In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8 +/- 25.2 nM and 167.39 +/- 60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4 +/- 15.9 nM to laminin and of 290.8 +/- 11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Infusion of Recombinant Human Tissue Plasminogen Activator Through the Superior Mesenteric Artery in the Treatment of Acute Mesenteric Venous Thrombosis

Acute mesenteric venous thrombosis is an uncommon condition that is usually treated with systemic anticoagulation. Catheter-directed thrombolysis through the superior mesenteric artery may be a viable adjunct to treat this morbid condition. In the present article, we have described a case of superior mesenteric venous thrombosis treated with catheter-directed infusion of tissue plasminogen activator through the superior mesenteric artery.

Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

Atividade de plasmina e plasminogênio no leite longa vida com alta e baixa contagem de células somáticas durante o armazenamento

O objetivo deste estudo foi avaliar o efeito da contagem de células somáticas (CCS) do leite na atividade de plasmina e plasminogênio durante o período de armazenamento do leite longa vida integral. Os leites crus foram categorizados em grupos de CCS de baixa (342.000-487.000 células mL-1) e alta contagem (603.000-808.000 células mL-1). Dois lotes de leite longa vida em cada categoria de CCS foram analisados para determinação de plasmina e plasminogênio após 10, 30, 60, 90 e 120 dias de armazenamento em temperatura ambiente. Para a fabricação do leite longa vida, o leite cru foi submetido à pasteurização rápida seguida da esterilização industrial do leite por injeção de vapor pelo método direto e embalagem asséptica do produto. A CCS não apresentou efeitos sobre as características físico-químicas do leite cru, e nem sobre a atividade de plasmina e plasminogênio nos leites cru e longa vida, armazenados por 120 dias. Entretanto, independentemente da CCS, a atividade de plasmina e plasminogênio aumentou no leite longa vida ao longo do armazenamento, indicando a possibilidade de aumento da proteólise no produto durante sua vida de prateleira.

Sedentary subjects have higher PAI-1 and lipoproteins levels than highly trained athletes

Physical exercise protects against the development of cardiovascular disease, partly by lowering plasmatic total cholesterol, LDL-cholesterol and increased HDL-cholesterol levels. In addition, it is now established that reduction plasmatic adiponectin and increased C-reactive protein (CRP) and plasminogen activator inhibitor-1 (PAI-1) levels play a role in the maintenance of an inflammatory state and in the development of cardiovascular disease. This study aimed to examine plasma lipid profile and inflammatory markers levels in individual with sedentary lifestyle and/or highly trained athletes at rest. Methods: Fourteen male subjects (sedentary lifestyle n = 7 and highly trained athletes n = 7) were recruited. Blood samples were collected after an overnight fast (similar to 12 h). The plasmatic lipid profile (Triglycerides, HDL-cholesterol, LDL-cholesterol, total cholesterol, LDL-oxidized and total cholesterol/HDL-c ratio), glucose, adiponectin, C - reactive protein and PAI-1 levels were determined. Results: Total cholesterol, LDL-cholesterol, TG and PAI-1 levels were lower in highly trained athletes group in relation to sedentary subjects (p < 0.01). In addition, we observed a positive correlation between PAI-1 and total cholesterol (r = 0.78; p < 0.0009), PAI-1 and LDL-c (r = 0.69; p < 0.006) and PAI-1 and TG levels (r = 0.56; p < 0.03). The plasma concentration of adiponectin, CRP, glucose, HDL-cholesterol and total cholesterol/HDL-c ratio levels were not different. These results indicate that lifestyle associated with high intensity and high volume exercise induces changes favourable in the lipid profile and PAI-1 levels and may reduce risk cardiovascular diseases.

Invasion of Ureaplasma diversum in Hep-2 cells

Background: Understanding mollicutes is challenging due to their variety and relationship with host cells. Invasion has explained issues related to their opportunistic role. Few studies have been done on the Ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. The invasion in Hep-2 cells of four clinical isolates and two reference strains of their ureaplasma was studied by Confocal Laser Scanning Microscopy and gentamicin invasion assay. Results: The isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. The adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% of the ATCC strains were inside the infected Hep-2 cells in contrast to 10% to the clinical isolates. A high level of phospholipase C activity was also detected in all studied ureaplasma. Conclusions: The results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

The Bypass Angioplasty Revascularization Investigation 2 Diabetes Randomized Trial of Different Treatment Strategies in Type 2 Diabetes Mellitus With Stable Ischemic Heart Disease Impact of Treatment Strategy on Cardiac Mortality and Myocardial Infarction

Background-The Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial in 2368 patients with stable ischemic heart disease assigned before randomization to percutaneous coronary intervention or coronary artery bypass grafting strata reported similar 5-year all-cause mortality rates with insulin sensitization versus insulin provision therapy and with a strategy of prompt initial coronary revascularization and intensive medical therapy or intensive medical therapy alone with revascularization reserved for clinical indication(s). In this report, we examine the predefined secondary end points of cardiac death and myocardial infarction (MI). Methods and Results-Outcome data were analyzed by intention to treat; the Kaplan-Meier method was used to assess 5-year event rates. Nominal P values are presented. During an average 5.3-year follow-up, there were 316 deaths (43% were attributed to cardiac causes) and 279 first MI events. Five-year cardiac mortality did not differ between revascularization plus intensive medical therapy (5.9%) and intensive medical therapy alone groups (5.7%; P = 0.38) or between insulin sensitization (5.7%) and insulin provision therapy (6%; P = 0.76). In the coronary artery bypass grafting stratum (n = 763), MI events were significantly less frequent in revascularization plus intensive medical therapy versus intensive medical therapy alone groups (10.0% versus 17.6%; P = 0.003), and the composite end points of all-cause death or MI (21.1% versus 29.2%; P = 0.010) and cardiac death or MI (P = 0.03) were also less frequent. Reduction in MI (P = 0.001) and cardiac death/MI (P = 0.002) was significant only in the insulin sensitization group. Conclusions-In many patients with type 2 diabetes mellitus and stable ischemic coronary disease in whom angina symptoms are controlled, similar to those enrolled in the percutaneous coronary intervention stratum, intensive medical therapy alone should be the first-line strategy. In patients with more extensive coronary disease, similar to those enrolled in the coronary artery bypass grafting stratum, prompt coronary artery bypass grafting, in the absence of contraindications, intensive medical therapy, and an insulin sensitization strategy appears to be a preferred therapeutic strategy to reduce the incidence of MI. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT00006305. (Circulation. 2009;120:2529-2540.)

Streptokinase and Enoxaparin as an Alternative to Fibrin-Specific Lytic-Based Regimens An ExTRACT-TIMI 25 Analysis

Background: Enoxaparin was superior to unfractionated heparin (UFH), regardless of fibrinolytic agent in ST-elevation myocardial infarction (STEMI) patients receiving fibrinolytic therapy in ExTRACT-TIMI 25 (Enoxaparin and Thrombolysis Reperfusion for Acute Myocardial Infarction Treatment Thrombolysis in Myocardial Infarction 25) trial. Objective: This post hoc analysis compared outcomes with streptokinase plus enoxaparin to the standard regimen of fibrin-specific lytic (FSL) plus UFH and to the newer combination of FSL plus enoxaparin. Methods: In ExTRACT-TIMI 25, STEMI patients received either streptokinase or a FSL (alteplase, reteplase or tenecteplase) at the physician`s discretion and were randomized to enoxaparin or UFH, stratified by fibrinolytic type. Thirty-day outcomes were adjusted for baseline characteristics, region, in-hospital percutaneous coronary intervention (PCI) and a propensity score for the choice of lytic. Results: The primary trial endpoint of 30-day death/myocardial infarction (MI) occurred in fewer patients in the streptokinase-enoxaparin cohort (n = 2083) compared with FSL-UFH (n = 8141) [10.2% vs 12.0%, adjusted odds ratio [OR(adj)] 0.76; 95% CI 0.62, 0.93; p = 0.008]. Major bleeding was significantly increased with streptokinase-enoxaparin compared with FSL-UFH (ORadj 2.74; 95% CI 1.81; 4.14; p < 0.001) but intracranial haemorrhage (ICH) was similar (OR(adj) 0.90; 95% CI 0.40, 2.01; p = 0.79). Net clinical outcomes, defined as either death/MI/major bleeding or as death/MI/ICH tended to favour streptokinase-enoxaparin compared with FSL-UFH (OR(adj) 0.88; 95% CI 0.73, 1.06; p = 0.17; and OR(adj) 0.77; 95% CI 0.63, 0.93; p = 0.008, respectively). Patients receiving FSL-enoxaparin (n = 8142) and streptokinase-enoxaparin therapies experienced similar adjusted rates of the primary endpoint (OR(adj) 1.08; 95% CI 0.87, 1.32; p = 0.49) and net clinical outcomes. Conclusions: Our results suggest that fibrinolytic therapy with the combination of streptokinase and the potent anticoagulant agent enoxaparin resulted in similar adjusted outcomes compared with more costly regimens utilizing a FSL.

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pl 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

Frequency of thrombophilia determinant factors in patients with livedoid vasculopathy and treatment with anticoagulant drugs - a prospective study

Background Livedoid vasculopathy (LV) is a chronic idiopathic disease characterized by painful purpuric macules on lower extremities. Its exact aetiology remains uncertain, but thrombotic and microcirculatory phenomena have been implicated as possible pathogenic factors. Objectives To assess prospectively the frequency of thrombophilia and to verify the effectiveness of anticoagulant therapy among LV patients. Methods Thirty-four LV patients were tested for prothrombin time, activated partial thromboplastin time, antithrombin activity, protein C and S activity, anticardiolipin antibodies, lupus anticoagulant, prothrombin gene mutation, factor V Leiden mutation, methylenetetrahydrofolate reductase mutation, plasma homocysteine and fibrinogen. Thirteen of these patients were treated with anticoagulant drugs (either warfarin or heparin). Results Of 34 patients, 18 (52%) presented laboratory abnormalities of procoagulant conditions. Positive treatment response to anticoagulant therapy was observed in 11 patients. Improvement of pain was obtained in 1-3 weeks, an average of 1.8 week. Complete healing of the lesions was observed in about 2.3 months. Remission was sustained even after treatment interruption and lasted an average 7.8 months. No severe adverse effects were noticed. Conclusion The authors suggest all patients with diagnosis of LV to be investigated for thrombophilic status. Anticoagulant drugs were well tolerated and seemed to be effective in treating not only LV symptoms but also its ulcerations.

Hemostatic changes and clinical sequelae after on-pump compared with off-pump coronary artery bypass surgery: a prospective randomized study

Objective To delineate the effects of extracorporeal bypass on biomarkers of hemostasis, fibrinolysis, and inflammation and clinical sequelae. Methods Patients were assigned prospectively and randomly to either on-pump (n=41) or off-pump (n=51) coronary bypass surgery. The concentrations of C-reactive protein, fibrinogen, D-dimer, and plasminogen activator inhibitor type-1 in blood were quantified before and after (1 and 24 h) surgery. Similar surgical and anesthetic procedures were used for both groups. Clinical events were assessed during initial hospitalization and at the end of I year. Results The concentrations of plasminogen activator inhibitor type-1 and D-dimer were greater compared with preoperative values 1 and 24 h after surgery in both groups, but their concentrations increased to a greater extent 24 h after surgery in the on-pump group (P<0.01). The concentration of C-reactive protein did not change appreciably immediately after surgery in either group but increased in a parallel manner 24 h after either on-pump or off-pump surgery (P<0.01). Bypass surgery in the on-pump group was associated with greater blood loss during surgery and more bleeding after surgery (P <= 0.01). The incidence of all other complications was similar in the two groups. Conclusion On-pump surgery was associated with biochemical evidence of a prothrombotic state early after surgery but no greater incidence of thrombotic events was observed. The prothrombotic state might be a consequence of extracorporeal bypass, compensation in response to more bleeding, or both in patients undergoing on-pump surgery. Coron Artery Dis 20:100-105 (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Altered endothelial function following the Fontan procedure

Objective: Thrombosis has been widely described after the Fontan procedure. The vascular endothelium plays a central role in the control of coagulation and fibrinolysis. The aim of this study was to investigate if patients undergoing a modified Fontan procedure have impaired endothelial function and fibrinolysis in the late postoperative course. Patients and methods: We compared 23 patients aged from 7 to 26 years with age-matched healthy volunteers, collecting blood samples prior to and following standardized venous occlusion testing. Plasma levels of von Willebrand factor antigen, tissue-type plasminogen activator antigen, plasminogen activator inhibitor-1, and D-dimer were measured with enzyme-linked immunosorbent assay. Results: We found increased plasma levels of von Willebrand factor antigen in patients when compared to controls (p = 0.003). At the basal condition, concentrations of tissue-type plasminogen activator antigen and plasminogen activator inhibitor-1 antigen in the plasma, as well as their activity, were not significantly different between patients and controls. Following venous occlusion, concentrations of tissue-type plasminogen activator antigen in the plasma were significantly increased both in patients and controls, compared to pre-occlusion values. D-dimer was within the reference range. Multivariate discriminant analysis differentiated patients and their controls on the basis of differences for plasminogen activator inhibitor-1 and von Willebrand factor antigen (p = 0.0016). Conclusions: Our data suggest that patients with the Fontan circulation may have endothelial dysfunction, as indicated by raised levels of von Willebrand factor. Fibrinolysis seems to be relatively preserved, as suggested by appropriate response to venous occlusion.

Role of Microparticles in the Hemostatic Dysfunction in Acute Promyelocytic Leukemia

Serious bleeding and thrombotic complications are frequent in acute promyelocytic leukemia (APL) and are major causes of morbidity and mortality. Microparticles (MP) have been used to study the risk and pathogenesis of thrombosis in many malignant disorders. To date, from published articles, this approach had not been applied to APL. In this article, the hemostatic dysfunction in this disorder is briefly reviewed. A study design to address this problem using MP is described. MP bearing tissue factor, profibrinolytic factors (tissue plasminogen activator and annexin A2), and the antifibrinolytic factor plasminogen activator inhibitor type 1 were measured using flow cytometry. The cellular origin of the MP was identified by specific cell surface markers. Comparison of the various populations of MP was made between samples collected at the time of diagnosis with those collected at molecular remission. Preliminary data suggest that this approach is feasible.