The structural integrity of anion binding exosite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII

The Journal of Biological Chemistry
Michael A BukysM Kalafatis

Abstract

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Ar...Continue Reading

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Citations

Sep 20, 2007·The Journal of Biological Chemistry·Kenneth SegersGerry A F Nicolaes
Oct 8, 2011·Thrombosis and Haemostasis·Jolanta M Siller-MatulaBernd Jilma
Jan 31, 2014·Thrombosis and Haemostasis·James A Huntington
Aug 1, 2007·Journal of Thrombosis and Haemostasis : JTH·P E BockI M A Verhamme
Oct 20, 2007·Journal of Thrombosis and Haemostasis : JTH·E ErdoganM Kalafatis
Sep 22, 2009·Journal of Thrombosis and Haemostasis : JTH·R M Camire, M H A Bos
Dec 24, 2018·The Journal of Biological Chemistry·Harlan N Bradford, Sriram Krishnaswamy
Nov 26, 2015·The Journal of Biological Chemistry·Joesph R WiencekMichael Kalafatis
Dec 25, 2019·Biochemistry. Biokhimii︠a︡·V A SpiridonovaA V Mazurov

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