Targeting Platelets in Multiple Organ Dysfunction Syndrome in Sepsis

Sepsis is a systemic inflammatory response to an infection. However, symptoms are produced by the host defense system, as opposed to the invading pathogen. Mortality rates remain high, despite advances in critical care medicine, and sepsis is the tenth leading cause of death in the United States. The frequent precursor to mortality from sepsis is multiple organ dysfunction syndrome (MODS), which is characterized by apoptosis in organs such as the lung, intestine, vascular endothelium, and lymphoid tissue. Notably, platelets have been shown to accumulate in the microvasculature of these tissues. Previous work in the laboratory has shown that sepsis induces an alteration in the megakaryocyte-platelet transcriptional axis, resulting in strongly cytotoxic platelets that express the serine protease granzyme B and are capable of inducing apoptosis in splenocytes ex vivo. Given these findings, we aimed to further investigate the role of platelet cytotoxicity in sepsis and the mechanism by which they induce cell death. We performed a mortality study comparing survival from sepsis in wild type and granzyme B null mice. We found that granzyme B null mice survived longer following induction of polymicrobial sepsis in comparison to wild type mice. Additionally, lung and spleen sections from septic granzyme B null mice showed decreased apoptosis, as measured by TUNEL staining, than sections from wild type mice. Given the apparent importance of granzyme B in cell death in sepsis, we sought to determine the mechanism by which platelet granzyme B enters target cells. Granzyme B is commonly released with perforin, which enables entry into target cells, but it can also enter cells independently of perforin. By co-incubating septic perforin null or wild type platelets with healthy splenocytes ex vivo, we determined platelet granzyme B does not require perforin to induce cell death, as splenocyte apoptosis was similar regardless of the septic platelet genotype. However, septic platelets do require contact to induce apoptosis in splenocytes ex vivo. Separation of septic platelets from splenocytes with a semi-permeable membrane significantly decreased splenocyte apoptosis. This finding suggested pharmacologically targeting platelet adhesion to other cell types could prevent platelet-induced apoptosis. To determine if this was the case, we exposed septic platelets to eptifibatide, a platelet GPIIb/IIIa platelet receptor inhibitor, before co-incubation with healthy splenocytes ex vivo. Splenocytes incubated with eptifibatide-exposed platelets had significantly less apoptosis in comparison to those without. To further test the potential of platelet adhesion inhibition as a therapeutic target in sepsis, we conducted an in vivo mortality study in which polymicrobial sepsis was induced in wild type mice that were subsequently treated with three intravenous injections of eptifibatide or PBS vehicle. Mice treated with eptifibatide had slower progression of sepsis and survived longer following induction of polymicrobial sepsis. However, apoptosis, as measured by TUNEL staining, was evident in lung and spleen sections from septic mice regardless of treatment. In conjunction with our other studies, this preclinical study emphasizes the importance of platelet cytotoxicity in sepsis. As such, inhibiting platelet adhesion could be a therapeutic target for the treatment of sepsis and MODS and warrants further study.