Hydrogenated Fat Intake during Pregnancy and Lactation Modifies Serum Lipid Profile and Adipokine mRNA in 21-day-old Rats

Objective We examined whether feeding pregnant and lactating rats hydrogenated fats rich in trans-fatty acids modifies the plasma lipid profiles and the expression of adipokines involved with insulin resistance and cardiovascular disease in their 21-d-old offspring. Methods Pregnant and lactating Wistar rats were fed with a control diet (C group) or one enriched with hydrogenated vegetable fat (T group). After delivery, male offspring were weighed weekly and killed at day 21 of life by decapitation. Blood and retroperitoneal, epididymal, and subcutaneous white adipose tissues were collected. Results Offspring of T-group rats had increased serum triacylglycerols and cholesterol, white adipose tissue plasminogen activator inhibitor-1, and tumor necrosis factor-α gene expression, and carcass lipid content and decreased blood leptin and adiponectin and adiponectin gene expression. Conclusion Ingestion of hydrogenated vegetable fat by the mother during gestation and lactation alters the blood lipid profiles and the expression of proinflammatory adipokynes by the adipose tissue of offspring aged 21 d.

Tenecteplase and alteplase in acute ischaemic stroke thrombolysis: clinical and imaging study

Introduction: Intravenous thrombolysis in acute ischaemic stroke with alteplase improves clinical outcomes, but it has limited efficacy and is associated with increased risk of intracranial haemorrhage. An improved tissue plasminogen activator, tenecteplase, was evidenced to be at least equally effective with lower risk of haemorrhage in acute myocardial infarction thrombolysis. To date, two completed phase II randomised controlled studies comparing tenecteplase and alteplase in acute ischaemic strokes showed variable results. Methods: A literature review of thrombolytic agents used in myocardial infarction and acute ischaemic stroke was performed, followed by a retrospective investigation of the bolus-to- infusion delay of alteplase administration. The main focus of this thesis is the report of our single centre phase II randomised controlled trial that compared tenecteplase (0.25mg/kg, maximum 25mg) and alteplase (0.9mg/kg, maximum 90mg, 10% as the initial bolus, following by one hour infusion with the rest of the dose) in acute ischaemic stroke thrombolysis using advanced imaging as biomarkers. Imaging comprised baseline computed tomography (CT), CT perfusion (CTP) and CT angiography (CTA), and CT+CTA at 24-48 hours. The primary end-point was penumbral salvage (CTP-defined penumbra volume minus follow-up CT infarct volume). A sub-study of coagulation and fibrinolysis analysis of the two agents was performed by comparing a group of coagulation variables measured pre-treatment, 3-12 hours, and 24±3 hours post thrombolysis. An individual patient data (IPD) meta-analysis was carried out using all three completed tenecteplase/alteplase comparison studies in stroke thrombolysis. We compared clinical outcomes including modified Rankin scale at 3 months, early neurological improvement at 24 hours, intracerebral haemorrhage rate and mortality at 3 months between all three tenecteplase doses (0.1mg/kg, 0.25 mg/kg, and 0.4mg/kg) examined and standard alteplase. Imaging outcomes including penumbra salvage, recanalisation rates were also compared using the data from the two studies that had advance imaging carried out. Results: Delay between the initial bolus and the subsequent infusion in administration of alteplase is common. This may reduce the likelihood of achieving a good functional outcome. Among the 104 patients recruited in ATTEST trial, 71 contributed to the imaging primary outcome. No significant differences were observed for penumbral salvage [68 (SD 28) % tenecteplase vs 68 (SD 23) % alteplase], mean difference 1% (95% confidence interval -10%, 12%, p=0·81) or for any secondary end-point. The SICH incidence (1/52, 2% vs 2/51, 4%, by SITS-MOST definition, p=0·55; by ECASS-2 definition, 3/52, 6% tenecteplase vs 4/51, 8% alteplase, p=0.59) did not differed significantly. There was a trend towards lower ICH risk in the tenecteplase group (8/52 tenecteplase, 15% vs 14/51 alteplase, 29%, p=0·091). Compared to baseline, alteplase caused significant hypofibrinogenaemia (p=0.002), prolonged Prothrombin Time (PT) (p=0.011), hypoplasminogenaemia (p=0.001) and lower Factor V (p=0.002) at 3-12 hours after administration with persistent hypofibrinogenaemia at 24h (p=0.011), while only minor hypoplasminogenaemia (P=0.029) was seen in the tenecteplase group. Tenecteplase consumed less plasminogen (p<0.001) and fibrinogen (p=0.002) compared with alteplase. In a pooled analysis, tenecteplase 0.25mg/kg had the greatest odds to achieve early neurological improvement (OR [95%CI] 3.3 [1.5, 7.2], p=0.093), excellent functional outcome (mRS 0-1) at three months (OR [95%CI] 1.9 [0.8, 4.4], p= 0.28), with reduced odds of ICH (OR [95%CI] 0.6 [0.2, 1.8], P=0.43) compared with alteplase. Only 19 patients were treated with tenecteplase 0.4mg/kg, which showed increased odds of SICH compared with alteplase (OR [95% CI] 6.2 [0.7, 56.3]). In the two studies where advanced imaging was performed, the imaging outcomes did not differ in the IPD analysis. Conclusion: Tenecteplase 0.25 mg/kg has the potential to be a better alternative to alteplase. It can be given as a single bolus, does not cause disruption to systemic coagulation, and is possibly safer and more effective in clot lysis. Further phase III study to compare tenecteplase and alteplase in acute ischaemic stroke is warranted.

Efficacy and safety of very early mobilisation within 24 h of stroke onset (AVERT): a randomised controlled trial

Background Early mobilisation after stroke is thought to contribute to the eff ects of stroke-unit care; however, the intervention is poorly defined and not underpinned by strong evidence. We aimed to compare the effectiveness of frequent, higher dose, very early mobilisation with usual care after stroke.MethodsWe did this parallel-group, single-blind, randomised controlled trial at 56 acute stroke units in five countries. Patients (aged ≥18 years) with ischaemic or haemorrhagic stroke, first or recurrent, who met physiological criteriawere randomly assigned (1:1), via a web-based computer generated block randomisation procedure (block size of six), to receive usual stroke-unit care alone or very early mobilisation in addition to usual care. Treatment with recombinant tissue plasminogen activator was allowed. Randomisation was stratified by study site and stroke severity. Patients, outcome assessors, and investigators involved in trial and data management were masked to treatment allocation. The primary outcome was a favourable outcome 3 months after stroke, defined as a modified Rankin Scale score of 0–2. We did analysis on an intention-to-treat basis. The trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12606000185561.FindingsBetween July 18, 2006, and Oct 16, 2014, we randomly assigned 2104 patients to receive either very early mobilisation (n=1054) or usual care (n=1050); 2083 (99%) patients were included in the 3 month follow-up assessment. 965 (92%) patients were mobilised within 24 h in the very early mobilisation group compared with 623 (59%) patients in the usual care group. Fewer patients in the very early mobilisation group had a favourable outcome than those in the usual care group (n=480 [46%] vs n=525 [50%]; adjusted odds ratio [OR] 0·73, 95% CI 0·59–0·90; p=0·004). 88 (8%) patients died in the very early mobilisation group compared with 72 (7%) patients in the usual care group (OR 1·34, 95% CI 0·93–1·93, p=0·113). 201 (19%) patients in the very early mobilisation group and 208 (20%) of those in the usual care group had a non-fatal serious adverse event, with no reduction in immobility-related complications with very early mobilisation.InterpretationFirst mobilisation took place within 24 h for most patients in this trial. The higher dose, very early mobilisation protocol was associated with a reduction in the odds of a favourable outcome at 3 months. Early mobilisation after stroke is recommended in many clinical practice guidelines worldwide, and our findings should affect clinical practice by refi ning present guidelines; however, clinical recommendations should be informed by future analyses of dose–response associations.

Activation of membrane-bound proteins and receptor systems: A link between tissue kallikrein and the KLK-related peptidases

The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and roles in a range of cellular processes, including proliferation, migration, invasion, differentiation, inflammation and angiogenesis that are required in both normal physiology as well as pathological conditions. These roles require cleavage of a range of substrates, including extracellular matrix proteins, growth factors, cytokines as well as other proteinases. In addition, it has been clear since the earliest days of KLK research that cleavage of cell surface substrates is also essential in a range of KLK-mediated cellular processes where these peptidases are essentially acting as agonists and antagonists. In this review we focus on these KLK-regulated cell surface receptor systems including bradykinin receptors, proteinase-activated receptors, as well as the plasminogen activator, ephrins and their receptors, and hepatocyte growth factor/Met receptor systems and other plasma membrane proteins. From this analysis it is clear that in many physiological and pathological settings KLKs have the potential to regulate multiple receptor systems simultaneously; an important issue when these peptidases and substrates are targeted in disease.

Interactions of Lipoprotein(a) with the Plasminogen System: Mechanisms and Pathophysiological Consequences

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are associated with increased risk of atherothrombotic disease. Lp(a) is a unique lipoprotein consisting of a low density lipoprotein-like moiety covalently linked to apolipoprotein(a) (apo(a)), a homologue of the fibrinolytic proenzyme plasminogen. Apo(a) is extremely heterogeneous in size with small isoforms being independently associated with increased cardiovascular risk. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator (tPA)-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu1-plasminogen to Lys78-plasminogen. Additionally, abnormal fibrin clot structures have been associated with both an increased risk of cardiovascular disease and elevated Lp(a) levels. Similarly, oxidized phospholipids have been implicated in the development of cardiovascular disease, and are not only preferentially carried by Lp(a) in the plasma but have also been shown to covalently-modify both apo(a) and plasminogen. In this thesis, we built upon the understanding of the role of apo(a) in plasminogen activation on the fibrin/degraded fibrin surface by determining that: (i) apo(a) inhibits plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion and identifying the critical domains in apo(a) responsible for this effect, (ii) apo(a) isoform size does not affect either the inhibition of tPA-mediated plasminogen activation or the inhibition of plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion, (iii) apo(a) modifies fibrin clot structure to form more dense clots with thinner fibers and reduced permeability, modifications that enhance the ability of apo(a) to inhibit tPA-mediated plasminogen activation and (iv) the phosphorus content of apo(a) affects its ability to inhibit tPA-mediated plasminogen activation and the phosphorus content of plasminogen affects its ability to be activated by tPA. By understanding these individual reactions, each of which has the potential to affect the broader fibrin clot lysis process, we have expanded our understanding of the overall effect of Lp(a)/apo(a) in the inhibition of plasminogen activation on the fibrin/degraded fibrin surface and thus broadened our understanding of how Lp(a)/apo(a) may mediate the inhibition of thrombolysis in vivo.

Mutant and chimeric recombinant plasminogen activators

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-Pa have been designed to direct the synthesis of new plasminogen activators.

Tissue factor gene expression in the adipose tissues of obese mice

Altered expression of proteins of the fibrinolytic and coagulation cascades in obesity may contribute to the cardiovascular risk associated with this condition. We previously reported that plasminogen activator inhibitor 1 (PAI-1) is dramatically up-regulated in the plasma and adipose tissues of genetically obese mice. This change may disturb normal hemostatic balance and create a severe hypofibrinolytic state. Here we show that tissue factor (TF) gene expression also is significantly elevated in the epididymal and subcutaneous fat pads from ob/ob mice compared with their lean counterparts, and that its level of expression in obese mice increases with age and the degree of obesity. Cell fractionation and in situ hybridization analysis of adipose tissues indicate that TF mRNA is increased in adipocytes and in unidentified stromal vascular cells. Transforming growth factor β (TGF-β) is known to be elevated in the adipose tissue of obese mice, and administration of TGF-β increased TF mRNA expression in adipocytes in vivo and in vitro. These observations raise the possibility that TF and TGF-β may contribute to the increased cardiovascular disease that accompanies obesity and related non-insulin-dependent diabetes mellitus, and that the adipocyte plays a key role in this process. The recent demonstration that TF also influences angiogenesis, cell adhesion, and signaling suggests that its exact role in adipose tissue physiology/pathology, may be complex.

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-l-cysteine, d-penicillamine, captopril, l-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.

Reverse biochemistry: Use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a “fold-specific” inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription–PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Roles of the matrix metalloproteinases in mammary gland development and cancer

Tissue remodeling is a key process involved in normal development, wound healing, bone remodeling, and embryonic implantation, as well as pathological conditions such as tumor invasion and metastasis, and angiogenesis. The degradation of the extracellular matrix that is associated with those processes is mediated by a number of families of extracellular proteinases. These families include the serine proteinases, such as the plasminogen-urokinase plasminogen activator system and leukocyte elastases, the cysteine proteinases, like cathepsin D and L, and the zinc-dependent matrix metalloproteinases (MMPs). Accumulating evidence has highlighted the central role of MMP-driven extracellular matrix remodeling in mammary gland development and breast cancer.

Acute resistance exercise increases the expression of chemotactic factors within skeletal muscle

Intense resistance exercise causes mechanical loading of skeletal muscle, followed by muscle adaptation. Chemotactic factors likely play an important role in these processes. Purpose We investigated the time course of changes in the expression and tissue localization of several key chemotactic factors in skeletal muscle during the early phase of recovery following resistance exercise. Methods Muscle biopsy samples were obtained from vastus lateralis of eight untrained men (22+-0.5 yrs) before and 2, 4 and 24 h after three sets of leg press, squat and leg extension at 80% 1 RM. Results Monocyte chemotactic protein-1 (95×), interleukin-8 (2,300×), IL-6 (317×), urokinase-type plasminogen activator (15×), vascular endothelial growth factor (2×) and fractalkine (2.5×) mRNA was significantly elevated 2 h post-exercise. Interleukin-8 (38×) and interleukin-6 (58×) protein was also significantly elevated 2 h post-exercise, while monocyte chemotactic protein-1 protein was significantly elevated at 2 h (22×) and 4 h (21×) post-exercise. Monocyte chemotactic protein-1 and interleukin-8 were expressed by cells residing in the interstitial space between muscle fibers and, in some cases, were co-localized with CD68+ macrophages, PAX7+ satellite cells and blood vessels. However, the patterns of staining were inconclusive and not consistent. Conclusion In conclusion, resistance exercise stimulated a marked increase in the mRNA and protein expression of various chemotactic factors in skeletal muscle. Myofibers were not the dominant source of these factors. These findings suggest that chemotactic factors regulate remodeling/adaptation of skeletal muscle during the early phase of recovery following resistance exercise.

Ethnic differences in lipid metabolism in two groups of obese South African women

There is a higher prevalence of ischemic heart disease (IHD) in South African white than black women. The objective of this study was to determine biochemical explanations for this prevalence. The study group contained 15 obese black women (OBW) and 14 obese white women (OWW), ah premenopausal, who were examined after an overnight fast. Anthropometric measurements and blood concentrations of glucose, non-esterified fatty acids (NEFAs), catecholamines, plasminogen activator inhibitor-1, C-peptide, proinsulin, lipograms, cortisol, growth hormone, and post-heparin Lipoprotein Lipase activity were measured during an oral glucose tolerance test (OGTT), Body composition was measured using bioelectrical impedance analysis, and subcutaneous and visceral fat mass were assessed with CT-scans. Visceral fat area was higher in OWW (139.7 +/- 10.7 cm(2)) than in OBW (72.3 +/- 3.9 cm(2)) (P < 0.01), as were fasting and 3 h triglyceride concentrations (P < 0.05 for all). OWW also had higher NEFA levels than OBW at 3 and 4 h compared, with OBW (P < 0.05 for both). Fasting cortisol (266 +/- 24 vs. 197 +/- 19 nmol/l; P < 0.05) was higher in OWW than in OBW. These data demonstrate that OWW have higher visceral fat mass than OBW, which may lead to a more atherogenic fasting and postprandial Lipid profile. The higher cortisol levels of the OWW may promote visceral fat deposition. - Punyadeera, C., M-T. van der Merwe, N.J. Crowther, M. Toman, C. P. Schlaphoff, and I. P. Gray. Ethnic differences in lipid metabolism in two groups of obese South African women.

Functional studies of purified transmembrane proteases, omptins, of Yersinia pestis and Salmonella enterica.

Surface proteolysis is important in migration of cells through tissue barriers. In the case of prokaryotes, surface proteolysis has been associated with invasiveness of pathogenic bacteria from the primary infection site into circulation and secondary infection sites in the host. This study addressed surface proteases of two important bacterial pathogens, Yersinia pestis which is the causative agent of the lethal systemic zoonosis, plague, and Salmonella enterica serovar Typhimurium which is an oral-faecal pathogen that annually causes millions of cases of gastoenteritis that may develop to septicaemia. Both bacterial species express an ortholog of the omptin family of transmembrane β-barrel, outer membrane proteases/adhesins. This thesis work addressed the functions of isolated plasminogen activator Pla of Y. pestis and the PgtE omptin of S. enterica. Pla and PgtE were isolated as His6-fusion proteins in denaturing conditions from recombinant Escherichia coli and activated by adding lipopolysaccharide (LPS). The structural features in LPS that enhance plasminogen activation by His6-Pla were determined, and it was found that the lack of O-specifi c chain, the presence of outer core oligosaccharide, the presence of phosphates in lipid A, as well as a low level of acylation in lipid A influence the enhancement of Pla activity by LPS. A conserved lipid A phosphate binding motif in Pla and PgtE was found important for the enhancement of enzymatic activity by LPS. The results help to explain the biological signifi cance of the genetic loss of the O-specifi c chain biosynthesis in Y. pestis as well as the variations in LPS structure upon entry of Y. pestis into the human host. Expression of Pla in Y. pestis is associated with adhesiveness to lamin of basement membranes. Here, isolated and LPS-activated His6-Pla was coated onto fluorescent microparticles. The coating conferred specifi c adhesiveness of the particles to laminin and reconstituted basement membrane, thus confi rming the intrinsic adhesive characteristics of the Pla protein. The adhesiveness is thought to direct plasmin proteolysis at tissue barriers, thus increasing tissue damage and bacterial spread. Gelatinase activity has not been previously reported in enteric bacteria. Expression of PgtE in S. enterica was associated with cleavage of porcine skin gelatin, denaturated human type I collagen, as well as DQ-gelatin. Purifi ed His6-PgtE also degraded porcine skin gelatin and human type I gelatin but did not react with DQ-gelatin, indicating that minor differences are seen in proteolysis by isolated and cell-bound PgtE. Pla was less effective in gelatin degradation. The novel gelatinase activity in S. enterica is likely to enhance bacterial dissemination during infection.

Atividade pró-trombótica da toxina ExoU de Pseudomonas aeruginosa detectada em modelos experimentais in vivo e in vitro

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-selectina em sua superfície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-α nos LBAs. A análise imunohistoquímica mostrou intensa deposição de fibrina nos alvéolos e septos interalveolares. A atividade pró-coagulante dependente do fator tissular detectada nos LBAs dos camundongos infectados com PA103 foi independente da produção do inibidor da via de ativação do fator tissular (TFPI), mas associada ao aumento da produção do inibidor do ativador do plasminogênio-1 (PAI-1). Para investigar a participação do fator de ativação plaquetária (PAF) na liberação de PAI-1, foi pesquisada a atividade da enzima PAF-acetil-hidrolase (PAF-AH) nos LBAs dos camundongos. A atividade de PAF-AH apresentou-se significativamente elevada nos LBA dos camundongos infectados com PA103. O tratamento dos animais com um inibidor do PAF, antes da infecção, resultou na diminuição significativa das concentrações de PAI-1 e de leucócitos totais, bem como da atividade pró-coagulante dos LBAs. In vitro, ExoU induziu maior expressão do RNA mensageiro de PAI-1 e maior liberação da proteína PAI-1 nos sobrenadantes de células epiteliais respiratórias da linhagem A549. O tratamento das células A549 com um anticorpo anti-receptor de PAF, antes da infecção, reduziu significativamente a concentração de PAI-1 nos sobrenadantes de células infectadas com a cepa selvagem. Estes resultados demonstraram um novo mecanismo de virulência de P. aeruginosa através da atividade pró-trombótica de ExoU e a possibilidade de utilização da identificação de ExoU em isolados clínicos de pacientes graves como um marcador prognóstico para estes pacientes.