Fig 1.
Platelets (Panel A) and pulmonary endothelial cells (Panel B) from wild-type and Wdr1-hypomorphic mice were isolated, solubilized in 1% SDS and subjected to PAGE followed by immunoblot with antibodies to Wdr1 or β actin.
Fig 2.
Platelet aggregation in Wdr1-hypomorphic mice.
Platelets were isolated from both Wdr1-hypomorphic mice and littermate wild-type controls and stimulated with various concentrations of collagen (Panels A-D) or thrombin (Panels E and F). Aggregations were measured using a turbidometric aggregometer. In the upper panel, the top tracings reflect platelet aggregation while the bottom tracings indicate ATP secretion. The traces shown are representative of at least 3 or more independent experiments.
Fig 3.
Collagen-induced calcium signaling and conformational changes in αIIbβ3.
Panel A and C. Intracellular Ca2+ levels in resting and activated platelets (n = 3/group). Platelets from Wdr1-hypomorphic and littermate wild-type mice were loaded with Fura-2, washed and suspended in Tyrode’s buffer for 5 minutes before the addition of collagen or thrombin. The maximal increase in calcium levels were quantified by Fura-2 fluorescence. Shown are the mean and standard deviations of three separate experiments are shown. Panel B and D. Isolated platelets were stimulated with various concentrations of collagen (0–10 μg/mL) and thrombin (0.1unit/ml). Conformational changes in αIIbβ3 were assessed by flow cytmetry using activation-specific antibody, JON/A-PE. The mean fluorescence index (MFI) and standard deviation of three separate experiments are shown. * denotes P <0.05
Table 1.
Expression of platelet glycoproteins in wild-type littermate control and Wdr1-hypomorphic mice.
Washed platelets were incubated with fluorescein-labeled antibodies to glycoproteins and the mean fluorescence (MFI) intensities are measured.
Fig 4.
Effect of Wdr1-deficiency on hemostasis in vivo.
Panel A. Tail bleeding time. The data represent 10 mice in each group. Panel B. Time for total occlusion. Flow cessation was determined by intravital microscopy following endothelial injury induced by ferric chloride in carotid arteries. The data represents Wdr1-hypomorphic (n = 12) and wild-type mice (n = 10). The p values were determined by nonparametric Mann-Whitney U test).
Fig 5.
Activation-induced redistribution of talin in platelets.
Left Panels. Washed platelets from wild-type (Panels A, C, E, G, I and K) or Wdr1-hypomorphic mice (Panels B, D, F, H, J and L) were immobilized on polylysine (resting) or collagen (activated) coated coverslips as described in the experimental section. The platelets were incubated with talin-1 antibody (Panels A-D) or phalloidin (Panels E-H), and β3 antibody (Panels I-L) examined under a fluorescence microscope with 1000x magnification. Panels M-P. β3 integrin and talin Interaction was assessed by proximity ligation assay in resting and collagen treated platelet from wild-type and Wdr1-hypomorphic mice. Right Panels. Washed platelets from wild-type or Wdr1-hypomorphic mice were treated with collagen (10 μg/ml) in suspension. The cytoskeletal fractions from resting (time 0 minute) and collagen-treated platelets (time 2 minutes) in suspension were isolated by centrifugation at 15600g, washed, solubilized, subjected to SDS-PAGE and immunoblotted with antibodies to talin, integrin β3, GAPDH, Wdr1 or actin. The purity of cytoskeleton fractions were assessed by the presence of GAPDH. actin, integrin β3 and talin of the total cell lysate are shown as loading control.
Fig 6.
Actin Reorganization in platelets.
Panels A and D. Washed platelets in suspension were stimulated with 10 μg/ml of collagen (Panel A) or 0.05 unit/ml of thrombin (Panel D), lysed and centrifuged. The supernatant containing the soluble G-actin and triton X-100-insoluble pellet containing F-actin were solubilized, and subjected to SDS-PAGE and immunoblotted using monoclonal anti-actin antibody. Panels B and E. The immunoblots of actin were quantified and a change in the ratio of insoluble F-actin to triton X-100-soluble G-actin is a considered an indicator of actin reorganization. The mean and standard deviations of three independent experiments are shown. Panels C and F. Washed platelets in suspension were stimulated with 10 μg/ml of collagen (Panel C) or 0.05 unit/ml of thrombin (Panel F), for 2 minutes, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with Alexa488-phalloidin. The fluorescence was measured by flow cytometry and expressed as fluorescence intensity in resting and collagen treated platelets. The mean and standard deviations of three independent experiments are shown.