β2 -Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions

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

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

BACKGROUND: This work was aimed at characterizing the interaction of β(2)-glycoprotein I (β(2)GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. METODS: The β(2)GPI-thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of β(2)GPI on the procoagulant (fibrin generation and platelet aggregation) and anticoagulant (protein C activation) functions of thrombin were investigated with turbidimetric, immunocytofluorimetric and enzymatic assays. RESULTS: SPR and fluorescence data indicated that β(2)GPI tightly bound thrombin (K(d) = 34 nM) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the β(2)GPI-thrombin complex. In particular, blockage of thrombin exosites with binders specific for exosite-1 (hirugen and HD1 aptamer) or exosite-2 (fibrinogen γ'-peptide and HD22 aptamer) impaired the β2 GPI-thrombin interaction. Identical results were obtained with thrombin mutants having one of the two exosites selectively compromised by mutation (Arg73Ala and Arg101Ala). Fluorescence measurements indicated that β(2)GPI did not affect the affinity of the enzyme for active site inhibitors, such as p-aminobenzamidine and the hirudin(1-47) domain, in agreement with the structural model. β(2)GPI dose-dependently prolonged the thrombin clotting time and ecarin clotting time in β(2)GPI-deficient plasma. β(2)GPI inhibited thrombin-induced platelet aggregation (IC50 = 0.36 μM) by impairing thrombin cleavage of protease-activated receptor 1 (PAR1) (IC50 = 0.32 μM), both on gel-filtered platelets and in whole blood. Strikingly, β(2) GPI did not affect thrombin-mediated generation of the anticoagulant protein C. CONCLUSIONS: β(2) GPI functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function.
Original languageEnglish
Pages (from-to)1093-1102
Number of pages10
JournalJournal of Thrombosis and Haemostasis
Publication statusPublished - 2013

Keywords

  • Beta2-glicoprotein I
  • Lupus anticoagilant

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