Signaling pathways regulating FAK activation downstream of G_12/13 pathways.

<p>Aspirin-treated, washed human platelets were pre-treated with different inhibitors (as indicated) for 5 minutes at 37°C followed by stimulation with AYPGKF (500 µM) for 90 seconds under stirring conditions at 37°C in an aggregometer. The lysates were then subjected to western blotting analysis and probed with anti- phospho FAK (Y-397) and total FAK antibodies as lane loading control (A &C) or anti- phospho- Src (416) and total c-Src antibodies as lane loading control (E). Quantitative data, normalized to the lane loading control, are representative of mean ± S.E.M (n = 3). The data was analyzed by ANOVA and * P≤0.05 was considered significant (B, D, and F).</p

Role of c-Src and Syk downstream of integrin αIIbβ3 in thromboxane generation in platelets.

<p>Aspirin–treated, washed platelets were stimulated with 2MeSADP (100 nM) at various time points (A) or with varying concentrations of 2MeSADP (B) for one minute under stirring conditions at 37°C. The lysates were then subjected to western blotting analysis and probed with anti-phospho-(Y416) and total c-Src antibodies as lane loading control. Washed murine (WT or c-Src KO) platelets, without aspirin-treatment, were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB₂ levels were analyzed (C) as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016586#pone-0016586-g001" target="_blank">Figure 1</a>. The data are represented as the % Fold increase over the control. Aspirin-treated washed platelets were stimulated with 2MeSADP (100 nM) for (30–120 seconds) or convulxin (100 ng/ml) for 30 seconds under stirring conditions at 37°C (D). The lysates were then subjected to western blotting analysis and probed with anti- phospho- Syk(Y 525/526) and total Syk antibodies as lane loading control. The data are representative of at least 3 separate experiments.</p

Focal Adhesion Kinase is activated downstream of integrins and G_12/13 pathways.

<p>Aspirin treated, washed platelets were stimulated with 2MeSADP (100 nM) in presence or absence of reagents (as indicated) for 60 seconds under stirring conditions at 37°C (A). The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control. The data are representative of mean ± S.E.M (n = 3). The data was analyzed by ANOVA and * P≤0.05 was considered significant (B). Aspirin-treated washed platelets were stimulated with AYPGKF (500 µM) in presence or absence of YM254890 (150 nM) (C), The lysates were then subjected to western blotting analysis and probed with anti- phospho- FAK (Y-397) and total FAK antibodies as lane loading control.</p

Model depicting the regulation of TXA₂ generation by G_12/13 pathways and integrins through FAK.

<p>Integrin clustering leads to FAK activation (1). Fibrinogen receptor antagonist SC57101 prevents integrin clustering hence inhibits FAK activation (2). FAK can be activated in an integrin-independent manner by G_12/13 pathways (3). FAK is activated downstream of Rho kinase and SFKs upon stimulation of G_12/13 pathways (4). FAK can be inhibited by TAE-226 (5). Common effector molecule downstream of integrins and G_12/13 pathways contributing to thromboxane generation are unknown (6).</p

Regulation of thromboxane generation by G_12/13 pathways.

<p>Non-aspirin-treated, washed human platelets were stimulated with different concentrations of AYPGKF in presence and absence of SC57101 (10 µM) (A) or other agonists (B) as indicated for 3.5 minutes at 37°C under stirring conditions (900 rpm) in an aggregometer. After 3.5 minutes the reaction was stopped by snap- freezing in dry ice-methanol bath. TXB₂ levels were analyzed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016586#s4" target="_blank">Materials and Methods</a>”. The values are representative of 3 independent experiments mean ± S.E.M (n = 3). The data were analyzed by student t-test and ANOVA, ** P≤0.005 was considered significant.</p

Evaluation of FAK as a common signaling effector molecule regulating thromboxane generation downstream of integrins and G_12/13 pathways.

<p>Non-aspirin-treated, washed human platelets were pre-treated with varying concentrations of TAE-226 for 5 minutes at 37°C (A) and murine platelets from WT and Pf4-Cre/Fak-Floxed mice (B) were stimulated with 2MeSADP (100 nM) for 3.5 minutes and TXB₂ levels were analyzed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016586#pone-0016586-g001" target="_blank">Figure 1</a>. Aggregation tracings were measured from WT and Pf4-Cre/Fak-Floxed mice and representative tracings are shown (C). The values are representative of 3 independent experiments mean ± S.E.M (n = 3). The data were analyzed by ANOVA and student t-test, * P≤0.05 was considered significant.</p