## 3 Results
### TCE Upregulated the cAMP Pathway in Collagen-Activated Platelets  
Elevated cellular cAMP levels inhibit platelet activation [12]. Therefore, we investigated the effects of TCE on the cAMP pathway in collagen-activated platelets. Collagen stimulation (2.5 \(\upmu\)g/mL) significantly decreased platelet cAMP levels from 2.88 \(\pm\) 0.41 pmol/mL to 2.16 \(\pm\) 0.01 pmol/mL. However, pretreatment with TCE at 100 \(\upmu\)g/mL elevated cAMP levels to 3.57 \(\pm\) 0.19 pmol/mL (Figure 3A). Protein kinase A (PKA) activity in platelets depends on cellular cAMP levels, and activated PKA phosphorylates key target proteins such as VASP (Ser\({}^{157}\)) and IP\({}_{3}\)R\({}_{1}\). To elucidate the effects of TCE on these cAMP-dependent proteins, we analyzed the phosphorylation status of IP\({}_{3}\)R\({}_{1}\), VASP (Ser\({}^{157}\)), and PKA. In collagen-treated platelets, PKA phosphorylation was reduced compared to unstimulated platelets, while IP\({}_{3}\)R\({}_{1}\) and VASP (Ser\({}^{157}\)) showed phosphorylation due to an inhibitory feedback mechanism involving NF-\(\upkappa\)B complex activity [29]. Remarkably, TCE pretreatment in collagen-treated platelets  
Figure 2: To examine the impact of TCE on platelet granule release, human platelets were preincubated with TCE for 2 min before stimulation with collagen (2.5 \(\upmu\)g/mL) for 5 min. The extent of granule secretion was assessed by quantifying the levels of (**A**) serotonin release, (**B**) ATP release, and (**C**) calcium mobilization. The data are expressed as mean \(\pm\) SD (\(n=3\)). Statistical significance was determined as * \(p<0.05\) and ** \(p<0.001\)** compared to collagen-treated platelets. significantly enhanced phosphorylation of IP\({}_{3}\)R\({}_{1}\), VASP (Ser157), and PKA by activating the cAMP pathway (Figure 3B). TCE Inhibits Platelet Integrin alIb\(\beta\)3 Activation, Delaying Clot Retraction Without Affecting Coagulation Parameters  
Clot retraction is essential for hemostasis and a hallmark of thrombosis [30]. Upon stimulation by agonists, platelets form a fibrin-platelet network and release potent coagulation triggers. To investigate the effects of TCE on clot retraction and integrin \(\alpha\)IIb\(\beta\)3 activation, thrombin (1 U/mL) was used to induce clot retraction, which reached 93.0% within 30 min (Figure 4A). However, pretreatment with TCE delayed thrombin-induced clot retraction in a dose-dependent manner, reducing it to 58.3% at 100 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro}\)/mL after 30 min. Similarly, collagen (2.5 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro}\)/mL) activated integrin \(\alpha\)IIb\(\beta\)3 up to 94.41%, but TCE significantly inhibited this activation, reducing it to 26.0% at 100 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro}\)/mL (Figure 4B). To evaluate whether TCE affects blood coagulation, PT and APTT values were measured. These tests assess the activity of coagulation factors in the extrinsic and intrinsic pathways, respectively. TCE had no effect on PT or APTT values (Figure 4C), confirming that its inhibitory effects are specific to platelet function rather than the coagulation cascade. These results indicate that TCE functions as an antiplatelet agent by suppressing clot retraction and integrin \(\alpha\)IIb\(\beta\)3 activation, without exerting anticoagulant effects. Figure 3: The impact of TCE on the cAMP signaling pathway. (**A**) Measurement of intracellular cAMP levels in human platelets pretreated with TCE for 2 min, followed by stimulation with collagen (2.5 \(\mathrm{\SIUnitSymbolMicro}\)/mL) for 5 min. (**B**) Assessment of PKA, VASP, and IP3R1 phosphorylation under the same experimental conditions. (**B**) Representative immunoblots displaying phosphorylation changes in PKA, VASP, and IP3R1. The data are expressed as mean \(\pm\) SD (\(n\) = 3). Statistical significance is indicated as * \(p<0.05\) and **\(p<0.001\)** compared to collagen-treated platelets. Figure 4: Effects of TCE on clot retraction, integrin \(\alpha\)IIb\(\beta\)3 activation, and coagulation parameters.