Immunoaffinity Purification of HTC Rat Hepatoma Cell Plasminogen Activator-Inhibitor-1

Ron Zeheb; Usha M Rafferty; Miguel A Rodriguez; Peter Andreasen; Thomas D Gelehrter

doi:10.1055/s-0038-1646047

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1987; 58(04): 1017-1023
DOI: 10.1055/s-0038-1646047

Original Article

Schattauer GmbH Stuttgart

Immunoaffinity Purification of HTC Rat Hepatoma Cell Plasminogen Activator-Inhibitor-1

Ron Zeheb

The Departments of Internal Medicine and Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A.

,

Usha M Rafferty

The Departments of Internal Medicine and Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A.

,

Miguel A Rodriguez

The Departments of Internal Medicine and Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A.

,

Peter Andreasen

¹Finsen Laboratory, Rigshospitalet and Institute of Biochemistry C, University of Copenhagen, Copenhagen, Denmark

,

Thomas D Gelehrter

The Departments of Internal Medicine and Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A.

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Abstract

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Summary

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45° C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.

Keywords

Plasminogen activator-inhibitor - HTC rat hepatoma cells - Purification - Antibody

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Referenzen

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