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Background: Dysregulations in cholesterol and lipid metabolism have been linked to human diseases like hypercholesterolemia, atherosclerosis or the metabolic syndrome. Many ABC transporters are involved in trafficking of metabolites derived from these pathways. Pseudoxanthoma elasticum (PXE), an autosomal-recessive disease caused by ABCC6 mutations, is characterized by atherogenesis and soft tissue calcification., Methods: In this study we investigated the regulation of cholesterol biosynthesis in human dermal fibroblasts from PXE patients and healthy controls., Results: Gene expression analysis of 84 targets indicated dysregulations in cholesterol metabolism in PXE fibroblasts. Transcript levels of ABCC6 were strongly increased in lipoprotein-deficient serum (LPDS) and under serum starvation in healthy controls. For the first time, increased HMG CoA reductase activities were found in PXE fibroblasts. We further observed strongly elevated transcript and protein levels for the proprotein convertase subtilisin/kexin type 9 (PCSK9), as well as a significant reduction in APOE mRNA expression in PXE., Conclusion: Increased cholesterol biosynthesis, elevated PCSK9 levels and reduced APOE mRNA expression newly found in PXE fibroblasts could enforce atherogenesis and cardiovascular risk in PXE patients. Moreover, the increase in ABCC6 expression accompanied by the induction of cholesterol biosynthesis supposes a functional role for ABCC6 in human lipoprotein and cholesterol homeostasis.

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.

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.

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.

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.

Introduction: 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. It inhibits activated factor X directly and factor VIIa/tissue factor via a quaternary complex. The composition of human semen is governed by the ejaculatory mixing of sperm-rich epididymal fluid, with secretion provided by the accessory sex glands. It is composed of more than 30 proteins including coagulation and liquefaction proteins. Ovarian follicular fluid plays an important biological role in folliculogenesis and oocyte maturation, and it remains in a hypocoagulable state until ovulation., Materials and Methods: TFPI levels were measured in ovarian follicular fluid gained from the punctured follicles of superovulated women (n=70), and, for the first time, in seminal plasma of 28 healthy ejaculate donors and 23 infertile patients with oligozoospermia, asthenozoospermia, or teratozoospermia., Results: TFPI concentrations determined in liquor folliculi (median 298 ng/ml, 90% range 109-648 ng/ml) were four times higher than the levels found in human blood of healthy individuals. TFPI concentrations in seminal plasma samples of infertile men were significantly reduced (median 2.20 ng/ml, 90% range 0.28-6.02 ng/ml, p<0.07) in comparison to healthy donors (median 3.55 ng/ml, 90% range 0.93-7.90 ng/ml)., Conclusions: The high TFPI levels measured in the ovarian follicular fluid underline the physiological importance of this inhibitor for maintaining the hypocoagulable state. The decreased TFPI concentrations in seminal plasma of infertile men support the possible correlation between the coagulation properties of ejaculated semen and male fertility.

Tissue factor pathway inhibitor (TFPI) is an important regulator in the extrinsic blood coagulation pathway. Although the regulatory biochemical role of TFPI is evident, the clinical significance of this proteinase inhibitor remains to be elucidated. The definition of a clinical TFPI deficiency seems to be more complex than that of other coagulation inhibitors because the activity and concentration of circulating TFPI can not be considered a true measure of in vivo levels. Its determination in plasma samples by immunological methods or functional assays has been shown to be inadequate in the detection of a clinical deficiency. Therefore, we screened genomic DNA samples of blood donors and thrombotic patients for alterations in the TFPI gene to assess the influence of a modified TFPI in venous thromboembolic diseases. We detected a single nucleotide substitution in exon 7 (536C-->T) leading to a proline to leucine exchange at amino acid position 151 of the protein ([P151L]TFPI) and found the prevalence of heterozygous carriers in German unrelated blood donors to be 0.2% (n = 5120). Four unrelated persons out of 14 probands carrying the genetic variation could be linked to venous thrombosis. For calculation of a potential risk for venous thrombosis for carriers of the mutation we investigated healthy blood donors about thrombotic events. 7 out of 308 blood donors were found to have a history of venous thrombosis, one of them carried the TFPI mutation. Statistical calculation showed a significant relative risk for venous thrombosis for individuals with the trait (odds ratio, 9.3; confidence interval, 1.8-48.6; p <0.01).