Prevention of Anastomotic Leak Via Local Application of Tranexamic Acid to Target Bacterial-mediated Plasminogen Activation: A Practical Solution to a Complex Problem.

OBJECTIVE: To investigate the role of bacterial- mediated plasminogen (PLG) activation in the pathogenesis of anastomotic leak (AL) and its mitigation by tranexamic acid (TXA). BACKGROUND: AL is the most feared complication of colorectal resections. The pathobiology of AL in the setting of a technically optimal procedure involves excessive submucosal collagen degradation by resident microbes. We hypothesized that activation of the host PLG system by pathogens is a central and targetable pathway in AL. METHODS: We employed kinetic analysis of binding and activation of human PLG by microbes known to cause AL, and collagen degradation assays to test the impact of PLG on bacterial collagenolysis. Further, we measured the ability of the antifibrinolytic drug TXA to inhibit this process. Finally, using mouse models of pathogen-induced AL, we locally applied TXA via enema and measured its ability to prevent a clinically relevant AL. RESULTS: PLG is deposited rapidly and specifically at the site of colorectal anastomoses. TXA inhibited PLG activation and downstream collagenolysis by pathogens known to have a causal role in AL. TXA enema reduced collagenolytic bacteria counts and PLG deposition at anastomotic sites. Postoperative PLG inhibition with TXA enema prevented clinically and pathologically apparent pathogen-mediated AL in mice. CONCLUSIONS: Bacterial activation of host PLG is central to collagenolysis and pathogen-mediated AL. TXA inhibits this process both in vitro and in vivo. TXA enema represents a promising method to prevent AL in high-risk sites such as the colorectal anastomoses.

Proteomic and mechanistic study of Qingxuan Tongluo formula and curcumin in the treatment of Mycoplasma pneumoniae pneumonia.

OBJECTIVE: Mycoplasma pneumoniae (MP) is the only pathogen in the Mycoplasma family that can cause respiratory symptoms, including acute upper respiratory tract infection and bronchitis, which are often attributed to Mycoplasma pneumoniae pneumonia (MPP). MPP is one of the diseases that commonly affects the pediatric respiratory system, but its pathogenesis is unclear. This study investigated the therapeutic effects and mechanisms of Qingxuan Tongluo formula and its main component, curcumin, on MPP. METHODS: A mouse model of MPP was obtained by nasal drip of the MP strain. The effects of Qingxuan Tongluo formula and curcumin on the treatment of MPP were studied. The proteomic profiles of the alveolar lavage fluid of mice in the model group, Qingxuan Tongluo formula group and curcumin group were evaluated by LC-MS/MS. ELISA and immunohistochemistry were used to verify the possible presence of MP infection biomarkers and drug target proteins. RESULTS: Compared with the mice in the model group, the MPP mice in the Qingxuan Tongluo formula group had significantly reduced fever and cough and prolonged the cough incubation period. Moreover, the pulmonary pathology of the MPP mice was significantly improved, and the lung histopathological score was decreased. After treatment with Qingxuan Tongluo formula and curcumin, the functional and pathway abnormalities caused by MP were mainly inhibited. Levels of HSP90AA1, GRP94, ENO1 and PLG expression were verified by ELISA and immunohistochemistry. CONCLUSION: Qingxuan Tongluo formula significantly reduced fevers and cough and prolonged the cough incubation period of MPP mice. Qingxuan Tongluo formula and curcumin significantly improved the pathological changes in lung tissue caused by MP infection. Proteomics analyses indicated that Qingxuan Tongluo formula and curcumin may have therapeutic effects on MPP by regulating energy metabolism, relieving oxidative stress and activating the fibrinolytic system. ENO1 and PLG were found to be potential drug targets.

Preventing Candida albicans from subverting host plasminogen for invasive infection treatment.

Candida albicans is a common fungal pathogen in humans that colonizes the skin and mucosal surfaces of the majority healthy individuals. How C. albicans disseminates into the bloodstream and causes life-threatening systemic infections in immunocompromised patients remains unclear. Plasminogen system activation can degrade a variety of structural proteins in vivo and is involved in several homeostatic processes. Here, for the first time, we characterized that C. albicans could capture and "subvert" host plasminogen to invade host epithelial cell surface barriers through cell-wall localized Eno1 protein. We found that the "subverted" plasminogen system plays an important role in development of invasive infection caused by C. albicans in mice. Base on this finding, we discovered a mouse monoclonal antibody (mAb) 12D9 targeting C. albicans Eno1, with high affinity to the 254FYKDGKYDL262 motif in α-helices 6, ß-sheet 6 (H6S6) loop and direct blocking activity for C. albicans capture host plasminogen. mAb 12D9 could prevent C. albicans from invading human epithelial and endothelial cells, and displayed antifungal activity and synergistic effect with anidulafungin or fluconazole in proof-of-concept in vivo studies, suggesting that blocking the function of cell surface Eno1 was effective for controlling invasive infection caused by Candida spp. In summary, our study provides the evidence of C. albicans invading host by "subverting" plasminogen system, suggesting a potential novel treatment strategy for invasive fungal infections.

High-fat diet-induced plasma protein and liver changes in obese rats can be attenuated by melatonin supplementation.

Obesity triggers changes in protein expression in various organs that might participate in the pathogenesis of obesity. Melatonin has been reported to prevent or attenuate such pathological protein changes in several chronic diseases. However, such melatonin effects on plasma proteins have not yet been studied in an obesity model. Using a proteomic approach, we investigated the effect of melatonin on plasma protein profiles after rats were fed a high-fat diet (HFD) to induce obesity. We hypothesized that melatonin would attenuate abnormal protein expression in obese rats. After 10weeks of the HFD, animals displayed increased body weight and fat accumulation as well as increased glucose levels, indicating an obesity-induced prediabetes mellitus-like state. Two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry revealed 12 proteins whose expression was altered in response to the HFD and the melatonin treatment. The altered proteins are related to the development of liver pathology, such as cirrhosis (α1-antiproteinase), thrombosis (fibrinogen, plasminogen), and inflammation (mannose-binding protein A, complement C4, complement factor B), contributing to liver steatosis or hepatic cell death. Melatonin treatment most probably reduced the severity of the HFD-induced obesity by reducing the amplitude of HFD-induced plasma protein changes. In conclusion, we identified several potential biomarkers associated with the progression of obesity and its complications, such as liver damage. Furthermore, our findings reveal melatonin's beneficial effect of attenuating plasma protein changes and liver pathogenesis in obese rats.

The consumption of acai pulp changes the concentrations of plasminogen activator inhibitor-1 and epidermal growth factor (EGF) in apparently healthy women / El consumo de pulpa acai cambia las concentraciones de activador del plasminógeno inhibidor 1 y factor de crecimiento epidérmico (EGT) en mujeres aparentemente sanas

Introduction: obesity, characterized by adiposity excess, is associated with endothelial dysfunction and possible inflammatory state with release of cytokines that determine endothelial function and can trigger chronic diseases. The dietary pattern are associated with the synthesis these cytokines. Fruits as the acai, which is rich in flavonoids, have a direct and beneficial effect on the control of this inflammatory process through the exercised antioxidant capacity. Objective: to evaluate the effect of acai pulp consumption on the inflammatory markers, anthropometric measurements, body composition, biochemical and dietary parameters in healthy women. Methods: forty women, were divided in 25 eutrophic and 15 with overweight. They intaked 200 g of acai pulp during 4 weeks. Anthropometric measurements, body composition, inflammatory markers, biochemical data, dietary intake and dietary antioxidants capacity were evaluated before and after the intervention. Results and discussion: after the intervention, there was significant increase of EGF (p=0.021) and PAI- 1(p=0.011) in overweight women. Moreover, there was increase in body weight (p=0.031), body mass index (p=0.028), percentage of truncal fat (p=0.003) and triceps skinfold thickness (p=0.046) in eutrophic women. However, the skinfold thickness (p=0.018) and total body fat (p=0.016) decreased in overweight women. There was reduction of total protein (p=0.049) due to the globulin reduction (p=0.005), but the nutritional status was maintained in eutrophic group. Conclusion: the intake of 200g acai pulp, modulated the EGF and PAI-1 expression, possibly by modulation of acai on the parameters of body composition, dietary, clinical, biochemical and inflammatory, led to a redistribution and resizing of body fat of the trunk area, and presumably increased visceral fat (AU)

Introducción: la obesidad, que se caracteriza por el exceso de adiposidad, se asocia con disfunción endotelial y posible estado inflamatorio con liberación de citoquinas que determinan la función endotelial y pueden desencadenar enfermedades crónicas. El patrón de dieta está asociado con la síntesis de estas citoquinas. Los frutos del acai, que es rico en flavonoides, tienen un efecto directo y positivo en el control de este proceso inflamatorio a través de los ejercicios de la capacidad antioxidante. Objetivo: evaluar el efecto del consumo de pulpa de acai en los marcadores inflamatorios, las medidas antropométricas, la composición corporal y los parámetros bioquímicos y dietéticos en mujeres sanas. Métodos: cuarenta mujeres fueron divididas en 25 eutróficas y 15 con sobrepeso. Se las administró 200 g de pulpa de acai durante 4 semanas. Antes y después de la intervención se evaluaron: medidas antropométricas, composición corporal, marcadores inflamatorios, datos bioquímicos, ingesta dietética y antioxidantes en la dieta. Resultados y discusión: después de la intervención, hubo un aumento significativo de EGF (p=0,021) y PAI-1 (p=0,011) en las mujeres con sobrepeso. Por otra parte, en las mujeres eutróficas hubo aumento del peso corporal (p=0,031), el índice de masa corporal (p=0,028), el porcentaje de grasa del tronco (p=0,003) y el espesor del pliegue cutáneo del tríceps (p=0,046). Sin embargo, el espesor del pliegue cutáneo (p=0,018) y la grasa corporal total (p=0,016) se redujeron en las mujeres con sobrepeso. Hubo una reducción de la proteína total (p=0,049) debida a la disminución de globulina (p=0,005), pero el estado nutricional se mantuvo en el grupo eutrófico. Conclusión: la ingesta de 200 g de pulpa de acai modula el EGF y PAI-1 de expresión, posiblemente por la modulación del acai en los parámetros de la composición corporal, la dieta, clínicos, bioquímicos e inflamatorios, lo que dio lugar a una redistribución y modificación del tamaño de la grasa corporal de la zona del tronco, y, presumiblemente, un aumento de la grasa visceral (AU)

Towards effective and safe thrombolysis and thromboprophylaxis: preclinical testing of a novel antibody-targeted recombinant plasminogen activator directed against activated platelets.

RATIONALE: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. OBJECTIVE: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. METHODS AND RESULTS: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5-scuPA as compared with the same dose of a mutated, nontargeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. CONCLUSIONS: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.

Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells.

OBJECTIVE: The rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used. RESULTS: Black cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1. CONCLUSIONS: BcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.

Potent fibrinolysis inhibitor discovered by shape and electrostatic complementarity to the drug tranexamic acid.

Protein-protein interfaces provide an important class of drug targets currently receiving increased attention. The typical design strategy to inhibit protein-protein interactions usually involves large molecules such as peptides and macrocycles. One exception is tranexamic acid (TXA), which, as a lysine mimetic, inhibits binding of plasminogen to fibrin. However, the daily dose of TXA is high due to its modest potency and pharmacokinetic properties. In this study, we report a computational approach, where the focus was on finding electrostatic potential similarities to TXA. Coupling this computational technique with a high-quality low-throughput screen identified 5-(4-piperidyl)-3-isoxazolol (4-PIOL) as a potent plasminogen binding inhibitor with the potential for the treatment of various bleeding disorders. Remarkably, 4-PIOL was found to be more than four times as potent as the drug TXA.

Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination: links between altered multifunction of enolase and neurodegenerative diseases.

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ϵ-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.

Localization of plasminogen in mouse hippocampus, cerebral cortex, and hypothalamus.

Although the tissue plasminogen activator/plasminogen system contributes to numerous brain functions, such as learning, memory, and anxiety behavior, little attention has as yet been given to the localization of plasminogen in the brain. We have investigated the localization of plasminogen in the adult mouse brain by using immunohistochemistry. In the hippocampus, plasminogen immunoreactivity was seen in the pyramidal cell layer as numerous punctate structures in neuronal somata. An electron-microscopic study further demonstrated that the plasminogen-immunoreactive punctate structures represented secretory vesicles and/or vesicle clusters. In the cerebral cortex, plasminogen immunoreactivity was evident in the somata of the layer II/III and V neurons. A quantitative analysis revealed that parvalbumin (PV)-positive neurons had more plasminogen-immunoreactive puncta compared with those of PV-negative neurons in the hippocampus and cerebral cortex. Plasminogen immunoreactivity was present throughout the hypothalamus, being particularly prominent in the neuronal somata of the organum vasculosum laminae terminalis, ventromedial preoptic nucleus, supraoptic nucleus, subfornical organ, medial part of the paraventricular nucleus (PVN), posterior part of the PVN, and arcuate hypothalamic nucleus. Thus, plasminogen is highly expressed in specific populations of hippocampal, cortical, and hypothalamic neurons, and plasminogen-containing vesicles are mainly observed at neuronal somata.

Protease purified from Schizophyllum commune culture broth digests fibrins without activating plasminogen.

Schizophyllum commune is a widely distributed mushroom used as an herbal medicine and an ingredient in healthy food. In this study, a protease from a fermented culture broth of S. commune demonstrated strong fibrinolytic and fibrinogenolytic activities. This fibrinolytic protease showed a suppression effect in blood coagulation in co-incubation with rat citrated blood through thromboelastographic analysis. The protease suppressed aggregation of fibrin (ogen), but not the platelets, in clotting formation and significantly decreased the clot strength. We also found very little potency in this protease to activate plasminogen, thus it exhibits the potential for an ideal fibrinolytic candidate for therapeutic applications in the future.

Genistein alters coagulation gene expression in ovariectomised rats treated with phytoestrogens.

Recent data has shown that hormone therapy (HT) increases the risk of cardiovascular and thromboembolic disease, particularly in users of oral HT. Phytoestrogens are popular alternatives to oestrogen therapy; however, their effects on cardiovascular risk are unknown. We investigated the effect of the phytoestrogen, genistein on the expression of genes and proteins from the haemostatic system in the liver in an ovariectomised rat model. Fifty-nine virgin female Sprague-Dawley rats were fed with soy-free chow supplemented with 17ß estradiol (E2) (daily uptake 0.19 or 0.75 mg/kg body weight), or genistein (daily uptake 6 or 60 mg/kg body weight), for three months and compared to soy-free control rats. Gene expression of prothrombin, factor VII, fibrinogen alpha and fibrinogen beta was increased with E2 and genistein compared to the soy-free control group (p<0.001). Genistein increased factor VII significantly more than E2 (p<0.005). Plasminogen mRNA was increased in both treatment groups compared to the soy-free control, with genistein expression significantly higher than E2 (p<0.001). Tissue plasminogen inhibitor (tPA), plasminogen activator inhibitor-1 (PAI-1) and C-reactive protein (CRP) expression were also increased in both groups relative the soy-free control. Results of protein analysis largely concurred with those of the mRNA. Oestrogen receptor ß (ERß) was undetected while oestrogen receptor α (ERα) was detected in each sample group. Genistein can increase the expression of coagulation and fibrinolytic genes. This effect was similar and in some cases higher than 17ß estradiol. These results suggest that genistein may not be neutral with respect to the haemostatic system.

Mechanism of the synergistic effect between oversulfated chondroitin-6-sulfate and lysine or 6-aminohexanoic acid in enhancing the in-vitro activation of glutamic plasminogen by tissue plasminogen activator or urokinase.

Earlier studies of addition of naturally sulfated polysaccharides including unfractionated heparin showed a significant enhancement of the in-vitro activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA). However, supplementing of physiological concentration of NaCl (0.9%) to the buffer reversed the enhancement. To overcome this reversal attempts were made to oversulfate the compounds and re-evaluate their biological properties. Chondroitin-6-sulfate (N-2) was oversulfated using chlorosulfonic acid-pyridine complex and isolated as the sodium salt. Infrared and H-NMR studies of the oversulfated compound showed introduction of new sulfate groups with the formation of 60% of chondroitin-4-6-disulfate. In-vitro studies were conducted on comparing the effect of oversulfated chondroitin-6-sulfate (S-2) with native compound (N-2) and unfractionated heparin in enhancing the activation of Glu-Plg by t-PA or u-PA using 0.05 mol/l Tris buffer (pH 7.3) containing 0.9% of NaCl. The enhancement of activation of Glu-Plg by t-PA or u-PA was measured by formation of plasmin using H-D-Glu-Phe-Lys-pNA (S-2403) as the substrate. The activation by t-PA was enhanced two-fold by 2.86 microg/ml of S-2, 4-6-fold by addition of 32.4 mmol/l of lysine or 5.4 mmol/l of 6-aminohexanoic acid (6-AH) and 14-16-fold enhancement by addition of both S-2 and lysine or S-2 and 6-AH showing a synergistic effect, whereas unfractionated heparin alone gave no enhancement and in conjunction with lysine or 6-AH gave no additional enhancement. Similar studies using u-PA in place of t-PA gave identical results. During the activation of Glu-Plg to plasmin, lysine plasminogen (Lys-Plg) is reported to be an intermediate. Therefore we investigated the role of S-2, lysine and 6-AH in the activation of Lys-Plg to plasmin. The results showed that S-2 enhanced this activation, whereas lysine or 6-AH which were active in enhancing the activation of Glu-Plg were not active using Lys-Plg indicating that the site of enhancement by lysine or 6-AH was during the initial phase. Double reciprocal plot of Glu-Plg activation by t-PA with or without S-2 and lysine showed no change in Km but a 10-fold increase of Kcat suggesting a template mechanism for the attenuation when both cofactors are used.

Hypochlorous acid is a potent inactivator of human plasminogen at concentrations secreted by activated granulocytes.

BACKGROUND: Myeloperoxidase (MPO) neutrophils have been considered an important pathophysiological factor in oxidative stress. Mainly through generation of hypochlorous acid in the phagosome, unchecked activity may lead to inactivation of important proteins through modification of tyrosine and other residues. This has been shown for low-density lipoprotein, apolipoprotein AI and paraoxonase 1 (PON-1). Notably, plasminogen has 29 tyrosine residues and we previously demonstrated that it can be inactivated by nitration of these residues. METHODS: We hypothesized that plasminogen can also be inactivated by HOCl/OCl(-) in a similar manner to PON-1, and that this inhibition can be counteracted by cysteine or taurine. In the present study we compared the effects of HOCl/OCl(-) on these two plasma proteins and explored the effects of inhibitors. RESULTS: Our study revealed that HOCl/OCl(-) inhibits streptokinase-induced plasmin activity at low micromolar concentrations that may well occur in vivo at sites of inflammation (IC(50)=40 micromol/L). This inhibitory effect occurs at much lower concentrations than those that inhibit PON-1 activity (IC(50) 120 micromol/L). The inhibition is paralleled by an increase in 3-chlorotyrosine adducts in the protein. HOCl/OCl(-) inhibition of plasminogen and 3-chlorotyrosine formation are blocked by cysteine; taurine shows much lower protection. HOCl/OCl(-) does not modify the activation of plasminogen by streptokinase, leaving inactivation of active site Tyr 614 (possibly directed by Lys 615) as a very attractive hypothesis to explain the effect and the high sensitivity found. CONCLUSIONS: Since neutrophils have been shown to secrete HOCl/OCl(-) up to 100 micromol/L, inactivation of plasmin molecules by neutrophil-generated HOCl/OCl(-) could partly explain the increased thrombo-genicity observed in inflammatory conditions, in smokers and in other diseases. Thus, dietary intervention or the use of thiols or large doses of taurine compounds may be useful as coadjuvant therapeutic measures.

Effects of Bulbus allii macrostemi on clinical outcomes and oxidized low-density lipoprotein and plasminogen in unstable angina/non-ST-segment elevation myocardial infarction patients.

Unstable angina (UA)/non-ST-segment elevation myocardial infarction (NSTEMI) is associated with an increased risk of cardiac death and an efficacious drug with few side effects is necessary. The study aimed to evaluate the effects of Bulbus allii macrostemi (B. macrostemi) on UA/NSTEMI patients as well as to elucidate possible mechanisms. 79 patients were randomly divided into two groups: the trial group received B. macrostemi plus baseline therapy, the control group was given placebo plus baseline therapy. The trial lasted 8 weeks. The evaluation involved main clinical symptoms, changes of electrocardiogram and biochemical examination. After treatment, the trial group showed more significant improvement on clinical manifestation. The plasma oxidized low-density lipoprotein (ox-LDL) level decreased significantly in the trial group (p < 0.01); the plasminogen activator inhibitor-1 (PAI-1) level decreased in both groups and it decreased more significantly in the trial group (p < 0.01). In contrast, the activity of plasminogen (PLG) increased in both groups and the change was more marked in the trial group (p < 0.01). The results suggested that B. macrostemi combined with baseline therapy could improve clinical symptoms of UA/NSTEMI patients by decreasing the ox-LDL and PAI-1 levels and enhancing the activity of PLG.

The role of urokinase in idiopathic pulmonary fibrosis and implication for therapy.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and frequently fatal form of interstitial lung disease for which there are no proven drug therapies. The pathogenesis of IPF is complex and the urokinase-type plasminogen activator (uPA)/plasminogen system participates in the repair process. The balance between the activating enzyme uPA, and its inhibitor PAI-1, is a critical determinant of the amount of scar development that follows. OBJECTIVE: To address the role of urokinase in the pathogenesis of pulmonary fibrosis and its implications for therapy. METHODS: We reviewed a spectrum of therapeutic strategies and focused on fibrinolytic and anticoagulant drugs for IPF patients. RESULTS/CONCLUSION: There is currently a search for new pharmacotherapeutic agents that may modulate the fibrogenic pathways in IPF. Either blocking PAI-1 or using uPA itself may be a promising new therapeutic strategy.

Ligneous conjunctivitis: a clinicopathological, immunohistochemical, and genetic study including the treatment of two sisters with multiorgan involvement.

Ligneous conjunctivitis (LC) is a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosae secondary to plasminogen (PLG) deficiency. In this paper, we report two cases of LC in two sisters of 57 and 62 years of age that presented with recurrent, bilateral pseudomembranes on conjunctiva and a history of consanguinity and deafness. Pseudomembranes showed superficial and/or subepithelial deposits of eosinophilic amorphous hyaline, amyloid-like material with a variable proportion of granulation tissue, and inflammatory cells. The eosinophilic deposits were negative for Congo red stain, immunoreactive for fibrinogen, and consistently negative for amyloid A component, transthyretin, beta(2)-microglobulin, albumin, fibronectin, collagen type IV, vimentin, and cytokeratins. Among inflammatory cells, a percentage of positivity of roughly 60% for lymphocytes T (CD3+) and 40% for lymphocytes B (CD8+), with a relation of cytotoxic/helper (CD8/4) T cells of 3:2, was found. In one case, nasal polyps and recurrent gastric peptic ulcer were also characterized by the same subepithelial hyaline deposits. A novel homozygous point mutation c.1856 C>T was found in exon 15 of the PLG gene in both patients. Amniotic membrane transplantation was done in one case with promising results.

Antithrombotic effects of odorless garlic powder both in vitro and in vivo.

Antithrombotic activities of odorless garlic powder were demonstrated in blood fibrinolytic and coagulation systems. Though the odorless garlic preparation did not influence tissue-type plasminogen activator (t-PA) or its inhibitor secretions from human umbilical vein endothelial cells, it enhanced plasmin generation by t-PA on fibrin film and in chromogenic assays by 1.8-fold and 8.7-fold respectively. The coagulation system was considerably reduced after the administration of the garlic in a rat in situ loop model, indicating that increased levels of thrombin-antithrombin III (TAT) complex in the control group were significantly reduced to normal (sham) in the garlic group (p<0.05), which was associated with decreasing tendencies towards prolonged or increased values of coagulation parameters in the control group. These findings suggest that odorless garlic not only activates fibrinolytic activity by accelerating t-PA-mediated plasminogen activation, but also suppresses the coagulation system by downregulating thrombin formation, suggesting a beneficial role in preventing pathological thrombus formation in such cardiovascular disorders.

Insights into the substrate specificity of a novel snake venom serine peptidase by molecular modeling.

The cDNA encoding BthaTL, a serine peptidase from the venom of the snake Bothrops alternatus, was cloned and sequenced. The deduced primary structure shows over 62% of identity with snake venom thrombin-like enzymes (SVTLEs), molecules with high substrate specificity toward different natural substrates. Indeed, a phylogenetic reconstruction by two different methods clustered this enzyme close to other SVTLEs. These enzymes generally affect the hemostatic system in several ways, and therefore are used as tools in pharmacology and clinical diagnosis. A three-dimensional model of BthaTL was built by homology modeling using TSV-PA (Trimeresurus stejnegeri venom plasminogen activator) crystal structure as template. BthaTL model showed that the typical catalytic triad conformation of serine peptidases was preserved. The calcium coordination ligands were absent or adopt an unfavorable conformation, preventing interactions with metals. On the other hand, the Asp97-Arg174 saline bridge of TSV-PA was not found and its specificity determinant Phe193 is replaced by a Gly in BthaTL. The substitution of essential residues in the neighborhoods of the catalytic site cleft of BthaTL indicates that these two proteins do not share the same enzymatic specificity, what means that BthaTL will probably not activate plasminogen. Such observations may be helpful in the understanding of the molecular mechanism for substrate specificity of these enzymes.

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: proteolysis during ripening.

Proteolysis in cheese is influenced by the state of proteins (protein-calcium-phosphate interactions), level of indigenous milk enzymes (plasmin), externally added milk-clotting enzymes (chymosin), and endogenous and exogenous enzymes from starter and non-starter lactic acid bacteria (NSLAB). The objective of this study was to determine how different levels of calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) in cheese influence proteolysis during ripening. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), 2 levels of lactose at pressing (2.4 vs. 0.78%), and 2 levels of S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for changes in pH 4.6-soluble N, and starter and NSLAB counts during 48 wk of ripening. Cheeses at d 1 were also analyzed for residual chymosin, plasmin, and plasminogen activity. A significant increase in soluble N was observed during ripening for all the treatments. Cheeses with low Ca and P, low lactose, and low S/M treatments exhibited higher levels of proteolysis as compared to their corresponding high treatments. Differences in the rate of proteolysis for cheeses with different levels of Ca and P might be due to changes in protein conformation and differences in residual chymosin in the cheeses. Cheeses with low Ca and P were manufactured by lowering the pH at set and drain, which led to higher chymosin retention in cheeses with low Ca and P compared with high Ca and P. Differences in proteolysis between treatments with different levels of lactose were also partly attributed to residual chymosin activity. In all treatments, a major fraction of plasmin existed as plasminogen, indicating minimal contribution of plasmin to proteolysis in Cheddar cheeses. The number of starter bacteria, in all treatments, decreased significantly during ripening. However, the decrease was larger in the case of high S/M treatments compared with low S/M treatments. In contrast, the number of NSLAB increased during ripening, and low S/M cheeses had higher counts compared with high S/M cheeses. The differences in proteolysis due to S/M were partially attributed to changes in protein conformation or bacterial proteolytic activity.