The effects of diquertin on the content of cyclic nucleotides in human platelets was studied. Diquertin in a concentration of 5 mM increased the content of cAMP and cGMP in native and thrombin-activated platelets probably due to inhibition of phosphodiesterase. Increasing the concentration of diquertin above 5 mM did not potentiate this effect. The antiaggregation effect of diquertin was probably associated with the increase in platelet levels of cyclic nucleotides. Key Words: flavonoids; diquertin; platelet; cyclic nucleotidesFlavonoids are natural plant compounds [8]. Diquertin or dihydroquercetin (DQC,3,3',4',5,) is a new preparation (Russia) obtained from shredded larch wood ofLarix dahurica T. and Larix sibirica L. [1]. Flavonoids inhibit enzymes involved in cell activation (protein kinase C, tyrosine kinase, and phospholipase A2). Some flavonoids are strong inhibitors of cyclic nucleotide phosphodiesterase [2,7]. The most potent phosphodiesterase inhibitors display an antiaggregation effect on human platelets in vitro.Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers suppressing platelet activation. Cyclic nucleotides inhibit phospholipase C [5], reduce platelet content of cytoplasmic Ca 2+ [4], and activate Ca 2+-ATPase [6]. cAMP reduces platelet content of cytoplasmic Ca 2+ enhancing its intracellular deposition and effiux from cells [4,6]. cGMP inhibits agonist-induced Ca 2+ mobilization from intracellular stores and Ca 2 § entry, attenuates phospholipase C activation, and modulates protein phosphorylation [3]. Department of General Pathology and Pathophysiology, Russian Academy of Postgraduate Education, MoscowTaking into account the role of cyclic nucleotides in platelet activation, we studied the effect of DQC on the content of cAMP and cGMP in human platelets. MATERIALS AND METHODSVenous blood from 20 fasting donors (12 women and 8 men) was collected into tubes with 3.8% sodium citrate (9:1 ratio). Platelets were isolated by centrifugation at 190g. DQC dissolved in DMSO to final concentrations of l, 5, and 10 mM was added to the platelet suspension and incubated at 37~ and constant agitation for 2 min. Absolute ethanol and HCI were added to the suspension, mixed, and stored at -20~ To analyse the effect of DQC on cAMP and cGMP contents in activated platelets, the suspension of native platelets was incubated with various concentrations of DQC at 37~ and constant agitation for 2 min, thrombin (0.5 U/ml) was added, and platelet aggregation was recorded for 5 min. The reaction was stopped with absolute ethanol and HC1, The samples were mixed and stored at -20~ Platelet contents of cAMP and cGMP were estimated in supematants after ethanol extraction using enzyme immunoassay test systems for quantitative analysis of cAMP and cGMP (Bioimmunogen, Russia).
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