Fig. 4

Detection of signaling phosphoproteins by Immunoblot analysis and Ca2+ release into human platelets. (a) p-Src family, (b) p-FAK, and (c) p-p38 washed platelets were treated with cat K (20 nM), papain (1.6 µM), and α-thrombin (0.001 UNIH/500µL) for 10 min at 37 °C; lysate proteins were separated by 10 % SDS-PAGE and electrotransferred to nitrocellulose membranes. Membranes were blocked and incubated with rabbit primary antibodies, anti-phospho-Src (Tyr-416), anti-Src, anti-phospho-FAK (Tyr-397), anti-FAK, anti- phospho-p38 MAPK, anti-p38 MAPK, and anti-ß-actin. Antibody binding was visualized by chemiluminescence and the relative levels of these proteins were determined by densitometric analysis. Papain (1.6 µM) slightly induced Src- and FAK-phosphorylation (2.1 ± 0.2 and 2.5 ± 0.4, respectively). Treatment with α-thrombin resulted in increased Src phosphorylation with the pSrc/total Src ratio of 6.2 ± 0.4, and increased FAK phosphorylation with the pFAK/total FAK ratio of 6.3 ± 0.2. The use of PAR-4 antagonist and PAR-3 antibody eliminated the Src Tyr416 phosphorylation by cat K (unpublished data). The phosphorylation of p38 was required when human platelets were stimulated with cat K. Pre-treatment of platelets with PAR-3 antibody and PAR-4 antagonist abolished the Src-Tyr-416 and FAKTyr- 397 phosphorylation. Data are expressed as mean ± SEM from 3 independent experiments (*p<0.01,**p<0.001, ***p<0.0001). See also Fig. 1a. (d) The [Ca2+]i mobilization was measured as described. The arrow indicates when α-thrombin was added to platelets (around 100 s). The washed platelets responded strongly with a transient rise in [Ca2+]i. (e) Cat K (20 nM) shows a calcium spike followed by a sustained elevation in the fluorescence ratio. The presence of α-thrombin (1.0 UNIH/500µL) led to an increase in [Ca2+]i indicating that the platelets induced Ca2+ release. (f) The papain (1.6 µM) response was similar to that from cat K, however, no substantial rise in [Ca2+]i was detected with the addition of α-thrombin. (g) Cat K did not block the papain effect. Sustained elevation in the fluorescence ratio was observed. (h) Cat L (0.2 µM) does not induce a detectable effect on Ca2+ mobilization; α-thrombin (1.0 UNIH/500µL) showed a strong response with a transient rise in [Ca2+]i. Platelets were treated with indomethacin (0.1 mg/mL) to prevent thromboxane synthesis, which may also be affected by PKC [Ca2+]. The mesurements were monitored by Fluo-4/AM (4 µM) intensity in a laser scanning confocal miscroscopy; scale bar = 10 µm