Characterization of thrombin-bound dabigatran effects on protease-activated receptor-1 expression and signaling in vitro.
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 thrombin-induced PAR1 cleavage, activation, internalization, and β-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.
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California (B.C., A.G.C., L.J.C., J.T.); Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut (A.G.); and Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma Gmbh, Biberach, Germany (J.R.).
Buxin
B
Chen
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California (B.C., A.G.C., L.J.C., J.T.); Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut (A.G.); and Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma Gmbh, Biberach, Germany (J.R.).
Antonio G
AG
Soto
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California (B.C., A.G.C., L.J.C., J.T.); Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut (A.G.); and Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma Gmbh, Biberach, Germany (J.R.).
Luisa J
LJ
Coronel
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California (B.C., A.G.C., L.J.C., J.T.); Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut (A.G.); and Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma Gmbh, Biberach, Germany (J.R.).
Ashley
A
Goss
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California (B.C., A.G.C., L.J.C., J.T.); Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut (A.G.); and Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma Gmbh, Biberach, Germany (J.R.).
Joanne
J
van Ryn
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California (B.C., A.G.C., L.J.C., J.T.); Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut (A.G.); and Department of CardioMetabolic Disease Research, Boehringer Ingelheim Pharma Gmbh, Biberach, Germany (J.R.) joanntrejo@ucsd.edu.
JoAnn
J
Trejo
D000991:Antithrombins / Q000494:pharmacology*; D019390:Arrestins / Q000378:metabolism*; D001562:Benzimidazoles / Q000494:pharmacology*; D000069604:Dabigatran; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D066298:In Vitro Techniques; D011485:Protein Binding; D044463:Receptor, PAR-1 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism*; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000071557:beta-Arrestins
D000991:Antithrombins; D019390:Arrestins; D001562:Benzimidazoles; D044463:Receptor, PAR-1; D000071557:beta-Arrestins; D015091:beta-Alanine; D013917:Thrombin; D000069604:Dabigatran