Sepsis alters the megakaryocyte- platelet transcriptional axis resulting in platelet lymphotoxicity M. Sharron, 1 A.S Benton, 1 A. A. Wiles, 1 N. Sabzevheri, 2 E. P. Hoffman, 1,2 R. J. Freishtat 1,2 1 Children's National Medical Center, Washington, DC; 2 George Washington University, Washington, DC

Disclosure The authors have documented that they have nothing to disclose.

Sepsis Whole-body inflammatory state and the presence of a known or suspected infection. 215,000 deaths annually in US (Angus et al 2001) Little change in morbidity and mortality over last 20 yrs ~30% to 40% mortality (Granja et al, 2004; Angus et al, 2004)

Sepsis, Lymphopenia and Platelets Lymphopenia during sepsis associated with poor outcomes (Hotchkiss, et al 1999,2003) lymphopenia in sepsis due to apoptosis (Hotchkiss, et al 2006) Platelets accumulate in spleen, lung, liver, other end organs in sepsis (Drake et al, 1993; Shibazaki et al 1996) Platelet microparticles are cytotoxic in a variety of cell types. (Cognasse, F, et al 2007, Von Hundelshausen P, et al, 2007, Danese S, et al 2004)

Contents of megakaryocytes and platelets Megakaryocyte Platelets Contain:DNA mRNA Protein Protein Protein Platelet microparticles

Experimental Hypothesis: Platelet transcriptome can change in response to systemic perturbations (ie sepsis)

Parallel studies in humans and mice Broad effort to define the progression of sepsis at the molecular level Mouse model of sepsis (CLP- Cecal Ligation and Puncture) Isolation of peripheral blood cell fractions throughout the progression of sepsis in human pediatric populations

Cecal Ligation and Puncture (CLP)

Platelet mRNA expression profiling in murine sepsis Differential regulation of 59 probe sets (56 genes) 6 cell death (GO: ) Septic mice via CLP Septic humans in PICU Granzyme B alone upregulated

GzmB-Induced Cell Death cytotoxic serine protease secreted within cytotoxic granules known player in well described apoptotic pathways

qT RT PCR validation Murine platelet granzyme B mRNA expression Human platelet granzyme B mRNA expression

Gz B upregulated in both platelets AND megakaryocytes during sepsis Megakaryocyte granzyme B mRNA expression Correlates with Platelet granzyme B mRNA expression

Intracellular granzyme B protein expression in platelets granzyme B protein in platelets from healthy and septic children. Day1 (49.7%) and Day 3 (44.3%) in a severely septic patient

Granzyme B upregulated in sepsis Megakaryocyte platelets Platelet microparticles GzB mRNA GzB Protein GzB Protein?

Granzyme B essential for platelet induced apoptosis Platelet/lymphocyte co-incubation Granzyme B necessary for platelets to induce lymphocyte apoptosis Presence of platelet activator (TNF α) not necessary for apoptosis

Discussion  Sepsis induces change in megakaryocyte- platelet transcriptional axis  Platelets up-regulate granzyme B in murine and human sepsis  Granzyme B from platelets induces lymphocyte apoptosis  Novel paradigm in sepsis: platelets are active lymphotoxic agents

Future Directions Apoptosis in additional cell types?- including lung, kidney, liver, spleen, heart Mechanism by which Granzyme B causes apoptosis – contact vs non contact dependent? Micro-particle mediated?

Acknowledgements Funding support provided to RJF by grants: –1K23RR and 1M01RR from the National Institutes of Health, Bethesda, Maryland –Board of Visitors and Research Advisory Council of Children’s National Medical Center, Washington, DC

Classification of sepsis severity by clustering Hierarchical Clustering Explorer (