## 3 Results
### Regulatory Activity of PI3K-Akt-GSK3\(\beta\) and MAPK Activation by Metformin  
A study indicated that high shear stress induces thrombus formation via PI3K/Akt/G-SK3\(\beta\)[20]. PI3K stimulation is greatly involved in platelet activation and plays a critical role in regulating Akt activation [20]. Akt (protein kinase B (Ser/Thr kinase)) is found to be stimulated by several platelet agonists; in platelets, GSK3\(\beta\), a classical enzyme, is controlled downstream of the PI3K/ Akt pathway [21]. In the present study, metformin at concentrations of 1.2 and 1.8 mM markedly reduced the phosphorylation of the PI3K/Akt/GSK3\(\beta\) pathway under collagen stimulation (Figure 5). The corresponding data are presented on the right-hand side of each figure. MAPK signals are involved in many cellular activities, such as inflammation, cell proliferation, apoptosis, and platelet activation; the MAPKs that are most often observed in platelets are ERK1/2, JNK1/2, and p38 MAPK [22]. Metformin (1.2 and 1.8 mM) reduced p38 MAPK but not ERK1/2 and JNK1/2 phosphorylation, indicating that p38 MAPK molecule plays a vital role in the metformin-mediated antiplatelet effects (Figure 6). These results indicate that inhibition of PI3K/Akt/GSK3\(\beta\) and p38 MAPK activation may be critical contributors to the antiplatelet effects of metformin.  
Figure 4: Effectiveness of metformin in protein kinase C activation in platelets. Washed platelets were preincubated with PBS or metformin (1.2 and 1.8 mM) and treated with collagen (1 ug/mL) for immunoblotting of (**A**) protein kinase C (PKC) activation (pleckstrin; p-p47) or (**B**) detecting confocal images of PKC activation (green fluorescence) and \(\alpha\)-tubulin (blue fluorescence) using goat anti-rabbit CFTM 488A and anti-mouse CFTM 405M Dyes, respectively. (**C**) For other study, washed platelets were preincubated with PBS or metformin (1.2 and 1.8 mM) and treated with phorbol 12,13-dibutyrate (PDBu, 150 nM) to stimulate platelet aggregation. Data are presented as mean \(\pm\) standard error of the mean (\(n=4\)). ***** \(p\) \(<\) 0.001 vs. [PERSON]’s solution group; # \(p\) \(<\) 0.05 vs. PBS + collagen group. The confocal images and profiles in (**B**) are the representative of four independent experiments. Bar: 5 μm.  
Figure 5: Regulatory effect of metformin in phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß. Washed platelets were preincubated with PBS or metforminin (1.2 and 1.8 mM) and treated with collagen (1 ug/mL) for immunoblotting of (**A**) phosphoinositide 3-kinase (PI3K), (**B**) Akt, and (**C**) glycogen synthase kinase-3ß (GSKß) phosphorylation. Results are expressed as mean \(\pm\) standard error of the mean (\(n\) = 4). \({}^{**}\)\(p\) < 0.01 and \({}^{***}\)\(p\) < 0.001 vs. [PERSON]’s solution; # \(p\) < 0.05 and ### \(p\) < 0.001 vs. PBS + collagen group.  
Figure 6: Regulatory activity of metformin in mitogen-activated protein kinase phosphorylation in platelets. Washed platelets were preincubated with PBS or metformin (1.2 and 1.8 mM) and treated with collagen (1 ug/mL) for immunoblotting of (**A**) p38 mitogen-activated protein kinase (p38 MAPK), (**B**) ERK1/2, and (**C**) JNK1/2 phosphorylation. Data are expressed as mean \(\pm\) standard error of the mean (\(n\) = 4). \({}^{**}\)\(p\) < 0.01 and \({}^{***}\)\(p\) < 0.001 vs. [PERSON]’s solution; # \(p\) < 0.05 vs. PBS + collagen group.