Trout thrombocytes adhere to and spread on collagen in a Src dependent manner.

<p>A) Trout blood was diluted by a factor of 100 to reduce the number of cells, and then allowed to adhere to either collagen or fibrinogen coated coverslips for 90 min at room temperature, either in the presence of 20 µM dasatinib or DMSO vehicle control. B) Anticoagulated whole blood was flowed through collagen coated glass capillary tubes at a range of shear rates (i) or at 100 s⁻¹ for inhibitor studies (ii). Blood was pre-treated with either 20 µM dasatinib or DMSO vehicle control.</p

Trout thrombocytes aggregate to collagen in a Src dependent manner.

<p>Anticoagulated whole blood from rainbow trout was diluted 1∶1 with normal saline (0.9%) and stirred for 3 min at room temperature in a Multiplate mini-cuvette. Dasatinib (5 µM) or DMSO vehicle control was pre-incubated at room temperature for 1 min followed by addition of collagen, 30 or 10 µg/ml, and aggregation was monitored for 12 min at room temperature. Aggregation was measured in arbitrary impedance units (AU).</p

G6fL responds to collagen in a cell line assay through its ITAM.

<p>DT40 cells were transfected with wild type zebrafish G6fL (A) or with point mutants of G6fL (Y499F, Y515F and Y527F) (B) and stimulated with either collagen (10 µg/ml) or CRP (3 µg/ml). Where stated, cells were pre-incubated with 20 µM PP2. 6 hrs following stimulation, cells were lysed and luciferase activity was measured as a readout of signalling. Data is expressed as fold over basal (dotted line). Statistical significance was calculated with a Student’s t-test (*P>0.05, ***P>0.005). Similar levels of expression of all G6fL constructs was confirmed by western blotting (not shown).</p

Presence of novel PKC isoforms in human and mouse platelets.

<p>Washed human (Hu) or mouse (Mo) platelets were lysed and samples separated by SDS-PAGE. The resulting membranes were blotted with antibodies raised against each isoform. Alongside each sample, the positive control (+) was run, as recommended by the antibody manufacturer. The results are representative of three experiments.</p

(A) Effect of PP2 on PKCδ tyrosine phosphorylation.

<p>Human washed platelets were treated with 0.1 or 1 U/ml thrombin for 1 min in the presence or absence of PP2 (10 µM, 5 min). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (B) Effect of PP2 on pleckstrin phosphorylation in thrombin-stimulated platelets. Washed human platelets were labelled with radioactive ³²P-orthophosphate and stimulated with 1 U/ml thrombin for 1 min. PP2 (10 µM, 5 min) or Ro31-8220 (10 µM, 1 min) were added where indicated. After separation by SDS-PAGE, the resulting gel was analysed using a phosphoimager to obtain radioactivity levels. Histogram shows the level of pleckstrin phosphorylation compared to basal levels. Data from one experiment, representative of two. (C) Tyrosine phosphorylation of PKCδ in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin in the absence or presence of Ro31-8220 (10 µM, 1 min) (left), or with 1 U/ml thrombin for the times shown (right) in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (D) Tyrosine phosphorylation of PKCε in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin for 1 min. Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. All studies described in this figure were performed in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Histograms show mean density of bands, normalised according to the basal value: *signifies values significant from basal, ^#signifies significant from equivalent time point without inhibitor. Data is from 3 separate experiments.</p

Platelet adhesion to collagen under flow in PKCε ^−/− mice.

<p>Blood from PKCε^−/− mice or wild-type (WT) littermate controls was flowed over collagen for 4 min at a shear rate of 1000 s⁻¹, then rinsed with Tyrode's buffer for 5 min. A. Adherent platelets and aggregates were imaged by DIC microscope and representative images are shown. B. The surface coverage was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a> and is shown as the mean±s.e.m. from four experiments.</p

Expression of PKC isoforms in PKCε-null platelets.

<p>Platelets were prepared from PKCε-deficient mice (PKCε−/−) and wild-type littermate controls (WT) and equal protein amounts of whole cell lysates analysed by western blot, using antisera specific for individual PKC isoforms, PKC ε, α, δ and θ, as indicated.</p

Effect of Ro 31-8220 (Ro) on PKCδ tyrosine phosphorylation.

<p>Human washed platelets in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM) were treated with (A) 1 U/ml thrombin or (B) 3 µg/ml convulxin in the presence or absence of Ro 31-8220 (10 µM, 1 min) for the times shown. Immunoprecipitations and reprobes were carried out as in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histograms show mean density of bands, normalised according to the basal value. *signifies statistically significant from basal, ^#signifies statistically significant from equivalent time point without inhibitor. Data is from 3 separate experiments.</p

Platelet function in PKCε-null mice.

<p>(A) Aggregation and ATP secretion. Washed platelets from PKCε-deficient (PKCε^−/−) mice or wild-type (WT) littermate controls were stimulated with the indicated concentrations of CRP, collagen or PAR4 peptide. Aggregation and dense granule secretion were investigated using light-scattering aggregometry and luciferin-luciferase luminescence, respectively. Traces are representative of between 3–7 mice. (B) Spreading. PKCε^−/−and wild-type (WT) washed platelets in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM) were exposed to surfaces of 100 µg/ml fibrinogen (FG), 1 U/ml thrombin (Thr), or 100 µg/ml collagen (COLL) for 45 min before fixing and mounting. Representative images from each condition are shown. Histograms depict levels of platelet adhesion to each surface and platelet mean surface area (spreading). The results are from one experiment that is representative of three. (C) Expression levels of surface glycoproteins. PKCε^−/− and WT washed platelets were incubated with FITC-labelled antisera against (upper panels) GPVI and integrin α_IIbβ₃ and (lower panels) integrin α₂β₁ and expression levels analysed by flow cytometry. The shaded area represents the signal from non-specific IgG control.</p