β2-Glycoprotein I Binds Thrombin and Selectively Inhibits the Enzyme Procoagulant Functions

Alessandro Arcovito, Raimondo De Cristofaro, Giovanni Luca Scaglione, N. Pozzi, L. Acquasaliente, R. Frasson, A. Cristiani, S. Moro, A. Banzato, V. Pengo, V. De Filippis

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18 Citazioni (Scopus)

Abstract

BACKGROUND: This work is aimed at characterising the interaction of β2-glycoprotein I (β2GpI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. METHODS: β2GpI-thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence and molecular modelling. The effect of β2GpI on the procoagulant (fibrin generation and platelet aggregation) and anticoagulant (protein C activation) functions of thrombin were investigated by turbidimetric, immunocytofluorimetric, and enzymatic assays. RESULTS: SPR and fluorescence data indicate that β2GpI tightly binds thrombin (Kd=34nM) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the β2GpI-thrombin complex. In particular, blockage of thrombin exosites with binders specific for exosite-1 (hirugen and HD1 aptamer) or exosite-2 (fibrinogen gamma'-peptide and HD22 aptamer) impairs β2GpI-thrombin interaction. Identical results were obtained with thrombin mutants having one of the two exosites selectively compromised by mutation (Arg73Ala and Arg101Ala). Fluorescence measurements indicate that β2GpI does not affect the affinity of the enzyme for active-site inhibitors, like p-aminobenzamidine and hirudin(1-47) domain, in agreement with the structural model. β2GpI dose-dependently prolongs thrombin clotting time and ecarin clotting time in β2GpI deficient plasma. β2GpI inhibits thrombin-induced platelet aggregation (IC50=0.36μM) by impairing thrombin cleavage of PAR1 (IC50=0.32μM) either on gel-filtered platelets and in whole blood. Strickingly, β2GpI does not affect thrombin-mediated generation of the anticoagulant protein C. CONCLUSIONS: β2GpI functions as a physiological anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function. This article is protected by copyright. All rights reserved.
Lingua originaleEnglish
pagine (da-a)N/A-N/A
RivistaJournal of Thrombosis and Haemostasis
DOI
Stato di pubblicazionePubblicato - 2013

Keywords

  • surface plasmon resonance
  • thrombin

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