| Tissue factor pathway inhibitor-2(TFPI-2),also named as placenta protein-5 (PP5) and matrix associated serine protease inhibitor(MSPI),is a protease inhibitor with Kunitz domains and belongs to serine protease inhibitor superfamily.The mature protein contains a short acidic amino-terminal region,three tandem Kunitz domains and a carboxyl-terminal tail highly enriched in basic amino acids.Previous studies showed that TFPI-2 could inhibit several proteases activity including trypsin,plasmin, chymotrypsin,plasma kallikrein,cathepsin G and a wide variety of matrix metalloproteases(MMPs).TFPI-2,with the similar structure and function of tissue factor pathway inhibitor-1(TFPI-1),is also be recognized as an important endogenous anticoagulant factor and shows the inhibition to FVⅡa/TF complex,FXa,t-PA (tissue-type plasminogen activator),uPA(urokinase-type plasminogen activator),and so on.But the biological functions of TFPI-2 are still unclear.Especially,the interactions of TFPI-2 with other proteins haven't been revealed well.We constructed the bait vector pGBKT7/TFPI-2 and screened the candidate proteins from hFB-cDNA library by use of Matchmaker two hybrid system3 and analysis method of bioinformatics.We fished 95 proteins which displayed different roles in the different biologic processes from hFB-cDNA library by use of pGBKT7/TFPI-2 as bait vector.Such proteins included 32 metablism associated proteins(8 proteometabolism proteins,13 nucleic acid metabolism proteins and 11 glycometabolism proteins),21 signal transduction associated proteins,five Cell growth and/or maintenance associated proteins,seven transport associated proteins, two immune response or apoptosis associated proteins and 28 proteins with unknown function.By advanced analysis from cell location,known protein function and the possibility to express the inserted protein correctly under such yeast two hybrid system,we finally harvested eight proteins associated with the potential interaction with TFPI-2.Furthermore,we investigated into the roles of TFPI-2 in the development of acute lung injury(ALI).ALI,characterized by diffuse damage of pulmonary alveolus-capillary membrane,catholic and leached pulmonary edema and microatelectasis,is a serious clinical syndrome induced by several factors such as infection,trauma,transfusion,shock,acidosis,inhalation of toxic gas,and so on.The pathogenesis of ALI is much complicated,which include an urgent on-set,and a persistent and gradually pathologic process.Acute respiratory distress syndrome (ARDS),a frequent serious clinic disease,is the most serious form of ALI and still has the high mortality up to 40-60%.Acute inflammation cascades,the first-revealed and important pathological changes in the development of ALI,have been evidenced to go through the whole processes of ALI and explained lots of pathological appearances in the development of ALI.Recent studies show that abnormal coagulation whose role has been extensively focused in the development of ALI is associated with kinds of ALI pathological changes such as thrombosis,fibrin deposition,hemorrhage,DIC,and so on.Even the formation of hyaline membrane as a pathological hallmark of ALI has been recognized to be associated with abnormal coagulation.Previous studies showed that many coagulation factors significantly increased in the development of ALI such as tissue factor(TF) and plasminogen activator inhibitor (PAI) while anticoagulant factors such as TFPI-1 decreased or changed no obviously. But the change and the role of TFPI-2 in the development of ALI is still unclear.So we investigated the dynamic changes of TFPI-2 in murine lung tissue during the development of ALI and the relationships between the major coagulation associated factors and inflammatory cytokines.We had established the murine ALI animal model by injecting mice with LPS via tail vein.Pathomorphology detection results evidenced that a serial of pathological appearances associated inflammatory cascades and abnormal coagulation were present at different time points after LPS treatment such as obvious congestion of blood capillary,proliferation and influx of neutrophils,leached pulmonary edema, thrombosis,the fibrin deposition and the formation of hyaline membrane lining the denuded alveolar surface,pulmonary consolidation,hemorrhage,DIC,and so on. Then we detected the dynamic changes of TFPI-2,TFPI-1,TF,IL-1βand TNF-αin murine lung tissue after LPS treatment both at mRNA level by real time PCR and at protein level by immunohistochemical staining.The results showed that TFPI-2 increased significantly in murine lung tissue in time dependent pattern in the development of ALI.TFPI-2 peaked at 2h and then decreased gradually.It almost returned to the normal at 12h.TFPI-2 had the similar dynamic expression pattern to the major inflammatory cytokines IL-1βand TNF-α.TFPI-2 increased following the up-regulation of IL-Iβand TNF-αin ALI and strong correlated with such both inflammatory cytokines.Besides,TF started to increase at 6h and peaked at 12h, while TFPI-1 trended to reduce and started to significantly lower than the control at 10h after LPS treatment.Although TFPI-1 started to return to up gradually after it reached the peak at 10h,it was still lower than the normal even at 24h.The expression pattern of TFPI-2 is different from that of other two coagulation associated factors (TF and TFPI-1).The molar ratios of TF/TFPI-2 at 2h and 6h after LPS treatment were significantly lower than that of control mice,which meant the amount of TFPI-2 expression was much higher than TF expression at these time points.However,the ratios of TF/TFPI-1 were much higher than that of control at 6h,10h and 12h after LPS treatment,these suggested that the coagulation activity of TF was significantly stronger than the anti-coagulation activity of TFPI-1 during these period.And more, we also had seen that TFPI-2 mainly located along alveolar wall as well as TF and TFPI.Our studies suggest that TFPI-2 may play an important role in the development of ALI. |
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