Soluble Expression Of TFPI And Its Fregments And Research Of Their Effects On Mesangial Proliferative Glomerulonephritis

TFPI is the physiological inhibitor on extrinsic coagulation pathway consisting of 276 amino acid residues.Its structure can be divided into an acidic N terminal,three tandem organized Kunitz-type domains(KD1,KD2 and KD3) and a basic C terminal.KD1 and KD2 are responsible for the anticoagulation effect of TFPI, in which KD2 binds to factor Xa(FXa) and inactivates it via the formation of a TFPI-FXa complex.This complex can further bind to the tissue factor-factor FVII complex(TF-FVIIa) induced by KD2 resulting in the formation of a TF-FVIIa-TFPI-FXa quaternary complex.Thereby,a negative feedback regulation is set on the extrinsic coagulation pathway.KD3 and C terminal is the position to combine with heparin,LDL,VLDL,LRP,TSP-1 and LPS etc.In addition,it's the area in TFPI that takes anti-proliferation effect on cultured human neonatal aortic smooth muscle cells and induces apoptosis to human endothelial cells.It has been proved that TFPI can be used in the treatment of a series of diseases. For example,it can decrease the platelet and fibrin deposition at the plaque rupture position in atherosclerosis;transgenic therapy with TFPI can inhibit the vascular intima hyperplasy after the sacculus proprius impairment;in the treatment of sepsis or DIC,it can lower the IL-6 and TNF-a inflammatory factors and increase the survival rate.The third clinical trial of TFPI in sepsis treatment indicates it can decrease the death rate in 28-day period.TFPI can also prevent deep vein thrombosis,reduce the respiratory distress syndrome and ischemical reperfusion injury,and inhibit tumor metastasia.Development of TFPI usage in clinic has been blocked by the protein preparation for a long time.Primarily,TFPI was isolated from serum or HepG2 cell culture supernatant.And then,the eukaryotic and prokaryotic geneengineering method followed up.However,neither of these methods can meet the clinic research needs. Isolation and expression with eukaryotic system are too expensive and lower productive.Expression with prokaryotic system includes complex purification steps and the products are with low activity.Here,we aim to fine a method to solublly express active TFPI.Firstly,we applied the prokaryotic system.With TFPI fused to the C terminal of MBP,we got the soluble expression of the fusion protein.However,the product was proved no activity,which might resulte from the mis-refolding.Then,we changed to express the protein in yeast cell.The TFPI gene was optimized through eliminate a hairpin structure in mRNA secondary structure or samesense mutate the rare codones to yeast cell preferring ones. At last,through the secondary structure optimization,we got a trace expressing stain. Immunoaffinity chromatography was applied in the purification.Though the protein was with a comfortable activity,the molecular was heterogeneously glycosylated and degradated.So this method had yet not solved the problem thoroughly.Distal to the 161 residue of TFPI is the structure including KD3 and C terminal mainly.In the prokaryotic system,we expressed several fragments of this area,with each fragment fused to the C terminus of MBP.These fragments are TFPI_162-188 corresponding to a fragment of the linker between KD2 and KD3,TFPI_187-241 to the third Kunitz domain and TFPI_240-276 to the C-terminal of TFPI,TFPI_162-241 including the linker of KD2 and KD3 and KD3,MBP-TFPI_187-276 including KD3 and C terminal, TFPI_162-276 including all of above.All of these fusion proteins are solublly expressed and purified with affinity chromatograph and ion exchange methods.In the activity analysis,we found the C terminal with anticoagulation activity and the KD3 none; however,KD3 can increase the activity of C terminal.In the respect this area might be concerning with mesangial cell apoptosis induced by TFPI,we added these fusion proteins to the culture media,and found apoptosis in cells which added with MBP-TFPI_162-276, MBP-TFPI_187-276 or MBP-TFPI_240-276 in the culture media. MBP-TFPI_187-241 had no this effect.It clues to that C terminal is the requisite structure to induce mesangial cell apoptosis.Similar to the anticoagulation effect,KD3 may increase the effect of C terminal in the apoptosis induction.Mesangial cells play a central role in many kind glomerulonephritises which always include amplification and activation of mesangial cell.Activated mesangial cell can secret some cytokines as well as Extracellular matrix(ECM),which makes the glomerulus disorganized.Mesangial cell apoptosis or amplification inhibition is a important mechanism in the treatment of these diseases.Since the C terminal of TFPI can induce mesangial cell apoptosis in vitro,we further investigated the effect of this peptide as well as TFPI and TFPI162-276 on mesangial cell in the rat anti-thy-1 nephritis model.Renal functions of each group are also under consideration.We found no mesangial cell apoptosis in vivo;however,the amplification and activation of mesangial cell are inhibited by the C terminal.No statistic differences were found in other two groups compared with the control.The differences in the results between in vivo and in vitro or the differences between C terminal and each of other two groups may due to the heterogeneity in the expression effective and chronergy of this transgenic system.Since the C terminal of TFPI affect mesangial cell both in vivo and in vitro, neither through inducing apoptosis or anti-proliferation and activation;as well as,the C terminal of TFPI presents a protective effect on the renal function,it has a potential usage in the clinic to treat glomerulonephritises that companied with mesangial cell amplification and activation.