Antiangiogenic Activity of the Cleaved Conformation of the Serpin Antithrombin-Reference-Cited by-同舟云学术

Antiangiogenic Activity of the Cleaved Conformation of the Serpin Antithrombin

Published:1999-09-17 Issue:5435 Volume:285 Page:1926-1928
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

O'Reilly Michael S.¹²,Pirie-Shepherd Steven¹,Lane William S.³,Folkman Judah¹

Affiliation:

1. Department of Surgery, Children's Hospital, Departments of Surgery and Cellular Biology,

2. Joint Center for Radiation Therapy, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA.

3. Harvard Microchemistry Facility, 16 Divinity Avenue, Cambridge, MA 02138, USA.

Abstract

Antithrombin, a member of the serpin family, functions as an inhibitor of thrombin and other enzymes. Cleavage of the carboxyl-terminal loop of antithrombin induces a conformational change in the molecule. Here it is shown that the cleaved conformation of antithrombin has potent antiangiogenic and antitumor activity in mouse models. The latent form of intact antithrombin, which is similar in conformation to the cleaved molecule, also inhibited angiogenesis and tumor growth. These data provide further evidence that the clotting and fibrinolytic pathways are directly involved in the regulation of angiogenesis.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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5. H69i and H69ni cell lines were established in vitro and grown as loosely adherent spheroids in 80 ml of Dulbecco's modified Eagle's medium with 2.5% heat-inactivated fetal bovine serum and 1% glutamine penicillin-steptomycin in 900-cm 2 roller bottles. After 96 hours at 37°C and 10% CO 2 media was collected centrifuged filtered (0.45 μm) and stored at 4°C. Pooled conditioned medium was diluted 1:3 with 10 mM tris-HCl (pH 7) and applied to a CM Sepharose column coupled to a DEAE Sepharose column. The columns were uncoupled and eluted with a step gradient of NaCl in 10 mM tris-HCl with 50 mM 0.2 M 0.6 M 1 M and 2 M steps. Fractions with evidence of protein by absorbance at 280 nanometers were pooled and a portion of each was applied to bovine capillary endothelial cells in vitro. The 0.2 M NaCl elution from the DEAE column inhibited proliferation and was diluted 1:2 and applied to a heparin Sepharose column equilibrated with 0.2 M NaCl and 10 mM tris-HCl. The column was eluted with a continuous gradient of 0.2 to 2 M NaCl in 10 mM tris-HCl. Fractions that inhibited capillary endothelial cell proliferation were pooled and concentrated with a NanoSpin 10K centrifugal concentrator. The sample was applied to a Sephacryl S200 HR column and eluted with phosphate-buffered saline (PBS). Inhibitory fractions were pooled concentrated and applied to a SynChropak RP-4 HPLC column equilibrated with H 2 O and 0.1% trifluoroacetic acid (TFA). Bound protein was eluted with a gradient of acetonitrile in TFA and a portion of each fraction was evaporated by vacuum centrifugation resuspended in PBS and applied to capillary endothelial cells. The inhibitory activity was purified to apparent homogeneity by subsequent cycles on the C4 column.

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