A Novel Anti-angiogenic Form of Antithrombin with Retained Proteinase Binding Ability and Heparin Affinity

Helena Larsson,Peter Åkerud,Kerstin Nordling,Elke Raub-Segall,Lena Claesson-Welsh,Ingemar Björk

doi:10.1074/jbc.m010170200

Helena Larsson, Peter Åkerud + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m010170200

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Abstract

Latent antithrombin, an inactive antithrombin form with low heparin affinity, has previously been shown to efficiently inhibit angiogenesis and tumor growth. We now show that heat treatment similar to that used for preparation of latent antithrombin also transforms antithrombin to another form, which we denote prelatent, with potent anti-angiogenic and anti-tumor activity but with retained proteinase- and heparin-binding properties. The ability of prelatent antithrombin to inhibit angiogenesis is presumably due to a limited conformational change, which may partially resemble that in latent antithrombin. Such a change is evidenced by a different cleavage pattern of prelatent than of native antithrombin by nontarget proteinases. Prelatent antithrombin exerts its anti-angiogenic effect by a similar mechanism as latent antithrombin, i.e. by inhibiting focal adhesion formation and focal adhesion kinase activity, thereby leading to decreased proliferation of endothelial cells. The proteinase inhibitory fractions in commercial antithrombin preparations, which have been heat treated during production, also have anti-angiogenic activity, comparable with that of the prelatent antithrombin form.

Highlights

Latent antithrombin, an inactive antithrombin form with low heparin affinity, has previously been shown to efficiently inhibit angiogenesis and tumor growth
We show that heat treatment similar to that used for preparation of latent antithrombin transforms antithrombin to another form, which we denote prelatent, with potent anti-angiogenic and anti-tumor activity but with retained proteinase- and heparin-binding properties
We show that heat treatment similar to that used for preparation of latent antithrombin converts native antithrombin to a form with potent anti-angiogenic and tumor inhibitory activity but, unlike latent antithrombin, with retained ability to bind proteinases and heparin

Summary

EXPERIMENTAL PROCEDURES

Antithrombin—Human ␣-antithrombin was purified from plasma by affinity chromatography on heparin-agarose, followed by anion-exchange chromatography [18]. CAM, on which were placed filter discs (Whatman, Maidstone, United Kingdom) that had been saturated with 3 mg/ml cortisone acetate (Sigma-Aldrich, St. Louis, MO) and soaked in 30 ␮l of 0.02 M sodium phosphate, 0.1 M NaCl, pH 7.4, with or without 0.2 ␮g of fibroblast growth factor-2 (FGF-2; Roche Molecular Biochemicals, Germany) and 0.03, 0.3, 1, or 3 ␮g of native or prelatent antithrombin. Stoichiometry and Kinetics of Proteinase Inactivation—Stoichiometries of inhibition of ␣-thrombin by native and prelatent antithrombin were determined by titrating the enzyme with the inhibitor forms and measuring the residual enzyme activity with a chromogenic substrate, as detailed previously [18, 32]. The membrane was stained with Coomassie Brilliant Blue R-250, appropriate bands were excised and the N-terminal sequences of the polypeptides were analyzed directly in an Applied Biosystems (Foster City, CA) 470A gas phase sequencer, connected on-line to a 120A PTH analyzer

RESULTS

28 Ϯ 6 41 Ϯ 4 52 Ϯ 5 57 Ϯ 9

DISCUSSION