TY - JOUR
T1 - β2 -Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions
AU - Pozzi, N.
AU - Acquasaliente, L.
AU - Frasson, R.
AU - Cristiani, A.
AU - Moro, S.
AU - Banzato, A.
AU - Pengo, V.
AU - Scaglione, G. L.
AU - Arcovito, Alessandro
AU - De Cristofaro, Raimondo
AU - De Filippis, V.
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
KW - Beta2-glicoprotein I
KW - Lupus anticoagilant
KW - Beta2-glicoprotein I
KW - Lupus anticoagilant
UR - http://hdl.handle.net/10807/56650
M3 - Article
SN - 1538-7933
SP - 1093
EP - 1102
JO - Journal of Thrombosis and Haemostasis
JF - Journal of Thrombosis and Haemostasis
ER -