Detection of the Three <i>Kunitz</i>-Type Single Domains of Membrane-Bound Tissue Factor Pathway Inhibitor (TFPI) by Flow Cytometry

Tiemann, Carsten; Brinkmann, Thomas; Kleesiek, Knut

doi:10.1515/cclm.1997.35.11.855

Cited by 6 publications

(6 citation statements)

References 26 publications

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“…Moreover, our findings support the growing evidence that the full length TFPIα is the exclusive TFPI isoform released upon heparin treatment as similar levels were detected using either ELISA kit. Heparin treatment released TFPI from breast cancer cells without reducing cell surface levels, which is consistent with what was observed in endothelial cells in this and other studies [6,8,9,30]. Thus, our findings indicate that the breast cancer cells possessed intracellular storage pools of TFPIα in a similar manner to that suggested for normal endothelial cells.…”

Section: Discussionsupporting

confidence: 91%

“…Moreover, a significant increase in both free and total TFPI was observed after PI-PLC treatment of the normal endothelial cells HCAEC in this study, although ELISA results indicated that TFPIβ was the most abundant GPI-attached isoform in both cell types. Endothelial cells have previously been shown to express TFPIα on the cell surface [30], and in a study by Piro and Broze the endothelial cells ECV304 (later identified as a bladder carcinoma cell line [31]) were shown to express both TFPIα and TFPIβ on the surface [12]. These results, together with our findings, may indicate cell type dependent differences in the expression of a GPI-attached cofactor for TFPIα.…”

Section: Discussionsupporting

confidence: 80%

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TFPIα and TFPIβ are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity

et al. 2013

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BackgroundTissue factor (TF) pathway inhibitor-1 (TFPI) is expressed in several malignant tissues- and cell lines and we recently reported that it possesses anti-tumor effects in breast cancer cells, indicating a biological role of TFPI in cancer. The two main splice variants of TFPI; TFPIα and TFPIβ, are both able to inhibit TF-factor VIIa (FVIIa) activity in normal cells, but only TFPIα circulates in plasma. The functional importance of TFPIβ is therefore largely unknown, especially in cancer cells. We aimed to characterize the expression and function of TFPIα, TFPIβ, and TF in a panel of tumor derived breast cancer cell lines in comparison to normal endothelial cells.MethodsTFPIα, TFPIβ, and TF mRNA and protein measurements were conducted using qRT-PCR and ELISA, respectively. Cell-associated TFPI was detected after phosphatidylinositol-phospholipase C (PI-PLC) and heparin treatment by flow cytometry, immunofluorescence, and Western blotting. The potential anticoagulant activity of cell surface TFPI was determined in a factor Xa activity assay.ResultsThe expression of both isoforms of TFPI varied considerably among the breast cancer cell lines tested, from no expression in Sum149 cells to levels above or in the same range as normal endothelial cells in Sum102 and MDA-MB-231 cells. PI-PLC treatment released both TFPIα and TFPIβ from the breast cancer cell membrane and increased TF activity on the cell surface, showing TF-FVIIa inhibitory activity of the glycosylphosphatidylinositol- (GPI-) anchored TFPI. Heparin treatment released TFPIα without decreasing the cell surface levels, thus indicating the presence of intracellular storage pools of TFPIα in the breast cancer cells.ConclusionGPI-attached TFPI located at the surface of breast cancer cells inhibited TF activity and could possibly reduce TF signaling and breast cancer cell growth locally, indicating a therapeutic potential of the TFPIβ isoform.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 80%

TFPIα and TFPIβ are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity

et al. 2013

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“…These data suggest that heparin-releasable TFPI represents a fraction of the GPI-anchored TFPI that has had its GPI anchor cleaved but remains nonspecifically associated with cell-surface glycosaminoglycans. Because heparinreleasable TFPI has not been detected on cultured endothelial cells 5,25 and was released in only very small amounts from the washed placental fragments, it is likely that the heparinreleasable pool of TFPI readily dissociates from the cell surface and is removed during preliminary washing steps in in vitro studies. Because of the presence of plasma TFPI in the placental blood, it is not possible to quantify the amount of TFPI removed from the placental fragments during washing steps in the experiments presented here.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Association of Tissue Factor Pathway Inhibitor With Human Placenta

Mast¹,

Acharya²,

Malecha³

et al. 2002

ATVB

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Objective-Tissue factor pathway inhibitor (TFPI) is an endothelial-associated inhibitor of blood coagulation. Because the mechanism for attachment of TFPI to endothelium is not clear, we investigated its association with human placenta. Methods and Results-Western blots demonstrate that treatment with phosphatidylinositol-specific phospholipase C (PIPLC) removes more placental TFPI than either PBS or heparin, a finding confirmed by immunohistochemistry. The amounts of heparin-releasable and PIPLC-releasable TFPI activity on placental endothelium were measured in placentas from 5 individuals. PIPLC removes Ͼ10-fold more TFPI activity from the placental fragments than 10 U/mL heparin and Ͼ100-fold more than 1 U/mL heparin. Pretreatment of the placental fragments with PIPLC increases the amount of heparin-releasable TFPI by Ϸ3-fold. An antibody specific for the C-terminal region of TFPI recognizes PIPLC-releasable TFPI in Western blots. Conclusions-GPI-anchored TFPI is the predominant form on placental endothelium. Heparin-releasable TFPI likely represents only a small portion of the total TFPI on endothelium that remains attached to cell-surface glycosaminoglycans after cleavage of the GPI anchor by endogenous enzymes. The predominance of GPI-anchored TFPI suggests that heparin infusion does not significantly redistribute TFPI within the vasculature. The intact C-terminus in GPI-anchored TFPI indicates it is not directly attached to a GPI anchor. Rather, it most likely associates with endothelium by binding to a GPI-anchored protein.

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“…The C-terminal region of TFPI most likely binds to the cell surface, allowing the first and second Kunitz domains to interact with other membrane-associated and soluble proteins located on or near the cell surface. The third Kunitz domain is also thought to be accessible to interactions with soluble or membrane proteins ( , ), yet it has no identified function as a proteinase inhibitor.…”

Section: Discussionmentioning

confidence: 99%

Contribution of Regions Distal to Glycine-160 to the Anticoagulant Activity of Tissue Factor Pathway Inhibitor

Lockett

Mast

2002

Biochemistry

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The functions of the first two Kunitz domains of tissue factor pathway inhibitor (TFPI) are well defined as active site-directed inhibitors of factor VIIa and factor Xa. The anticoagulant properties of the third Kunitz domain and C-terminal region were probed using altered forms of TFPI. TFPI-160 contains the first two Kunitz domains. K1K2C contains the first two Kunitz domains and the basic C-terminus. Neither TFPI-160 nor K1K2C contains the third Kunitz domain. In amidolytic assays containing calcium, TFPI-160 is a less potent inhibitor of factor Xa than TFPI. However, addition of the C-terminus in K1K2C nearly restores inhibitory activity to that of TFPI, indicating that the third Kunitz domain is not required for direct inhibition of factor Xa. When compared in assays containing phospholipids and factor Va, K1K2C and TFPI-160 are poor inhibitors compared to TFPI, demonstrating that the third Kunitz domain is required for the full anticoagulant activity of TFPI. TFPI was further characterized in amidolytic assays performed with Gla-domainless factor Xa and in prothrombin activation assays using submicellar concentrations of short-chain phospholipids (C6PS). TFPI and K1K2C are worse inhibitors of Gla-domainless factor Xa, compared to wild-type factor Xa, while TFPI-160 inhibits both forms of factor Xa equally, suggesting a C-terminus/Gla domain interaction. TFPI is a potent inhibitor of thrombin generation by prothrombinase assembled with C6PS, while TFPI-160 and K1K2C are not. Conversely, TFPI does not inhibit prothrombin activation by prothrombinase assembled on a two-dimensional lipid bilayer. Together, the data indicate that the region between Gly-160 and the end of the third Kunitz domain contributes to TFPI function by orienting the second Kunitz domain so that it can bind the active site of phospholipid-associated factor Xa prior to prothrombinase assembly and/or by slowing formation of the prothrombinase complex.

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Detection of the Three Kunitz-Type Single Domains of Membrane-Bound Tissue Factor Pathway Inhibitor (TFPI) by Flow Cytometry

Cited by 6 publications

References 26 publications

TFPIα and TFPIβ are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity

TFPIα and TFPIβ are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity

Characterization of the Association of Tissue Factor Pathway Inhibitor With Human Placenta

Contribution of Regions Distal to Glycine-160 to the Anticoagulant Activity of Tissue Factor Pathway Inhibitor

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