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Expression of hirudin fusion proteins in mammalian cells : A strategy for prevention of intravascular thrombosis

Riesbeck, Kristian LU orcid ; Chen, Daxin ; Kemball-Cook, Geoffrey ; McVey, John H. ; George, Andrew J.T. ; Tuddenham, Edward G.D. ; Dorling, Anthony and Lechler, Robert I. (1998) In Circulation 98(24). p.2744-2752
Abstract

Background - Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. Methods and Results - An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins... (More)

Background - Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. Methods and Results - An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti- hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P- selectin fusion proteins accumulated in adrenocorticotropic hormone- containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. Conclusions - Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Anticoagulants, Coagulation, Thrombosis, Transplantation
in
Circulation
volume
98
issue
24
pages
2744 - 2752
publisher
Lippincott Williams & Wilkins
external identifiers
  • scopus:0032534470
  • pmid:9851962
ISSN
0009-7322
DOI
10.1161/01.CIR.98.24.2744
language
English
LU publication?
yes
id
a372514d-4cc9-4aac-a341-a37142b1596f
date added to LUP
2019-06-07 15:20:41
date last changed
2024-04-02 07:08:22
@article{a372514d-4cc9-4aac-a341-a37142b1596f,
  abstract     = {{<p>Background - Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. Methods and Results - An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti- hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P- selectin fusion proteins accumulated in adrenocorticotropic hormone- containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. Conclusions - Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.</p>}},
  author       = {{Riesbeck, Kristian and Chen, Daxin and Kemball-Cook, Geoffrey and McVey, John H. and George, Andrew J.T. and Tuddenham, Edward G.D. and Dorling, Anthony and Lechler, Robert I.}},
  issn         = {{0009-7322}},
  keywords     = {{Anticoagulants; Coagulation; Thrombosis; Transplantation}},
  language     = {{eng}},
  month        = {{12}},
  number       = {{24}},
  pages        = {{2744--2752}},
  publisher    = {{Lippincott Williams & Wilkins}},
  series       = {{Circulation}},
  title        = {{Expression of hirudin fusion proteins in mammalian cells : A strategy for prevention of intravascular thrombosis}},
  url          = {{http://dx.doi.org/10.1161/01.CIR.98.24.2744}},
  doi          = {{10.1161/01.CIR.98.24.2744}},
  volume       = {{98}},
  year         = {{1998}},
}