Functional Assays for Protein C Activity and Protein C Inhibitor Activity in Plasma

Margarethe Geiger; Teresa M White; John H Griffin

doi:10.1055/s-0038-1646532

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1989; 61(01): 086-092
DOI: 10.1055/s-0038-1646532

Original Article

Schattauer GmbH Stuttgart

Functional Assays for Protein C Activity and Protein C Inhibitor Activity in Plasma

Authors

Margarethe Geiger

The Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA, USA
Teresa M White

The Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA, USA
John H Griffin

The Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA, USA

Abstract

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Summary

An assay system for protein C (PC) activity and PC-inhibitor in plasma was developed. The assay was based on: (1) binding of PC to wells of a microtiter plate coated with a murine monoclonal anti-PC antibody (C3) that did not interfere with the activity or activation of PC; (2) activation of immobilized PC with Protac C; (3) incubation with or without a source of activated PC inhibitor; and (4) measurement of amidolytic activity using the substrate S-2366. The activity assay was specific for PC and sensitive to <1 μl of plasma or 4 ng PC. Inhibition of activated PC by plasma followed pseudo first order kinetics. Heparin caused a dose dependent increase in the inhibition rate with half maximal stimulation at approximately 3 U/ml and maximal stimulation at heparin concentrations ≥10 U/ml. This assay is suitable not only for determination of functional plasma levels of PC and PC inhibitor activities but also for kinetic studies of inhibition of activated PC in complex systems, such as plasma. Studies showed that urokinase interfered with the inhibition of APC by plasma inhibitor(s).

Key words

Protein C - Protein C inhibitor - Functional protein C assay - Functional protein C inhibitor assay

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References

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