FIGURE 2.

Effects of Col003 on platelet aggregation induced by various agonists and platelet adhesion and Ca²⁺ intra-platelet calcium flux induced by collagen. Washed rat platelets (3 × 10⁸/ml) were pre-incubated 5 minutes with the indicated concentrations of Col003 or vehicle followed by the addition of collagen (Ai), arachidonic acid (Bi), or thrombin (Ci). The aggregation rates (%) were quantified (Aii, Bii, Cii; ***p < 0.001, vs vehicle). The aggregation rates (%) in collagen-induced group were 70.33 ± 3.93% (Vehicle), 57.00 ± 4.58% (Col003 25uM), 27.67 ± 1.76% (Col003 50uM), respectively. The aggregation rates (%) in arachidonic acid-induced group were 77.33 ± 3.28% (Vehicle), 87.00 ± 3.79% (Col003 25uM), 85.33 ± 3.84% (Col003 50uM), respectively. The aggregation rates (%) in thrombin-induced group were 69.67 ± 6.89% (Vehicle), 73.00 ± 2.31% (Col003 25uM), 66.33 ± 5.70% (Col003 50uM), respectively. (D) Representative images of platelet adhesion on collagen-coated round coverslips. (E) The number of adherent platelets per vision was quantified. The adherence number of platelets in the five groups were 8.68 ± 0.58 platelets/vision (BSA), 56.44 ± 3.55 platelets/vision (Collagen), 46.44 ± 5.69 platelets/vision (Vehicle), 27.56 ± 1.88 platelets/vision (Col003 25uM), 21.67 ± 2.30 platelets/vision (Col003 50uM), respectively. **p < 0.01, ***p < 0.001, and ****p < 0.0001. (F) The intracellular Ca²⁺ concentration was measured by labeled Fluo-3 AM, and the Fluo-3 fluorescence of platelets was presented normalized to the fluorescence of resting platelets. The [Ca2+]i levels in the three groups were 1.53- ± 0.04-fold of resting (Vehicle), 1.47- ± 0.17-fold of resting (Col003 25uM), 1.26- ± 0.08-fold of resting (Col003 50uM), respectively. *p < 0.05 (vs vehicle). Statistical methods for the above experiments using unpaired two-tailed Student’s t-test.