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Thromb Res. 2014 Aug;134(2):418-25. doi: 10.1016/j.thromres.2014.06.001. Epub 2014 Jun 11.

The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca(2+) signaling pathways.

Author information

1
Department of Nephrology, Qingyuan City Hospital of Jinan University, Guangdong, China; Department of Medical and Health Sciences, Linköping University, Linköping, Sweden(1). Electronic address: peng.xiang@liu.se.
2
Department of Experimental and Clinical Medicine, Linköping University, Linköping, Sweden.
3
Department of Medical and Health Sciences, Linköping University, Linköping, Sweden(1).
4
Department of Medical and Health Sciences, Linköping University, Linköping, Sweden(1); School of Medicine, Örebro University, Örebro, Sweden.

Abstract

INTRODUCTION:

Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated.

METHODS:

The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry.

RESULTS:

PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca(2+) mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca(2+) chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it.

CONCLUSION:

The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca(2+) signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.

KEYWORDS:

ANCA-associated vasculitis; Ca(2+) signaling pathway; Platelet shape change; Proteinase PR3; Rho/Rho kinase signaling pathway

PMID:
24993595
DOI:
10.1016/j.thromres.2014.06.001
[Indexed for MEDLINE]
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