Construction and characterization of a chimeric protein consisting of tissue-type plasminogen activator and the epidermal growth factor-like regions of thrombomodulin

M. A. Myers, H. H. Salem, P. Bird

Research output: Contribution to journalArticleResearchpeer-review

2 Citations (Scopus)

Abstract

We have constructed and characterized a chimeric anticoagulant and fibrinolytic protein by inserting the portion of thrombomodulin (TM) carrying protein C (PC) activating cofactor activity into tissue-type plasminogen activator (t-PA). This was done by replacing the epidermal growth factor (EGF)-like domain of t-PA with the six EGF-like repeats from TM. The 105 kDa chimeric molecule was secreted from transfected COS-7 cells as a single-chain molecule that was recognised by both anti-TM and anti-t-PA antisera. It was shown to have similar PC activating cofactor activity to other soluble derivatives of TM (Km for Protein C of 2.2±0.4 μM), as well as amidolytic and plasminogen activating activity. However, the chimeric protein is not converted to a two chain molecule and is stimulated less by fibrin, due to a lower affinity for lysine. The demonstration that TM activity can be incorporated into a fibrinolytic enzyme is a step towards enhanced fibrinolysis in patients with thrombosis.

Original languageEnglish
Pages (from-to)229-237
Number of pages9
JournalFibrinolysis and Proteolysis
Volume8
Issue number4
DOIs
Publication statusPublished - 1 Jan 1994

Cite this

@article{64d835c4e688484187d4b2782891ad02,
title = "Construction and characterization of a chimeric protein consisting of tissue-type plasminogen activator and the epidermal growth factor-like regions of thrombomodulin",
abstract = "We have constructed and characterized a chimeric anticoagulant and fibrinolytic protein by inserting the portion of thrombomodulin (TM) carrying protein C (PC) activating cofactor activity into tissue-type plasminogen activator (t-PA). This was done by replacing the epidermal growth factor (EGF)-like domain of t-PA with the six EGF-like repeats from TM. The 105 kDa chimeric molecule was secreted from transfected COS-7 cells as a single-chain molecule that was recognised by both anti-TM and anti-t-PA antisera. It was shown to have similar PC activating cofactor activity to other soluble derivatives of TM (Km for Protein C of 2.2±0.4 μM), as well as amidolytic and plasminogen activating activity. However, the chimeric protein is not converted to a two chain molecule and is stimulated less by fibrin, due to a lower affinity for lysine. The demonstration that TM activity can be incorporated into a fibrinolytic enzyme is a step towards enhanced fibrinolysis in patients with thrombosis.",
author = "Myers, {M. A.} and Salem, {H. H.} and P. Bird",
year = "1994",
month = "1",
day = "1",
doi = "10.1016/0268-9499(94)90048-5",
language = "English",
volume = "8",
pages = "229--237",
journal = "Fibrinolysis",
issn = "0268-9499",
number = "4",

}

TY - JOUR

T1 - Construction and characterization of a chimeric protein consisting of tissue-type plasminogen activator and the epidermal growth factor-like regions of thrombomodulin

AU - Myers, M. A.

AU - Salem, H. H.

AU - Bird, P.

PY - 1994/1/1

Y1 - 1994/1/1

N2 - We have constructed and characterized a chimeric anticoagulant and fibrinolytic protein by inserting the portion of thrombomodulin (TM) carrying protein C (PC) activating cofactor activity into tissue-type plasminogen activator (t-PA). This was done by replacing the epidermal growth factor (EGF)-like domain of t-PA with the six EGF-like repeats from TM. The 105 kDa chimeric molecule was secreted from transfected COS-7 cells as a single-chain molecule that was recognised by both anti-TM and anti-t-PA antisera. It was shown to have similar PC activating cofactor activity to other soluble derivatives of TM (Km for Protein C of 2.2±0.4 μM), as well as amidolytic and plasminogen activating activity. However, the chimeric protein is not converted to a two chain molecule and is stimulated less by fibrin, due to a lower affinity for lysine. The demonstration that TM activity can be incorporated into a fibrinolytic enzyme is a step towards enhanced fibrinolysis in patients with thrombosis.

AB - We have constructed and characterized a chimeric anticoagulant and fibrinolytic protein by inserting the portion of thrombomodulin (TM) carrying protein C (PC) activating cofactor activity into tissue-type plasminogen activator (t-PA). This was done by replacing the epidermal growth factor (EGF)-like domain of t-PA with the six EGF-like repeats from TM. The 105 kDa chimeric molecule was secreted from transfected COS-7 cells as a single-chain molecule that was recognised by both anti-TM and anti-t-PA antisera. It was shown to have similar PC activating cofactor activity to other soluble derivatives of TM (Km for Protein C of 2.2±0.4 μM), as well as amidolytic and plasminogen activating activity. However, the chimeric protein is not converted to a two chain molecule and is stimulated less by fibrin, due to a lower affinity for lysine. The demonstration that TM activity can be incorporated into a fibrinolytic enzyme is a step towards enhanced fibrinolysis in patients with thrombosis.

UR - http://www.scopus.com/inward/record.url?scp=0028358912&partnerID=8YFLogxK

U2 - 10.1016/0268-9499(94)90048-5

DO - 10.1016/0268-9499(94)90048-5

M3 - Article

VL - 8

SP - 229

EP - 237

JO - Fibrinolysis

JF - Fibrinolysis

SN - 0268-9499

IS - 4

ER -