Evaluation Of Interactions Between Platelet Or Thrombin And Small Molecules By Capillary Electrophoresis

Interactions between biomacromolecules and biomacromolecules or small molecules can be found in various physiological processes,such as gene transcription,signal transduction and enzymatic reaction.In the receptor theory,drugs produce their therapeutic effects by binding with specific receptors.Therefore,it is of great significance to evaluate these affinity interactions.Capillary electrophoresis(CE)has been widely applied in the study of interactions between nucleic acid,protein,cell membrane,cell and small molecules.Platelet membrane receptors and thrombin,as main therapeutic targets in antithrombotic treatment,have been used in the evaluation and screening of antithrombotic active component.In this thesis,frontal analysis capillary electrophoresis(FACE)and affinity capillary electrophoresis(ACE)were used to evaluate the interactions between platelet and small molecules,thrombin and small molecules,respectively.Besides,online immobilized thrombin microreaction based on CE was developed and used to screen thrombin inhibitors.This thesis mainly includes five parts(chapters).The first part is literature review.The important roles of platelet and thrombin in thrombosis were introduced;and the screening of antithrombotic active components by using platelet membrane receptors and thrombin as targets was summarized.Afterwards,the applications in the study of interactions between biomacromolecules(such as nucleic acid and protein)or biomacromolecules assemblies(such as cell membranes,bacteria and cell)and ligands,and research progress of immobilized enzyme microreaction(IMER)based on CE were also summarized.In the second part,a FACE method was developed to measure the binding site constants and stoichiometries of interactions between platelets and 8 alkaloids(including glaucine,magnoflorine,dehydrocorydaline,palmatine,berberine,isorhynchophylline,rhynchophylline and brucine)using PVA dynamic coating capillary.The FACE results showed that glaucine,dehydrocorydaline and isorhynchophylline have relative strong affinity with platelets according to the binding site constant values.In the third part,an ACE method was developed to measure the binding constants of interactions between thrombin and 10 phenolic compounds(p-hydroxybenzoic acid,protocatechuic acid,vanillic acid,gallic acid,catechin,epicatechin,dihydroquercetin,naringenin,apigenin and baicalein).In the calculation of binding constants,“mobility ratio” was introduced into the calculation equation for eliminating the influence of buffer viscosity on calculation of binding constant.Additionally,the optimum conformations of 6 flavonoids(catechin,epicatechin,dihydroquercetin,naringenin,apigenin and baicalein)bind with thrombin active centre and contributing residues were obtained by molecular docking.The ACE results showed that 6 flavonoids and gallic acid have relative strong affinity with thrombin.On the other hand,the docking results showed that the optimum conformation of 6 flavonoids docking thrombin located in active centre of thrombin and have interaction with SER195 or HIS57 of catalytic triad.In the fourth part,a CE based online thrombin IMER was developed firstly and used to evaluate the thrombin inhibitory activity of catechins including epicatechin(EC),epigallocatechin(EGC),epicatechin gallate(ECG),and epigallocatechin gallate(EGCG).Besides,the inhibition action mechanism of catechins on thrombin was investigated from molecule level using molecular docking.The results showed that thrombin IMER was established successfully and can be used to screen the thrombin inhibitors.Thrombin inhibition assay results showed that ECG and EGCG had good thrombin inhibition activity.On the other hand,the docking results showed that the benzopyran groups of ECG and EGCG were inserted into the thrombin catalytic active pocket and interacted with residues LYS60 F,TRP60D,TRY60 A,IEU99,GLY216,HIS57 and SER195.The last part is conclusion and prospects of the present study.CE plays an important role in the research of interaction between cell or protein and small molecules due to its unique advantages in separation and analysis of biological samples.Combining with molecular docking analysis,CE could further provide more reference information for drug action mechanism and activity prediction.Furthermore,based on CE online IMER with the advantages,such as low enzyme consumption and good stability,could be directly used to screen enzyme inhibitors.