ABSTRACT
Platelet activation and subsequent accumulation at sites of vascular injury are central to thrombus formation, which is considered to be a trigger of several cardiovascular diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicinal herb for promoting blood circulation by removing blood stasis. In our previous study, several compounds extracted from this herb, including luteolin4'OßDglucopyranoside (LGP), were revealed to exert inhibitory effects on adenosine diphosphate (ADP)induced platelet aggregation. The aim of present study was to confirm these antiplatelet effects and elucidate the potential mechanisms. Using a plateletaggregation assay, it was revealed that LGP significantly inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid. It was also found that LGP exhibited marked inhibitory effects on the activation of αIIbß3 integrin, the secretion of serotonin from granules, and the synthesis of thromboxane A2. In addition, the results showed that LGP suppressed Ras homolog family member A and phosphoinositide 3kinase/Akt/glycogen synthase kinase 3ß signal transduction. Data from a radiolabeled ligandbinding assay indicated that LGP exhibited apparent competing effects on thromboxane receptor (TP) and P2Y12 receptors. In conclusion, the data presented here demonstrated that LGP, a natural compound from C. nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dualreceptor inhibition on P2Y12 and TP receptors.
Subject(s)
Glucosides/pharmacology , Luteolin/pharmacology , Platelet Activation/drug effects , Receptors, Purinergic P2Y12/metabolism , Receptors, Thromboxane A2, Prostaglandin H2/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Adenosine Diphosphate/pharmacology , Animals , Arachidonic Acid/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Fatty Acids, Unsaturated/metabolism , Female , Glucosides/chemistry , Glycogen Synthase Kinase 3 beta/metabolism , Hydrazines/metabolism , Luteolin/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats, Sprague-Dawley , Serotonin/metabolism , Signal Transduction/drug effects , Thromboxane A2/biosynthesis , Tritium , rhoA GTP-Binding Protein/metabolismABSTRACT
BACKGROUND: Platelet activation and subsequent accumulation at sites of vascular injury perform a central role in thrombus formation, which is believed to be the trigger of several cardiovascular diseases, such as atherosclerosis, myocardial infarction and strokes. In this sense, the search for agents that are capable of blocking platelets aggregation has important implications for these diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicine herb for eliminating stasis to subdue swelling and hemostasis. Our previous study found several compounds extracted from this herb, including 1, 6-di-O-caffeoyl-ß-D-glucopyranoside (CGP), showed inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation. PURPOSE: The aim of current study is confirmation of the anti-platelet effects and elucidation of the probable mechanisms. METHODS: The experiments were performed on platelet rich plasma freshly isolated from SD rat. ADP, U46619 or arachidonic acid (AA) induced platelet aggregation assay were performed to evaluate the anti-platelet properties of CGP. Activated αIIbß3 integrin abundance, serotonin (5-HT) secretion, thromboxane A2 (TXA2) synthesis was determined to assess the effects of CGP on platelet activation. Furthermore, RhoA and PI3K/Akt/GSK3ß signal transduction were analyzed by Western Blotting assay. In addition, radiolabelled ligand binding assay was involved to evaluate the ability of CGP binding to thromboxane prostanoid (TP) and P2Y12 receptors. RESULTS: CGP inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid (AA), significantly. Furthermore, it is also found that LGP exhibited obvious inhibitory effects on αIIbß3 integrin activation, serotonin (5-HT) secretion from granule and thromboxane A2 (TXA2) synthesis. Next, we found that CGP suppressed RhoA and PI3K/Akt/GSK3ß signal transduction. Data from radiolabelled ligand binding assay showed that CGP displayed apparent competing effects on TP and P2Y12 receptors. CONCLUSION: Collectively, the data presented here demonstrated that CGP, a natural compound from Callicarpa nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual-receptor inhibition on P2Y12 and TP receptors.
