Published online before print February 27, 2006, doi: 10.1161/CIRCULATIONAHA.105.587758
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(Circulation. 2006;113:1244-1254.)
© 2006 American Heart Association, Inc.
Vascular Medicine |
Blocking the Protease-Activated Receptor 1-4 Heterodimer in Platelet-Mediated Thrombosis
From the Hemostasis and Thrombosis Laboratory, Division of Hematology/Oncology, Molecular Oncology Research Institute, Tufts–New England Medical Center (A.J.L., S.L.J., J.B., N.C.K., L.C., A.K.), and Department of Biochemistry (A.J.L., A.K.), Tufts University School of Medicine, Boston, Mass; and Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, Spring House, Pa (C.K.D., P.A.-G.).
Correspondence to Athan Kuliopulos, MD, PhD, Tufts–New England Medical Center, 750 Washington St, Box 7510, Boston, MA 02111. E-mail athan.kuliopulos{at}tufts.edu
Received September 6, 2005; revision received December 27, 2005; accepted January 3, 2006.
Background— Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors.
Methods and Results— Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin–PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1.
Conclusions— We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.
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