asp-Ser, 200 μM), N-acetyl-D-glucosamine (GlcNAc, 100 mM) and O-sialo-glycoprotein endopeptidase (OSGE, 80 μg/mL) prior to ristocetin stimulation (3 mg/mL) and membrane association of (A) NEU1 and (B) NEU2 were measured by flow cytometry using NEU1 or NEU2 antibodies followed by fluorescently conjugated secondary antibodies. *P<0.05 was considered significant when compared to unstimimulated (unstim)*, †: significant against risto (one-way ANOVA, n=3). (C) Secondary antibody-only controls (unstim platelets) were used to set a gate (~1%) and everything above this was considered positive (% posve). Washed platelets were stimulated with VWF/risto (10 μg/mL VWF+ 1.2 mg/mL risto), collagen (10 μg/mL), thrombin (0.1 U/mL), AA (50 μM), ADP (20 μM), and U46619 (1 μM) prior to measurement of (D) NEU1 (n=4) or (E) NEU2 (n=4) membrane association by flow cytometry. Prior to VWF/risto stimulation, platelets were treated with the indicated inhibitors (n=4) for calcium (BAPTA-AM, 10 μM), TXA2 (indomethacin, indo, 30 μM) and ADP (apyrase, 0.1 U/mL). As indicated calcium (1 mM), fibrinogen (500 μg/mL), GM3 (10 μM) were also used. (F) NEU1 or (G) NEU2 membrane association was measured. Results are shown as mean fluorescent intensities (MFI) ± standard error of mean (SEM). *P<0.05 significant against unstimulated (unstim); †: significant against VWF/risto, §: signif- icant against vehicle (dmso, one-way ANOVA); VWF: von Willebrand factor; GPIbα: glycoprotein Ibα.
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