Inhibitory effect of disodium qurecetin-7, 4′-disulfate on aggregation of pig platelets induced by thrombin and its mechanism

AIM: To study the inhibitory effect of semi-synthesized quercetin derivatives - disodium quercetin-7,4'-disulfate (DQD) on the platelet aggregation induced by thrombin and its mechanism. METHODS: Platelet aggregation was analysed by turbidimetry. Cytosolic free calcium concentration ([Ca2+](i)) was determined by Fura-2 fluorescence technique. Activity of Ca2+/PL dependent protein kinase C (PKC) was assayed by incubating PKC with histone III S and [gamma-P-32]ATP. The cytoskeletal proteins were precipitated by Triton and separated by SDS-PAGE. RESULTS: DQD inhibited the platelet aggregation induced by thrombin (500 U/L), when DQD concentrations were 100, 200, and 400 mu mol/L, the inhibition rates were 77 %, 86 %, and 82 % respectively. DQD inhibited Ca2+ influx in platelets induced by thrombin (500 U/L) in the presence of extracellular Ca2+ 1 mmol/L in a concentration-dependent manner (10 - 80 mu mol/L); DQD also had inhibitory effect on intracellular Ca2+ mobilization in the absence of extracellular Ca2+. DQD (10 - 160 mu mol/L) inhibited the cytosolic Ca2+/ PL dependent PKC from platelets in a concentration-dependent manner, but had no effect on membrane PKC. DQD (20 - 200 mu mol/L) inhibited the actin polymerization induced by thrombin (500 U/L) in platelets in a concentration-dependent manner. CONCLUSION: DQD inhibited pig platelet aggregation induced by thrombin and its molecular mechanism was due to its inhibition of Ca2+ influx, intracellular Ca2+ mobilization, Ca2+/ PL dependent PKC activity, and actin polymerization.