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note
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2O 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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0344674841
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M. S. O'Reilly, S. Pine-Shepherd, W. S. Lane, J. Folkman, data not shown
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0344674840
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Bovine calf serum was stored at 4°C for 14 days to allow for degradation of antithrombin to its R form. Alternatively, outdated human plasma was centrifuged (10,000 rpm for 30 min) and filtered (0.45 μm). Serum or plasma was diluted 1:3 with 10 mM tris-HCl (pH 7.0) and applied to a CM Sepharose column coupled to a DEAE Sepharose column equilibrated with the dilution buffer. The DEAE column was washed with 50 mM NaCl in 10 mM tris-HCl and then coupled to a heparin Sepharose column. Bound protein from the DEAE column was eluted directly onto the heparin Sepharose column with 0.2 M NaCl and 10 mM tris-HCl. The heparin Sepharose column was washed with 0.5 M NaCl and 10 mM tris-HCl and then eluted with a continuous gradient of 0.6 to 2 M NaCl in 10 mM tris-HCl. The purity of the final samples was assessed by SDS-polyacrylamide gel electrophoresis (PAGE) with silver staining, and concentration was determined with a Bio-Rad protein assay.
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16
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0345105692
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note
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Stressed bovine and human antithrombin were obtained from Sigma or Calbiochem, respectively, or purified as described. Recombinant human antithrombin was obtained as a generous gift from a joint venture between Genryme and Genzyme Transgenics Corporations. Stressed human antithrombin was cleaved by incubation with porcine pancreatic elastase (Calbiochem) for 12 hours at 37°C in PBS (pH 7.8) at a 1:50 molar ratio. Cleaved and latent antithrombin were purified with heparin Sepharose and eluted at 0.4 M NaCl as a distinct peak. Purity was assessed by SDS-PAGE, and samples were concentrated with a Nanospin 30K centrifugal concentrator or lyophilized after dialysis.
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0022168229
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0345105681
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note
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We thank J. M. Neveu for Edman microsequencing, the staff of the blood bank of Children's Hospital for providing the outdated plasma, Genzyme and Genzyme Trangenics Corporations for recombinant human antithrombin, L. DeSantis and K. Gullage for photography, E. Flynn for immunohistochemistry, and Advanced Medical Graphics for help with the figures. Supported in part by NIH grants RO1-CA64481 and PO1-CA45548.
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