Figure 4
Figure 4. Apoptotic platelets demonstrate reduced integrin αIIbβ3 activation, aggregation, and recruitment to forming thrombi under flow conditions. (A) Human whole blood was “spiked” with fluorescently labeled washed platelets treated with either vehicle (DMSO) or ABT-737 (1μM). The recruitment of vehicle or ABT-737–incubated platelets to forming thrombi was quantified as described in supplemental Methods. (i) Images are taken from 1 experiment representative of 3 independent experiments, with the mean ± SEM (n = 3) depicted in subpanel ii. (B-C) Effect of ABT-737 on integrin αIIbβ3 activation and platelet aggregation. Washed platelets resuspended in Tyrode buffer in the presence of BSA (5 mg/mL) were pretreated with vehicle (DMSO) or ABT-737 (1μM) for the indicated time periods, or 180 minutes. In some experiments, platelets were pretreated with Q-VD-Oph (Q-VD: 50μM) or calpeptin (CP: 100 μg/mL). (B) Integrin αIIbβ3 activation was measured after stimulation with ADP (25μM, 30 seconds), thrombin (0.1 or 1.0 U/mL, 5 minutes), or CRP (10 μg/mL, 5 minutes) in the presence of Oregon green–labeled fibrinogen (OG-FGN; 20 μg/mL) or FITC-PAC1, and quantified by flow cytometry, as indicated in supplemental Methods. Line graphs represent the mean ± SEM (n = 3). (C) Aggregation in response to the indicated concentration of agonist was monitored as described in supplemental Methods. Histograms represent maximal aggregation (mean ± SEM, n = 4). ns indicates not significant. **P < .01. ***P < .001.

Apoptotic platelets demonstrate reduced integrin αIIbβ3 activation, aggregation, and recruitment to forming thrombi under flow conditions. (A) Human whole blood was “spiked” with fluorescently labeled washed platelets treated with either vehicle (DMSO) or ABT-737 (1μM). The recruitment of vehicle or ABT-737–incubated platelets to forming thrombi was quantified as described in supplemental Methods. (i) Images are taken from 1 experiment representative of 3 independent experiments, with the mean ± SEM (n = 3) depicted in subpanel ii. (B-C) Effect of ABT-737 on integrin αIIbβ3 activation and platelet aggregation. Washed platelets resuspended in Tyrode buffer in the presence of BSA (5 mg/mL) were pretreated with vehicle (DMSO) or ABT-737 (1μM) for the indicated time periods, or 180 minutes. In some experiments, platelets were pretreated with Q-VD-Oph (Q-VD: 50μM) or calpeptin (CP: 100 μg/mL). (B) Integrin αIIbβ3 activation was measured after stimulation with ADP (25μM, 30 seconds), thrombin (0.1 or 1.0 U/mL, 5 minutes), or CRP (10 μg/mL, 5 minutes) in the presence of Oregon green–labeled fibrinogen (OG-FGN; 20 μg/mL) or FITC-PAC1, and quantified by flow cytometry, as indicated in supplemental Methods. Line graphs represent the mean ± SEM (n = 3). (C) Aggregation in response to the indicated concentration of agonist was monitored as described in supplemental Methods. Histograms represent maximal aggregation (mean ± SEM, n = 4). ns indicates not significant. **P < .01. ***P < .001.

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