Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis.
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.


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.
Leger
Andrew J
AJ
Division of Hematology/Oncology, Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111, USA.
Jacques
Suzanne L
SL
Badar
Jehangir
J
Kaneider
Nicole C
NC
Derian
Claudia K
CK
Andrade-Gordon
Patricia
P
Covic
Lidija
L
Kuliopulos
Athan
A
D000818:Animals; D001792:Blood Platelets; D002460:Cell Line; D002633:Chemotaxis; D019281:Dimerization; D004195:Disease Models, Animal; D004359:Drug Therapy, Combination; D006168:Guinea Pigs; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology* / Q000627:therapeutic use; D010974:Platelet Aggregation; D011485:Protein Binding; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology / Q000627:therapeutic use; D013917:Thrombin / Q000378:metabolism; D013927:Thrombosis / Q000188:drug therapy* / Q000209:etiology; D014162:Transfection; D010975:Platelet Aggregation Inhibitors
D006629:Hirudins; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D013917:Thrombin; C112908:protease-activated receptor 4; C074619:bivalirudin; D010975:Platelet Aggregation Inhibitors