A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent

Kenichi Fujise, Bryan Mitch Revelle, Lowell Stacy, Edwin L. Madison, Edward T H Yeh, James T. Willerson, Pamela J. Beck

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Background: P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that a tissue plasminogen activator (TPA)/P-selectin fusion protein would have not only thrombolytic activity but also might target TPA to the thrombi. In addition, it seemed possible that this chimeric protein would competitively inhibit the binding of native P-selectin on endothelial cells and platelets to leukocytes and thus further promote thrombolysis. Methods and Results: The full-length, plasminogen activator inhibitor-1-resistant form of TPA (TPA(JR)) together with two TPA(IR)/P-selectin fusion constructs (P280(IR) and P121(IR)) were expressed with the use of baculovirus vectors. After infection of Sf-21 cells with the recombinant baculovirus, recombinant TPA(IR) and P-selectin/TPA(IR) fusion proteins were purified with the use of metal ion chromatography. The intact protease activity of TPA(IR) and the ligand binding capability of P- selectin were confirmed through indirect chromogenic and cell binding assays, respectively. These molecules were assessed both in vitro and in vivo for thrombolytic activity. In vitro clot lysis assays indicated equal efficacy of TPA(JR), P280(IR), and P121(IR) (P>.5). The in vivo efficacy was tested in a cyclic flow variation model with the use of the rat mesenteric artery. Compared with saline control treatment, reduction in cyclic flow variations was significant (P<.05) and similar (P>.5) among TPA(IR), P280(IR), and P121(IR). No significant bleeding was noted among treated animals. Conclusions: Chimeric proteins P280(IR) and P121(IR) have clot lysis activities that are similar to TPA(IR) both in vitro and in vivo. These chimeric proteins also bind to P-selectin ligand in vitro. Thus, these proteins may provide an efficient method of targeting TPA to the thrombotic region. Further experimental analysis with the use of larger animal coronary occlusion models should help determine the future value of these proteins as clinical therapeutic agents.

Original languageEnglish (US)
Pages (from-to)715-722
Number of pages8
JournalCirculation
Volume95
Issue number3
StatePublished - 1997
Externally publishedYes

Fingerprint

P-Selectin
Fibrinolytic Agents
Tissue Plasminogen Activator
Proteins
Baculoviridae
Blood Platelets
Endothelial Cells
Ligands
Mesenteric Arteries
Coronary Occlusion
Plasminogen Activator Inhibitor 1
Chromatography
Thrombosis
Leukocytes
Peptide Hydrolases
Metals

Keywords

  • acute coronary syndromes
  • cyclic flow variations
  • P-selectin
  • plasminogen activators
  • thrombolysis

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Fujise, K., Revelle, B. M., Stacy, L., Madison, E. L., Yeh, E. T. H., Willerson, J. T., & Beck, P. J. (1997). A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent. Circulation, 95(3), 715-722.

A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent. / Fujise, Kenichi; Revelle, Bryan Mitch; Stacy, Lowell; Madison, Edwin L.; Yeh, Edward T H; Willerson, James T.; Beck, Pamela J.

In: Circulation, Vol. 95, No. 3, 1997, p. 715-722.

Research output: Contribution to journalArticle

Fujise, K, Revelle, BM, Stacy, L, Madison, EL, Yeh, ETH, Willerson, JT & Beck, PJ 1997, 'A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent', Circulation, vol. 95, no. 3, pp. 715-722.
Fujise K, Revelle BM, Stacy L, Madison EL, Yeh ETH, Willerson JT et al. A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent. Circulation. 1997;95(3):715-722.
Fujise, Kenichi ; Revelle, Bryan Mitch ; Stacy, Lowell ; Madison, Edwin L. ; Yeh, Edward T H ; Willerson, James T. ; Beck, Pamela J. / A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent. In: Circulation. 1997 ; Vol. 95, No. 3. pp. 715-722.
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T1 - A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent

AU - Fujise, Kenichi

AU - Revelle, Bryan Mitch

AU - Stacy, Lowell

AU - Madison, Edwin L.

AU - Yeh, Edward T H

AU - Willerson, James T.

AU - Beck, Pamela J.

PY - 1997

Y1 - 1997

N2 - Background: P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that a tissue plasminogen activator (TPA)/P-selectin fusion protein would have not only thrombolytic activity but also might target TPA to the thrombi. In addition, it seemed possible that this chimeric protein would competitively inhibit the binding of native P-selectin on endothelial cells and platelets to leukocytes and thus further promote thrombolysis. Methods and Results: The full-length, plasminogen activator inhibitor-1-resistant form of TPA (TPA(JR)) together with two TPA(IR)/P-selectin fusion constructs (P280(IR) and P121(IR)) were expressed with the use of baculovirus vectors. After infection of Sf-21 cells with the recombinant baculovirus, recombinant TPA(IR) and P-selectin/TPA(IR) fusion proteins were purified with the use of metal ion chromatography. The intact protease activity of TPA(IR) and the ligand binding capability of P- selectin were confirmed through indirect chromogenic and cell binding assays, respectively. These molecules were assessed both in vitro and in vivo for thrombolytic activity. In vitro clot lysis assays indicated equal efficacy of TPA(JR), P280(IR), and P121(IR) (P>.5). The in vivo efficacy was tested in a cyclic flow variation model with the use of the rat mesenteric artery. Compared with saline control treatment, reduction in cyclic flow variations was significant (P<.05) and similar (P>.5) among TPA(IR), P280(IR), and P121(IR). No significant bleeding was noted among treated animals. Conclusions: Chimeric proteins P280(IR) and P121(IR) have clot lysis activities that are similar to TPA(IR) both in vitro and in vivo. These chimeric proteins also bind to P-selectin ligand in vitro. Thus, these proteins may provide an efficient method of targeting TPA to the thrombotic region. Further experimental analysis with the use of larger animal coronary occlusion models should help determine the future value of these proteins as clinical therapeutic agents.

AB - Background: P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that a tissue plasminogen activator (TPA)/P-selectin fusion protein would have not only thrombolytic activity but also might target TPA to the thrombi. In addition, it seemed possible that this chimeric protein would competitively inhibit the binding of native P-selectin on endothelial cells and platelets to leukocytes and thus further promote thrombolysis. Methods and Results: The full-length, plasminogen activator inhibitor-1-resistant form of TPA (TPA(JR)) together with two TPA(IR)/P-selectin fusion constructs (P280(IR) and P121(IR)) were expressed with the use of baculovirus vectors. After infection of Sf-21 cells with the recombinant baculovirus, recombinant TPA(IR) and P-selectin/TPA(IR) fusion proteins were purified with the use of metal ion chromatography. The intact protease activity of TPA(IR) and the ligand binding capability of P- selectin were confirmed through indirect chromogenic and cell binding assays, respectively. These molecules were assessed both in vitro and in vivo for thrombolytic activity. In vitro clot lysis assays indicated equal efficacy of TPA(JR), P280(IR), and P121(IR) (P>.5). The in vivo efficacy was tested in a cyclic flow variation model with the use of the rat mesenteric artery. Compared with saline control treatment, reduction in cyclic flow variations was significant (P<.05) and similar (P>.5) among TPA(IR), P280(IR), and P121(IR). No significant bleeding was noted among treated animals. Conclusions: Chimeric proteins P280(IR) and P121(IR) have clot lysis activities that are similar to TPA(IR) both in vitro and in vivo. These chimeric proteins also bind to P-selectin ligand in vitro. Thus, these proteins may provide an efficient method of targeting TPA to the thrombotic region. Further experimental analysis with the use of larger animal coronary occlusion models should help determine the future value of these proteins as clinical therapeutic agents.

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