Expression of the V264M TFPI mutant in endothelial cell cultures may involve mRNA stability

Abstract

Introduction 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). Materials and 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. Results and 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.