Effects Of Tanshinone ⅡA On Calcium Signals In Activated Platelets Using Time-lapse Imaging

Objective: Using time-lapse imaging, record the dynamic change of intracellularcalcium level which is activated by exogenous platelet membrane receptor’s activators.We can observe the effects of Tanshinone ⅡA (TSN) on platelet intracellular calciumion concentration ([Ca2+]i) and research the transmission mechanism of calcium signal inorder to provide experimental basis for clinical medication.Methods: We separated platelet-rich plasma and non-active platelet which was dyedwith fura-2/AM that is calcium signal and indicator, in order to observe the effects ofTSN on platelet [Ca2+]i. The environment temperature was set to37℃. We used plateletactivation agent adenosine diphosphate (ADP),adenosine triphosphate (ATP) andIonomycin (IMC), observed and recorded within5minutes under electromagneticstirring platelet aggregation and fura-2/AM, using for340/380nm ultraviolet excitationwavelength, analyzed image with Andor iQ2image software, analysis the fluorescenceratio of340/380, so as to determine effects of TanshinoneⅡA of [Ca2+]ipeak variationand time to peak (TTP).Results:1Measurement of Platelet AggregationTSN group compared with the negative control group have the significant effects ofreducing the rate of maximum aggregation (P﹤0.05), except0.5μM TSN group;between the two TSN group1μM and4μM, there was no statistically significantdifference (P=1.00﹥0.05);2μM TSN group has significant difference compared withOther groups (P﹤0.05). 2Monitoring of platelet intracellular calcium ion concentration2.1The activity of platelet test, characterized by fluorescence mode, the target cellfluorescence was lighten in the center of the cell and the pseudopodium could be seen,while in ordinary light mode appears integrity quasi-circular platelet. When the test wascarried out in positive control group, the target cell fluorescence disappeared andordinary light mode appears fragmented platelet.2.2Monitoring of calcium signals in activated plateletsWhen using different activating agent and the test was carry on in calcium buffer,the significant change of fluorescence was detected.2μM TSN group could reducefluorescence variation. In a short period of time,2μM TSN make fluorescent stabilityand lower than other groups and significantly prolong TTP.Conclusion:1. TSN can inhibit the platelet aggregation induced by ADP and ATP;2. TSN can inhibit control the deformation process of platelet, keep the activity ofplatelet and its structural integrity;3. TSN may make the levels of intracellular calcium ion concentration lower causedby ADP and ATP. the mechanism may be relate to inhibit the activity of ADP receptorsor store-operated calcium channels;4. TSN can reduce platelet membrane permeability to inhibit extracellular calciuminternal and lower levels of intracellular calcium ion concentration caused by calcium iontransport agent IMC.