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

Published

July 29, 2020

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

NIST Pub Series

Journal of Research (NIST JRES)

Pub Type

NIST Pubs

Download Paper

https://doi.org/10.6028/jres.125.021

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Keywords

blood clotting, clotting activity, exosite, fibrinogen, fibrinopeptide, heparin, peptide p- nitroanilide, thrombin, α thrombin, β thrombin, γ thrombin

Citation

Jackson, C. , Esnouf, M. and Duewer, D. (2020), Thrombin: An Approach to Developing a Higher-Order Reference Material and Reference Measurement Procedure for Substance Identity, Amount, and Biological Activities, Journal of Research (NIST JRES), National Institute of Standards and Technology, Gaithersburg, MD, [online], https://doi.org/10.6028/jres.125.021, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=921154 (Accessed April 20, 2024)