Figure 1.
Figure 1. ADAM10/17-mediated glycoprotein shedding upon platelet stimulation with strong agonists. Washed platelets, which were preincubated for 15 minutes with DMSO (vehicle) or ADAM17/10 inhibitor GW280264X (GW; 5 μM), were stimulated with 2MeS-ADP, CRP-XL, thrombin (Thr), or CRP-XL/Thr (GPIbα, n = 8) for 180 minutes or with ionomycin (GPIbα, n = 8) for 60 minutes (all at 37°C). Unstimulated platelets were used as control (Ctrl). Staining for GPIbα (A,D), GPVI (B,E), and GPIX (C,F) was measured with optimized concentrations of FITC anti-GPIbα mAb, PE anti-GPVI mAb, or FITC anti-GPIX mAb using flow cytometry (mean fluorescence intensities). (A-C) Effect of agonist stimulation on glycoprotein expression levels. Data are normalized to those of unstimulated platelets. (D-F) Effect of GW treatment on glycoprotein expression level. Data are mean ± SD, n = 3-5 (≥3 donors). *P < .05 vs unstimulated platelets, #P < .05 vs vehicle. n.s., not significant.

ADAM10/17-mediated glycoprotein shedding upon platelet stimulation with strong agonists. Washed platelets, which were preincubated for 15 minutes with DMSO (vehicle) or ADAM17/10 inhibitor GW280264X (GW; 5 μM), were stimulated with 2MeS-ADP, CRP-XL, thrombin (Thr), or CRP-XL/Thr (GPIbα, n = 8) for 180 minutes or with ionomycin (GPIbα, n = 8) for 60 minutes (all at 37°C). Unstimulated platelets were used as control (Ctrl). Staining for GPIbα (A,D), GPVI (B,E), and GPIX (C,F) was measured with optimized concentrations of FITC anti-GPIbα mAb, PE anti-GPVI mAb, or FITC anti-GPIX mAb using flow cytometry (mean fluorescence intensities). (A-C) Effect of agonist stimulation on glycoprotein expression levels. Data are normalized to those of unstimulated platelets. (D-F) Effect of GW treatment on glycoprotein expression level. Data are mean ± SD, n = 3-5 (≥3 donors). *P < .05 vs unstimulated platelets, #P < .05 vs vehicle. n.s., not significant.

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