## 3 Results  
### Mitigation of Human Platelet Aggregation by Metformin  
As previously reported [10], metformin (1 mM) significantly diminished platelet aggregation stimulated by ADP (20 uM), thrombin (0.1 U/mL), and AA (900 uM) in rat platelets. However, with the exception of the aforementioned, no other study has reported on platelet activation, especially in humans. This study is the first report to state that metformin (1.2-1.8 mM) suppressed collagen-induced (1 ug/mL) platelet aggregation in washed human platelets in a concentration-dependent manner (Figure 1A); however, metformin had only a slight or nonsignificant effect under stimulation by AA (60 uM), U46619 (1 uM; a prostaglandin endoperoxide analogue compound), and thrombin (0.05 U/mL; Figures 1B-D and S1). In addition, metformin at 2-4 mM also exhibited a concentration-dependent inhibitory effect in platelet aggregation stimulated by thrombin (Figure S2). These results showed that the effectiveness of metformin in human platelets differed from that of other agonists. Therefore, collagen was used as an ideal agonist to unravel the inhibitory mechanisms of metformin in human platelet activation. In addition, the LDH assay revealed that metformin (2-10 mM) did not change the release of LDH when it pre-treated with platelets for 20 min, which indicates that metformin did not have a cytotoxic effect on the platelets (Figure 1E). Figure 1: Effect of metformin on platelet aggregation upon stimulation with agonists and lactate dehydrogenase (LDH) release. Washed human platelets (3.6 \(\times\) 10\({}^{8}\) cells/mL) were preincubated with a solvent control (PBS) or metformin (1.2, 1.6, and 1.8 mM) and treated with collagen ((**A**), 1 ug/mL), arachidonic acid (AA; (**B**), 60 uM), U46619 (**C**), 1 uM), and thrombin ((**D**), 0.05 U/mL) to trigger platelet aggregation. (**E**) To evaluate the cytotoxic effect of the metformin on the human platelets, the platelets were pretreated with solvent control (PBS) or metformin (2, 5, and 10 mM) or the positive control (0.5% Triton) for 20 min, and 10 uL of the supernatant was dropped on a Fuji Dri-Chem slide LDH-PIII. Data are presented as mean \(\pm\) standard error of the mean (\(n\) = 4). \(\mathbf{\mathrm{\char 37}}\)\(p\) < 0.001 vs. the resting group (Tyrode’s solution); \(\mathbf{\mathrm{\#}}\)\(p\) < 0.01 and \(\mathbf{\mathrm{\#}}\)\(p\) < 0.001 vs. PBS + collagen group. Effects of Metformin on ATP Release, Relative [Ca\({}^{2+}\)]i Mobilization, and Surface P-selectin Expression  
Several lines of evidence have indicated that platelet activation is directly associated with the release of granular contents, such as ATP and Ca\({}^{2+}\). This release results in an upsurge in platelet aggregation, which will result in strong platelet activation. As presented in Figure 2A,B, metformin significantly suppressed both the ATP release reaction and [Ca\({}^{2+}\)]i mobilization stimulated by collagen. The relative data are expressed as inhibition percentages on the right-hand side of each figure (A, ATP release reaction: metformin 1.2 mM, 81.1 \(\pm\) 5.5%; 1.8 mM, 80.7 \(\pm\) 9.8%, \(n\) = 4; B, [Ca\({}^{2+}\)]i mobilization: metformin 1.2 mM, 84.1 \(\pm\) 4.1%; 1.8 mM, 37.8 \(\pm\) 11.0%, \(n\) = 4). Furthermore, P-selectin is a key biomarker for platelet activation. For instance, P-selectin is expressed on the inner face of \(\alpha\)-granules in a normal condition; however, when platelets become activated, they expose the inner face of the granules to the outer parts of the cells [18]. As indicated in Figure 2C, metformin significantly decreased the collagen-stimulated FITC-P-selectin expression in the flow cytometry study; the results are presented in the right panel of Figure 2C (a, resting control ([PERSON]'s solution), 95.7 \(\pm\) 14.5; b, collagen-activated platelets, 262.3 \(\pm\) 14.1; c, metformin 1.2 mM, 221.3 \(\pm\) 16.8; d, 1.8 mM, 198.7 \(\pm\) 7.2; \(n\) = 4).