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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets / cytology*
  • Blood Platelets / immunology*
  • Calcium / immunology*
  • Cell Shape
  • Humans
  • Myeloblastin / immunology*
  • P-Selectin / immunology
  • Platelet Activation*
  • Signal Transduction
  • rho-Associated Kinases / immunology*


  • P-Selectin
  • rho-Associated Kinases
  • Myeloblastin
  • Calcium