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J. Biol. Chem., Vol. 283, Issue 40, 26886-26893, October 3, 2008

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Coordinate Activation of Human Platelet Protease-activated Receptor-1 and -4 in Response to Subnanomolar -Thrombin^*

Frederick A. Ofosu¹, Lori Dewar, Sharon J. Craven, Yingqi Song, Aisha Cedrone, John Freedman^¶, and John W. Fenton, II^||

From the Canadian Blood Services, Hamilton, Ontario, Canada, the Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario L8N 3Z5, Canada, the ^¶Department of Medicine, St. Michael's Hospital, University of Toronto, Toronto, Ontario M5B 1W8 Canada, and the ^||Pharmaceutical Research Institute, Albany College of Pharmacy, Albany, New York 12144

We previously demonstrated that human platelets activated with SFLLRN release PAR-1 activation peptide, PAR-1-(1–41), even in the presence of hirudin. This observation suggests that during their activation, platelets generate a protease that activates PAR-1. In this study, PAR-1 and -4 activation peptides were detected 10 s after 1.0 nM -thrombin, 10 µM SFLLRN, or 100 µM AYPGKF were added to platelets. When SFLLRN or AYGPKF were added to platelets, generation of PAR-1 and -4 activation peptides was complete at 10 s. Generation of both PAR-1 and -4 activation peptides in response to 1 nM -thrombin was significantly inhibited by affinity-purified anti-PAR-1-(35–62) IgY, anti-PAR-4-(34–54) IgY, and by the specific PAR-1 antagonist BMS 200261, but not by the PAR-4 antagonist YD3. Effective inhibition of platelet aggregation in response to 1.0 nM -thrombin occurred only in the presence of both anti-PAR span antibodies. We conclude that platelet activation initiated with 1.0 nM -thrombin proceeds via simultaneous PAR-1 and -4 activation. Inhibiting the activation of either PAR inhibits activation of the other. Both PAR-1 and -4 activation must be inhibited to prevent platelet activation subsequent to thrombin binding to platelets. The more efficient generation of PAR activation peptides by platelets activated with SFLLRN or AYGPKF, compared with -thrombin, indicates that a platelet-derived serine protease that is inactivated by soybean trypsin inhibitor propagates PAR-1 and -4 activation.

Received for publication, March 20, 2008 , and in revised form, July 17, 2008.

^* This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Ontario and operational support funds from the Canadian Blood Services, Research and Development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: 1200 Main St. West, HSC 3N26, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22535); Fax: 905-521-2613; E-mail: ofosuf{at}mcmaster.ca.

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