Activated protein C (APC) is a systemic anti-coagulant and anti-inflammatory factor. It reduces organ damage in animal models of sepsis, ischemic injury and stroke and substantially reduces mortality in patients with severe sepsis. It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect is secondary to its anti-coagulant and anti-inflammatory effects. We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells. Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR. This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells.

IntroductionThe ability of our innate immune system to rapidly recognize and respond to invading microbes is essential for controlling infections. This is accomplished by the expression of pattern recognition receptors (PRRs) on macrophages and other leukocytes, which recognize and respond to microbial components. 1,2 The PRRs recognize a diverse set of molecules generally classified as pathogen-associated molecular patterns (PAMPs), which include proteins, glycolipids, and glycoproteins, as well as DNA and RNA. 3 The importance of this interaction has been highlighted in numerous human and animal studies, but has been most extensively studied in mice deficient in the PRRs Toll-like receptor 2 (TLR2) or TLR4, which show increased susceptibility to Gram-positive and Gram-negative bacterial infections, respectively. 4 Therefore, based on their importance in both the innate and acquired immune responses, increased interaction between PAMPs and PRRs would likely lead to improved control of an infection. Although it is well established that intracellular pathogens are relatively inaccessible to certain components of the immune system, it is also likely that PAMPs expressed by intracellular pathogens would have only limited contact with immune cells compared with PAMPs released by extracellular pathogens. However, there may be mechanisms by which microbial proteins and lipids may be released from infected cells and thus overcome, at least in part, this sequestration of PAMPs.One recently described mechanism for release of proteins and lipids from cells is through the fusion of multivesicular bodies (MVBs) with the plasma membrane and subsequent exocytosis of their cargo. The membranes of the MVBs have their origin within the endocytic network, and thus this secretion system would consist of components present within this network. This would include foreign molecules present within phagosomes that have access to the endosomal pathway and which are subsequently trafficked to the MVB. The fusion of the MVB with the plasma membrane results in the release of the internal vesicles known as exosomes into the extracellular space. Exosomes are small 50-to 100-nm diameter vesicles that are released from many different cell types. 5,6 Originally characterized as a mechanism to remove transferrin from maturing reteculoyctes, 7 exosomes derived from dendritic cells (DCs) and B cells have recently garnered considerable interest since they express both major histocompatibility class (MHC) class I and II molecules as well as costimulatory molecules, and have been shown to promote T-cell activation. [8][9][10] Treatment of patients with cancer with exosomes primed with specific tumor antigens are now in clinical trials, 11 and show promise as novel therapeutic agents. 12 Therefore, recent evidence suggests that exosomes have biological significance. Nevertheless, there remain major gaps in our knowledge of exosome biology. In the present study, we show that exosomes released from macrophages infected with different intracellular patho...

Activated protein C (APC) reduces mortality of severe sepsis patients but increases the risk of serious bleeding. APC exerts anticoagulant activity by proteolysis of factors Va/VIIIa. APC also exerts antiinflammatory and antiapoptotic effects and stabilizes endothelial barrier function by APC-initiated cell signaling that requires two receptors, endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR1). The relative importance of APC's various activities for efficacy in sepsis is unknown. We used protein engineering of mouse APC and genetically altered mice to clarify mechanisms for the efficacy of APC in mouse sepsis models. Mortality reduction in LPS-induced endotoxemia required the enzymatic active site of APC, EPCR, and PAR-1, highlighting a key role for APC's cytoprotective actions. A recombinant APC variant with normal signaling but <10% anticoagulant activity (5A-APC) was as effective as wild-type APC in reducing mortality after LPS challenge, and enhanced the survival of mice subjected to peritonitis induced by gram-positive or -negative bacteria or to polymicrobial peritoneal sepsis triggered by colon ascendens stent implantation. Thus, APC's efficacy in severe sepsis is predominantly based on EPCR- and PAR1-dependent cell signaling, and APC variants with normal cell signaling but reduced anticoagulant activities retain efficacy while reducing the risk of bleeding.

Background To determine the prevalence of platelet dysfunction, using an end-point of assembly into a stable thrombus, following severe injury. Background: Although the current debate on acute traumatic coagulopathy (ATC) has focused on the consumption or inhibition of coagulation factors, the question of early platelet dysfunction in this setting remains unclear. Study Design Prospective platelet function in assembly and stability of the thrombus was determined within 30 minutes of injury using whole blood samples from trauma patients at the point of care employing thrombelastography (TEG)-based platelet functional analysis. Results There were 51 patients in the study. There were significant differences in the platelet response between trauma patients and healthy volunteers such that there was impaired aggregation to these agonists. In trauma patients, the median ADP inhibition of platelet function was 86.1% (IQR: 38.6–97.7%), compared to 4.2 % (IQR 0–18.2%) in healthy volunteers. Following trauma, the impairment of platelet function in response to AA was 44.9% (IQR 26.6–59.3%), compared to 0.5% (IQR 0–3.02%) in volunteers (Wilcoxon non parametric test p<0.0001 for both tests). Conclusions In this study, we show that platelet dysfunction is manifest following major trauma, before significant fluid or blood administration. These data suggest a potential role for early platelet transfusion in severely injured patients at risk for postinjury coagulopathy.