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Thromb Haemost 2008; 99(04): 729-738
DOI: 10.1160/TH07-06-0403
Endothelium and Vascular Development
Schattauer GmbH

Mutation of human plasminogen kringle 1–5 enhances antiangiogenic action via increased interaction with integrin αvβ3

Po-Chiao Chang
1   Department of Biochemistry and Molecular Biology
2   The Institute of Basic Medical Sciences, College of Medicine
,
Yu-Jia Chang
1   Department of Biochemistry and Molecular Biology
,
Hua-Lin Wu
1   Department of Biochemistry and Molecular Biology
3   Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan
,
Chin-Wei Chang
1   Department of Biochemistry and Molecular Biology
,
Chung-I. Lin
1   Department of Biochemistry and Molecular Biology
,
Wei-Chih Wang
1   Department of Biochemistry and Molecular Biology
,
Guey-Yueh Shi
1   Department of Biochemistry and Molecular Biology
3   Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan
› Author AffiliationsFinancial support: This work was supported by grants NSC-95–2320-B-006–077-MY3, NSC-95–2752-B-006–003-PAE, and NSC-95–2752-B-006–005-PAE from the National Science Council, Taiwan.
Further Information

Publication History

Received: 12 June 2007

Accepted after major revision: 04 March 2008

Publication Date:
25 November 2017 (online)

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Summary

Angiogenesis plays a primary role in tumor growth and metastasis. Angiostatin, a proteolytic fragment containing the first four kringle domains of human plasminogen, can inhibit angiogenesis. The anti-angiogenic activities of kringle 1–5 (K1–5) and kringle 5 fragments of plasminogen are greater than angiostatin in inhibiting angiogenesis and angiogenesis-dependent tumor growth. To further optimize kringle fragment anti-angiogenic activities, mutations were created at the potential glycosylation sites Asn-289 and Thr-346 and the Lys binding site, Leu-532, at kringle 5, including K1–5N289A (replacing Asn by Ala at residue 289), K1–5T346A, K1–5L532R, K1–5N289A/T346A, K1–5T346A/ L532R, K1–5N289A/L532R, and K1–5N289A/T346A/L532R. Wild-type and mutant K1–5 proteins were expressed successfully by the Pichia pastoris expression system. Native K1–5 from proteolytic cleavage and wild-type K1–5 have similar activity in inhibiting basic fibroblast growth factor-induced endothelial cell proliferation. Among these mutated proteins, K1–5N289A/ T346A/L532R exhibited the greatest effect in inhibiting endothelial cell proliferation and in inducing endothelial cell apoptosis. Integrin αvβ3-mediated adhesion of K1–5N289A/ T346A/L532R to endothelial cells was more greatly enhanced when compared to wild type K1–5. Furthermore, K1–5N289A/ T346A/L532R was most potent in inhibiting basic fibroblast growth factor-induced angiogenesis in Matrigel assay in vivo. Angiogenesis-dependent tumor growth was inhibited by systemically injected K1–5N289A/T346A/L532R into mice. These results demonstrate that alteration of glycosylation and Lys binding properties could increase the anti-angiogenic action of K1–5, possibly via enhanced interaction with integrin αvβ3 in endothelial cells.

Keywords

Angiogenesis and inhibitors - endothelial cells - integrins - plasminogen (-fragments)
 

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