Figure 1.
Phenotyping of Myo5a−/− mice and platelets.
(A) Schematic of the targeting vector used to disrupt the Myo5a genomic locus between exons 2 and 3. The cassette contains the lacZ reporter gene, the neomycin (neo) resistance gene, and a polyadenylate (pA) sequence which causes transcription termination. Flp recombination target (FRT) sites are included on either side of the vector along with a loxP site at the 3′ end. In (B), the difference in coat pigmentation of Myo5a−/− mice compared to wild-type (WT) is shown. (C) Offspring ratios of knockouts (−/−), heterozygotes (+/−) and wild-types (+/+) from heterozygote breeding pairs were compared from 162 mice. Blood platelet count (D) and mean platelet volume (E) were measured in Myo5a−/− and wild-type (WT) mice (n = 18 and 25, respectively). (F) Immunoblots showing the expression of myosin Va protein in lysates from human, wild-type (WT) mouse and Myo5a−/− mouse platelets. Note the absence of a detectable myosin Va band in the knockout platelets. (G) Platelet surface levels of the indicated proteins were analysed by flow cytometry. Data (mean +/− SEM, n = 4–7) are mean fluorescence intensity levels.
Figure 2.
Myo5a−/− platelets have normal subcellular morphology.
(A) TEM images (4800×, scale bar: 1 µM) show representative images of WT and Myo5a−/− mouse platelets (n = 4). Black arrows: dense granules. White arrows: α-granules. (B) Platelet dense and α-granules were quantified per field of view (10 fields of view per preparation) and data (mean +/− 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.
Figure 3.
Loss of myosin Va does not affect platelet dense, α-granule or lysosome secretion.
Wild-type and Myo5a−/− 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 +/− SEM, n = 4–7) are levels of released ATP. (B) P-selectin expression as a result of α-granule secretion was measured by flow cytometry, after agonist stimulation for 10 min. Data (mean +/− SEM, n = 5–7) are shown as fold increase over basal. (C) Time course of P-selectin expression induced by AYPGKF (300 µM). Data (mean +/− 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 +/− SEM, n = 4) are shown as fold increase over basal.
Figure 4.
No enhanced expression of myosin Vb, Vc and VI in Myo5a−/− platelets.
Immunoblots showing the expression of myosin Vb, Vc, and VI in lysates from human, wild-type mouse (WT) and Myo5a−/− 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.
Figure 5.
No difference in integrin αIIbβ3 activation in myosin Va-deficient platelets.
Wild-type and Myo5a−/− 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 αIIbβ3 was measured by flow cytometry. Data (mean +/− SEM, n = 5) are shown as fold increase over basal.
Figure 6.
Normal Ca2+ signalling in Myo5a−/− platelets.
Wild-type and Myo5a−/− platelets were loaded with the Ca2+-sensitive dye Fura-PE3. (A) Ionomycin (1 µM) was added in presence of the calcium chelator EGTA (200 µM). (B) Platelets were stimulated with the indicated concentrations of CRP in the presence of 1 mM CaCl2. The left hand panels show representative traces (n = 3). Data in the right hand panels (mean +/− SEM, n = 3) are expressed as area under the curve (AUC).
Figure 7.
Loss of myosin Va does not affect platelet spreading on fibrinogen.
Wild-type and Myo5a−/− mouse platelets were dispensed onto fibrinogen-coated coverslips for 1 h. Where indicated, platelets were stimulated with the PAR4 agonist AYPGKF (300 µM) for 1 min prior to adhesion. Cells were stained with TRITC-phalloidin. In (A), representative images are shown (n = 3). (B) Platelet morphological subtypes were counted and fractions (mean +/− SEM, n = 3) were calculated.