Figure 6.
Figure 6. Vps34 kinase activity regulates platelet secretion. (A) Kinetics of ATP secretion of washed platelets under resting or stimulated conditions (CRP, 1 µg/mL; thrombin, 0.1 IU/mL) were recorded by measuring the luminescence from the firefly luciferin-luciferase reaction by lumi-aggregometry using the Chrono-log aggregometer. Graphs represent the percentage of WT maximal secretion at 80 seconds (mean ± SEM; n = 6-15 mice of each genotype depending on the agonist; *P < .05; **P < .01; ***P < .001 vs WT according to 2-way ANOVA). (B) Kinetics of vWF secretion of washed platelets under resting or CRP (1 µg/mL) or thrombin (0.1 IU/mL) stimulated conditions was analyzed by enzyme-linked immunosorbent assay . The results are expressed as the fold increase compared with resting WT platelets (mean ± SEM; n = 6 mice of each genotype; *P < .05 vs resting WT according to 2-way ANOVA). The kinetics of ATP secretion (C) and vWF secretion (D) of washed platelets treated 1 hour with vehicle (dimethyl sulfoxide) or 1 μM VPS34-IN1 and stimulated with CRP (1 µg/mL) or thrombin (0.1 IU/mL) were analyzed as described above (mean ± SEM; n = 6; *P < .05 vs resting WT according to 2-way ANOVA). (E) Washed platelets were spread on a fibrinogen-coated surface for 20 minutes in the presence or absence of apyrase (2 IU/mL), and the platelet surface was measured using ImageJ software (mean ± SEM; n = 3 mice per genotype; **P < .01 vs WT according to 2-way ANOVA). (F) Whole blood from WT mice treated for 1 hour with dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 or from Pf4-Cre-Pik3c3lox/lox mice was perfused through a collagen-coated microcapillary in the presence of fluoxetine (25 µM) at a physiological shear rate of 1500 s−1. The serotonin content was quantified in plasma by immunoassay (mean ± SEM normalized by thrombus volume; n = 3-5 per condition; ***P < .001 vs WT according to 1-way ANOVA). (G) Unlabeled whole blood from WT (WT > WT) or Pf4-Cre-Pik3c3lox/lox (WT > Vps34) mice were perfused through collagen-coated microcapillaries at 1500 s−1 for 1 minute, and were then replaced by DiOC6-labeled WT whole blood perfused at the same shear rate. The surface covered by fluorescent platelets was analyzed by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; ***P < .001 vs WT > WT according to 2-way ANOVA test).

Vps34 kinase activity regulates platelet secretion. (A) Kinetics of ATP secretion of washed platelets under resting or stimulated conditions (CRP, 1 µg/mL; thrombin, 0.1 IU/mL) were recorded by measuring the luminescence from the firefly luciferin-luciferase reaction by lumi-aggregometry using the Chrono-log aggregometer. Graphs represent the percentage of WT maximal secretion at 80 seconds (mean ± SEM; n = 6-15 mice of each genotype depending on the agonist; *P < .05; **P < .01; ***P < .001 vs WT according to 2-way ANOVA). (B) Kinetics of vWF secretion of washed platelets under resting or CRP (1 µg/mL) or thrombin (0.1 IU/mL) stimulated conditions was analyzed by enzyme-linked immunosorbent assay . The results are expressed as the fold increase compared with resting WT platelets (mean ± SEM; n = 6 mice of each genotype; *P < .05 vs resting WT according to 2-way ANOVA). The kinetics of ATP secretion (C) and vWF secretion (D) of washed platelets treated 1 hour with vehicle (dimethyl sulfoxide) or 1 μM VPS34-IN1 and stimulated with CRP (1 µg/mL) or thrombin (0.1 IU/mL) were analyzed as described above (mean ± SEM; n = 6; *P < .05 vs resting WT according to 2-way ANOVA). (E) Washed platelets were spread on a fibrinogen-coated surface for 20 minutes in the presence or absence of apyrase (2 IU/mL), and the platelet surface was measured using ImageJ software (mean ± SEM; n = 3 mice per genotype; **P < .01 vs WT according to 2-way ANOVA). (F) Whole blood from WT mice treated for 1 hour with dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 or from Pf4-Cre-Pik3c3lox/lox mice was perfused through a collagen-coated microcapillary in the presence of fluoxetine (25 µM) at a physiological shear rate of 1500 s−1. The serotonin content was quantified in plasma by immunoassay (mean ± SEM normalized by thrombus volume; n = 3-5 per condition; ***P < .001 vs WT according to 1-way ANOVA). (G) Unlabeled whole blood from WT (WT > WT) or Pf4-Cre-Pik3c3lox/lox (WT > Vps34) mice were perfused through collagen-coated microcapillaries at 1500 s−1 for 1 minute, and were then replaced by DiOC6-labeled WT whole blood perfused at the same shear rate. The surface covered by fluorescent platelets was analyzed by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; ***P < .001 vs WT > WT according to 2-way ANOVA test).

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