Study On The Antiplatelet And Antithrombotic Effects And Molecular Mechanisms Of The Flavanoid Derivative XJ-8 From Dragon's Bloo

Thrombotic disease is a kind of disease that is resulted from thrombosis in blood vessels for a variety of reasons,which leads to ischemia or infarction of the heart,brain,lungs or other important tissues and organs,and even leads to organ function damage,disability or death.It is characterized by high morbidity,high disability and high mortality and seriously endangers human life and health.Antithrombotic drug therapy has become the main management method for thrombotic diseases.However,the commonly used antithrombotic drugs have the disadvantages of unstable efficacy,higher risk of massive hemorrhage and repeated analysis of coagulation function.In China,Traditional Chinese Medicine（TCM）has a long history in the treatment of thrombotic diseases.Daemonorops Draco is one of TCM drugs with the founction of activating blood and removing blood stasis.It was called "Huo Xue Sheng Yao" in the book "Compendium of Materia Medica".Both clinical and basic studies have demonstrated its antithrombotic effects.However,the active components and mechanism of its antithrombotic effects are still unknown.Activated platelets release adenosine trisphosphate（ATP）and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants.However,bioluminescence analysis of ATP possesses poor linearity and quickly signal attenuation,and is hard to accurately determine ATP release of platelets and be used in high throughput screening of platelet inhibitors.The present study was designed to optimize bioluminescence analysis of ATP released by platelets and then screen the specific antiplatelets inhibitors from Daemonorops Draco using the optimized ATP analysis,investigate its mechanism and discover new antiplatelet molecular targets,and find antiplatelet drugs that have strong activaty and less adverse reaction.Firstly,the bioluminescence analysis method of platelet ATP release were optimized:The results showed that accuracy of ATP analysis was significantly improved by adding coenzyme A（CoA）and signal attenuation of ATP analysis was greatly postponed by adding bovine serum albumin（BSA）both in Hank’s balanced salt solution（HBSS）and Tyrode’s buffer.Furthermore,optimized bioluminescence analysis of ATP accurately determined ATP release of activated platelets and evaluated inhibitory effects of Ly294002 and Staurosporine on the activation of platelets.Thus,we have successfully constructed an optimized bioluminescence analysis of ATP which can be used in high throughput screening of platelet inhibitors.Secondly,the antiplatelts compunds from Daemonorops Draco were screened.The antiplatelet activity of the 4 crude extracts and 21 compounds isolated from Daemonorops Draco were examined using the modified ATP analysis.The results showed that the total,water,petroleum ether and ethyl acetate extracts showed strong inhibitory effects on thrombin induced ATP release,and the IC50 were 0.92±0.10,0.98 ± 0.10,2.35 ± 0.07 and 3.27 ± 0.30μg/mL,respectively.9 of the 21 compounds showed inhibitory effects on thrombin induced ATP release in platelets.Among them,compound 11 had the strongest inhibitory effect（IC507.8 ± 0.7 μM）,followed by compounds 1,7 and 8（IC50 11.2 ± 0.9,18.7 ± 0.5 and 22.4± 5.1μM）,and then were compounds 4,5,9,13 and 16（IC50 59.7 ± 2.1,31.1 ± 6.5,51.0± 0.6,40.2 ± 14.7 and 41.1 ± 4.2 μM）.Next,effects of the 4 crude extracts and compounds（1,4,5,7,8,9,11 and 16）on platelets aggregation were anaylzed.The results showed that the total,petroleum ether,and ethyl acetate extracts and compound 4,7,8,11 and 16 inhibited collagen induced platelets aggregation.More interestingly,compound 8（XJ-8）showed strong inhibitory effect on collagen induced platelets aggregation,comparing with other compounds and the crude extracts.This indicates that compound 8（XJ-8）has the potential to be developed as an antiplatelets drug candidate.Finally,the anti-thrombotic effect of XJ-8 and its mechanism were studied.In vitro,effects of XJ-8 on granule release,membrane protein expression,platelets aggregation,platelets-fibrin clot retraction,APTT,PT and TT were analyzed.In vivo,effects of XJ-8 on thrombosis were analysised using the tail bleeding time mouse model and FeCl₃ induced common carotid thrombosis rats model.Subsequently,55 protein kinases that play important role in the processes of platelets activation were selected and the direct inhibition of XJ-8 on them was determined using kinase assay.At last,effects of XJ-8 on MAPK pathway activation were analysized by western blotting.The results showed that XJ-8 significantly inhibited collagen and thrombin induced ATP release in platelets and platelets aggregation and had no effects on collagen and thrombin induced integrin αⅡbβ3 and CD62P activation and clot retraction.In vivo,XJ-8 significantly prolonged tail bleeding time and increased hemoglobin content in mice.XJ-8 significantly inhibited the formation of common carotid artery thrombosis induced by FeCl₃,The results of protein kinase assay showed that XJ-8 selectively inhibit MAP3K3 and the inhibition ratio was only slowly increased with the increase of the concentration of XJ-8,indicating XJ-8 was a moderate and selected MAP3K3 inhibitor.And XJ-8 selectively inhibit collagen and thrombin induced phosphorylation of MEK1/2,ERK1/2 and AKTS473.In the present study,we have successfully established an optimized bioluminescence ATP assay which can be used in high screening of platelets inhibitors.9 anti-platelet compounds were screened from Daemonorops Draco,and XJ-8 has a strong inhibitory effect on platelet activation and has the potential to be developed as a anti-thrombotic drug.XJ-8 has been identified as a moderately potent and highly selective MAP3K3 inhibitor for the first time,which can be a lead compound for the development of MAP3K3 inhibitors for the prevention and treatment of thrombotic diseases.And also,the anti-platelets and anti-thrombotic effects of XJ-8 were through downregulating the phosphorylation of MEK1/2-ERK1/2 and AKTS473 signaling.