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Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface

Riesbeck, Kristian LU orcid ; Dorling, Anthony ; Kemball-Cook, Geoffrey ; McVey, John H. ; Jones, Mick ; Tuddenham, Edward G.D. and Lechler, Robert I. (1997) In Thrombosis and Haemostasis 78(6). p.1488-1494
Abstract

Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition,... (More)

Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Thrombosis and Haemostasis
volume
78
issue
6
pages
1488 - 1494
publisher
Schattauer GmbH
external identifiers
  • pmid:9423800
  • scopus:0030723879
ISSN
0340-6245
language
English
LU publication?
yes
id
c8dd7737-a4ec-4a81-8470-ab8f6b28a270
date added to LUP
2019-06-09 16:36:59
date last changed
2024-01-01 09:36:33
@article{c8dd7737-a4ec-4a81-8470-ab8f6b28a270,
  abstract     = {{<p>Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.</p>}},
  author       = {{Riesbeck, Kristian and Dorling, Anthony and Kemball-Cook, Geoffrey and McVey, John H. and Jones, Mick and Tuddenham, Edward G.D. and Lechler, Robert I.}},
  issn         = {{0340-6245}},
  language     = {{eng}},
  month        = {{12}},
  number       = {{6}},
  pages        = {{1488--1494}},
  publisher    = {{Schattauer GmbH}},
  series       = {{Thrombosis and Haemostasis}},
  title        = {{Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface}},
  volume       = {{78}},
  year         = {{1997}},
}