## 4 Discussion  
Thrombotic cardiovascular diseases cause 65% death in diabetic patients. Research has demonstrated that 10 to 40% of patients with diabetes are biochemically resistant to aspirin treatment. Therefore, a clearer understanding of existing new therapies targeting the underlying mechanisms on platelet activation must be established [1; 2]. Studies have reported that metforminin is a potential drug candidate as it is associated with a decreased rate of mortality in diabetes-associated CVDs and cerebrovascular diseases in those with T2D [23; 24; 25]. The findings of this study reveal that metformin possesses significant antiplatelet activity against collagen-stimulated human platelets, in addition to its other well-known properties. In this study, metforminin was only effective on inhibiting platelet aggregation induced by collagen but it exerted a very slight or even no effect on other agonist (i.e., AA, U46619, and thrombin)-induced platelet activation. Platelet activation is triggered by different physiological agonists, which show their effects through interactions with specific receptors on platelet membranes. Among these platelet agonists, collagen plays a key role in platelet activation. Platelets adhere to the connective tissue protein in collagen, resulting in a change in the shape and release of granules. Adhesion is partly dependent on [Ca2+]i mobilization, ADP/ATP release, and thromboxane A2 formation, and aggregation is entirely dependent on this release [7]. Collagen is present in vascular subendothelium and vessel walls, acting as a substrate for platelet adhesion; it is also an endogenous strong platelet activator. Metformin decreased collagen-induced platelet aggregation, which suggests that metformin affects a unique PLC-dependent mechanism. PLC activation leads to IP3 and DAG formation, which, in turn, activates PKC and induces p47 protein phosphorylation [26]. The PLCy family comprises isozymes 1 and 2, and PLCy2 contributes in collagen-dependent signaling in platelets [27]. The PKC family plays a vital role in serine/threonine kinase phosphorylation, which considerably affects numerous signal transduction pathways in  
Figure 8: Influence of metforminin in acute pulmonary thromboembolism and tail bleeding time in vivo. (**A**) For acute pulmonary thrombosis, PBS or metforminin (150 and 250 mg/kg) or heparin (1.5 U/g) was intraperitoneally administered to mice, and ADP (0.7 mg/g) was injected into the tail veins. (**B**) The bleeding time was measured through mouse tail transaction after 30 min of intraperitoneal administration of either PBS or metforminin (150 and 250 mg/kg) or heparin (1.5 U/g). Results are expressed as mean \(\pm\) standard error of the mean ((**A**), \(n=12\); (B), \(n=8\)). ***\(p<0.001\) vs. PBS-treated group. Data in (**A**) are presented as the percentage of mortality. most cells [28]. This study found that metformin reduced collagen-induced PLCy2/PKC activation. However, it did not directly affect PKC activation because PDBu-induced platelet aggregation was not reduced, suggesting that PLCy2-mediated signaling plays a vital role in metformin antiplatelet activity. Platelet activation is usually associated with an increase in [Ca\({}^{2+}\)]i mobilization and ATP release, as well as surface P-selectin expression due to granule secretion; \(\alpha\)-granules are mostly located in platelet protein storage compartments and contain P-selectin, membrane-associated proteins and fibrinogen, and platelet-derived growth factor, soluble proteins. In platelet activation, \(\alpha\)-granules exocytosis represents as a marker and, therefore, we evaluated \(\alpha\)-granule exocytosis via flow cytometric analysis of P-selectin expression (Figure 2C). Almost all the isoforms of PI3K (i.e., \(\alpha\), \(\beta\), \(\gamma\), and \(\delta\)) expressed in platelet contribute extensively in platelet activation.