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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.

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 The physiological function of ADTRP (androgen‐dependent tissue factor pathway inhibitor regulating protein) is unknown. We previously identified ADTRP as coregulating with and supporting the anticoagulant activity of tissue factor pathway inhibitor in endothelial cells in vitro. Here, we studied the role of ADTRP in vivo, specifically related to vascular development, stability, and function. Methods and Results Genetic inhibition of Adtrp produced vascular malformations in the low‐pressure vasculature of zebrafish embryos and newborn mice: dilation/tortuosity, perivascular inflammation, extravascular proteolysis, increased permeability, and microhemorrhages, which produced partially penetrant lethality. Vascular leakiness correlated with decreased endothelial cell junction components VE ‐cadherin and claudin‐5. Changes in hemostasis in young adults comprised modest decrease of tissue factor pathway inhibitor antigen and activity and increased tail bleeding time and volume. Cell‐based reporter assays revealed that ADTRP negatively regulates canonical Wnt signaling, affecting membrane events downstream of low‐density lipoprotein receptor‐related protein 6 ( LRP 6) and upstream of glycogen synthase kinase 3 beta. ADTRP deficiency increased aberrant/ectopic Wnt/β‐catenin signaling in vivo in newborn mice and zebrafish embryos, and upregulated matrix metallopeptidase ( MMP )‐9 in endothelial cells and mast cells ( MCs ). Vascular lesions in newborn Adtrp −/− pups displayed accumulation of MCs , decreased extracellular matrix content, and deficient perivascular cell coverage. Wnt‐pathway inhibition reversed the increased mmp9 in zebrafish embryos, demonstrating that mmp9 expression induced by Adtrp deficiency was downstream of canonical Wnt signaling. Conclusions Our studies demonstrate that ADTRP plays a major role in vascular development and function, most likely through expression in endothelial cells and/or perivascular cells of Wnt‐regulated genes that control vascular stability and integrity.

Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 Escherichia coli We found that, albeit with different timing, both FXa/PCPS and E coli infusion led to robust thrombin and plasmin generation. Conversely, only E coli challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates.

Cell exposed tissue factor (TF) is generally in a low procoagulant ("cryptic") state, and requires an activation step (decryption) to exhibit its full procoagulant potential. Recent data suggest that TF decryption may be regulated by the redox environment through the oxidoreductase activity of protein disulfide isomerase (PDI). In this article we review PDI contribution to different models of TF decryption, namely the disulfide switch model and the phosphatidylserine dynamics, and hypothesize on PDI contribution to TF self-association and association with lipid domains. Experimental evidence debate the disulfide switch model of TF decryption and its regulation by PDI. More recently we showed that PDI oxidoreductase activity regulates the phosphatidylserine equilibrium at the plasma membrane. Interestingly, PDI reductase activity could maintain TF in the reduced monomeric form, while also maintaining low exposure of PS, both states correlated with low procoagulant function. In contrast, PDI inhibition or oxidants may promote the adverse effects with a net increase in coagulation. The relative contribution of disulfide isomerization and PS exposure needs to be further analyzed to understand the redox control of TF procoagulant function. For the moment however TF regulation remains cryptic.

Objective— Extracellular matrix (ECM) remodeling during angiogenesis is accomplished through plasmin-dependent pericellular proteolysis and through the action of matrix metalloproteinases (MMPs). Because tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type protease inhibitor with prominent ECM localization, inhibits plasmin and MMPs activity, we investigated the role of TFPI-2 in endothelial cell (EC) migration and angiogenesis. Methods and Results— Real-time polymerase chain reaction and immunostaining showed that the expression of TFPI-2 mRNA and protein was upregulated in migrating ECs. The effect of TFPI-2 on angiogenesis was studied in mouse models of Matrigel and polyvinylalcohol sponge implants by overexpressing TFPI-2 through infection with a replication-deficient adenovirus (AdTFPI-2). Using (immuno)fluorescence and confocal microscopy we observed that TFPI-2 reduced neovascularization and promoted ECM deposition. Lateral cell migration and capillary tube formation in vitro also were impaired by TFPI-2, a process reversed by anti–TFPI-2 antibodies. Increased apoptosis occurred both in AdTFPI-2–treated ECs and in the mouse implants. Zymography and assays in the absence of plasminogen confirmed plasmin inhibition as a main mechanism through which TFPI-2 inhibits EC migration. Conclusions— Our data suggest that TFPI-2 may be an important regulator of aberrant angiogenesis associated with tumor growth/metastasis, cardiovascular diseases, chronic inflammation, or diabetes.

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.

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa) activation of factors X and IX through the formation of the TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of caveolar TFPI are not yet known. Here we show that expression of caveolin-1 (Cav-1) in 293 cells keeps TFPI exposed on the plasmalemma surface, decreases the membrane lateral mobility of TFPI, and increases the TFPI-dependent inhibition of TF-FVIIa. Caveolae-associated TFPI supports the co-localization of the quaternary complex with caveolae. To investigate the significance of these observations for EC we used RNA interference to deplete the cells of Cav-1. Functional assays and fluorescence microscopy revealed that the inhibitory properties of TFPI were diminished in EC lacking Cav-1, apparently through deficient assembly of the quaternary complex. These findings demonstrate that caveolae regulate the inhibition by cell-bound TFPI of the active protease production by the extrinsic pathway of coagulation.

We used in vitro and in vivo models to characterize the physiological role of the novel protein encoded by C6ORF105. This gene's expression is androgen-responsive, and the encoded protein is predicted to be palmitoylated and membrane multi-spanning. Previously we showed that C6ORF105 expression co-regulates with tissue factor pathway inhibitor (TFPI)in human endothelial cells (EC); hence we named this protein "androgen-dependent TFPI-regulating protein" (ADTRP). Using in vitro cell-based TOP-Flash reporter assay we identified ADTRP as a negative regulator of canonical Wnt signaling in human cells. Overexpressing ADTRP in HEK293T cells inhibited the activity of beta-catenin/TCF-dependent transcriptional reporter, while silencing ADTRP increased the expression of Wnt target genes LEF-1, AXIN-2, IL-8 and DKK-2 in EA.hy926 EC line and HUVEC. Addition of LiCl showed that the effect of ADTRP was upstream of GSK3, therefore we focused the investigations on the Wnt signalosome proteins. ADTRP expression in HEK293T cells led to decreased phosphorylation of Wnt co-receptor LRP6, suggesting that ADTRP can affect this critical membrane-located event of Wnt signaling. Furthermore, ADTRP expression in reporter cells transfected with a constitutively phosphorylated form of LRP6 (LRP6DN mutant) inhibited Wnt3a- induced signaling, which suggests that ADTRP can interfere with events downstream of LRP6 phosphorylation, such as Axin-2 binding. Altogether, these data indicate that the Wnt signaling inhibitory activity of ADTRP takes place at the plasma membrane level. Site directed mutagenesis of the predicted palmitoylation site Cys61 showed that Wnt inhibitory effects of ADTRP require palmitoyl-mediated anchoring, highlighting the importance of proper membrane location of ADTRP for Wnt pathway inhibition. In vivo morpholino-based knockdown of adtrp in zebrafish embryos produced aberrant angiogenesis, defective branching and ruptured vessels, hemorrhage spots, pericardial edema and slow heart-beat, all reminiscent of defects caused by activation of canonical Wnt signaling. Indeed, adtrp knock down increased Wnt mediated lef-1 and pax-2a as well as mmp2 and mmp9 mRNA expression. Co-injection of ADTRP mRNA partially recovered the adtrp morpholino- induced morphologic abnormalities. Also, knock down of adtrp in a Wnt reporter zebrafish showed increased expression of ectopic Wnt signaling. Furthermore, our recently established Adtrp-/- mice also display some typical Wnt-mediated vascular defects, including: (i) abnormal patterning, increased capillary tortuosity, abnormal branching and increased density of the capillary network; (ii) dilated vessels, especially venules and veins; (iii) increased leakeage of permeability tracers (Evans blue and fluorescent dextran) without evident changes in endothelial junctions; (iv) hemorrhage spots in the skin, meningeal layers, heart, bladder and kidneys; (v) intravascular and interstitial fibrin deposition in the lung, liver and kidney. ADTRP deficiency decreased plasma TFPI antigen by ~2-times. Furthermore, TFPI antigen and anticoagulant activity in lung extracts and isolated lung EC were similarly decreased, which confirms our previous in vitro data. We aslo noticed increased tail bleeding time (>500 sec vs. 200 sec in WT littermates) and blood volume loss, which likely was caused by increased dilation of the tail vein. Gene expression analysis of whole organs showed upregulation of Wnt target genes involved in vascular contractility (Nos3), and extracellular matrix remodeling (Mmp2). Similarly, skin fibroblasts and lung EC isolated from Adtrp-/- mice showed increased expression of Wnt target genes (Lef-1, Cyclin D, Dkk2, c-Myc), which indicates constitutive activation of canonical Wnt signaling. In conclusion, we used genetic animal models and cell culture systems to show for the first time that the novel protein ADTRP plays major roles in vascular development and function. Lack of, or low levels of ADTRP associate with activation of coagulation and vascular development defects, which may be due, at least in part, to intrinsic high levels of ectopic canonical Wnt signaling. Disclosures No relevant conflicts of interest to declare.

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.

Abstract —Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor–dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm 2 (minimal flow), 4.1 dyne/cm 2 (venous flow), and 19 dyne/cm 2 (moderate arterial flow). Step increase of the shear stress from 0.27 to 19 dyne/cm 2 induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro.

Abstract —Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagulant activity of the tissue factor–factor VIIa complex (TF · FVIIa), is synthesized and constitutively secreted by endothelial cells (ECs). Here we describe the in vitro effects of heparin on the cellular localization, gene expression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-dependently induced a significant enhanced secretion of TFPI, paralleled by a redistribution and increase of TFPI on the cell surface and a decrease of intracellular TFPI. Immunogold electron microscopy showed the presence of clusters of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF · FVIIa on ECs treated with endotoxin was inhibited by both heparins but to a higher extent by LMWH. Inhibition of protein synthesis by cycloheximide did not reduce the release of TFPI induced by heparin. Long-term incubation (48 hours) resulted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly with UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1.4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs with phorbol ester (PMA) significantly enhanced the secretion of TFPI and increased its activity on the cell surface, probably by preventing invagination of caveolae. Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2.4 times lower than the expected additive value for PMA and UFH separately. Pretreatment of ECs with PMA suppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mechanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx. We assume that the increased secretion and redistribution of cellular TFPI induced by heparins in ECs in culture can play an important role in the modulation of the anticoagulant properties of the endothelium.

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.

Complement is a "dual-edged sword" that is critical for the innate immunity against pathogens, but its uncontrolled activation contributes to disseminated intravascular coagulation (DIC), inflammation, cell death, immune paralysis and cardiac dysfunction, leading to multiple organ failure and death. E. coli bacteremia is accompanied by robust complement activation triggered by the pathogen. The end products of the complement cascade are the cytolytic terminal complement complex C5b-9 and the C5a anaphylatoxin, both known mediators of sepsis pathology. We hypothesized that complement activation during bacteremia is the culprit of sepsis-induced inflammation and DIC by inducing rapid bacteria lysis and massive LPS release. Thus, C5 complement inhibition could block LPS release and downstream signaling through toll-like receptor 4 (TLR4) leading to nuclear factor (NF)-kappa B activation and production of inflammatory cytokines and procoagulant proteins. We used in vitro whole blood (WB) assays and a baboon model of E. coli sepsis to test the effects of a novel C5 inhibitor, the macrocyclic peptide RA101295, on sepsis-induced complement activation, LPS release, inflammation, oxidative stress and phagocytosis of the bacteria. WB was incubated with 5x107 cfu/ml live E. coli in presence/absence of RA101295. The treatment abolished complement activation measured as C5b-9 and C5a generation. RA101295 decreased the "oxidative burst" induced by E. coli in neutrophils and monocytes, a C5a dependent process that leads to rapid generation of highly toxic peroxide. C3b opsonization and bacteria phagocytosis were not affected. Concordantly, plasma samples treated with E. coli and RA101295 showed significantly lower TLR4 signaling in HEK-TLR4 reporter cells (Invivogen). These data indicate that RA101295 blocks the lysis of bacteria and release of LPS in plasma, without affecting E. coli phagocytosis. To test the in vivo effects of C5 inhibition we used a baboon model of sepsis. Animals challenged with E. coli (1-2x1010 cfu/kg; LD100) by iv infusion show robust generation of soluble C5b-9. Treatment with RA101295 fully inhibited complement activation and improved the clinical parameters and provides significant survival benefit. Notably, RA101295 treatment during the bacteremic sepsis did not impede bacteria clearance; on the contrary, the colony counts in the blood at 8 hours were slightly lower in the treated than in non-treated septic baboons. Plasma LPS levels were also significantly lower in treated animals suggesting that complement inhibition can block bacterial lysis without impairing clearing by phagocytosis. Remarkably, RA101295 abolished six sepsis-induced proinflammatory cytokines (TNF-α, IL6, IL8, IL1RA, MCP1 and G-CSF), reduced hypotension/shock and decreased the febrile response. Distinctly, LPS-driven expression of TNF-α is >57 fold decreased in RA101295 treated group, suggesting that the treatment blocks E. coli induced cytokine storm by inhibiting bacteria lysis and LPS release. The expression of IL6 and IL8, two cytokines induced via C5a signaling was almost fully inhibited. Moreover, RA101295 treatment significantly lowered consumptive coagulopathy, reflected by APTT, PT, and fibrin/fibrinogen degradation products tests. Furthermore, plasma levels of plasminogen activator inhibitor 1, a LPS and TNF regulated fibrinolysis inhibitor, were decreased by >2.4 fold in the treated versus non-treated septic animals. Altogether, our data show that RA101295 has protective effects when administered during the bacteremic stage by inhibiting C5b-9 induced lysis of the bacteria and explosive release of LPS. Equally important from a therapeutic point of view, C5 inhibition does not interfere with the phagocytosis and intracellular digestion of the pathogen, which ensures that efficient removal of the bacteria is unaffected. These data suggest that targeting complement activation can be an effective therapy to prevent damaging inflammation and DIC in sepsis patients. Disclosures DeMarco: Ra Pharmaceuticals: Employment. Lupu:Ra Pharmaceuticals: Research Funding; National Institutes of Health: Research Funding.

Thrombosis and cardiovascular disease (CVD) represent major causes of morbidity and mortality. Low androgen correlates with higher incidence of CVD/thrombosis. Tissue Factor Pathway Inhibitor (TFPI) is the major inhibitor of tissue factor-factor VIIa (TF-FVIIa)–dependent FXa generation. Because endothelial cell (EC) dysfunction leading to vascular disease correlates with low EC-associated TFPI, we sought to identify mechanisms that regulate the natural expression of TFPI. Data mining of NCBI's GEO microarrays revealed strong coexpression between TFPI and the uncharacterized protein encoded by C6ORF105, which is predicted to be multispan, palmitoylated and androgen-responsive. We demonstrate that this protein regulates both the native and androgen-enhanced TFPI expression and activity in cultured ECs, and we named it androgen-dependent TFPI-regulating protein (ADTRP). We confirm ADTRP expression and colocalization with TFPI and caveolin-1 in ECs. ADTRP-shRNA reduces, while over-expression of ADTRP enhances, TFPI mRNA and activity and the colocalization of TF-FVIIa–FXa-TFPI with caveolin-1. Imaging and Triton X-114–extraction confirm TFPI and ADTRP association with lipid rafts/caveolae. Dihydrotestosterone up-regulates TFPI and ADTRP expression, and increases FXa inhibition by TFPI in an ADTRP- and caveolin-1-dependent manner. We conclude that the ADTRP-dependent up-regulation of TFPI expression and activity by androgen represents a novel mechanism of increasing the anticoagulant protection of the endothelium.

Tissue factor (TF) is the cellular receptor for plasma protease factor VIIa (FVIIa), and the TF-FVIIa complex initiates coagulation in both hemostasis and thrombosis. Cell surface-exposed TF is mainly cryptic and requires activation to fully exhibit the procoagulant potential. Recently, the protein disulfide isomerase (PDI) has been hypothesized to regulate TF decryption through the redox switch of an exposed disulfide in TF extracellular domain. In this study, we analyzed PDI contribution to coagulation using an in vitro endothelial cell model. In this model, extracellular PDI is detected by imaging and flow cytometry. Inhibition of cell surface PDI induces a marked increase in TF procoagulant function, whereas exogenous addition of PDI inhibits TF decryption. The coagulant effects of PDI inhibition were sensitive to annexin V treatment, suggesting exposure of phosphatidylserine (PS), which was confirmed by prothrombinase assays and direct labeling. In contrast, exogenous PDI addition enhanced PS internalization. Analysis of fluorescent PS revealed that PDI affects both the apparent flippase and floppase activities on endothelial cells. In conclusion, we identified a new mechanism for PDI contribution to coagulation on endothelial cells, namely, the regulation of PS exposure, where PDI acts as a negative regulator of coagulation.

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.

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.

Abstract —Tissue factor (TF) pathway inhibitor (TFPI) is the major downregulator of the procoagulant activity of the TF–factor VIIa (FVIIa) complex (TF · FVII). The active TF present in the atherosclerotic vessel wall is proposed to be responsible for the major complication of primary atherosclerosis, namely, acute thrombosis after plaque rupture, but our knowledge of the sites of TFPI expression in relation to TF remains fragmentary. The aim of this study was to investigate the expression, localization, and activity of TFPI and its relation to the activity and distribution of TF in the normal and atherosclerotic vessel wall. We applied a novel approach in which serial cross sections of human vascular segments were used to perform a complete set of assays: immunolabeling for TFPI and/or TF, in situ hybridization for the expression of TFPI mRNA, ELISA for the determination of TFPI antigen, and functional assay for the activity of TFPI and TF. In healthy vessels, TFPI protein and mRNA are present in luminal and microvascular endothelial cells (ECs) and in the medial smooth muscle cells (SMCs). In atherosclerotic vessels, TFPI protein and mRNA frequently colocalized with TF in ECs overlying the plaque and in microvessels, as well as in the medial and neointimal SMCs, and in macrophages and T cells in areas surrounding the necrotic core. At the ultrastructural level, immunogold electron microscopy confirmed the localization of TFPI in ECs, macrophages/foam cells, and SMCs. In ECs and SMCs, the gold particles decorated the plasmalemma proper and the caveolae. ELISA on cross sections revealed that atherosclerotic tissues contain more TFPI than do the healthy vessels. TFPI was functionally active against TF · FVIIa-induced coagulation, and its activity was higher in those tissues that display less TF. The largest amount of TFPI and TF were detected in complicated arterial plaques. By immunofluorescence, TFPI colocalized with platelet- and fibrin-rich areas within the organized thrombi. Atherosclerotic vessel sections promote activation of factor X, which is dependent on the presence of TF and enhanced by preincubation of the sections with anti-TFPI IgG. Taken altogether, our results suggest that TFPI is largely expressed in the normal vessel wall and enhanced in the atherosclerotic vessel, in a manner suggesting a significant role of TFPI in the regulation of TF activity.

Abstract Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor•factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1- 3 H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol–phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface–associated inhibitory activity toward tissue factor•factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.

Abstract Tissue factor pathway inhibitor (TFPI) is a vascular anticoagulant that regulates the tissue factor (TF)–dependent pathway of coagulation. The majority of intravascular TFPI is thought to be noncovalently bound to the vessel wall. Our immunolocalization studies in cultures of human umbilical vein endothelial cells (HUVEC) and immortalized EA.hy926 cells show that TFPI is located in well-defined granules evenly spread over the cell surface and with apical polarization within the cytoplasm. These granules are smaller than and distinct from Weibel-Palade bodies. Upon treatment of cultured cells with low concentrations of thrombin (0.01 to 1 NIH U/mL), a marked redistribution of TFPI, occurred with patching in focal points and increased exposure of both TFPI antigen and anticoagulant activity on the surface of the stimulated endothelial cells. This redistribution was paralleled by an acute release of TFPI in the cell medium. EA.hy926 cells responded more readily to thrombin stimulation than HUVECs. The process was inhibited by both hirudin and anti-thrombin receptor antibody. Our findings demonstrate a novel mechanism by which thrombin may exert a negative feedback control on blood coagulation. Therefore, this pathway can be of physiological importance in controlling TF-mediated thrombin generation.

Abstract 1131 Cell exposed tissue factor (TF), the physiologic initiator of blood coagulation, is normally expressed in a low procoagulant, or cryptic conformation, and requires activation, or decryption, to fully exhibit its procoagulant potential. TF decryption is not fully understood and multiple decrypting mechanisms have been proposed including phosphatidylserine (PS) exposure, TF monomerization, association with lipid rafts and redox modulation of TF. Calcium ionophores have been extensively used as TF decrypting agents, and both PS-dependent and independent mechanisms have been associated with ionophore-induced TF decryption. In the present study we analyzed the changes that occur in the lateral mobility of cell exposed TF during calcium ionophore-induced decryption, using a TF chimera with monomeric yellow fluorescent protein (YFP-TF). The YFP-TF expressed by endothelial cells (EC) retains TF procoagulant activity, is mainly exposed on the cell surface and can be decrypted similarly with endogenous TF by the calcium ionophore ionomycin. We analyzed the changes in TF membrane mobility during decryption using live cell imaging of YFP-TF expressed in EC. Fluorescence recovery after photobleaching (FRAP) analysis revealed a decreased mobility of TF in EC treated with the decrypting agent ionomycin. The YFP-TF fluorescence in the region of interest was more easily bleached in ionomycin–treated cells as compared with controls. The observed maximum recovery (Rmax) of YFP-TF fluorescence in the bleached region of interest was significantly higher in control cells (80.84% recovery) as compared with ionomycin treated EC (39.29% recovery). These correlated with a decrease in YFP-TF mobile fraction from 50% for the control cells to 18% for the ionomycin treated EC. The lateral diffusion of the YFP-TF mobile fraction was similar between the two conditions, with halftime of fluorescence recovery of 7.69 sec in ionophore-treated cells and 10.69 sec in controls. These results suggest an immobilization of YFP-TF during decryption, which can be achieved by either lipid raft translocation or cytoskeleton floating. Similar to previous observations where TF cytoplasmic domain did not influence TF decryption, deletion of the TF cytoplasmic domain did not affect the lateral mobility of YFP-TF in FRAP analysis. To analyze decryption-induced changes in TF association with lipid domains, membrane fractions were isolated on a discontinuous Opti-Prep density gradient. Ionomycin treatment induced YFP-TF translocation from higher density, non-raft membrane fractions toward higher-buoyancy, raft fractions. Furthermore, the observed TF translocation into lipid rafts occurs without the formation of the quaternary complex with coagulation factors FVIIa, FXa and tissue factor pathway inhibitor (TFPI), as previously described. To address the functional modulation of TF procoagulant potential in response to lipid raft translocation, cell membrane cholesterol was either depleted with methyl-β-cyclodextrine (MβCD) or supplemented from an aqueous mixture of cholesterol-MβCD. Membrane cholesterol depletion decrypted TF in EC, likely through PS exposure, while also enhancing the procoagulant potential of ionomycin-decrypted TF. In contrast, cholesterol supplementation decreases the procoagulant potential of ionomycin-decrypted TF. Taken together, these observations support the model of tonic inhibition of TF procoagulant activity by the lipid raft environment. In conclusion, by live cell imaging we show that TF membrane mobility changes during calcium-ionophore induced decryption resulting in an immobilization of TF in lipid rafts. The immobilization is not influenced by the cytoplasmic domain of TF and does not require the formation of the TF-FVIIa-FXa-TFPI quaternary complex. Translocation into lipid rafts provides tonic inhibition of TF procoagulant potential and, as a consequence, we show for the first time that decrypting agents can also initiate negative regulation of TF procoagulant function. This negative feedback loop may help convert the decrypted TF back to its cryptic, low coagulant form. Disclosures: No relevant conflicts of interest to declare.

Caveolin-1 (CAV1) is a scaffolding protein that is essential for the formation of caveolae membrane domains, functions as a master-regulator of signaling molecules in caveolae, and has major role in the regulation of endothelial nitric oxide synthase (eNOS). Although it is thought that caveolins play some immunomodulatory roles, the actual function of CAV1 with respect to innate immunity in response to bacterial challenge is not clear. The aim of our study was to examine the in vivo role of CAV1 in a mouse model of endotoxemia. CAV1 knock out (ko) and wild-type (WT) mice were intravenously injected with LD80 of E. coli lipopolysaccharide (LPS, 1 mg/kg). Assessment of mortality during 7 days showed that the survival rate of CAV1 ko mice was significantly higher than WT mice (46% vs. 19%). Another group of mice similarly injected with LPS were sacrificed after 2, 8 and 24 hrs, at which times we analyzed multiple parameters of the inflammatory, NO production and coagulation responses in plasma and tissues. Non-challenged CAV1 ko mice have slightly increased numbers of leukocytes comparing to WT mice. After LPS challenge, the amount of circulating leukocytes decreased at 2 hrs in both genotypes, but were significantly lower in CAV1 ko vs. WT mice and correspondingly increased into the tissues (e.g. lung). Interestingly, non-challenged CAV1 ko mice displayed slightly but significantly higher number of neutrophils in the lung than WT mice. After LPS challenge, neutrophils migration increased dramatically at 2 and 24 hrs in WT, but not in CAV1 ko mice. This was paralleled by an increase in myeloperoxidase (a neutrophil product) in WT vs. CAV1 ko mice at 2 hrs and 24 hrs post-challenge. The levels of several cytokines (GM-CSF, IL-1b, IL-5, IL-6, IL-12) were significantly lower in LPS-treated CAV1 ko versus the corresponding WT mice. Only TNF-alpha peaked in CAV1 ko vs. WT mice at 2 hrs post challenge, then decreased at 24 hrs. Several chemokines (KC, IP-10, MCP1, MIG, MIP1a) were transiently upregulated at 2 hrs and diminished at 24 hrs during endotoxemia, but no significant difference between groups was observed. NO generation in response to LPS was evaluated by measuring nitrite/nitrate production in plasma and tissues. While CAV1 ko mice generated higher NO levels during the early stages of sepsis, likely due to increased eNOS function, WT mice displayed 4-fold increase in nitrite/nitrate at 24 hrs, due to a significant upregulation of inducible NOS (iNOS) in tissue leukocytes, as visualized by immunocytochemistry. We observed a temporal correlation between WT mice morbidity and NO generation during the late stages of endotoxemia. Consistent with their increased survival, CAV1 ko mice had lower nitrite/nitrate levels. Non-challenged CAV1 ko mice already have increased tissue factor activity in the lung comparing to WT mice, which further increased at 8 hrs post LPS injection. The resulting procoagulant state was also reflected by elevated levels of thrombin-antithrombin complexes, decreased fibrinogen in plasma, increased fibrin deposition in the lung, increased D-dimmer (peak at 24 hrs after LPS administration) and soluble thrombomodulin plasma levels, as compared with the WT animals. In conclusion, our data reveal that CAV1 ko mice have a more procoagulant but less inflammatory phenotype, which may confer them better survival during endotoxemic challenge. The mechanisms underlying the increased procoagulant and anti-inflammatory functions in CAV1-deficient mice remain to be determined.

The assembly of tissue factor-factor VIIa (TF-FVIIa) complex results in the proteolytic activation of factors X (FX) and IX and ultimate thrombin generation. The inhibition of this pathway by the Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI), involves the formation of a stable TF-FVIIa-FXa-TFPI complex. TFPI in endothelial cells (EC) locates primarily in rafts and caveolae, which are membrane microdomains enriched in cholesterol, glycosphingolipids (GSL) and caveolins, and which regulate the function of TFPI. Since caveolin-1 supports the TFPI-dependent inhibition of TF-FVIIa, we aimed to decipher the role played by the individual components of rafts in the anticoagulant function of cell surface TFPI. To this end, we studied the distribution of TFPI, TF and caveolin-1 by immunofluorescence microscopy, and we assayed the functional activity of TFPI after cholesterol-complexation on EC (EA. hy926 and HUVEC) and HEK293 expressing TFPI or TFPI+caveolin-1, or we used GSL-deficient CHO mutant cell lines. In EC, cholesterol complexation with filipin led to patching of TFPI over the cell surface and reduced inhibition of TF-FVIIa. Extraction of cholesterol from the external leaflet of the membrane with methyl-β-cyclodextrin (M-β-CD) shifted the partition of TFPI from predominantly raft-associated to the non-raft cellular fractions isolated through temperature-induced phase separation of Triton X-114 lysates. Although activation of FX by TF-FVIIa was significantly enhanced by M-β-CD and reversed after cholesterol replenishment, the effect was only modestly affected by the TFPI activity reduction. By immunofluorescence we observed that M-β-CD produced redistribution of both TFPI and TF over the EC and 293 cell surface with apparent segregation into separate domains and complete lack of co-localization. Such accumulations of TF will likely promote strong procoagulant activity when not inhibited by TFPI. Since M-β-CD selectively disrupts the glycerophospholipid-rich regions of the membrane while leaving the caveolar cholesterol virtually intact, we also tested progesterone, which extracts cholesterol specifically from caveolae. Treatment of HEK293 cells with progesterone for 2 hrs reduced significantly the inhibition of TF-FVIIa-dependent activation of FX by TFPI for TFPI+Cav+ cells but not for TFPI+ cells, suggesting that the process was specific for cells that have caveolae. To study the role of GSL for the activity of TFPI, we used Ly-B cells, a GSL-deficient mutant derived from CHO-K1, which have a defect in the LCB1 subunit of serine palmitoyltransferase. Characterization of endogenous TFPI in CHO-K1, Ly-B and its genetically corrected revertant Ly-B/cLCB1 (cLCB) revealed strong similarities between CHOs and EC with regard to the expression and function of TFPI. Whereas not affecting cLCB cells, incubation of Ly-B for 2 days in sphingolipid-deficient medium shifted the partition of cellular TFPI from the detergent-soluble (rafts) to the water-soluble (non-raft) fraction, which suggests that GSL play a major role in the distribution and function of the membrane TFPI. The fundamental knowledge developed by these studies will improve our understanding of the mechanisms by which TFPI functions against TF-FVIIa procoagulant activity on cell surfaces. In the long term, they may guide novel therapeutic approaches to prevent inflammation and thrombosis.

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.