Shh induces noncanonical signaling in platelets. (A) Expression of pMLC and pMYPT1 in platelets treated for 10 to 15 minutes with the following reagents (without stirring) as indicated in the figure: Shh, 3 μg/mL; thrombin (Thr), 0.5 U/mL; cyclopamine (Cyclo), 10 μM; compound C (CC), 50 μM; Y27632, 10 μM. PVDF membranes developed for pMLC and pMYPT1 were stripped and reprobed for total MLC and β-actin, respectively. (D) Expression of RhoA in platelets treated with Shh, thrombin, and cyclopamine (without stirring) as indicated in the figure. RhoA-GTP was pulled down from pretreated platelets employing a bead-based assay system and analyzed on Western blot. Whole cell lysate was separately prepared and probed for total RhoA. (F) Expression of pAMPK and pACC in platelets treated with different reagents for 15 minutes (without stirring) as indicated in the figure. PVDF membranes developed for pAMPK and pACC were stripped and reprobed for total AMPK and β-actin, respectively. (B-C,E,G-H) Corresponding bar diagrams after densitometric analyses of blots normalized against total MLC, RhoA, AMPK, or β-actin averaging 3 to 5 different experiments. (I) Intracellular calcium measurements in Fura-2–loaded platelets preincubated either with 10 μM cyclopamine (tracing 2) or vehicle (tracing 3), followed by the addition of thrombin (indicated by arrow). Tracing 1 represents unstimulated Fura-2–loaded platelets. (J) Represents intracellular calcium concentrations (mean ± standard error of the mean [SEM]) averaging 3 individual experiments. Figures are representative of ≥3 individual experiments (mean ± SEM). *P < .05 as compared with resting platelets; #P < .05 as compared with thrombin-stimulated platelets; and §P < .05 as compared with Shh-pretreated platelets.