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Thromb Haemost 2000; 84(02): 299-306
DOI: 10.1055/s-0037-1614011
Review Article
Schattauer GmbH

Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

Kristian Bangert
1   From the Section for Hemostasis and Thrombosis, Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Denmark
,
Sixtus Thorsen
1   From the Section for Hemostasis and Thrombosis, Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Denmark
› Author AffiliationsThe expert technical assistance of Ms. Birgitte Lillethorup, Ms. Merete Lindholm Faurholt and Mr. Allan Kastrup is gratefully acknowledged. Dr. Anders H. Johnsen is thanked for performing HPLC purification and N-terminal amino sequence analysis. This study was supported by grants from Faculty of Health Sciences at University of Copenhagen, Danish Medical Research Council, Novo Nordic Foundation and Danish Foundation for the Advancement of Medical Science.
Further Information

Publication History

Received 05 August 1999

Accepted after resubmission 06 March 2000

Publication Date:
14 December 2017 (online)

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Summary

An improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the K m value (≈20 µM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and α-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-α-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

Key words

Rat - plasminogen - assay
 

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