Figure 3.

Effects of TQ-6 in integrin α_IIbβ₃ and GP VI activation as well as cyclic nucleotide formation in human platelets. (A) Washed platelets (3.6 × 10⁸/ml) were preincubated with the (b) solvent control (0.5% DMSO) or TQ-6 (c, 0.3 µM; d, 0.5 µM) and FITC-PAC-1 (2 µg/ml) for 3 min in the (a) absence or (b–d) presence of collagen (1 μg/ml). (B) Solid line represents the fluorescence profiles of FITC-triflavin (2 µg/ml) serving only as a positive control (a); treatment with EDTA (b, 5 mM; as a negative control), TQ-6 (c, 0.3 µM; d, 0.5 µM), followed by the addition of FITC-triflavin (2 µg/ml). (C) Washed platelets were preincubated with the solvent control (0.5% DMSO) or TQ-6 (0.3 and 0.5 µM), and subsequently activated by immobilized fibrinogen (100 μg/ml). Platelets were collected, and their subcellular extracts were analyzed to determine the levels of integrin β₃ phosphorylation. (D) The fluorescence profiles of (a) FITC only as a background control; washed platelets were preincubated with the (b) solvent control (0.5% DMSO) or TQ-6 (c, 0.3 µM; d, 0.5 µM) for 3 min, followed by the addition of FITC-JAQ1 (1 µg/ml). (E) For other experiments, washed platelets (3.6 × 10⁸ cells/mL) were preincubated with 10 µM nitroglycerin (NTG), 1 µM prostaglandin E₁ (PGE₁), or 0.5 µM TQ-6 with or without 10 µM ODQ or 100 µM SQ22536 and were subsequently treated with 1 µg/ml collagen to induce platelet aggregation. (F) Washed platelets were incubated with 0.1 μM PGE₁, 10 μM NTG, or 0.3 or 0.5 μM TQ-6 for 6 min. The cells were collected, and subcellular extracts were analyzed to determine the levels of cyclic AMP and cyclic GMP, respectively. Profiles in (C and E) are representative of four independent experiments. Data are presented as means ± standard errors of the means (n = 4). **p < 0.01 and ***p < 0.001, compared with the resting control; ^#p < 0.05, compared with the collagen-treated group.