Effect Of Tumour-derived Microparticles On Blood Coagulation Status And Its Mechanism

ObjectiveTo investigate the effects of tumor-derived microparticles(TMPs)on the blood coagulation status,and explored its mechanism,providing new ideas and new targets for study and prevent the mechanism of tumor-related thrombosis.methods1.H22-hepatocarcinoma cells passage in mouse abdominal cavity,H22-hepatocarcinoma cells with 200?l were inoculated into the abdominal cavity of Balb/c mice.When ascitic fluid grows,extracted aseptically,washed with PBS and resuspended,and continue inoculated until the third generation.The tumour cell can be used when recovery of cell activity.2.Extraction and identification of Tumour-derived microparticles,extract H22-hepatocarcinoma cells and was induced without serum for 48 hours,microparticles were extracted by differential high-speed centrifugation,Identification by transmission electron microscope,flow cytometry and Nanoparticle tracking analysis.3.Establishmentoftransplantationtumormodelofhepatocellularcarcinoma,H22-hepatocarcinoma cells with 200?L were inoculated subcutaneously in the right axillary fossa of Balb/c mice,wait for it grows into a tumour.After inoculation,the body weight and tumor size of the mice were monitored.At the end of the experiment,the blood was taken to determine the prothrombin time,the activated partial thromboplastin time and the number of tumor-derived microparticles in the plasma,eventually detect the relationship between the counts of TMPs and coagulation time.4.Different structure of Tumor-derived microparticles,10%trypsin-treated TMPs can remove the surface protein,but its membrane lipid structure still exists.10%SDS treatment TMPs the phospholipid structure was removed,leaving only the protein components in the microparticles.5.Coagulation index test,In this experiment,Prothrombin time(PT)and Activated partial thromboplastin time(APTT)were used as indicators reflecting the state of blood coagulation,four-channel coagulometer detects its value.Result1.With the increase of tumor diameter in tumor-bearing mice,the number of TMPs in plasma gradually increased,while PT and APTT decreased gradually(P<0.05),and there was a negative correlation between TMPs and coagulation time.2.The proportion of TMPs in extracted MPs was 70%.3.In vitro experiments,with the increase of the counts of TMPs,PT,APTT gradually shortened(P<0.05).4.When removed the surface protein of TMPs,transmission electron microscopy revealed that the vesicle structure of TMPs were still presence,the results of Nanoparticle Tracking Analysis showed no significant change in particle size,flow cytometry results showed that the TF levels of TMPs decreased significantly,while the procoaglant activity compared with the intact structure TMPs group were weaken(P<0.05),but it was still had procoaglant activity than the PBS control group(P<0.05)5.After removed the TMPs phospholipid structure,transmission electron microscopy results showed that the vesicle structure of TMPs was absent,and only flake-like aggregates remained.The results of Nanoparticles Tracking Analysis showed that the particle size distribution changed significantly.Flow cytometry results showed that the PS levels of TMPs significantly decreased,while the procoaglant activity compared with the intact structure TMPs group were weaken(P<0.05),but it was still had procoaglant activity than the PBS control group(P<0.05)6.Co-removed the surface protein and phospholipid structure of TMPs,while the procoaglant activity compared with the intact structure TMPs group were significant weaken(P<0.05),but it was not had procoaglant activity than the PBS control group(P>0.05)Conclusion1.The extraction of substances in vitro can be identified as MPs by transmission electron microscopy,flow cytometry and Nanoparticle Tracking Analysis.At the same time,Annexin-V~+CD326~+MPs are defined as TMPs,Flow cytometry results show that TMPs account for 70%of MPs.2.In vitro and in vivo experiments shows that with the increase the number of TMPs,the values of PT and APTT gradually decreasing,suggesting that TMPs have procoagulant activity and may mediate blood hypercoagulation in patients with malignant tumors..3.TMPs play a procoagulant activity dependent on surface protein and phospholipid.