title: β2 -Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions.
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.
  
  
  METHODS
  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.
authors.affiliation: Laboratory of Protein Chemistry, School of Medicine, University of Padua, Padova, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Pozzi
authors.forename: L
authors.initials: L
authors.lastname: Acquasaliente
authors.forename: R
authors.initials: R
authors.lastname: Frasson
authors.forename: A
authors.initials: A
authors.lastname: Cristiani
authors.forename: S
authors.initials: S
authors.lastname: Moro
authors.forename: A
authors.initials: A
authors.lastname: Banzato
authors.forename: V
authors.initials: V
authors.lastname: Pengo
authors.forename: G L
authors.initials: GL
authors.lastname: Scaglione
authors.forename: A
authors.initials: A
authors.lastname: Arcovito
authors.forename: R
authors.initials: R
authors.lastname: De Cristofaro
authors.forename: V
authors.initials: V
authors.lastname: De Filippis
mesh_terms: D000925:Anticoagulants / Q000737:chemistry; D016736:Antiphospholipid Syndrome / Q000188:drug therapy; D001550:Benzamidines / Q000737:chemistry; D001777:Blood Coagulation; D020134:Catalytic Domain; D002850:Chromatography, Gel; D003029:Coagulants / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D005337:Fibrin / Q000737:chemistry; D005434:Flow Cytometry; D006487:Hemostasis; D006629:Hirudins / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D009154:Mutation; D009391:Nephelometry and Turbidimetry; D011485:Protein Binding; D044463:Receptor, PAR-1 / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D053482:beta 2-Glycoprotein I / Q000737:chemistry*
chemical_list: D000925:Anticoagulants; D001550:Benzamidines; D003029:Coagulants; D004791:Enzyme Inhibitors; D006629:Hirudins; D044463:Receptor, PAR-1; D053482:beta 2-Glycoprotein I; C006500:4-aminobenzamidine; D005337:Fibrin; D013917:Thrombin
keywords: antiphospholipid syndrome; hemostasis; surface plasmon resonance; thrombin; β2-glycoprotein I