Study On The Mechanisms Of Potassium2-(1-hydroxypentyl)-benzoate Inhibiting Platelet Aggregation

Thrombosis is a common disease, which is seriously harmful to human life and health. The etiology and pathogenesis have not yet been fully elucidated because of its complexity. It is known that the abnormal platelet function and endothelial cell injury are very important factors of thrombosis, which is one of the major risk factors for cardiovascular and cerebrovascular ischemic diseases. Series of studies have shown that platelets lost its normal function, being over activated, and blood was in a hypercoagulable state when cerebral ischemia, which is important to secondary brain injury. Thus, the development and application of antiplatelet drugs are effective means of treatment of cerebral vascular disease, and to find effective antithrombotic drugs are of great important clinical significance.Potassium2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a newly synthesized compound which is under development as a therapeutic drug for cerebral ischemia. Previous studies showed that dl-PHPB had an antithrombotic effect, could increase cerebral blood flow markedly and reduce infarct volume in a rat model of transient focal cerebral ischemia. However, the detailed mechanisms of dl-PHPB antiplatelet and antithrombotic activity remain obscure. In this study, the effects of dl-PHPB on platelet activation and aggregation, coagulation system, fibrinolysis system and endothelial cell were explored. In addition, underlying mechanisms were also investigated. At the same time, we also compared Pueraria and salvianolic acid B with dl-PHPB in order to explore the antiplatelet mechanisms of traditional Chinese medicine, providing further theoretical and experimental basis for the development of more effective antiplatelet medicine.Hyper-activation of platelet function is an important factor in thrombosis, we first examined the effect of dl-PHPB on platelet function. The detection of platelet aggregation activity is the gold standard for evaluation of platelet function in vitro, we observed the effect of dl-PHPB on platelet aggregation. The results showed that dl-PHPB significantly inhibited ADP-, Collagen-and arachidonic acid-induced rat platelet aggregation in a dose-dependent manner, and dl-PHPB had a relatively greater inhibitory effect on rat aggregation induced by ADP than by other agonists. Then we used ADP as the inducer to further explore the mechanism of dl-PHPB in order to find the molecular target of dl-PHPB.ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes, P2Y1and P2Y12-The P2Y1receptor couples to Gq, activates phospholipase C, hydrolyzes phosphatidylinositol4,5-bisphosphates to diacylglycerol and inositol1,4,5-triphosphate, increases intracellular calcium. The P2Y12receptor couples to Gi, inhibits adenylyl cyclase, decreases cyclic AMP, finally induces platelet aggregation. Our results showed that dl-PHPB inhibited ADP-induced [Ca2+]i increasing in the presence or absence Ca2+, and had a more inhibition effect on calcium release than inflow.dl-PHPB significantly increased the expression of protein PLC-(3(Ser1105) phosphorylation induced by ADP, without change in total PLC-β3. Moreover, PLC activator attenurated the inhibition effect of dl-PHPB on ADP-induced platelet aggregation. dl-PHPB significantly inhibited the accumulation of IP1induced by ADP, but had no effect on IP1level that induced by PLC activator. As indicated above, dl-PHPB might display its antiplatelet effect through the P2Y1receptor pathway. Prostaglandin E1resulted in a significant increase in cyclic AMP levels, while ADP completely abolished the stimulatory effects of prostaglandin E1on cyclic AMP formation. Ticlopidine, a P2Y12antagonist, attenuated the ADP inhibition of PGE1-induced cAMP elevation. But dl-PHPB did not change the cAMP level, which different from Ticlopidine. The protein expression of PI3K, Akt and p-Akt were not altered by dl-PHPB. Above results indicated that P2Y12receptor might not play a role in the antiplatelt effect of dl-PHPB.dl-PHPB decreased PAI-1level, improved6-keto-PGF1α release, supressed CD62P expression. dl-PHPB did not affect t-PA and thromboxane B2level, prothrombin time, aPTT, and thrombin time.The integrity of vascular endothelial cell function plays a very important role to maintain normal blood flow and antithrombotic function. We use in vitro cultured human umbilical vein endothelial cells as the experimental object, observed the protective effect of dl-PHPB on LPS treated HUVEC, and detected the adhesion molecule ICAM-1and VCAM-1expression in the cell supernatant by ELISA. The results showed that HUVEC survival rate was significantly decreased after treated with different concentrations of LPS for24h. dl-PHPB could increase the survival rate of HUVEC damaged by LPS(10μg/ml). LPS injured endothelial cells, resulting in significantly increased adhesion molecule ICAM-1and VCAM-1release. dl-PHPB downregulated the expression of ICAM-1, but had no significant effect on VCAM-1level.Based on the above study, we demonstrated that dl-PHPB inhibited platelet aggregation, enhanced fibrinolytic activity, supressed platelet activation, improved PGI2/TXA2ratio, protected injured endothelial cells, decreased intercellular adhesion molecule ICAM-1release, without affecting coagulation system. The mechanisms underlying antiplatelet activity may be mediated through P2Y1receptor by suppressing PLC-β activity, decreasing cytoplasmic calcium, inhibiting platelet activation. The antiplatelet target of dl-PHPB is different from Ticlopidine, an antiplatelet drug in clinical.Many Traditional Chinese Herbs have antiplatelet effect, but the phase of study usually at an early stage, lacking of in-depth study of antiplatelet mechanism of action. Traditional Chinese medicine, Puerarin and salvianolic acid B, have significant antiplatelet and antithrombotic activity, and widely used in ischemic heart and cerebrovascular diseases. We observed the antiplatelet effect of puerarin and salvianolic acid B induced by various inducers on rat platelet, and compared the antiplatelet mechanism of puerarin and salvianolic acid B with dl-PHPB. Our aim was to find its target of commonness, and provide the basis for the further development of Chinese medicine and in-depth study.The results showed that puerarin and salvianolic acid B significantly inhibited ADP-induced platelet aggregation. Puerarin inhibited ADP-induced IP1accumulation, without affecting IP1accumulation induced by PLC activator. This result indicated that puerarin may be acting on the P2Y1receptor, inhibition of PLC activity like dl-PHPB. Salvianolic acid B also significantly inhibited platelet aggregation induced by ADP, but had no effect on ADP and PLC activator-induced IP1accumulation. Which indicate salvianolic acid B might not play a role in this pathway through PLC. Above these results suggest that different traditional Chinese medicine monomer have different structure, the target of its antiplatelet aggregation is also diverse. Therefore, the depth study of antiplatelet mechanism of traditional Chinese medicine has important guiding significance to the development of the antiplatelet new drugs from natural products...