Research On The Effect Of Salvianolate On Platelet Inhibition, A Water-soluble Component From Danshen

Salviae miltiorrhiza (Danshen) consists of water-soluble ingredients and fat-soluble ingredients. Salvianolate is the mixture of salvianolic acid B (Sal-B>80%) purified from Salviae miltiorrhiza, has been used to treat atherothrombotic diseases in clinical practice. So far, there is no evidence clearly showing the underlying mechanism. According to the preliminary study, we surmise that this effect is related to inhibition of platelet activation.Objective-This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome. Meanwhile we investigated the antiplatelet effects of salvianolic acid B (Sal-B), the major component which composes80%of salvianolate to explore the underlying mechanism.Methods-Sixty three ACS Patients were sequent divided into two groups: salvianolate group (n=32) was given Salvianolate200mg/day intravenously in addition to standard cornonary heart disease treatment, control group (n=31) was given standard treatment, drug therapy including aspirin (Bayaspirin) and clopidogrel (Plavix)(for the first time given a loading dose of300mg, followed by a regimen therapy of0.1g/d and75mg/d each), statins, LMWH. We evaluated the effects of salvianolate on platelets by flow cytometry, in patients with acute coronary syndrome by measuring ADP-induced PAC-1binding and P-selectin expression on platelets. The antiplatelet aggregative ability of Sal-B was examined in laboratory. Human platelets in suspension were preincubated with Sal-B, stimulated with the agonist thrombin, adenosine diphosphate (ADP), collagen, U46619or arachidonic acid (AA). Studies for platelets Gq-coupled P2Y1receptor was examined by intracellular Ca2+release with fluorescent Ca2+indicator. Gi-coupled P2Y12receptor, examined by phosphorylated form of vasodilator-stimulated phosphoprotein (VASP) in western blot. Radioimmunoassay was used to measure intracellular cyclic adenosine monophosphate (cAMP) content. Interaction between ADP and P2Y12receptor were monitored under direct physical methods with atomic force microscopy (AFM).Results-A total of63patients which presented to the emergency room with chest pain were consecutively enrolled, In ACS patients, additional treatment normalized platelet hyper function and reduced PAC-1expression activated by ADP (salvianolate group47.05±10.04%VS control group52.18±6.18%, P<0.05), and it also reduced CD62p expression activated by ADP (salvianolate group39.48±8.33%vs control group45.04±6.68%, P<0.01). In vitro, platelets aggregation was inhibited after preincubated with Sal-B induced by AA, U46619, thrombin or collagen in human washed platelets, especially with ADP as agonist.In the range of8-280μM, Sal-B concentration-dependently inhibited platelet aggregation induced by10μM ADP. It had no obvious effect on the initial rise in P2Yi downstream intracellular Ca2+release but accelerated the decay of Ca2+slightly, in the absence of extracellular Ca2+stimulated by ADP.Sal-B antagonized intracellular cAMP decrease induced by ADP and further increased forskolin-elicited increase in human platelets. Basal and sodium prusside-stimulated intracellular cAMP levels in human washed platelets were also enhanced by Sal-B. Similar to phosphodiesterase (PDE) inhibitor IBMX, Sal-B inhibited platelet PDE activity as measured by HPLC. But the maximal inhibition of280μM Sal-B (42%inhibition) on PDE extracts was lower than that of100μM IBMX (70%inhibition), a non-specific PDE inhibitor, which suggested that Sal-B might have minor affect on platelet PDE.With AFM analysis, we observed Sal-B exhibited P2Y12antagonism activity as evidenced by inhibiting the interaction between ADP and P2Y12receptor expressed in CHO-K1cells directly.Conclusions-For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12antagonism which may account for the clinical antiplatelet effects of salvianolate.