## 5 Conclusions  
Our results provide new insights into the role and mechanisms of metformin in human platelet activation. Metformin efficiently diminished platelet activation by interfering with the PLCy2/PKC, PI3K/Akt/GSK3\(\beta\), and p38 MAPK signaling pathways through a cyclic nucleotide/VASP-independent mechanism (Figure 9). The present results highlight a novel therapeutic drug for platelet abnormalities in diabetes mellitus and a general strategy for preventing cardiovascular complications. Metformin could be a new class of antiplatelet agent that is effective at inhibiting platelet activation.  
Figure 9: Summary scheme graphic showing suppressive signaling by metformin in human platelet activation, acute pulmonary thromboembolism, and tail bleeding time in mice. Collagen binds to its receptors, and then stimulates PLCy2-PKC cascade, PI3K-Akt-GSK3\(\beta\), and p38 MAPK, followed by the suppression of platelet activation, ultimately reducing the death rate associated with acute pulmonary thromboembolism without increasing the bleeding time.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12157426/s1](https://www.mdpi.com/article/10.3390/app12157426/s1)]([https://www.mdpi.com/article/10.3390/app12157426/s1](https://www.mdpi.com/article/10.3390/app12157426/s1)), Figure S1: Inhibitory curves of metforminin in platelet aggregation stimulated by various agonists. Washed human platelets (3.6 \(\times\) 10\({}^{8}\) cells/mL) were preincubated with a solvent control (PBS, black line) or metforminin (1.2 mM, red line; 1.6 mM, green line; 1.8 mM, blue line) and subsequently treated with (A) collagen (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), (B) thrombin (0.05 U/mL), (C) U46619 (1 \(\mathrm{\SIUnitSymbolMicro u}\)M), or (D) arachidonic acid (AA; 60 \(\mathrm{\SIUnitSymbolMicro u}\)M) to stimulate platelet aggregation. The data are the representative of four independent experiments.; Figure S2: Inhibitory activity of metforminin in thrombin-induced platelet aggregation. Washed human platelets (3.6 \(\times\) 10\({}^{8}\) cells/mL) were preincubated with a solvent control (PBS) or metforminin (2-4 mM) and subsequently treated with thrombin (0.05 U/mL) to stimulate platelet aggregation. The corresponding statistical data is displayed in the below panel. Data are expressed as mean \(\pm\) standard error of the mean (\(n\) = 4). \(\mathbf{***}\)\(p\) \(<\) 0.001 vs. [PERSON]'s solution; \(\mathbf{***}\)\(p\)\(<\) 0.001 vs. PBS + thrombin group.  
[PERSON] performed the research and drafted the paper; [PERSON], [PERSON]. ([PERSON]), and [PERSON] performed the research and contributed to the experiments; [PERSON] and [PERSON] ([PERSON]). contributed to the investigation and analyzed the data; [PERSON] and [PERSON] perceived and planned the study. All authors have read and agreed to the published version of the manuscript.  
Ministry of Science and Technology of Taiwan (MOST 108-2320-B-038-031-MY3), Taipei Medical University (DP2-107-21121-N-02), Shin Kong Wu Ho-Su Memorial Hospital (SKH-8302-106-DR-22), Cathay General Hospital (CGH-MR-A11005 and CGH-MR-A11104), Shin Kong Wu Ho-Su Memorial Hospital-Taipei Medical University (SKH-TMU-108-03), and Cathay General Hospital-Taipei Medical University (108 CGH-TMU-07) financially supported this work.  
This study was approved by the Institutional Review Board of Taipei Medical University (TMU-JIRB-N201812024) and conformed to the directives of the Declaration of Helsinki. All animal experiments and care procedures conformed to the Guide for the Care and Use of Laboratory Animals (LAC-2020-0294) and were approved by the Institutional Animal Care and Use Committee of Taipei Medical University.  
Informed Consent Statement: Informed consent was obtained from all study participants.  
Data Availability Statement: All data generated or analyzed in this study are included in this article.  
The authors declare no conflict of interest.