Β2‐Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions. Issue 6 (3rd July 2013)

• Record Type: Journal Article
• Authors: Pozzi, N.; Acquasaliente, L.; Frasson, R.; Cristiani, A.; Moro, S.; Banzato, A.; Pengo, V.; Scaglione, G. L.; Arcovito, A.; De, R.; De, V.
• Is Part Of: Journal of thrombosis and haemostasis. Volume 11:Issue 6(2013)
• Page Start: 1093
• Page End: 1102
• Publication Date: 2013-07-03
• Subjects: Thrombosis -- Periodicals; Hemostasis -- Periodicals; Blood coagulation disorders -- Periodicals; 616.1
• DOI: 10.1111/jth.12238
• Languages: English
• ISSNs: 1538-7933

Abstract

<abstract abstract-type="main" id="jth12238-abs-0001"> <title>Summary</title> <sec id="jth12238-sec-0001" sec-type="section"> <title>Background</title> <p>This work was aimed at characterizing the interaction of β<sub>2</sub>‐glycoprotein I (β<sub>2</sub>GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade.</p> </sec> <sec id="jth12238-sec-0002" sec-type="section"> <title>Methods</title> <p>The β<sub>2</sub>GPI–thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of β<sub>2</sub>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.</p> </sec> <sec id="jth12238-sec-0003" sec-type="section"> <title>Results</title> <p>SPR and fluorescence data indicated that β<sub>2</sub>GPI tightly bound thrombin (<italic>K</italic><sub>d</sub> = 34 n<sc>m</sc>) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the β<sub>2</sub>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 β<sub>2</sub>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<sub>2</sub>GPI did not affect the affinity of the enzyme for active site inhibitors, such as <italic>p</italic>‐aminobenzamidine and the hirudin(1–47) domain, in agreement with the structural model. β<sub>2</sub>GPI dose‐dependently prolonged the thrombin clotting time and ecarin clotting time in β<sub>2</sub>GPI‐deficient plasma. β<sub>2</sub>GPI inhibited thrombin‐induced platelet aggregation (IC<sub>50</sub> = 0.36 μ<sc>m</sc>) by impairing thrombin cleavage of protease‐activated receptor 1 (PAR1) (IC<sub>50</sub> = 0.32 μ<sc>m</sc>), both on gel‐filtered platelets and in whole blood. Strikingly, β<sub>2</sub>GPI did not affect thrombin‐mediated generation of the anticoagulant protein C.</p> </sec> <sec id="jth12238-sec-0004" sec-type="section"> <title>Conclusions</title> <p>β<sub>2</sub>GPI functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function.</p> </sec> </abstract>