Teins and receptors involved in other patho-physiological processes, such as inflammation. As Rab27 is known to be associated with myosin Va cargo vesicles and myosin Va is highly expressed in both human and mouse platelets [14], it is of great interest to determine the role of this motor protein in platelet granule secretion and formation. In the present study, we used a novel targeted Myo5a gene deletion mouse model, which is non-lethal and shows no overt neurological phenotype. As shown by immunoblotting, myosin Va was undetectable in platelets from Myo5a2/2 mice. Levels of surface proteins and granule counts were normal in Myo5a2/2 platelets. Contrary to our expectations, loss of myosin Va resulted in normal agonist-induced dense and a-granule secretion and unchanged numbers of dense and a-granules expressed. Furthermore, integrin aIIbb3 activation, Ca2+ signalling, and spreading on fibrinogen were not affected in Myo5a2/2 platelets. These resultsMyosin Va in Plateletsestablish that myosin Va 18325633 is not required for most platelet get PHCCC responses, including dense and a-granule secretion.Methods MaterialsThe myosin Va antibody (#3402) was from Cell Signaling Technology (Danvers, MA, USA). The myosin Vb antibody (18), the myosin Vc antibody (Y-19), and the GAPDH antibody (6C5) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The myosin VI antibody (ABT42) was from Millipore (Temecula, CA, USA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit, anti-mouse and anti-goat secondary antibodies were from Jackson ImmunoResearch Laboratories (Newmarket, UK). FITC-P-selectin and PE-JON/A antibodies were from Emfret Analytics (Eibelstadt, Germany). FITC-LAMP1 (1D4B) antibody was from Abcam (Cambridge, UK). NuPAGE LDS sample buffer was obtained from Invitrogen (Carlsbad, CA, USA). 106 blocking buffer and TRITC-phalloidin were from SigmaAldrich (Poole, UK). Fura-PE3 was from Teflabs (Austin, TX, USA). Luciferin-luciferase was from Chronolog (Labmedics, Stockport, UK). AYPGKF-NH2 (PAR4 activating peptide) was from Bachem (Weil-am-Rhein, Germany). CRP (cross-linked collagen-related peptide) was synthesized by Prof Richard Farndale (Department of Biochemistry, University of Cambridge, UK).Mouse platelet preparationA colony of Myo5a2/2 mice was obtained from the Wellcome Trust Sanger Institute (Cambridge, UK), which were mated with C57/Bl6j mice (Charles River, UK) to generate Myo5a+/2. These mice were crossed to generate Myo5a2/2 mice and littermate Myo5a+/+ mice, which were used as SC1 site control (wild-type, WT). Use of mouse platelets was approved by local research committee at the University of Bristol, UK, and mice were bred for this purpose under UK Home Office Licence PPL30/2908 held by Alastair W. Poole. Blood was drawn and washed platelets were prepared as described previously [15]. In brief, blood was drawn by cardiac puncture under terminal anesthesia into sodium citrate (4 ; 1:10 v/v). Blood was diluted with modified Tyrode’s-HEPES buffer (134 mM NaCl, 2.9 mM KCl, 20 mM HEPES, 5 mM glucose, and 1 mM MgCl2, pH 7.3) and centrifuged at 180 g for 6 minutes at room temperature. Platelet-rich plasma was removed, and platelets were isolated by centrifugation at 550 g for 6 minutes in the presence of PGE1 (140 nM) and apyrase (0.02 U/ml). Pelleted platelets were resuspended to the required density in modified Tyrode’s-HEPES buffer and rested for 30 minutes at 37uC in the presence of 0.02 U/ml apyrase prior to stimulation. No indomethaci.Teins and receptors involved in other patho-physiological processes, such as inflammation. As Rab27 is known to be associated with myosin Va cargo vesicles and myosin Va is highly expressed in both human and mouse platelets [14], it is of great interest to determine the role of this motor protein in platelet granule secretion and formation. In the present study, we used a novel targeted Myo5a gene deletion mouse model, which is non-lethal and shows no overt neurological phenotype. As shown by immunoblotting, myosin Va was undetectable in platelets from Myo5a2/2 mice. Levels of surface proteins and granule counts were normal in Myo5a2/2 platelets. Contrary to our expectations, loss of myosin Va resulted in normal agonist-induced dense and a-granule secretion and unchanged numbers of dense and a-granules expressed. Furthermore, integrin aIIbb3 activation, Ca2+ signalling, and spreading on fibrinogen were not affected in Myo5a2/2 platelets. These resultsMyosin Va in Plateletsestablish that myosin Va 18325633 is not required for most platelet responses, including dense and a-granule secretion.Methods MaterialsThe myosin Va antibody (#3402) was from Cell Signaling Technology (Danvers, MA, USA). The myosin Vb antibody (18), the myosin Vc antibody (Y-19), and the GAPDH antibody (6C5) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The myosin VI antibody (ABT42) was from Millipore (Temecula, CA, USA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit, anti-mouse and anti-goat secondary antibodies were from Jackson ImmunoResearch Laboratories (Newmarket, UK). FITC-P-selectin and PE-JON/A antibodies were from Emfret Analytics (Eibelstadt, Germany). FITC-LAMP1 (1D4B) antibody was from Abcam (Cambridge, UK). NuPAGE LDS sample buffer was obtained from Invitrogen (Carlsbad, CA, USA). 106 blocking buffer and TRITC-phalloidin were from SigmaAldrich (Poole, UK). Fura-PE3 was from Teflabs (Austin, TX, USA). Luciferin-luciferase was from Chronolog (Labmedics, Stockport, UK). AYPGKF-NH2 (PAR4 activating peptide) was from Bachem (Weil-am-Rhein, Germany). CRP (cross-linked collagen-related peptide) was synthesized by Prof Richard Farndale (Department of Biochemistry, University of Cambridge, UK).Mouse platelet preparationA colony of Myo5a2/2 mice was obtained from the Wellcome Trust Sanger Institute (Cambridge, UK), which were mated with C57/Bl6j mice (Charles River, UK) to generate Myo5a+/2. These mice were crossed to generate Myo5a2/2 mice and littermate Myo5a+/+ mice, which were used as control (wild-type, WT). Use of mouse platelets was approved by local research committee at the University of Bristol, UK, and mice were bred for this purpose under UK Home Office Licence PPL30/2908 held by Alastair W. Poole. Blood was drawn and washed platelets were prepared as described previously [15]. In brief, blood was drawn by cardiac puncture under terminal anesthesia into sodium citrate (4 ; 1:10 v/v). Blood was diluted with modified Tyrode’s-HEPES buffer (134 mM NaCl, 2.9 mM KCl, 20 mM HEPES, 5 mM glucose, and 1 mM MgCl2, pH 7.3) and centrifuged at 180 g for 6 minutes at room temperature. Platelet-rich plasma was removed, and platelets were isolated by centrifugation at 550 g for 6 minutes in the presence of PGE1 (140 nM) and apyrase (0.02 U/ml). Pelleted platelets were resuspended to the required density in modified Tyrode’s-HEPES buffer and rested for 30 minutes at 37uC in the presence of 0.02 U/ml apyrase prior to stimulation. No indomethaci.
