Combined blockade of thrombin anion binding exosite-1 and PAR4 produces synergistic antiplatelet effect in human platelets.
Thrombin exosite-1 mediates the specific binding of thrombin with fibrinogen and protease-activated receptor (PAR) 1. Exosite-1 inhibitors have been shown to effectively decrease the clotting activity of thrombin, while their antiplatelet effects are relatively weak. In the present study, the inhibitory effects of two exosite-1 inhibitors, hirugen and HD1, but not the exosite-2 inhibitor HD22, on thrombin-induced platelet aggregation and P-selectin expression were dramatically enhanced by a PAR4 antagonist, YD-3. In contrast, the PAR1 antagonist SCH-79797 did not affect the antiplatelet effects of exosite-1 inhibitors. The exosite-1 inhibitors and YD-3 prevented the Ca2+ spike and the prolonged Ca2+ response in thrombin-stimulated platelets, respectively; and combination of these two classes of agents led to abolishment of Ca2+ signal. Unlike exosite-1 inhibitors, the antiplatelet effects of the active site inhibitor PPACK and the bivalent inhibitor bivalirudin were not significantly enhanced by YD-3. In addition, the platelet-stimulating activity of γ-thrombin, an autolytic product of α-thrombin which lacks exosite-1, was inhibited by YD-3. These results suggest that the synergistic antiplatelet effects of exosite-1 inhibitor and PAR4 antagonist are resulted from combined blockade of PAR1 and PAR4 in platelets. In fibrinogen or plasma clotting assay, YD-3 neither prolonged the clotting time on its own nor enhanced the anticoagulant activity of exosite-1 inhibitors. Therefore, the combined blockade of exosite-1 and PAR4 may offer a potential strategy for improving the balance of benefits and risks of antithrombotic therapy.
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan. ccwu@kmu.edu.tw
Chin-Chung
CC
Wu
Wei-Ya
WY
Wang
Chien-Kei
CK
Wei
Che-Ming
CM
Teng
D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000627:therapeutic use; D011516:Prothrombin / Q000037:antagonists & inhibitors*; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors*
C412964:1-benzyl-3-(ethoxycarbonylpheny)-indazole; D006629:Hirudins; D007191:Indazoles; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D018179:Receptors, Thrombin; C062510:hirugen; D011516:Prothrombin; C112908:protease-activated receptor 4