Eastern Regional Meeting - 2008 Program & Abstracts
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PLATELETS AS CYTOTOXIC MEDIATORS IN SEPSIS: PLATELET GRANZYME B INDUCES LYMPHOCYTE APOPTOSIS
R. J. Freishtat1, 2, 3, J. Natale4, J. S. Cohen1, 3, B. Mojgani2, A. S. Benton2, W. M. Ngor2, M. Bradbury2, C. Bunaj2, E. P. Hoffman2, 3; 1. Division of Emergency Medicine, Children's National Medical Center, Washington, DC, USA. 2. Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC, USA. 3. School of Medicine and Health Sciences, The George Washington University, Washington, DC, USA. 4. Department of Pediatric Critical Care, University of California at Davis, Sacramento, CA, USA.
Purpose of Study: Poor outcomes in sepsis are associated with lymphopenia secondary to profound apoptosis. Based on data showing platelets can induce apoptosis through reactive oxygen species, we hypothesized that platelets are cytotoxic contributers to sepsis-associated lymphopenia.
Methods Used: Platelets were collected from mice (n=80) 0, 24, and 48h status post cecal ligation and puncture. Platelet mRNA was expression profiled. A validation set of critically ill children with sepsis (n=20) was also studied. Functional platelet testing was performed on fresh citrated murine platelets using flow cytometry and quantitative colorimetry.
Summary of Results: We found significant (p=0.01) up-regulation of mRNA for 20 cell death mediators in sepsis-activated platelets by microarray. These included the potent apoptosis inducing granzymes A and B (both p<0.001). Granzyme B up-regulation was validated by RT-PCR (24h:0h relative expression ratio (mean+SE)=10.1+0.1, p=0.04). Granzyme A up-regulation was not significant by RT-PCR. In a further validation step, we found up-regulation of platelet granzyme B in children with severe sepsis (72h:0h relative expression ratio=2.92, p=0.018) while those with milder disease showed no such increase. Flow cytometry of murine platelets showed intracellular platelet GzmB positivity increased from 4.4+1.3% to 19.6+6.3% (p=0.039) in septic mice over 24 hours. Quantitative colorimetry of control CD4+ splenocytes co-incubated with murine sepsis-activated platelets showed significantly increased percentage splenocyte apoptosis over controls (52.2+17.3 vs 18.6+5.7%, p=0.045).
Conclusions: Our data establish a novel paradigm in sepsis: platelets as active cytotoxic agents. Sepsis-activated platelets express and exocytose granzyme B which may account for a significant portion of sepsis-associated lymphocyte apoptosis. Since lymphocyte apoptosis is associated with sepsis severity, inhibiting platelet granzymes in vivo may provide a novel treatment for sepsis.
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