ADAMTS13 And Variants Structure Function Study In Vitro And In Vivo

PartⅠPurification and identification of Recombinant ADAMTS13 and Variants ProteinsObjective:To get highly purified recombinant ADAMTS13 and variants proteins for further study.Methods:Design specific primers for ADAMTS13 FL, delCUB and MDTCS gene sequences. Amplify the correct DNA sequence and clone into pcDNA3.1-V5-His TOPO vector. Transfect the correct plasmids into HEK293 cells, respectively. Detect the proteins expression by Western blot. Obtain sable cell lines with G418 selection. Collect the conditioned medium secreted from each stable cell line. Purify proteins with Q-fast flow ion binding column, Ni-NTA affinity column and gel filtration column. Verify the molecular weight and purity of each proteins by commassie blue staining.Results:Successfully express recombinant ADAMTS13 and variants proteins. All proteins are verified as high purity and right molecular weight.Conclusion:Successfully express and purify recombinant ADAMTS13 and variants proteins for further study. Part II ADAMTS13 and Vatiants Function in vitroObjective:Under the shear stress condition, to compare the the cleavage activity between ADAMTS 13 and variants in time-dependent, enzyme concentration-dependent and substrate concentration-dependent way.Methods:Measure the purified ADAMTS 13 and vatiants recombinant proteins specific activities by FRETS-VWF73 assay. Based on the same specific activity, identify the cleavage ability under 2500rpm shear stress, which are separately under different time (0,10,20,30 and 60 min), different enzyme concentrations (specific activity 0,12.5,25, 50,100,200 and 300mU/ml) and different substrate concentrations (0,18.75,37.5,75, 150,300 nM).Results:Based on the same specific activities of ADAMTS 13 and variants, the cleavage activity with VWF are increased with the increasing vortex time, enzyme concentrations and substrate concentrations, which are increased rapidly at the beginning and slowly later. There are no statistic differences on cleaving activity with ADAMTS 13 and variants (p>0.05).Conclusion:There are similar cleavage activity on VWF cleaved by same specific activity of ADAMTS 13 and variants under shear stress condition. PartⅢADAMTS13 and vatiants function in vivoObjective:To elucidate the difference of recombinant ADAMTS13 and variants proteins function in thrombosis formation in vivo.Methods:1.Recombinant ADAMTS13 FL and MDTCS proteins were separately injected into ADAMTS13 knock-out (ADAMTS13-/-) mice. Right carotid artery was immersed with 10% FeCl3 on filter paper infused right carotid artery. The time to complete occlusion was recorded with a Doppler detector underneath the vessel injury site. PBS was injected or C domain deleting 6 amino acid residues (del6aa) was injected as the controls. Recombinant TSP5-8CUB protein was injected into wild-type C57BL/6 mice. Right carotid artery was also immersed with 10% FeCl3 on filter paper infused right carotid artery. The time to complete occlusion was recorded with a Doppler detector underneath the vessel injury site. Compare all recombinant proteins of time to complete occlusion.2.ADAMTS13 FL or MDTCS recombinant proteins were injected into ADAMTS13-/-mice after injecting fluorescent labeled platelet infusion.10% FeCl3 injured mesenteric artery for 5min. complete occlusion time is recorded with introvital microscopy and camora. To compare the different complete occlusion times.Results:Time to complete occlusion for PBS, FL, MDTCS or del6aa group was 5.3±0.4min,12.7±1.7min,22.0±2.1min,5.9±1.9min, for WT group was 10.0±1.3min, for TSP5-8CUB was 5.5±2.1min. There was statistic significance between PBS group and WT group, FL group and PBS group, MDTCS group and PBS group, FL group and MDTCS group (p<0.01). There was no statistic significance between del6aa group and PBS group, TSP5-8CUB group and WT group (p>0.05).2. FeCl3 induced mesenteric artery injury mice model, time to occlusion are, WT 12.5±1.2 min, PBS injected ADAMTS13-/- mice are 8.5±0.6 min. There are statistic difference between the two group (p<0.01). The time to occlusion for FL and MDTCS injected ADAMTS13-/- mice are 17.1±1.9 min and 17.2±1.8 min. There are statistic difference betweenFL and PBS or MDTCS and PBS group (p<0.01). Conclusion:MDTCS is sufficient to cleave newly released VWF and can protect the FeCl3 induced-artery thrombosis formation. S domain was critical for ADAMTS13 function in vivo. TSP5-8CUB can resist thrombosis formation in vivo.