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Disseminated intravascular coagulation (DIC) is a syndrome triggered by infectious and noninfectious pathologies characterized by excessive generation of thrombin within the vasculature and widespread proteolytic conversion of fibrinogen. Despite diverse clinical manifestations ranging from thrombo-occlusive damage to bleeding diathesis, DIC etiology commonly involves excessive activation of blood coagulation and overlapping dysregulation of anticoagulants and fibrinolysis. Initiation of blood coagulation follows intravascular expression of tissue factor or activation of the contact pathway in response to pathogen-associated or host-derived, damage-associated molecular patterns. The process is further amplified through inflammatory and immunothrombotic mechanisms. Consumption of anticoagulants and disruption of endothelial homeostasis lower the regulatory control and disseminate microvascular thrombosis. Clinical DIC development in patients is associated with worsening morbidities and increased mortality, regardless of the underlying pathology; therefore, timely recognition of DIC is critical for reducing the pathologic burden. Due to the diversity of triggers and pathogenic mechanisms leading to DIC, diagnosis is based on algorithms that quantify hemostatic imbalance, thrombocytopenia, and fibrinogen conversion. Because current diagnosis primarily assesses overt consumptive coagulopathies, there is a critical need for better recognition of nonovert DIC and/or pre-DIC states. Therapeutic strategies for patients with DIC involve resolution of the eliciting triggers and supportive care for the hemostatic imbalance. Despite medical care, mortality in patients with DIC remains high, and new strategies, tailored to the underlying pathologic mechanisms, are needed.

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

The novel protein ADTRP, identified and described by us in 2011, is androgen-inducible and regulates the expression and activity of Tissue Factor Pathway Inhibitor, the major inhibitor of the Tissue Factor-dependent pathway of coagulation on endothelial cells. Single-nucleotide polymorphisms in ADTRP associate with coronary artery disease and myocardial infarction, and deep vein thrombosis/venous thromboembolism. Some athero-protective effects of androgen could exert through up-regulation of ADTRP expression. We discovered a critical role of ADTRP in vascular development and vessel integrity and function, manifested through Wnt signaling-dependent regulation of matrix metalloproteinase-9. ADTRP also hydrolyses fatty acid esters of hydroxy-fatty acids, which have anti-diabetic and anti-inflammatory effects and can control metabolic disorders. Here we summarize and analyze the knowledge on ADTRP and try to decipher its functions in health and disease.

Background During sepsis, gram‐negative bacteria induce robust inflammation primarily via lipopolysacharride (LPS) signaling through TLR4, a process that involves the glycosylphosphatidylinositol (GPI)‐anchored receptor CD14 transferring LPS to the Toll‐like receptor 4/myeloid differentiation factor 2 (TLR4/MD‐2) complex. Sepsis also triggers the onset of disseminated intravascular coagulation and consumptive coagulopathy. Objectives We investigated the effect of CD14 blockade on sepsis‐induced coagulopathy, inflammation, organ dysfunction, and mortality. Methods We used a baboon model of lethal Escherichia (E) coli sepsis to study two experimental groups (n = 5): (a) E coli challenge; (b) E coli challenge plus anti‐CD14 (23G4) inhibitory antibody administered as an intravenous bolus 30 minutes before the E coli. Results Following anti‐CD14 treatment, two animals reached the 7‐day end‐point survivor criteria, while three animals had a significantly prolonged survival as compared to the non‐treated animals that developed multiple organ failure and died within 30 hours. Anti‐CD14 reduced the activation of coagulation through inhibition of tissue factor‐dependent pathway, especially in the survivors, and enhanced the fibrinolysis due to strong inhibition of plasminogen activator inhibitor 1. The treatment prevented the robust complement activation induced by E coli, as shown by significantly decreased C3b, C5a, and sC5b‐9. Vital signs, organ function biomarkers, bacteria clearance, and leukocyte and fibrinogen consumption were all improved at varying levels. Anti‐CD14 reduced neutrophil activation, cell death, LPS levels, and pro‐inflammatory cytokines (tumor necrosis factor, interleukin (IL)‐6, IL‐1β, IL‐8, interferon gamma, monocyte chemoattractant protein‐1), more significantly in the survivors than non‐surviving animals. Conclusions Our results highlight the crosstalk between coagulation/fibrinolysis, inflammation, and complement systems and suggest a protective role of anti‐CD14 treatment in E coli sepsis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease affecting primarily premature infants. The disease is characterized by intestinal inflammation and leucocyte infiltration, often progressing to necrosis, perforation, systemic inflammatory response and death. Neutrophil extracellular traps (NETs), denoting nuclear DNA, histone and antimicrobial protein release, have been suggested to play a role in NEC. This study aimed to determine the role of NETs in NEC and explore the effect of chloramidine, a NET inhibitor, on a murine NEC‐like intestinal injury model. Blood and intestinal tissues were collected from infants diagnosed with ≥ Stage II NEC, and levels of nucleosomes and NETs, respectively, were compared with those of case‐matched controls. In mice, NEC was induced with dithizone/Klebsiella, and mice in the treatment group received 40 mg/kg chloramidine. Bacterial load, intestinal histology, plasma myeloperoxidase and cytokine levels, and immunofluorescent staining were compared with controls. Nucleosomes were significantly elevated in both human and mouse NEC plasma, whereas NET staining was only present in NEC tissue in both species. Chloramidine treatment increased systemic inflammation, bacterial load, organ injury and mortality in murine NEC. Taken together, our findings suggest that NETs are critical in the innate immune defence during NEC in preventing systemic bacteraemia.

Background Sepsis triggers dysfunction of coagulation and fibrinolytic systems leading to disseminated intravascular coagulation (DIC) that contributes to organ failure and death. Fondaparinux (FPX) is a synthetic pentasaccharide that binds to antithrombin (AT) and selectively inhibits factor (F) Xa and other upstream coagulation proteases but not thrombin (T). Objectives We used a baboon model of lethal Escherichia coli sepsis to investigate the effects of FPX treatment on DIC, organ function, and outcome. Methods Two experimental groups were studied: (a) E. coli challenge (n = 4); and (b) E coli plus FPX (n = 4). Bacteremia was modeled by intravenous infusion of pathogen (1-2 × 1010 CFU/kg). Fondaparinux (0.08 mg/kg) was administered subcutaneously, 3 h prior to and 8 h after bacteria infusion. Results Bacteremia rapidly increased plasma levels of inhibitory complexes of AT with coagulation proteases. Activation markers of both intrinsic (FXIa-AT), and extrinsic (FVIIa-AT) pathways were significantly reduced in FPX-treated animals. Factor Xa-AT and TAT complexes were maximal at 4 to 8 h post challenge and reduced >50% in FPX-treated animals. Fibrinogen consumption, fibrin generation and degradation, neutrophil and complement activation, and cytokine production were strongly induced by sepsis. All parameters were significantly reduced, while platelet count was unchanged by the treatment. Fondaparinux infusion attenuated organ dysfunction, prolonged survival, and saved two of four challenged animals (log-rank Mantel-Cox test, P = .0067). Conclusion Our data indicate that FPX-mediated inhibition of coagulation prevents sepsis coagulopathy; protects against excessive complement activation, inflammation, and organ dysfunction; and provides survival benefit in E. coli sepsis.

Source at https://doi.org/10.1073/pnas.1706818114. Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli-induced “oxidative burst,” as well as leukocyte activation, without affecting host phagocytosis of E. coli. RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death.

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long-lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS-induced fibroproliferation. Baboons challenged with 10(9) cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post-challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post-bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down-regulated sepsis-induced fibrosis genes, including transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C-C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF-β, phospho-Smad-2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis-induced fibrosis of the lung.

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.

Bacterial sepsis induces strong activation of coagulation, complement and fibrinolytic systems that contribute to disseminated intravascular coagulation, organ damage and death. While the contact of the blood with pathogens or pathogen-associated molecular patterns (PAMPs) can trigger the activation of both systems, a bidirectional complement-coagulation crosstalk is believed to occur. Although the role of complement activation products as positive regulators of coagulation is documented, direct activation of the complement proteins by thrombin or other hemostatic proteases was alluded but not demonstrated in vivo. Here we aimed to: (i) determine if in vivo generation of thrombin and other hemostatic proteases can activate the complement proteins and (ii) discriminate between the direct effect of the pathogen/PAMPs vs. hemostatic proteases on complement activation in a clinically relevant model of sepsis. We have compared the time-course of complement activation markers (C3b, C5a and C5b-9 terminal complex) in plasma of baboons exposed to 1010 cfu/kg (LD100) E. coli vs. intravenous infusion of factor Xa/PC:PS, a potent procoagulant stimulus. In baboons challenged with LD100 E. coli, complement activation markers C3b, C5a and C5b-9 reached maximum levels after 2 hrs (see figure). Complement activation coincided with the peak of bacteremia and LPS, but not with markers of thrombin generation (TAT and fibrinogen consumption; see figure) or fibrinolysis (FDP, D-dimers), which reached peak levels after 6 hours. Differently, infusion of FXa/PC:PS (36.6 pmol/L FXa and 56.3 nmol/L PC/PS per kg body weight) induced a rapid burst of thrombin and almost full consumption of fibrinogen during the first 10 min post-infusion, with no increase of complement activation markers. Based on these data we conclude that in vivo activation of the coagulation cascade does not support complement activation as was postulated by previous in vitro studies. Therefore, we conclude that pathogens and PAMPs are the main activators of the complement during sepsis while direct activation by hemostatic proteases is minor or absent. Figure Figure. Disclosures No relevant conflicts of interest to declare.

SummaryTissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. We used a baboon model of Escherichia coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. We performed extensive biochemical, histological, and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Similar to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial-to-mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6–27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared with age-matched control animals. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of transforming growth factor-β and platelet-derived growth factor signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, up-regulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps our understanding of the pathophysiology of fibrosis and the testing of new therapies.

Pulmonary fibrosis is a major and common medical condition, characterized by progressive scaring and decline in lung function. Persistent inflammation and acute lung injury in response to sepsis are potential triggers of the fibrotic response. Recently, we have reported that Escherichia coli sepsis in baboons strongly induces procoagulant responses and affects the integrity of the lung. These effects are diminished by the treatment with compstatin, a C3 convertase complement inhibitor [1].

Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis.

Background Bacterial invasion during sepsis induces disregulated systemic responses that could lead to fatal lung failure. The purpose of this study was to relate the temporal dynamics of gene expression to the pathophysiological changes in the lung during the first and second stages of E. coli sepsis in baboons. Results Using human oligonucleotide microarrays, we have explored the temporal changes of gene expression in the lung of baboons challenged with sublethal doses of E. coli. Temporal expression pattern and biological significance of the differentially expressed genes were explored using clustering and pathway analysis software. Expression of selected genes was validated by real-time PCR. Cytokine levels in tissue and plasma were assayed by multiplex ELISA. Changes in lung ultrastructure were visualized by electron microscopy. We found that genes involved in primary inflammation, innate immune response, and apoptosis peaked at 2 hrs. Inflammatory and immune response genes that function in the stimulation of monocytes, natural killer and T-cells, and in the modulation of cell adhesion peaked at 8 hrs, while genes involved in wound healing and functional recovery were upregulated at 24 hrs. Conclusion The analysis of gene expression modulation in response to sepsis provides the baseline information that is crucial for the understanding of the pathophysiology of systemic inflammation and may facilitate the development of future approaches for sepsis therapy.

Bacterial sepsis triggers robust early activation of the complement system that leads to the formation of the C5b-9 terminal complex (TCC) and potent proinflammatory anaphylatoxins, C3a and C5a, which significantly contribute to organ failure and death. Complement inhibition at C3 level provides organ protection in low bacteremia experimental sepsis but may impair the clearance of the bacteria. Here we used a baboon model of E. coli sepsis and in vitro whole-blood assays to test the protective effects of a novel C5 inhibitor (RA101295, a 2036 Da cyclic peptide). In vitro whole blood assays were performed to study the effects of RA101295 on sepsis-induced complement activation, inflammation, oxidative stress and phagocytosis of the bacteria. Lepirudin-anticoagulated blood was incubated with 5x107 cfu/ml E. coli or 500 µg/ml lipopolysaccharide (LPS) for 30-240 min in the presence/absence of the inhibitor. Results showed that RA101295 strongly inhibited E. coli and LPS–dependent C5b-9 and C5a generation. Similarly, RA101295 inhibited the E. coliand LPS induced oxidative burst in neutrophils and monocytes by 40-50% with minimal decrease ( Next, we used a baboon model of E. coli sepsis to investigate the in vivo effects of RA101295. E. coli challenge (1-2x1010 cfu/kg; LD100) in baboons strongly induced the generation of soluble C5b-9 complexes. Treatment with RA101295 C5 inhibitor (four subcutaneous 10 mg/kg doses at 8-12 hrs intervals) fully inhibited complement activation. RA101295 inhibitor remarkably improved the clinical parameters of all treated animals. Two of three baboons survived the lethal dose; while the survival time of the third animal was increased by 300%. Animals treated with complement inhibitor displayed better heart and lung performance and had lower febrile response than non-treated septic baboons. RA101295 treatment of E. coli sepsis led to significantly lower consumptive coagulopathy as reflected by the APTT/PT, fibrinogen consumption and fibrin/fibrinogen degradation products. These data highlight the tight crosstalk between the coagulation and complement systems and suggest that the observed protective effects are due in part to dampening of sepsis-induced coagulopathy. The treatment during the bacteremic stage had no negative effects on bacterial clearance. Similar to the in vitro data, these results suggest that C5 inhibition with this peptide does not increase the risk of secondary infections and reoccurrence of sepsis, probably because is not interfering with the generation of C3b opsonin. Histologic analysis of organs confirmed that treatment with RA101295 provided substantial organ protection. In contrast to the non-treated group, RA101295 treated animals showed no obvious signs of thrombosis and capillary leak in the lungs; no follicular necrosis in the spleen and no tubular necrosis and glomerular thrombosis in the kidneys. Overall, our data demonstrate that RA101295 strongly inhibits LPS and E. coli induced complement activation and TCC without affecting the bacterial clearance. These results suggest that complement inhibition at C5 level represents a promising therapeutic target in the fight against sepsis-induced MOF. Disclosures Keshari: Oklahoma Medical Research Foundation: Employment. Silasi-Mansat:Oklahoma Medical Research Foundation: Employment. Popescu:Oklahoma Medical Research Foundation: Employment. Chaaban:Oklahoma University Health Sciences Center: Employment. Lupu:Oklahoma Medical Research Foundation: Employment. DeMarco:Ra Pharmaceuticals: Employment. Lupu:American Heart Association: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; NIH: Research Funding; Oklahoma Medical Research Foundation: Employment.

Sepsis - the most common cause of death in hospitalized patients -is characterized by severe inflammatory response and profound systemic vasculitic pathology accompanied by activation of blood coagulation. We observed previously that infusion of E. coli in baboons produced severe disruption of the microcirculation, such as capillary leakage, intravascular coagulation and tissue injury, which may lead to global tissue hypoxia, shock, organ failure, and death. Here, we report data on the temporal dynamics of tissue specific changes of hypoxia, nitric oxide (NO) and apoptosis pathways in response to sublethal or lethal E. coli challenge. Animals were administered sublethal doses of E. coli (LD30–50; 108 CFU/kg) and sacrificed after 2, 8 and 24 hours or lethal doses (LD100; 9x109 −1x1010 CFU/kg) for 8 hours. Both groups were compared to non challenged controls (n=3 per experimental condition). Six organs (lung, liver, heart, kidney, spleen and lymph nodes) were analyzed by immunofluorescence/confocal imaging for Hypoxia Inducible Factor 1 (HIF1α), NOS2, NOS3 nitrotyrosine (NT), inflammatory cell markers (myeloperoxidase and CD68 for neutrophils and macrophages, respectively), and apoptosis markers (active caspase-3, TUNEL, Bcl2, Bax). Tissue extracts were analyzed by ELISA (NT and caspase-3), dot and western blot (NT, Bcl2 and Bax) and RT-PCR and microarray assays for mRNA expression. The results show that HIF1α mRNA was increased by 2–3 fold, reaching maximum values at 2 hours in the lung and 24 hours in the liver. HIF1α nuclear translocation was detected by immunostaining at 8 hours in lung and 24 hours in the liver, especially in the LD100 E. coli challenged baboons. HIF1α functions as a master transcriptional regulator of the adaptive response to hypoxia, and controls the expression of over 40 genes including NOS2, an inducible enzyme that induces NO production and thus decreases the vascular tone, and VEGF, a very potent permeability factor. NOS2 mRNA and protein were induced after 8 hours mainly in inflammatory cells, while endothelial NOS3 showed a transient decrease at 2–8 hours, and increased at 24 hours as compared to controls. The overall increase in NO production was reflected in transient high levels of nitrosylated proteins at 8 hours in sublethal animals and persistent high levels in lethal baboons. Ultrastructural analysis of lung and kidney performed by electron microscopy showed signs of defective permeability, such as accumulation of plasma proteins in the lung alveolae and in the subendothelium of the kidney peritubular capillaries, concomitant with intra- and/or extravascular fibrin deposition. Dose-dependent induction of apoptosis was detected by TUNEL and caspase-3 staining, especially in the proximal contort tubes of the kidney, hepatocytes, lymphoid and pericapsular cells of spleen and lymph nodes. Mitochondrial manganese superoxide dismutase (SOD2), a protective enzyme produced in reaction to oxidative stress had a latter response, reaching the maximum mRNA expression at 8 hour in lung and 24 hours in the liver. Taken together, these results place hypoxia as a key factor in the development of organ dysfunction and multiple organ failure in sepsis and suggest that targeting hypoxia controlled transcription factors, such as HIF1α, or upregulating the antioxidant mechanisms may represent valuable therapeutic approaches.

Activation of tissue factor (TF)-dependent coagulation is an early event in the pathogenesis of sepsis, responsible for microvascular thrombosis and consequent organ injury. We hypothesized that sepsis-induced TF procoagulant activity may be paralleled by a decreased expression or function of its natural inhibitor, tissue factor pathway inhibitor (TFPI). To test this hypothesis we used a non-human primate sepsis model in which adult baboons were infused with live E. coli at 109 CFU/kg (sublethal dose), 1010 CFU/kg (lethal dose) or saline (control group) and the animals were sacrified after 2, 8, or 24 hours. Lung tissue was snap frozen for protein and mRNA extraction or fixed and processed for light and electron microscopy. Tissue cryosections were immunostained using double/multiple fluorescence labeling approaches for TF, TFPI, factor VII/FVIIa and fibrin, in conjunction with cell markers for endothelial cells (CD31 or von Willebrand factor), leukocytes (PSGL-1), macrophages (CD68), PMN (myeloperoxidase) and platelets (gpIIb/IIIa). Large amounts of TF were detected in leukocytes, endothelial cells and platelet-rich microthrombi, starting from 2 hours and throughout the examined period. Concomitantly, confocal and electron microscopy analysis revealed increased leukocyte infiltration, platelet aggregates and fibrin deposition in the intravascular and interstitial compartments. In addition, TF induction was documented by semiquantitative RT-PCR, ELISA, western blot and factor Xa activation assays. Whereas TFPI mRNA showed only a modest increase, tissue-associated TFPI protein was found considerably decreased, especially during the first eight hours post E. coli infusion. Moreover, TFPI inhibitory activity of lung extracts from septic animals was 6–8 fold lower comparing to controls. TF activity measurements in the presence of inhibitory anti TFPI antibodies showed that only a very small fraction of endogenous TF was inhibited by tissue-associated TFPI, suggesting that most of the active TFPI available in the vascular compartment was depleted. The decrease of TFPI inhibitory potency cannot be exclusively explained by its proteolytic degradation, as we did not find significant amounts of truncated TFPI on western blots. In conclusion, our studies demonstrate that the exposure to septic and inflammatory stimuli lead to a decrease of TFPI-dependent endothelial anticoagulant potential, simultaneous with a strong TF-dependent procoagulant response. Activation of TF-dependent coagulation pathway not adequately countered by TFPI may have important roles in the pathogenesis of sepsis-associated disseminated intravascular coagulation. Strategies aimed to restore the physiological anticoagulant function of TFPI may help preventing sepsis-induced multiple organ dysfunction syndrome and death.

The effect of hypercholesterolemia on the platelet surface charge was examined in rabbits fed a lipid-rich diet (0.5% cholesterol and 5% butter). The strong anionic sites were detected with cationized ferritin (CF) pI 8.4, and the sialic acid concentration was evaluated by biochemical assays. In normal rabbits (average plasma cholesterol 0.36 +/- 0.05 mg/ml, and total platelet sialic acid 30.03 +/- 6 micrograms/mg protein) the platelet surface displayed a homogeneous distribution of CF, which also labeled the open canalicular system. Beginning with the third week of diet, at a plasma cholesterol level of 4.6 +/- 0.3 mg/ml, a reduction in the overall platelet negative charge was observed. As the diet progressed and the plasma cholesterol level increased, the CF binding to platelet surface diminished up to an almost total disappearance when the plasma cholesterol reached 18 mg/ml (the 20th week of diet). At the same time a progressive decrease in the sialic acid content up to 5.1 micrograms/mg protein was detected. These results suggest that diet-induced hyperlipidemia causes significant alterations in the platelet surface negative charge, especially in the sialic acid content.