Granzyme K Activates Protease-Activated Receptor-1 Dawn M. Cooper1,2, Dmitri V. Pechkovsky 1,3 , Tillie L. Hackett1,3, Darryl A. Knight1,3, David J. Granville1,2* 1 Institute for Heart and Lung Health, St. Paul’s Hospital, Vancouver, British Columbia, Canada, 2 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada, 3 Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada Abstract Granzyme K (GrK) is a trypsin-like serine protease that is elevated in patients with sepsis and acute lung inflammation. While GrK was originally believed to function exclusively as a pro-apoptotic protease, recent studies now suggest that GrK may possess other non-cytotoxic functions. In the context of acute lung inflammation, we hypothesized that GrK induces pro- inflammatory cytokine release through the activation of protease-activated receptors. The direct effect of extracellular GrK on PAR activation, intracellular signaling and cytokine was assessed using cultured human lung fibroblasts. Extracellular GrK induced secretion of IL-6, IL-8 and MCP-1 in a dose- and time-dependent manner in lung fibroblasts. Heat-inactivated GrK did not induce cytokine release indicating that protease activity is required. Furthermore, GrK induced activation of both the ERK1/2 and p38 MAP kinase signaling pathways, and significantly increased fibroblast proliferation. Inhibition of ERK1/2 abrogated the GrK-mediated cytokine release. Through the use of PAR-1 and PAR-2 neutralizing antibodies, it was determined that PAR-1 is essential for GrK-induced IL-6, IL-8 and MCP-1 release. In summary, extracellular GrK is capable of activating PAR-1 and inducing fibroblast cytokine secretion and proliferation. Citation: Cooper DM, Pechkovsky DV, Hackett TL, Knight DA, Granville DJ (2011) Granzyme K Activates Protease-Activated Receptor-1. PLoS ONE 6(6): e21484. doi:10.1371/journal.pone.0021484 Editor: Silvano Sozzani, University of Brescia, Italy Received March 8, 2011; Accepted June 2, 2011; Published June 30, 2011 Copyright: 2011 Cooper et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by a grants from the British Columbia Lung Association (DJG:20R64011). DMC is the recipient of Natural Sciences Engineering Research Council (NSERC), Canadian Institutes for Health Research (CIHR) IMPACT, and Heart & Stroke Foundation of Canada postdoctoral fellowships. TLH is supported by CIHR/Candian Lung Association/GSK, IMPACT, and Michael Smith Foundation for Health Research (MSFHR) postdoctoral fellowships. DAK is a Canada Research Chair in Airway Disease and a MSFHR Career Investigator. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts. DJG is a Founder and Consultant to viDA Therapeutics, Inc, however, DJG is not an employee of viDA, nor did viDA have any involvement in any aspect of this study (ie. viDA played no role in funding, initiation, design, data, any materials or interpretation of the data). No commercial/therapeutic products from viDA were used in this study. As such, in accordance with journal policies, the authors are in complete adherence to all PLoS One policies on sharing data and materials. * E-mail: david.granville@hli.ubc.ca Introduction Granzymes (granule-secreted enzymes) are a family of serine proteases that were once thought to function exclusively in immune- mediated target cell death through a perforin-dependent mecha- nism. In humans, there are 5 granzymes that differ in both function and substrate specificity: Granzyme A (GrA; tryptase), Granzyme B (GrB; aspartase), Granzyme H (GrH; chymase), Granzyme K (GrK; tryptase) and Granzyme M (GrM; metase). Despite their initial discovery and prediction to act as both intracellular and extracellular proteases, traditional views have limited granzyme function to the intracellular, perforin-dependent induction of cell death. However, over the past few years, evidence has emerged to challenge this view and strongly implies that granzymes exert other non-cytotoxic roles in health and disease [1,2,3,4,5,6,7,8] Elevated levels of GrA, GrB and GrK are observed in a wide array of inflammatory diseases such as atherosclerosis, arthritis, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and asthma (reviewed in [4,9]). However, while several studies have focused on the perforin-independent functions of GrA and GrB, little is known concerning the function of extracellular GrK [10,11,12,13,14]. GrK is found at low levels in the plasma of healthy patients but is markedly elevated in the plasma of patients suffering from viral infections and sepsis [11,15]. GrK is also elevated in the bronchoalveolar lavage (BAL) fluid of patients suffering from allergic asthma and viral pneumonias [10,11]. Although our understanding of the proteo- lytic regulation of extracellular GrK remains poorly understood, recent studies have identified inter-alpha inhibitor proteins (IAIP) as physiological inhibitors of GrK and have shown that a reduction in plasma IAIP levels and free, unbound GrK correspond to increased disease severity [16,17]. GrK is a highly cationic protease that displays tryptase-like activity, which cleaves after the basic amino acids Lys or Arg and is most closely related to GrA [18,19,20,21]. Despite sharing many substrates with GrA, proteomic profiling has demonstrated that GrK can target a unique set of substrates suggesting it likely functions distinctly from that of GrA [19,22]. GrA, also a tryptase- like protease, is capable of inducing cell detachment, cytokine release, neurite retraction and activation of Protease-Activated Receptor (PARs) [8,23,24,25]. PARs are a family of G-protein coupled receptors (GCPRs) that mediate the physiological responses to serine proteases (reviewed in [26,27]). PARs share a unique mechanism of activation that involves the cleavage of an N-terminal extracellular domain which leads to the unmasking of a tethered ligand that, in turn, activates the receptor by intramolecular binding followed by intracellular signaling [27,28]. PAR-1 is activated by thrombin and trypsin, PAR-2 is a PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21484