Fferent between WT and Myo5a2/2 platelets, indicating that lysosome secretion is not affected by loss of myosin Va. Taken together, these data show that myosin Va has no non-redundant role in granule secretion in mouse platelets. There are several possible explanations for these data. It is possible that myosin Va genuinely plays no role in granuleStatisticsWhere presented, mean data are given 6 SEM. Statistical significance was determined by 2-way ANOVA with Bonferroni post-test, performing using Prism 4.0 (GraphPad Software). P,0.05 was considered significant.Results and Discussion Selective depletion of myosin Va from mouse plateletsTo evaluate the function of myosin Va in platelets, Myo5a2/2 mice were obtained from Wellcome Trust Sangar Institute (Cambridge, UK), in which the gene encoding this protein (Myo5a) had been disrupted by homologous recombination in embryonic stem cells, and the cells were used to generate gene-targeted mice by standard techniques (Fig. 1A). Myo5a2/2 mice were viable 18325633 and healthy, but showed a lighter grey coat JI 101 cost colour compared to wildtype controls (Fig. 1B), similar to the hypopigmentation which is seen in the dilute mice. This suggests that our mice are likely to have a similar defect in Myo5a-dependent melanosome trafficking to dilute mice [5]. Mice were not obtained in their expected Mendelian ratios (Fig. 1C). Of 130 offspring genotyped, 48 were wild-type (37.5 ), 63 were heterozygous (49.2 ) and only 17 were Myo5a2/2 (13.3 ). Low generation of Myo5a2/2 mice was seen in both male and female offspring. Myo5a2/2 mice exhibited normal platelet count and mean platelet volume (Fig. 1D and E). Other haematological parameters, specifically the red blood cell count, haematocrit, mean corpuscular volume and white blood cell count, were also within the normal range (data not shown). As demonstrated by immunoblotting, myosin Va (207 kDa) was not detected in Myo5a2/2 platelets, whereas it was expressed in wild-type mouse platelets as well as in human platelets (Fig. 1F). Myo5a2/2 platelets had normal levels of surface-expressed CD41, glycoprotein (GP) VI and GPIb (Fig. 1G). Thus, Myo5a2/2 platelets provided us an ideal opportunity to study platelet function in the selective absence of myosin Va.Figure 4. No enhanced expression of myosin Vb, Vc and VI in Myo5a2/2 platelets. Immunoblots showing the expression of myosin Vb, Vc, and VI in lysates from human, wild-type mouse (WT) and Myo5a2/2 mouse platelets. Lysates from mouse liver, pancreas or kidney were used as positive controls for myosin Vb, Vc, and VI, respectively. GAPDH served as loading control. Images shown are representative of three independent experiments. doi:10.1371/journal.pone.0053239.gMyosin Va in PlateletsFigure 5. No difference in integrin aIIbb3 activation in myosin Va-deficient platelets. Wild-type and Myo5a2/2 mouse platelets were stimulated for 10 min with the indicated concentrations of collagen-related peptide (CRP) and the PAR4 agonist AYPGKF. JON/A binding to the activated form of integrin aIIbb3 was GNF-7 measured by flow cytometry. Data (mean +/2 SEM, n = 5) are shown as fold increase over basal. doi:10.1371/journal.pone.0053239.gFigure 6. Normal Ca2+ signalling in Myo5a2/2 platelets. Wild-type and Myo5a2/2 platelets were loaded with the Ca2+-sensitive dye Fura-PE3. (A) Ionomycin (1 mM) was added in presence of the calcium chelator EGTA (200 mM). (B) Platelets were stimulated with the indicated concentrations of CRP in the presence of 1 mM CaCl.Fferent between WT and Myo5a2/2 platelets, indicating that lysosome secretion is not affected by loss of myosin Va. Taken together, these data show that myosin Va has no non-redundant role in granule secretion in mouse platelets. There are several possible explanations for these data. It is possible that myosin Va genuinely plays no role in granuleStatisticsWhere presented, mean data are given 6 SEM. Statistical significance was determined by 2-way ANOVA with Bonferroni post-test, performing using Prism 4.0 (GraphPad Software). P,0.05 was considered significant.Results and Discussion Selective depletion of myosin Va from mouse plateletsTo evaluate the function of myosin Va in platelets, Myo5a2/2 mice were obtained from Wellcome Trust Sangar Institute (Cambridge, UK), in which the gene encoding this protein (Myo5a) had been disrupted by homologous recombination in embryonic stem cells, and the cells were used to generate gene-targeted mice by standard techniques (Fig. 1A). Myo5a2/2 mice were viable 18325633 and healthy, but showed a lighter grey coat colour compared to wildtype controls (Fig. 1B), similar to the hypopigmentation which is seen in the dilute mice. This suggests that our mice are likely to have a similar defect in Myo5a-dependent melanosome trafficking to dilute mice [5]. Mice were not obtained in their expected Mendelian ratios (Fig. 1C). Of 130 offspring genotyped, 48 were wild-type (37.5 ), 63 were heterozygous (49.2 ) and only 17 were Myo5a2/2 (13.3 ). Low generation of Myo5a2/2 mice was seen in both male and female offspring. Myo5a2/2 mice exhibited normal platelet count and mean platelet volume (Fig. 1D and E). Other haematological parameters, specifically the red blood cell count, haematocrit, mean corpuscular volume and white blood cell count, were also within the normal range (data not shown). As demonstrated by immunoblotting, myosin Va (207 kDa) was not detected in Myo5a2/2 platelets, whereas it was expressed in wild-type mouse platelets as well as in human platelets (Fig. 1F). Myo5a2/2 platelets had normal levels of surface-expressed CD41, glycoprotein (GP) VI and GPIb (Fig. 1G). Thus, Myo5a2/2 platelets provided us an ideal opportunity to study platelet function in the selective absence of myosin Va.Figure 4. No enhanced expression of myosin Vb, Vc and VI in Myo5a2/2 platelets. Immunoblots showing the expression of myosin Vb, Vc, and VI in lysates from human, wild-type mouse (WT) and Myo5a2/2 mouse platelets. Lysates from mouse liver, pancreas or kidney were used as positive controls for myosin Vb, Vc, and VI, respectively. GAPDH served as loading control. Images shown are representative of three independent experiments. doi:10.1371/journal.pone.0053239.gMyosin Va in PlateletsFigure 5. No difference in integrin aIIbb3 activation in myosin Va-deficient platelets. Wild-type and Myo5a2/2 mouse platelets were stimulated for 10 min with the indicated concentrations of collagen-related peptide (CRP) and the PAR4 agonist AYPGKF. JON/A binding to the activated form of integrin aIIbb3 was measured by flow cytometry. Data (mean +/2 SEM, n = 5) are shown as fold increase over basal. doi:10.1371/journal.pone.0053239.gFigure 6. Normal Ca2+ signalling in Myo5a2/2 platelets. Wild-type and Myo5a2/2 platelets were loaded with the Ca2+-sensitive dye Fura-PE3. (A) Ionomycin (1 mM) was added in presence of the calcium chelator EGTA (200 mM). (B) Platelets were stimulated with the indicated concentrations of CRP in the presence of 1 mM CaCl.
