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Thromb Haemost 2011; 105(01): 88-95
DOI: 10.1160/TH10-05-0305
Platelets and Blood Cells
Schattauer GmbH

Combined blockade of thrombin anion binding exosite-1 and PAR4 produces synergistic antiplatelet effect in human platelets

Chin-Chung Wu
1   Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan
,
Wei-Ya Wang
1   Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan
,
Chien-Kei Wei
1   Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan
,
Che-Ming Teng
2   Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
› Author Affiliations Financial support: This work was supported by grants from National Science Council of Taiwan (NSC 98–2320-B-037–013-MY3).
Further Information

Publication History

Received: 20 May 2010

Accepted after major revision: 13 September 2010

Publication Date:
22 November 2017 (online)

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Summary

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.

Keywords

Platelet activation - thrombin - exosite - protease-activated receptors
 

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