Study On Optimization Of Purification Of Atrase A And Its Effects On Endothelial Cells

Objective:Atrase A is a potential anticoagulant thrombolytic drug candidate molecule,a three-step purification process of Atrase A has been established in the early stage.In order to establish a scale-up separation and preparation process of Atrase A,this study carried out an optimization of the purification process based on the two-step method,and carried out a study on the effect of Atrase A on human microvascular endothelial cells from the perspective of safety.Methods:Crude cobra venom was prepared by a combination of different two-step purification methods（cation chromatography and heparin affinity chromatography,cation and molecular sieve chromatography,heparin affinity and cation affinity chromatography,heparin affinity chromatography and cation chromatography）,the isolation and preparation of Atrase A was optimized by changing buffer system,flow rate,washing and desalination concentration,gradient and other conditions.In addition,the preparation process of the two-step Atrase A obtained in the previous stage was amplified.The isolated samples were identified by SDS-PAGE,and the activity of Atrase A was studied by blood coagulation four items and TEG.Human microvascular endothelial cells were exposed to different concentrations of Atrase A in vitro cell culture experiments,the effect of Atrase A on the growth of human microvascular endothelial cells was detected by MTT and morphological observation.The effects of Atrase A on ROS,Ca²⁺influx,mitochondrial membrane potential,apoptosis,cycle of human microvascular endothelial cells and the expression of ICAM-1 on the surface of HMEC-1 were determined by flow cytometry.The change of ATP content was detected by chemiluminescence.The contents of inflammatory mediators IL-6,IL-8 and MCP-1 in the supernatant of cell culture were detected by ELISA.The m RNA expression levels of ICAM-1,VCAM-1,IL-6,IL-8,MCP-1 and IL-1βwere detected by RT-q PCR.Western blot was used to detect apoptosis and the expression levels of NF-κB and MAPK signaling pathways.The inactivated Atrase A was prepared by incubation of EDTA to observe its effect on endothelial cells.Results:Through the combination of different two-step purification methods,there was a small amount of miscellaneous protein in the Atrase A obtained,in which the crude poison of cobra venom passed Heparin Sepharose 6 Fast Flow heparin affinity chromatography and Superdex 75 prep grade molecular sieve chromatography,with the highest purity of 85.22%.A further study was made on the preparation process of Atrase A obtained by SP Sephadex C-25 cationic chromatography and Hitrap Heparin HP heparin affinity chromatography.The scale-up of this process were preliminarily confirmed.The prepared Atrase A activity assay showed that it exhibited anticoagulant activity after incubation with plasma.The results of TEG showed Atrase A has significant anticoagulant activity.In vitro cell assays,MTT results showed that 0.003 mg/m L Atrase A had little effect on the viability of endothelial cells,while 0.03 mg/m L and 0.3 mg/m L Atrase A showed significantly inhibit the growth of endothelial cells.At the same time,the microscopic results showed that Atrase A could significantly change the morphology of endothelial cells.Flow cytometry showed that Atrase A could affect the cycle of endothelial cells.The percentage of G1 phase cells decreased significantly at 6 h,and the percentage of G2/M phase cells increased significantly at 12 h.After HMEC-1 cells were exposure to Atrase A,ROS and Ca²⁺levels were increased,mitochondrial membrane potential and ATP contents were decreased,ICAM-1,IL-6,IL-8 and MCP-1 expressions were up-regulated,NF-кB p65 and ERK phosphorylation levels were significantly up-regulated.Atrase A induced HMEC-1 cells apoptosis at 48 h,and apoptosis-related protein Bax/Bcl-2 ratio and caspase3 were up-regulated.However,after the treatment of inactivated Atrase A on endothelial cells,the morphology of endothelial cells did not change significantly,the ROS level was not up-regulated,and apoptosis did not occur.Conclusion:1.The purification method of Atrase A obtained by crude cobra venom after Heparin Sepharose 6 Fast Flow heparin affinity chromatography and Superdex 75prep grade molecular sieve chromatography was the most reasonable and the highest purity of Atrase A was 85.22%,2.The magniability the two-step method of SP Sephadex C-25 cation chromatography and Hitrap Heparin HP heparin affinity chromatography was preliminarily confirmed.3.In vitro cell studies showed that cobra venom metalloproteinase Atrase A induced inflammatory response and cell apoptosis in endothelial cells via its metalloproteinase domain.