Relative quantification of glycosaminoglycan-induced upregulation of TFPI-mRNA expression in vitro.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial (Ea.hy 926) and in chondrosarcoma (SW 1353) cells. METHODS: We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene. RESULTS: Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect. CONCLUSION: Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Expression and characterization of wild-type TFPI and the [P151L]TFPI mutant in insect cells.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation. In earlier studies we could identify the [P151L]TFPI mutant, and we could also demonstrate that heterozygous carriers of this mutant show a nine-fold increased risk for deep venous thrombosis (DVT). To express greater amounts of both proteins and to enable their characterization, we expressed wild-type TFPI as well as [P151L]TFPI in High Five insect cells with expression rates of up to 215 ng/ml for wild-type TFPI and 214 ng/ml for [P151L]TFPI. The specific inhibitory activities for the recombinant proteins were determined as 11.3 and 11.5 mU/ng, respectively. Both proteins were detected via Western blot analysis and ELISA. The recombinant proteins' inhibitory activities were characterized by a chromogenic assay and by the determination of a modified activated thromboplastin time (aPTT) in which both of them proved to be inhibitorily active. We also examined both recombinant proteins' binding properties to glycoproteins, glycosaminoglycans, lipoproteins and tissue factor. Our results show that we have developed an efficient model system for the recombinant expression of inhibitorily active wild-type TFPI as well as [P151L]TFPI in insect cells, and we were able to characterize both proteins' inhibitory properties by determination of their influence on the aPTT and also their binding properties. Although both recombinant proteins did not show a significant difference in their effect on the aPTT, their binding properties differed significantly between the wild type and mutant protein.

Analysis of sequence variations in the promoter region of the human tissue factor pathway inhibitor 2 gene in apoplectic patients and blood donors.

Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine protease inhibitor with homology to TFPI-1, an important regulator of the extrinsic pathway of blood coagulation. Recent studies have focused on TFPI-2 and its implications for atherosclerosis. The promoter region and the exons of the human TFPI-2 gene were screened for sequence variations in 41 apoplectic patients and 140 blood donors with no history of ischemic stroke. The sequence variations -567T>C, -546T>C, -353A>G, -161G>C, -167G>A, -47C>A, and -18C>A, which are located in the TFPI-2 promoter, were discovered in both cohorts with allelic frequencies ranging from 0.3 to 2.4%. The influence of these sequence variations on the transcriptional activity of the TFPI-2 gene was investigated in HEK-293 cells using a promoter test system. A wild-type TFPI-2 promoter fragment 716 bp upstream of the translation start site was cloned into a secreted alkaline phosphatase expression vector, and the sequence variations were introduced by site-directed mutagenesis. Interestingly, the promoter activity of the tested mutants was reduced by 1.3- to 2.8-fold compared to that of wild-type control. The variation -18C>A, where a putative binding site of the transcription factor Sp-1 is located, had the strongest effect on transcriptional activity. In conclusion, our present study shows that the transcription of TFPI-2 is changed by single nucleotide polymorphisms and that the sequence variations in transcription factor binding sites of the TFPI-2 promoter may influence the regulation of this gene.

Elevated TFPI levels in female hyperhomocysteinemic patients.

OBJECTIVES: Hyperhomocysteinemia is a risk factor for cardiovascular diseases, while tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. We investigated the in vivo influence of elevated homocysteine concentrations on TFPI levels. METHODS: Total TFPI levels of 82 hyperhomocysteinemic patients were determined. RESULTS: The patients' TFPI levels were significantly elevated compared to blood donors, with a more pronounced effect in females than in males. CONCLUSIONS: We have shown a gender-dependent TFPI elevation in hyperhomocysteinemic patients.

Age dependent assessment of TFPI levels in follicular fluid of women undergoing IVF.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor which plays a central role in the extrinsic pathway of blood coagulation. A significant role of TFPI for the follicular development has been discussed in recent studies, and thrombotic complications during IVF procedure are a common problem. To elucidate the pathophysiological mechanisms underlying these problems, we have measured TFPI levels in human follicular fluid (hFF) of women undergoing in vitro fertilisation (IVF). METHODS: Total TFPI concentrations were determined in hFF of 28 women undergoing IVF treatment, 6 of whom developed an ovarian hyperstimulation syndrome (OHSS). RESULTS AND CONCLUSIONS: This is the first study to demonstrate an age-dependance of TFPI concentrations in hFF. Additionally, TFPI levels in hFF of women developing OHSS were determined as 323+/-66.8 ng/mL (mean+/-SD) in comparison with 279+/-137 ng/mL for non-OHSS patients. Our findings demonstrate that, unlike the decreased TFPI levels found in OHSS patients' blood, there is no statistically significant difference in hFF TFPI levels between OHSS and non-OHSS patients. Furthermore, we could show that the outcome of the IVF procedure is not correlated with TFPI levels in hFF.