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Mutation of human plasminogen kringle 1-5 enhances anti-angiogenic action via increased interaction with integrin

Po-Chiao Chang1, Yu-Jia Chang, Hua-Lin Wu

  • 1Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, No. 1, University Rd, Tainan, Taiwan, 701.

Thrombosis and Haemostasis
|April 9, 2008
PubMed

Insights

Mutating kringle 1-5 (K(1-5)) fragments of plasminogen at glycosylation and Lys binding sites enhanced their anti-angiogenic properties. The triple mutant K(1-5)N289A/T346A/L532R showed superior inhibition of angiogenesis and tumor growth in vivo.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cancer Research

Background:

  • Angiogenesis is crucial for tumor growth and metastasis.
  • Angiostatin and kringle fragments of plasminogen inhibit angiogenesis.
  • Kringle 1-5 (K(1-5)) shows greater anti-angiogenic activity than angiostatin.

Purpose of the Study:

  • To optimize the anti-angiogenic activity of K(1-5) fragments.
  • To investigate the effects of mutations at glycosylation sites (Asn-289, Thr-346) and Lys binding site (Leu-532) on K(1-5) function.
  • To evaluate the in vitro and in vivo efficacy of mutated K(1-5) fragments.

Main Methods:

  • Site-directed mutagenesis of K(1-5) at Asn-289, Thr-346, and Leu-532 residues.
  • Expression and purification of wild-type and mutant K(1-5) proteins using Pichia pastoris.
  • Assays for endothelial cell proliferation, apoptosis, and adhesion.
  • In vivo Matrigel angiogenesis assay and tumor growth inhibition studies in mice.

Main Results:

  • Mutations were successfully introduced into K(1-5) proteins.
  • The triple mutant K(1-5)N289A/T346A/L532R demonstrated the most potent inhibition of endothelial cell proliferation and induction of apoptosis.
  • K(1-5)N289A/T346A/L532R showed enhanced integrin alpha(v)beta(3)-mediated adhesion to endothelial cells.
  • This mutant significantly inhibited angiogenesis in vivo and suppressed angiogenesis-dependent tumor growth in mice.

Conclusions:

  • Altering glycosylation and Lys binding properties of K(1-5) enhances its anti-angiogenic potential.
  • Enhanced interaction with integrin alpha(v)beta(3) may contribute to the increased efficacy of the mutated K(1-5) fragment.
  • The K(1-5)N289A/T346A/L532R mutant represents a promising therapeutic candidate for inhibiting angiogenesis-dependent tumors.

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