[beta]2-Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions

Summary Background This work was aimed at characterizing the interaction of [beta]2-glycoprotein I ([beta]2GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. Methods The [beta]2GPI-thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of [beta]2GPI 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 [beta]2GPI tightly bound thrombin (Kd = 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 [beta]2GPI-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) impaired the [beta]2GPI-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 [beta]2GPI 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. [beta]2GPI dose-dependently prolonged the thrombin clotting time and ecarin clotting time in [beta]2GPI-deficient plasma. [beta]2GPI 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, [beta]2GPI did not affect thrombin-mediated generation of the anticoagulant protein C. Conclusions [beta]2GPI functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function. [PUBLICATION ABSTRACT]