Characterization of Thrombin-Bound Dabigatran Effects on Protease-Activated Receptor-1 Expression and Signaling In Vitro

Abstract

Thrombin, the key effector protease of the coagulation cascade, drives fibrin deposition and activates human platelets through protease-activated receptor-1 (PAR1). These processes are critical to the progression of thrombotic diseases. Thrombin is the main target of anticoagulant therapy, and major efforts have led to the discovery of new oral direct inhibitors of thrombin. Dabigatran is the first oral anticoagulant licensed for the prevention of thromboembolisms associated with orthopedic surgery and stroke prevention in atrial fibrillation. Dabigatran is a direct thrombin inhibitor that effectively blocks thrombin's catalytic activity but does not preclude thrombin's exosites and binding to fibrinogen. Thus, we hypothesized that catalytically inactive thrombin retains the capacity to bind to PAR1 through exosite-I and may modulate its function independent of receptor cleavage and activation. Here, we report that dabigatran at clinically relevant concentrations is an effective and acute inhibitor of thrombininduced PAR1 cleavage, activation, internalization, and b-arrestin recruitment in vitro. Interestingly, prolonged exposure to catalytic inactive thrombin incubated with dabigatran at 20-fold higher therapeutic concentration resulted in increased PAR1 cell-surface expression, which correlated with higher detectable levels of ubiquitinated receptor. These findings are consistent with ubiquitin function as a negative regulator of PAR1 constitutive internalization. Increased PAR1 expression also enhanced agonist-induced phosphoinositide hydrolysis and endothelial barrier permeability. Thus, catalytically inactive thrombin appears to modulate PAR1 function in vitro by stabilizing receptor cell-surface expression; but given the high clearance rate of thrombin, the high concentration of dabigatran required to achieve this effect the in vivo physiologic relevance is unknown.

Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation: comment

“…In contrast, our group, which aimed to explore differential effects of oral anticoagulants on platelet aggregation in patients treated with oral anticoagulants for atrial fibrillation, documented that patients treated with dabigatran exilate 150 mg twice daily for at least 1 month in the absence of concomitant antiplatelet agents showed significantly higher levels of platelet reactivity induced by TRAP than healthy volunteers [8], suggesting that this high platelet reactivity may contribute to an increased risk of platelet-mediated events, as thrombin is known to be the major pathway for platelet activation in vivo. Consistent with the results of Achilles et al [1] and of our group [8], an interesting in vitro study characterized the thrombin-bound dabigatran effect on protease-activated receptor-1 (PAR-1) expression and signaling, and showed that catalytically inactive thrombin was able to modulate PAR-1 function by increasing PAR-1 surface expression [9]. It is of note that this effect was only observed for dabigatran concentrations 20-fold higher than therapeutic concentrations [9].…”

“…Consistent with the results of Achilles et al [1] and of our group [8], an interesting in vitro study characterized the thrombin-bound dabigatran effect on protease-activated receptor-1 (PAR-1) expression and signaling, and showed that catalytically inactive thrombin was able to modulate PAR-1 function by increasing PAR-1 surface expression [9]. It is of note that this effect was only observed for dabigatran concentrations 20-fold higher than therapeutic concentrations [9].…”

Direct Thrombin Inhibitors Prevent Left Atrial Remodeling Associated With Heart Failure in Rats

“…Furthermore, we showed that 10 nmol/l thrombin, a concentration reached during the initiation phase of coagulation cascade (30), was sufficient to induce hypertrophic genes in atrial myocardium in vitro. Interestingly, maximal inhibition of PAR-1 cleavage is obtained in vitro with 30 to 100 nmol/l dabigatran and 10 nmol/l thrombin, whereas higher DTI concentrations have activating effects (19). It is known that PAR-1 is activated at subnanomolar thrombin concentrations (31).…”

Noncanonical Effects of Oral Thrombin and Factor Xa Inhibitors in Platelet Activation and Arterial Thrombosis

“…33 Chen et al revealed in first in vitro analyses that dabigatran attenuates thrombin-induced PAR-1 activation, internalization, and cleavage dose-dependently. 34 In accordance, PAR-1 surface expression increased again by prolonged incubation with inactivated thrombin. 34 In addition, we could show that in vivo inhibition of thrombin by dabigatran led to increased surface expression of PAR-1 and PAR-4 on platelets measured by flow cytometry.…”

“…34 In accordance, PAR-1 surface expression increased again by prolonged incubation with inactivated thrombin. 34 In addition, we could show that in vivo inhibition of thrombin by dabigatran led to increased surface expression of PAR-1 and PAR-4 on platelets measured by flow cytometry. 32 In an animal model of diabetes, it could furthermore be shown that long-term dabigatran treatment enhances PAR-4 expression.…”