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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
1   Finsen 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.
› Author Affiliations
Further Information

Publication History

Received 05 March 1987

Accepted after revision 17 August 1987

Publication Date:
29 June 2018 (online)

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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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