![Figure 2. The activation of integrin αIIbβ3, secretion of ADP, and early signaling downstream of GPVI are not regulated by PKCδ. (A) Platelets from either wild-type (WT) or PKCδ–/– mice were labeled with JON/A antibody, stimulated with CRP (5 μg/mL) for 15 minutes, and surface labeling measured by flow cytometry. The bar graph shows the geometric mean of the intensity of PE-antibody labeling for WT or PKCδ–/– platelets nonstimulated or stimulated with CRP. The data represent mean ± SEM from 3 independent experiments, and the statistical significance of differences between PKCδ–/– and wild-type was analyzed by 1-way ANOVA (nonsignificant). The top inset shows the forward-scattering (FSC-Height) versus side-scattering dot plot for a representative sample of mouse platelets. The bottom inset shows the frequency distribution of the labeling values for the platelet population in the different experimental conditions. (B) Platelets from wild-type (WT) or PKCδ–/– mice were preincubated for 15 minutes with 0.2 U/mL apyrase, 1 mM A3P5P, and 1 μM ARC and stimulated with collagen (30 μg/mL). Platelet aggregation responses were monitored by turbidimetric aggregometry, and data shown are representative of 3 independent experiments. (C) Wild-type (WT) or PKCδ–/– mouse platelets were loaded with [3H]5-HT. Subsequently, the percentage of total [3H]5-HT content released by platelets stimulated for 5 minutes with 30 μg/mL or 3 μg/mL collagen in the presence or absence of rottlerin (5 μM) was determined by liquid scintillation counting. Results shown are mean ± SEM from 3 independent experiments. The difference between WT and PKC–/– platelets was analyzed by 1-way ANOVA, and differences were statistically nonsignificant (P = .42). The differences between rottlerin-treated and nontreated controls in both wild-type and PKC–/– platelets stimulated with 3 μg/mL collagen were significant (P < .01). (D) PLCγ2 was immunoprecipitated from PKCδ–/– and wild-type (WT) mouse platelets after activation by collagen (30 μg/mL) for the time periods shown. Samples were analyzed by immunoblotting using antiphosphotyrosine antibody 4G10 (top panels). The efficiency of immunoprecipitation was analyzed by reblotting with anti-PLCγ2 antibody (bottom panels). The arrow indicates the molecular weight of PLCγ2. The immunoblots are representative of 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/108/13/10.1182_blood-2006-05-023739/8/zh80240605220002.jpeg?Expires=1730500832&Signature=MVEVV~Ubblmh~qUn7--eEtGhlvGV7qSJEYFaJ5zEc9Ksga3gU~yS1G~9t8I1DaiBq0mcBODKgKe6Lkz-9oIhP6M5Gt~Y0xNSkMLEJlBwFtmUPKJR7cQ0cm1XVdrS9eRJnX3sczreTFIGMEmbpTgfNsTNVYOM94G9A99wt4Z-fE7453PjMSIAxAnN1ibGLnFCjezeZ3ii0GGGm~I4dC5HBZswtMH9ClbhYktLHQvmG~Hxgum8LWxY8xEk8f0-AyjWXho6BvVJ~5FrSxMYWp9v4ZnYWSG22Sj-hoKsGIfY4X7wPcqq4EZSd1SC3W9OyHFeoSbggv~~B03gYbZcJhgacg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The activation of integrin αIIbβ3, secretion of ADP, and early signaling downstream of GPVI are not regulated by PKCδ. (A) Platelets from either wild-type (WT) or PKCδ–/– mice were labeled with JON/A antibody, stimulated with CRP (5 μg/mL) for 15 minutes, and surface labeling measured by flow cytometry. The bar graph shows the geometric mean of the intensity of PE-antibody labeling for WT or PKCδ–/– platelets nonstimulated or stimulated with CRP. The data represent mean ± SEM from 3 independent experiments, and the statistical significance of differences between PKCδ–/– and wild-type was analyzed by 1-way ANOVA (nonsignificant). The top inset shows the forward-scattering (FSC-Height) versus side-scattering dot plot for a representative sample of mouse platelets. The bottom inset shows the frequency distribution of the labeling values for the platelet population in the different experimental conditions. (B) Platelets from wild-type (WT) or PKCδ–/– mice were preincubated for 15 minutes with 0.2 U/mL apyrase, 1 mM A3P5P, and 1 μM ARC and stimulated with collagen (30 μg/mL). Platelet aggregation responses were monitored by turbidimetric aggregometry, and data shown are representative of 3 independent experiments. (C) Wild-type (WT) or PKCδ–/– mouse platelets were loaded with [3H]5-HT. Subsequently, the percentage of total [3H]5-HT content released by platelets stimulated for 5 minutes with 30 μg/mL or 3 μg/mL collagen in the presence or absence of rottlerin (5 μM) was determined by liquid scintillation counting. Results shown are mean ± SEM from 3 independent experiments. The difference between WT and PKC–/– platelets was analyzed by 1-way ANOVA, and differences were statistically nonsignificant (P = .42). The differences between rottlerin-treated and nontreated controls in both wild-type and PKC–/– platelets stimulated with 3 μg/mL collagen were significant (P < .01). (D) PLCγ2 was immunoprecipitated from PKCδ–/– and wild-type (WT) mouse platelets after activation by collagen (30 μg/mL) for the time periods shown. Samples were analyzed by immunoblotting using antiphosphotyrosine antibody 4G10 (top panels). The efficiency of immunoprecipitation was analyzed by reblotting with anti-PLCγ2 antibody (bottom panels). The arrow indicates the molecular weight of PLCγ2. The immunoblots are representative of 3 independent experiments.
