Nules were quantified per field of view (10 order RE-640 fields of view per preparation) and data (mean +/2 SEM, n = 4) are shown as the number of granules per platelet visible in the section. This number is for comparison between genotypes only, as granules above or below the thin section plane will not be visible, and so the number will be an underestimate of the total granule count per platelet. doi:10.1371/journal.pone.0053239.gMyosin Va in PlateletsMyosin Va in PlateletsFigure 3. Loss of myosin Va does not affect platelet dense, a-granule or lysosome secretion. Wild-type and Myo5a2/2 platelets were stimulated with the indicated concentrations of collagen-related peptide (CRP) and the PAR4 agonist AYPGKF. (A) ATP release from dense granules was assessed luminometrically. Data (mean +/2 SEM, n = 4?) are levels of released ATP. (B) P-selectin expression as a result of a-granule secretion was measured by flow cytometry, after agonist stimulation for 10 min. Data (mean +/2 SEM, n = 5?) are shown as fold increase over basal. (C) Time course of P-selectin expression induced by AYPGKF (300 mM). Data (mean +/2 SEM, n = 4) are levels of FITC fluorescence intensity. (D) Lysosome secretion, as assessed by LAMP1 flow cytometry, was determined after agonist stimulation for 10 min. Data (mean +/2 SEM, n = 4) are shown as fold increase over basal. doi:10.1371/journal.pone.0053239.gimage. The proportions of each morphology in each of the ten images were then averaged. This analysis was performed separately on platelets from 3 WT mice and 3 Myo5a2/2 mice.Platelet granule biogenesis and secretion are unaffected by loss of myosin VaSince Rab27 regulates dense granule formation and secretion in platelets [10] and has been shown to interact with myosin Va in melanocytes [8], we investigated whether myosin Va is also involved in platelet dense granule formation and secretion. Subcellular morphology of platelets from WT and Myo5a2/2 mice was examined by TEM (Fig. 2A), and visible granules counted (and normalized to the number of platelets in the field of view; Fig. 2B). Both dense and a-granule counts in each thin section were similar to wild-type, suggesting that myosin Va is not involved in platelet granule biogenesis. Dense granule secretion of ATP, monitored by luminometry, was stimulated by a range of concentrations of the GPVI agonist, collagen-related peptide (CRP) or the thrombin receptor PAR4 agonist, AYPGKF. However, no difference in ATP secretion was observed between wild-type and Myo5a2/2 platelets at any concentration tested (Fig. 3A). This indicates that myosin Va is not required for the secretion of dense granule content. Next, we addressed whether myosin Va has a role in a-granule secretion. By flow cytometric analysis, P-selectin expression on the platelet surface was assessed. P-selectin surface expression induced by various concentrations of CRP and AYPGKF was not significantly affected in Myo5a2/2 platelets compared to wild-type (Fig. 3B), suggesting that myosin Va is not required also for agranule secretion in platelets. In addition, analysis of the time course of a-granule secretion indicated that the rate of P-selectin expression was not different between wild-type and Myo5a2/2 platelets (Fig. 3C). Finally, we investigated whether myosin Va regulates lysosome secretion by assessing agonist-induced surface expression of LAMP1, which correlates with 11089-65-9 lysosomal enzyme release. Platelet activation induced an increase in surface LAMP1. This was not di.Nules were quantified per field of view (10 fields of view per preparation) and data (mean +/2 SEM, n = 4) are shown as the number of granules per platelet visible in the section. This number is for comparison between genotypes only, as granules above or below the thin section plane will not be visible, and so the number will be an underestimate of the total granule count per platelet. doi:10.1371/journal.pone.0053239.gMyosin Va in PlateletsMyosin Va in PlateletsFigure 3. Loss of myosin Va does not affect platelet dense, a-granule or lysosome secretion. Wild-type and Myo5a2/2 platelets were stimulated with the indicated concentrations of collagen-related peptide (CRP) and the PAR4 agonist AYPGKF. (A) ATP release from dense granules was assessed luminometrically. Data (mean +/2 SEM, n = 4?) are levels of released ATP. (B) P-selectin expression as a result of a-granule secretion was measured by flow cytometry, after agonist stimulation for 10 min. Data (mean +/2 SEM, n = 5?) are shown as fold increase over basal. (C) Time course of P-selectin expression induced by AYPGKF (300 mM). Data (mean +/2 SEM, n = 4) are levels of FITC fluorescence intensity. (D) Lysosome secretion, as assessed by LAMP1 flow cytometry, was determined after agonist stimulation for 10 min. Data (mean +/2 SEM, n = 4) are shown as fold increase over basal. doi:10.1371/journal.pone.0053239.gimage. The proportions of each morphology in each of the ten images were then averaged. This analysis was performed separately on platelets from 3 WT mice and 3 Myo5a2/2 mice.Platelet granule biogenesis and secretion are unaffected by loss of myosin VaSince Rab27 regulates dense granule formation and secretion in platelets [10] and has been shown to interact with myosin Va in melanocytes [8], we investigated whether myosin Va is also involved in platelet dense granule formation and secretion. Subcellular morphology of platelets from WT and Myo5a2/2 mice was examined by TEM (Fig. 2A), and visible granules counted (and normalized to the number of platelets in the field of view; Fig. 2B). Both dense and a-granule counts in each thin section were similar to wild-type, suggesting that myosin Va is not involved in platelet granule biogenesis. Dense granule secretion of ATP, monitored by luminometry, was stimulated by a range of concentrations of the GPVI agonist, collagen-related peptide (CRP) or the thrombin receptor PAR4 agonist, AYPGKF. However, no difference in ATP secretion was observed between wild-type and Myo5a2/2 platelets at any concentration tested (Fig. 3A). This indicates that myosin Va is not required for the secretion of dense granule content. Next, we addressed whether myosin Va has a role in a-granule secretion. By flow cytometric analysis, P-selectin expression on the platelet surface was assessed. P-selectin surface expression induced by various concentrations of CRP and AYPGKF was not significantly affected in Myo5a2/2 platelets compared to wild-type (Fig. 3B), suggesting that myosin Va is not required also for agranule secretion in platelets. In addition, analysis of the time course of a-granule secretion indicated that the rate of P-selectin expression was not different between wild-type and Myo5a2/2 platelets (Fig. 3C). Finally, we investigated whether myosin Va regulates lysosome secretion by assessing agonist-induced surface expression of LAMP1, which correlates with lysosomal enzyme release. Platelet activation induced an increase in surface LAMP1. This was not di.
