Functional and immunologic methods for the measurement of human tissue factor pathway inhibitor

Bognacki, John; Hammelburger, J W

doi:10.1097/00001721-199506001-00011

Cited by 31 publications

(18 citation statements)

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“…This assay measures the generation of FXa by TF/FVIIa complex in TFPI-depleted plasma relative to a TFPI standard 32 . TFPI activity is shown in plasma U/ml (with 1 U/ml corresponding to 55 ng/ml or 1.4 nM).…”

Section: Resultsmentioning

confidence: 99%

Factor VII–Activating Protease Promotes the Proteolysis and Inhibition of Tissue Factor Pathway Inhibitor

Kanse

Declerck

Ruf

et al. 2012

ATVB

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Objectives Factor VII activating protease (FSAP) activates FVII as well as pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating haemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk for carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. Results and Methods Using screening procedures to determine the influence of FSAP on various haemostasis-related processes on endothelial cells we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anti-coagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP, that specifically inhibited FSAP binding to TFPI, reversed the inhibitory effect of FSAP on TFPI. Conclusions The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating haemostasis and proliferative remodeling events in the vasculature.

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Section: Resultsmentioning

confidence: 99%

Factor VII–Activating Protease Promotes the Proteolysis and Inhibition of Tissue Factor Pathway Inhibitor

Kanse

Declerck

Ruf

et al. 2012

ATVB

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“…This assay measures the inhibition by TFPI of factor Xa generation after activation by TF/VIIa catalytic complex and was carried out at 37°C. 13 Duplicate samples were measured from 3 separate experiments.…”

Section: Measurement Of Tfpi Activitymentioning

confidence: 99%

Expression of Tissue Factor Pathway Inhibitor in Vascular Smooth Muscle Cells and Its Regulation by Growth Factors

Caplice

Mueske

Kleppe

et al. 1998

Circulation Research

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Abstract-Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours (PϽ0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4-to 5-fold and 2-to 3-fold, respectively (PϽ0.05), and these growth factors accounted for Ϸ50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B. (Circ Res. 1998;83:1264-1270.)Key Words: tissue factor Ⅲ inhibitor Ⅲ smooth muscle Ⅲ regulation A rterial thrombosis is a significant complication of primary atherosclerosis. The complex of tissue factor (TF) and factor VIIa is one of the most potent initiators of in vivo arterial thrombosis, leading to activation of factors IX and X. 1 Several studies have shown that TF is expressed in the adventitia of normal human arteries, 2,3 intima of human atherosclerotic plaque, and neointima of the balloon-injured rat arteries. 4 An important inhibitor of TF-mediated coagulation is tissue factor pathway inhibitor (TFPI), a multivalent protease inhibitor with 3 Kunitz-type domains that inactivates factor Xa generation by binding initially via its Kunitz II domain to factor Xa and subsequently by its Kunitz I domain to TF-VIIa catalytic complex. 5 The formation of a quaternary TF-VIIa-TFPI-Xa complex dampens ongoing coagulation and may allow modulation of thrombosis in vivo. TFPI was purified initially from conditioned medium of a hepatoma cell line (HepG2) 6 and since has been described in platelets, microvascular endothelium, and stimulated blood monocytes. 7-10 The endothelium currently is thought to be the principle site of in...

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“…Our chromogenic test system was calibrated with recombinant truncated tissue factor pathway inhibitor and was sufficient for the measurement of relative tissue factor pathway inhibitor concentration in transplant patients and normal individuals. However, the tissue factor pathway inhibitor concentrations determined with our chromogenic substrate assay were lower than published elsewhere (21,22). In contrast to other chromogenic assays we used recombinant tissue factor pathway inhibitor for calibration.…”

Section: Discussionmentioning

confidence: 70%

Effect of Cyclosporine A on the Release of Tissue Factor Pathway Inhibitor from Endothelial Cells in Heart Transplant Patients and Cell Culture

Tiemann

Prohaska²,

Körfer³

et al. 1997

Clinical Chemistry and Laboratory Medicine

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Summary: We investigated the influence of cyclosporine A on the concentration of tissue factor pathway inhibitor and von Willebrand factor antigen in plasma of heart transplant outpatients.Tissue factor pathway inhibitor was quantified in plasma of blood donors (n = 50) and heart transplant outpatients (n = 50) by a chromogenic substrate assay with a mean of 32.4 μg/l and 98.2 μ^, respectively. Von Willebrand factor antigen was determined with an enzyme-linked immunoassay with a mean of 90.9% for blood donors and 184.5% in plasma of heart transplant recipients.In addition, we investigated the effect of cyclosporine A on endothelial cell cultures over an incubation period of four days. A dose-dependent effect of cyclosporine A on the release of endothelial tissue factor pathway inhibitor and von Willebrand factor antigen was determined in a concentration range from 100 to 200 μg/l cyclosporine A. The tissue factor pathway inhibitor and von Willebrand factor antigen concentrations in the cell culture supernatant increased during the incubation time according to the cyclosporine A concentration 2-3 fold and 2 fold, respectively.For a further elucidation of the cyclosporine A effect we investigated the influence of cremophor EL, the vehicle of cyclosporine A. Cremophor EL alone did not increase the tissue factor pathway inhibitor release. However, the release was enhanced 2-4 fold after co-stimulation with the calcium ionophore A 23187 (10~4mol/l) in a concentration-dependent mode.We conclude that a generalized endothelial damage or activation is most probably caused by cyclosporine A and its vehicle cremophor EL. This process probably depends upon the increase of cytosolic free calcium, as described for the liberation of von Willebrand factor by endothelial cells.

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Functional and immunologic methods for the measurement of human tissue factor pathway inhibitor

Cited by 31 publications

References 0 publications

Factor VII–Activating Protease Promotes the Proteolysis and Inhibition of Tissue Factor Pathway Inhibitor

Factor VII–Activating Protease Promotes the Proteolysis and Inhibition of Tissue Factor Pathway Inhibitor

Expression of Tissue Factor Pathway Inhibitor in Vascular Smooth Muscle Cells and Its Regulation by Growth Factors

Effect of Cyclosporine A on the Release of Tissue Factor Pathway Inhibitor from Endothelial Cells in Heart Transplant Patients and Cell Culture

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