## 4 Discussion
PI3K isoforms are crucial downstream actors of many platelet receptors, such as GPVI (collagen receptor) and the P\({}_{2}\)Y\({}_{12}\) (ADP receptors) and integrin \(\alpha_{\text{IIb}}\beta_{3}\)[29]. PI3K\(\beta\) is a downstream regulator of the GPVI in terms of the control of PLCy2 phosphorylation and Ca\({}^{2+}\) mobilization [30]. Therefore, PI3K\(\beta\) may be a key target in preventing arterial thrombosis. Akt is an important effector of PI3K, and its three isoforms (Akt1, Akt2, and Akt3) are ubiquitously expressed in human platelets [20]. Akt-knockout mice reportedly showed platelet activation defects [31]. Therefore, the contribution of protein kinases, particularly PI3K\(\beta\), upon Akt activation, may establish as targets for antithrombotic therapy. Although the downstream regulators of Akt that participate in platelet activation remain unknown, several candidates of GSK3, such as \(\alpha\) and \(\beta\) isoforms, have been identified [32]. Both the \(\alpha\) and \(\beta\) isoforms are expressed in platelets, although GSK3\(\beta\) is the most abundant [33]. GSK3 inhibition may be required for full platelet response under stimulation by some agonists. Platelet-specific PI3K\(\beta\) knockout mice showed arterial thrombus instability under high shear stress due to diminished Akt activation and GSK3 inhibition [20]. Nevertheless, the mechanisms underlying GSK3 regulation of platelet activation remain unclear. Therefore, identifying the platelet substrates of GSK3 may enable identification of new antithrombotic candidates. In summary, PI3K/Akt/GSK3 signaling plays a dominant role in platelet activation and arterial thrombus growth in vivo. MAPKs are serine/threonine kinases that exchange extracellular stimuli into a wide range of cellular responses. MAPKs comprise three major subgroups, such as ERK1/2, p38 MAPK, and JNK1/2. ERK1/2 isoform is reported to involve in proliferation, adhesion, and cell progression [34]. p38 MAPK and JNK1/2 are involved in apoptosis [34]. MAPK-specific inhibitors and their knockout in mice demonstrate that ERK1/2, JNK1/2, and p38 MAPK are involved in platelet activation [35]. Although the mechanisms of JNK1/2 and ERK1/2 in platelets remain unclear, research demonstrates that the inhibition of integrin \(\alpha_{\text{IIb}}\beta_{3}\) activation may be involved [36]. In addition, p38 MAPK provides a crucial signal in aggregation caused by collagen [37]. This may explain why metformin exhibits more potent activity in reducing collagen-stimulated platelet activation than other agonists do. Of the several reported downstream targets of p38 MAPK, the most physiologically relevant in platelets is cytosolic phospholipase A\({}_{2}\), which is stimulated by various platelet agonists to catalyze AA release to produce thromboxane A\({}_{2}\)[38]. Moreover, both PI3K/Akt and MAPKs are equally activated in platelets, while PKC acts as an upstream regulator of these molecules [31]. In this study, metformin (1.8 mM) exerted no significant effects on VASPSer157 and VASPSer239 phosphorylation in human platelets. Elevation of intracellular cAMP and cGMP in platelets activates their individual protein kinase A and protein kinase G, which control platelet activation through phosphorylation of intracellular protein substrates, namely VASP (Ser157) and (Ser239), respectively [39]. Cyclic nucleotide elevation decreases the Ca\({}^{2+}\) influx and reduces the activity of cell membrane-bound calcium transporters, which suppresses PLC/PKC signaling activation. [PERSON] et al. [40] reported that metformin stimulates protein kinase A activation in human umbilical vein endothelial cell; however, this study eliminates the possibility of cyclic nucleotide/VASP signal involvement in metformin-mediated antiplatelet activity. Antiplatelet therapy has reportedly been effective in reducing the mortality and morbidity of CVDs. However, the antiplatelet drugs prescribed in clinical practice create serious side effects, including bleeding, gastrointestinal toxicity, neutropenia, and thrombocytopenia.