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Thrombin: An Approach to Developing a Higher-Order Reference Material and Reference Measurement Procedure for Substance Identity, Amount, and Biological Activities

Published

Author(s)

Craig M. Jackson, M. P. Esnouf, David L. Duewer

Abstract

Thrombin, the proteolytic enzyme that catalyzes the transformation of soluble fibrinogen to the polymerized fibrin clot, participates in multiple reactions in blood coagulation in addition to the clotting reaction. Although reference materials have existed for many years, structural characterization and measurement of biological activity have never been sufficient to permit claims of clear metrological traceability for the thrombin preparations. Our current state-of-the-art methods for protein characterization and determination of the catalytic properties of thrombin now make it practical to develop and characterize a metrologically acceptable reference material and reference measurement procedure for thrombin. Specifically, α-thrombin, the biologically produced protease formed during prothrombin activation, is readily available and has been extensively characterized. Dependences of thrombin proteolytic and peptide hydrolytic activities on a variety of substrates, pH, specific ions, and temperature are established, although variability remains for the kinetic parameters that describe thrombin enzymatic action. The roles of specific areas on the surface of the thrombin molecule (exosites) in substrate recognition and catalytic efficiency are described and characterized. It is opportune to develop reference materials of high metrological order and technical feasibility. In this article, we review the properties of α- thrombin important for its preparation and suggest an approach suitable for producing a reference material and a reference measurement procedure that is sensitive to thrombin's catalytic competency on a variety of substrates.
Citation
Journal of Research (NIST JRES) -
Volume
125

Keywords

blood clotting, clotting activity, exosite, fibrinogen, fibrinopeptide, heparin, peptide p- nitroanilide, thrombin, α thrombin, β thrombin, γ thrombin
Created July 28, 2020, Updated July 29, 2020