## 3 Results  
Effect of CBN in Platelet Plug Formation in Human Whole Blood and Tail Bleeding in Experimental Mice  
[PERSON] et al. [12] reported that 10 mg/kg CBN treatment markedly prolonged occlusion time in the mesenteric microvessels of mice. In the current study, we further confirmed the effective antithrombotic activity of CBN by using the platelet function analyzer (PFA-100) (Figure 1A), which consists of an instrument and test cartridges in which the process of platelet adhesion and aggregation following a vascular injury is stimulated in vitro. The single-use cartridges consists of a number of integrated parts including a capillary, a sample reservoir and a biochemically active membrane with a central circular aperture (Figure 1A). Citrated whole blood is aspirated from the sample reservoir through the capillary and aperture, which expose platelets to high shear flow conditions (5000-6000/s). The membrane is coated with collagen, a subendothelial protein generally believed to be the initial matrix for platelet attachment. In addition, the membrane is coated with ADP, which is other physiological agonist, along with collagen, are extensively used to trigger platelets in aggregometry testing. The time needed to occlude the aperture is described as closure time (CT). In the present study, the CT of the CADP membrane in whole blood treated with solvent control (0.1% DMSO) was \(81.5\pm 5.1\) s (\(n=8\)); CBN (90 \(\upmu\)M) significantly prolonged CT (45 \(\upmu\)M, \(99.8\pm 5.6\) s, \(n=8\); \(p>0.05\); 90 \(\upmu\)M, \(114.0\pm 9.8\) s, \(n=8\); \(p<0.05\)) compared with the solvent control (Figure 1A). We evaluated the effects of CBN on bleeding time, since it is a general side effect of antiplatelet drugs when they are using in clinical trials. This effect was also compared with antiplatelet drug aspirin. The bleeding time was \(165.4\pm 15.9\) s (\(n=8\)) in the solvent control group (Figure 1B). After 30 min of intraperitoneal treatment of CBN at 10 and 20 mg/kg, the bleeding time was \(180.4\pm 20.1\) and \(191.0\pm 26.1\) s (\(n=8\)), respectively; therefore, the bleeding time was not significantly affected by those doses. In addition, aspirin was administered at 50 mg/kg, and the bleeding time was markedly prolonged after 30 min from \(165.4\pm 15.9\) s (0.1% DMSO-treated group) to \(225.7\pm 6.3\) s (\(n=8\), \(p<0.05\)) (Figure 1B). Figure 1: Activity of columbianadin on the closure and bleeding time in mice. (**A**) High shear flow-induced platelet plug formation in human whole blood was determined by recording the closure time of collagen/ADP (CADP) membrane were preincubated with 0.1% DMSO, 45 or 90 \(\mu\)M columbianadin (CBN) as described in the Materials and Methods. (**B**) For the animal study, the bleeding time was measured by transection of mouse tails, 30 min after administering either 0.1% DMSO, 10 and 20 mg/kg CBN, or 50 mg/kg aspirin (positive control to compare with CBN) intraperitoneally. * \(p<0.05\), compared with the 0.1% DMSO-treated group (\(n=8\)). Inhibition of Platelet Aggregation and ATP-Release by BAY11-7082, MG-132, and CBN in Collagen-Stimulated Platelets  
BAY11-7082 is an IKK inhibitor that possesses several biological activities, including anticancer, neuroprotective, and anti-inflammatory effects [16]. MG-132 belongs to a proteasome inhibitor that blocks the activation of NF-\(\kappa\)B by inhibiting \(\mathrm{I\kappa B}\) degradation [17]. In the present study, we compared the relative activities of BAY11-7082 and MG-132 in the inhibition of platelet aggregation and determined that in washed human platelets, both BAY11-7082 (4 and 8 \(\mathrm{\SIUnitSymbolMicro m}\)M) and MG-132 (60 and 120 \(\mathrm{\SIUnitSymbolMicro m}\)M) inhibited collagen (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL)-stimulated platelet aggregation in a concentration-dependent manner, similar to CBN (45 and 90 \(\mathrm{\SIUnitSymbolMicro m}\)M) (Figure 2A). BAY11-7082 exhibited a higher potency than MG-132 in on molar basis (Figure 2A).