Isolation, Identification And Mechanism Study Of The Antithrombotic Component DPf3 Of P. Wilhelmi Based On A Multi-omics Model

Background:Thrombotic diseases have been regarded as one of the first leading cause of human death,and antithrombotic agents,including antiplatelet agents,anticoagulants and thrombolytic agents,have always been applied to treat these diseases in clinic,whereas the majority of medicines accompanied with bleeding side effects.In the view of traditional Chinese medicine,thrombotic diseases are closely related to the syndrome of blood stasis,and the type of blood-activation and stasis-removal medicines have always been applied to treat blood stasis syndrome.Among these,animal drugs（such as leeches and earthworms）have generated great attention.Earthworms and their preparations have been used as anticoagulants and thrombolytic agents for several centuries without bleeding side effects,and orally administrated lumbrokinase preparations have been applied to protect cardiovascular among many countries,and have been marketed as a second class new drug in China.In traditional Chinese medicine research,animal drugs have always been a knotty problem.As a Chinese Pharmacopoeia-recorded species,Pheretima guillemi（Michaelsen）is insufficient in research.The antithrombotic substance,the targets,and corresponding mechanisms of P.guillemi are still unclear,which limits its clinical application.Therefore,it is meaningful to study the bioactive components in P.guillemi and its antithrombotic mechanisms.Considering that protein is the major antithrombotic components in P.guillemi,a transcriptomic-proteomic integrated strategy was applied to illustrate the bioactive components in P.guillemi and its antithrombotic mechanisms.Objective:An omics analysis strategy was used to investigate the proteins in P.guillemi,its antithrombotic components were gathered by purification,and corresponding protein sequences,structure,and properties were identified.The animal model was applied to evaluate its thrombus prevention effect,thrombolysis ability,and vascular endothelium protection effect.The combination of molecular docking and ex vivo experiments were used to explore its antithrombotic targets and mechanisms,and the underlying mechanisms of vascular endothelium protection effect.The biological process and signaling pathways involved in its antithrombotic effect and vascular protection ability were holistically analyzed by proteomics.Methods and Results:Part 1 Study on the non-reference transcriptome construction of P.guillemiSequencing analysis was carried out over Illumina to precisely and integratedly establish a set of mRNA transcripts in P.guillemi,and then translated into protein sequences database which supports further research on this species.In general,over twenty million clean reads were generated,in which high-quality clean reads（Q20）accounted for over 97%.An average of over one hundred thousand of transcripts and unigenes were Trinity assembled from these high-quality clean reads to construct non-reference transcriptome of P.guillemi,and protein sequences were generated by translation to construct local protein database.A total of 11259 coding sequences were predicted via ESTScan,which could be regarded as a database to design primers.These unigenes were searched and annotated against a public database and a total of 38.21%unigenes were annotated.Part 2 Study on the identification and characterization of Pheretima guillemiIn this part,a transcriptomic-proteomics-anticoagulant bioactivity integrated approach was applied to appraise and characterize the anticoagulant specific bioactive proteins of P.guillemi extract applying the local databased constructed in Part 1.Besides,a fibrinogen-thrombin time（Fib-TT）method was established to evaluate the anticoagulant activity of P.guillelmi and to assess the stability of anticoagulant activity in different incubation systems;and the protein patterns of P.guillemi extract in different incubation systems were used to speculate its specific bioactive components.Thirty-one proteins were identified against established transcriptome.The results indicated that the established Fib-TT method expressed to be with satisfactory methodological evaluation results,and can be used to evaluate the bioactivity of P.guillemi.The bioactivity of P.guillemi extract was quite stable at near-neutral conditions,and the optimal temperature was 30-50℃.The protein band near 26 kDa was speculated to be the vital bioactivity component.Part 3 Study on the purification and identification of antithrombotic proteins in P.guillemiThe strong antithrombotic activity of P.guillemi extract was verified in Part 2.In this part,an ion-exchange chromatography method was used to isolate and purify the antithrombotic proteins,named DPf3,with Fib-TT as a criterion,and SDS-PAGE and MALDI-TOF-MS were combined to evaluate its protein pattern and molecular weight.A transcriptomic and bottom-up proteomics combined approach was used to identify the constituents and categories of the main proteins in DPf3,DPf3 ID NO.1 and NO.2,and both full protein sequences were revealed by de novo sequencing.The secondary and tertiary structures of DPf3 were identified by circular dichroism and I-TASSER in order,and the speculated three-dimensional（3D）structure was evaluated with SAVES.The hemolytic method was applied to study the blood compatibility of DPf3.The study indicated that the molecular weight of two dominated proteins in DPf3,named DPf3 ID NO.1 and NO.2,were 36121.745 Da and 24485.004 Da consisting of 329 and 241 amino acids in order,and their full protein sequences were uncovered by de novo sequencing,with 100%coverage compared with>Ac44553_g1_i1₁ and>Dc43026_gl_i1₂ in the constructed local database.The secondary structure of DPf3 is the mixture of α-helix（19%）,β-sheet（30%）,and random coil（51%）.Besides,DPf3（under 4.21 mg·mL-1）was with good blood compatibility.Part 4 Study on the effect of DPf3 on zebrafish vascular damaged thrombus modelThis part aimed to evaluate the antithrombotic activity and vascular endothelium protection effect of DPf3 in vivo using ponatinib-induced vascular endothelial injury zebrafish thrombus model.DPf3（4.2-12.5 ng/fish）was found to possess strong thrombus prevention ability which was similar to that of aspirin（45 μg·mL-1）.DPf3（4.2 ng/fish）exerted robust thrombolytic ability which was comparable with urokinase（5 ng/fish）.Besides,DPf3 exerted a robust ability to protect vascular damage.Part 5 Study on the in vitro bioactivity and mechanisms of DPf3In this part,docking analysis together with in vitro experiments were supplied to study the in vitro antithrombotic bioactivity and mechanisms of DPf3.ZDOCK online software was used to investigate the interaction between DPf3 ID NO.1,NO.2 and four targets including fibrin,fibrinogen,plasminogen,and thrombin.Then,zymography,light scattering assay,morphological study,fibrin-plate assay,in vitro thrombolysis assay,and four coagulation tests were combined to investigate the bioactivity and mechanisms of DPf3.By docking analysis,DPf3 ID NO.1 and NO.2 were predicted to have direct interaction with fibri（noge）n and plasminogen.The in vitro experiments indicated that DPf3 had a trivial influence on plasminogen,whereas DPf3 displayed a robust hydrolytic manner on fibrin,fibrinogen,and generated thrombus.Compared with fibrin clots,fibrinogen might be the initial target of DPf3,and DPf3 had a stronger effect on fibrinogen than fibrin clots;by four indexes of coagulation assay,DPf3 could prolong APTT,decrease Fib content and was able to slightly prolong TT.Part 6 Study on the vascular protection bioactivity of DPf3 via cell modelIn this part,HUVECs was applied to evaluate vascular protection effect of DPf3 with（hi）ox-LDL as an inducer,and the indications,including cell viability,cell cytotoxicity,ROS generation,cell migration,VCAM 1 expression and monocytes adhesion and angiogenesis were all applied to investigate corresponding mechanisms.The results indicated that DPf3 could prevent HUVECs from damage.The mechanisms were concerning improving the cell viability and protecting the integrity of cell membranes;decreasing intracellular ROS generation in damaged HUVECs;reducing VCAM 1 expression and monocytes adhesion;impairing migration ability and angiogenesis of damaged cells.Part 7 Study on the proteomics analysis of DPf3 on zebrafish vascular-damaged thrombotic modelThis study aimed to holistically illustrate the biomarkers,biological process,and signaling pathways involved in the antithrombotic and vascular protection ability of DPf3 via proteomics analysis.By comparing the acquired data of the model group with the normal group,a total of 1149 biomarkers were identified,582 of them were significantly up-regulated proteins,and 567 of them were significantly down-regulated proteins.By compared the acquired data of DPf3-treated group with model group,a total of 66 biomarkers were identified,46 of them were significantly up-regulated proteins,and 20 of them were significantly down-regulated proteins;among them,a total of 23 biomarkers had significant callback effect.By summarizing and analyzing the biological processes and signaling pathways involved in biomarkers,the mechanisms of DPf3 exerting antithrombotic and vascular protection effects were mainly related to the regulation of lipid metabolism,regulation of energy metabolism,complement and coagulation system,ECM receptor interaction,and MAPK signaling pathway.Conclusion:This thesis aims to purify,identify,and characterize specific antithrombotic proteins in P.guillemi and to investigate corresponding mechanisms based on transcriptomic-proteomics integrated omics analysis.P.guillemi extract was investigated to possess good antithrombotic property,and 31 of proteins were identified in P.guillemi.Among them,a group of antithrombotic specific proteins was purified,named DPf3.DPf3 exerted excellent thrombus prevention,thrombolysis,and vascular protection ability,and was found to have good blood comparability.The antithrombotic mechanisms of DPf3 involved（1）robust fibrin and fibrinogen hydrolysis ability,activating plasminogen to form plasmins,（2）obvious thrombolysis ability,（3）exerting antithrombosis effect via intrinsic/common coagulation pathway.The mechanisms of vascular protection manner included（1）improving cell viability,protecting the integrity of cell membrane of impaired HUVECs,and decreasing the intracellular ROS generation of impaired HUVECs,（2）lowering the expression of VCAM 1 and monocytes adhesion ability,（3）weakening the migration ability of impaired cells and preventing the spread of inflammatory cells,（4）inhibiting angiogenesis and further inhibiting the formation of vascular intima.Proteomics analysis was applied to holistically illustrate the pathological process of the antithrombotic and vascular protection mechanism of DPf3;and the related biological process and KEGG pathways involved regulation of lipid metabolism,regulation of energy metabolism,complement and coagulation system,ECM receptor interaction,and MAPK signaling pathway.In summary,this study identified the antithrombotic constituents in P.guillemi and preliminarily clarified its targets and mechanisms.This thesis provides a scientific basis for clinical application of P.guillemi,and provide the foundation and basis for further molecular mechanisms evaluation of its antithrombotic properties,and also provides a strategy to investigate animal drugs of Chinese materia medica.