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

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 TFPIV264M mutant in comparison with the wild type protein (TFPIWT). Materials and Methods: We have overexpressed the TFPIV264M mutant and TFPIWT in human coronary artery endothelial cells and compared the expression and activity levels of the mutated protein relative to the TFPIWT. 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 RTPCR. MFOLD was used to predict mRNA secondary structure. Results and Conclusions: TFPIV264M displayed increased protein levels and activity compared to TFPIWT accompanied by an increase in mRNA levels of TFPIV264M due to prolonged stability of TFPIV264M mRNA. The specific activity of the TFPIV264M was similar to TFPIWT, indicating that the mutation does not affect the enzymatic function of the protein. (C) 2008 Elsevier Ltd. All rights reserved.