Expression of the V264M TFPI mutant in endothelial cell cultures may involve mRNA stability.
BACKGROUND
Tissue factor (TF) pathway inhibitor (TFPI) is the endogenous inhibitor regulating TF-induced blood coagulation. Several polymorphisms have been identified in the TFPI gene and some of them have been correlated with variations in plasma TFPI levels. The aim of the present study was to characterize the TFPI(V264M) mutant in comparison with the wild type protein (TFPI(WT)).


METHODS
We have overexpressed the TFPI(V264M) mutant and TFPI(WT) in human coronary artery endothelial cells and compared the expression and activity levels of the mutated protein relative to the TFPI(WT). The protein levels were determined by ELISA, the inhibitory activity of the proteins was assessed with a chromogenic substrate assay. The mRNA level of the two TFPI variants was determined using real time RT-PCR. MFOLD was used to predict mRNA secondary structure.


CONCLUSIONS
TFPI(V264M) displayed increased protein levels and activity compared to TFPI(WT) accompanied by an increase in mRNA levels of TFPI(V264M) due to prolonged stability of TFPI(V264M) mRNA. The specific activity of the TFPI(V264M) was similar to TFPI(WT), indicating that the mutation does not affect the enzymatic function of the protein.
Skretting
Grethe
G
Department of Hematology, Ullevål University Hospital Trust, Oslo, Norway. grethe.skretting@medisin.uio.no
Lien
Trude
T
Sandset
Per Morten
PM
Iversen
Nina
N
D001483:Base Sequence; D002478:Cells, Cultured; D003198:Computer Simulation; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008074:Lipoproteins / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011110:Polymorphism, Genetic; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D008074:Lipoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor