Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).

Standardized protocol for a depletion of intramyocellular lipids (IMCL)

Intramyocellular lipids (IMCL) are flexible fuel stores that are depleted by physical exercise and replenished by fat intake. IMCL or their degradation products are thought to interfere with insulin signaling thereby contributing to insulin resistance. From a practical point of view it is desirable to deplete IMCL prior to replenishing them. So far, it is not clear for how long and at which intensity subjects have to exercise in order to deplete IMCL. We therefore aimed at developing a standardized exercise protocol that is applicable to subjects over a broad range of exercise capacity and insulin sensitivity and allows measuring reliably reduced IMCL levels.Twelve male subjects, including four diabetes type 2 patients, with wide ranges of exercise capacity (VO(2)peak per total body weight 27.9-55.8 ml x kg(-1) x min(-1)), insulin sensitivity (glucose infusion rate per lean body mass 4.7-15.3 mg x min(-1) x kg(-1)), and BMI (21.7-31.5 kg x m(-2)), respectively, were enrolled. Using (1)H magnetic resonance spectroscopy ((1)H-MRS), IMCL was measured in m.tibialis anterior and m.vastus intermedius before and during a depletion protocol of a week, consisting of a moderate additional physical activity (1 h daily at 60% VO(2)peak) and modest low-fat (10-15%) diet.Absolute IMCL-levels were significantly reduced in both muscles during the first 3 days and stayed constant for the next 3 days of an identical diet/exercise-scheme. These reduced IMCL levels were independent of insulin sensitivity, yet a tendency to lower depleted IMCL levels has been observed in subjects with higher VO(2)peak.The proposed protocol is feasible in subjects with large differences in exercise capacity, insulin sensitivity, and BMI, leading to reduced IMCL levels that neither depend on the exact duration of the depletion protocol nor on insulin sensitivity. This allows for a standardized preparation of IMCL levels either for correlation with other physiological parameters or for replenishment studies.

Studying the fate of non-volatile organic compounds in a commercial plasma air purifier

Degradation of non-volatile organic compounds-environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)-in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0m s(-1) (3200Lmin(-1)), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10Lmin(-1). Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10Hz or 50Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative "degradation" efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules.

Impaired cortical energy metabolism but not major antioxidant defenses in experimental bacterial meningitis.

The loss of soluble brain antioxidants and protective effects of radical scavengers implicate reactive oxygen species in cortical neuronal injury caused by bacterial meningitis. However, the lack of significant oxidative damage in cortex [J. Neuropathol. Exp. Neurol. 61 (2002) 605-613] suggests that cortical neuronal injury may not be due to excessive parenchymal oxidant production. To see whether this tissue region exhibits a prooxidant state in bacterial meningitis, we examined the state of the major cortical antioxidant defenses in infant rats infected with Streptococcus pneumoniae. Adenine nucleotides were co-determined to assess possible changes in energy metabolism. Arguing against heightened parenchymal oxidant production, the high NADPH/NADP(+) ratio ( approximately 3:1) and activities of the major antioxidant defense and pentose phosphate pathway enzymes remained unchanged at the time of fulminant meningitis. In contrast, cortical ATP, ADP and total adenine nucleotides were on average decreased by approximately 25%. However, energy depletion did not lead to a significant decrease in adenylate energy charge (AEC). ATP depletion was likely a consequence of metabolic degradation, since it correlated with both the loss of total adenine nucleotides and accumulation of purine degradation products. Furthermore, the loss of ATP and decrease in AEC correlated significantly with the extent of neuronal injury. These results strongly suggest that energy depletion rather than parenchymal oxidative damage is involved in the observed cortical neuronal injury.

A computer-assisted microscopic analysis of bone tissue developed inside a polyactive plymer implanted into an equine articular surface

One of the most promising applications for the restoration of small or moderately sized focal articular lesions is mosaicplasty (MP). Although recurrent hemarthrosis is a rare complication after MP, recently, various strategies have been designed to find an effective filling material to prevent postoperative bleeding from the donor site. The porous biodegradable polymer Polyactive (PA; a polyethylene glycol terephthalate - polybutylene terephthalate copolymer) represents a promising solution in this respect. A histological evaluation of the longterm PA-filled donor sites obtained from 10 experimental horses was performed. In this study, attention was primarily focused on the bone tissue developed in the plug. A computer-assisted image analysis and quantitative polarized light microscopic measurements of decalcified, longitudinally sectioned, dimethylmethylene blue (DMMB)- and picrosirius red (PS) stained sections revealed that the coverage area of the bone trabecules in the PA-filled donor tunnels was substantially (25%) enlarged compared to the neighboring cancellous bone. For this quantification, identical ROIs (regions of interest) were used and compared. The birefringence retardation values were also measured with a polarized light microscope using monochromatic light. Identical retardation values could be recorded from the bone trabeculae developed in the PA and in the neighboring bone, which indicates that the collagen orientation pattern does not differ significantly among these bone trabecules. Based on our new data, we speculate that PA promotes bone formation, and some of the currently identified degradation products of PA may enhance osteo-conduction and osteoinduction inside the donor canal.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A? followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot

Latex glycoprotein (LGP) from Synadenium grantii latex was purified by the combination of heat precipitation and gel permeation chromatography. LGP is a heat stable protein even at 80 degrees C showed a sharp single band both in SDS-PAGE as well as in native (acidic) PAGE. LGP is a monomeric protein appears as single band under reducing condition. It is a less hydrophobic protein showed sharp single peak in RP-HPLC with retention time of 13.3 m. The relative molecular mass of LGP is 34.4 kDa. CD spectrum of LGP explains less content of alpha-helix (7%), and high content of beta-pleated sheets (48%) and random coils (46%). The N-terminal sequence of LGP is D-F-P-S-D-W-Y-A-Y-E-G-Y-V-I-D-R-P-F-S. Purified LGP is a fibrinogen degrading protease hydrolyses all the three subunits in the order of Aalpha, Bbeta and gamma. The hydrolytic pattern is totally different from plasmin as well as thrombin. LGP reduces recalcification time from 165 to 30 s with citrated human plasma but did not show thrombin like as well as factor Xa-like activity. Although LGP induces procoagulant activity, it hydrolyses partially cross-linked fibrin clot. It hydrolyses all the subunits of partially cross-linked fibrin clot (alpha- chains, beta-chain and gamma-gamma dimer). LGP is a serine protease, inhibited by PMSF. Other serine protease inhibitors, aprotinin and leupeptin did not inhibit the caseinolytic activity as well as fibrinogenolytic activity. We report purification and characterization of a glycoprotein from Synadenium grantii latex with human fibrino(geno)lytic activity.

Standard vs. point-of-care measurement of fibrinogen: potential impact on clinical decisions

Intraoperative major bleeding is a common complication during surgery and can lead to the transfusion of blood products and/or procoagulant drugs. This is a therapeutic challenge, and adherence to guidelines is desirable to preserve blood product resources. The intraoperative administration of fibrinogen concentrate, a pro-coagulant drug, in bleeding patients might reduce the use and therefore the risks associated with blood products.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.

Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering

The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.

Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Valorization of agrobiodiversity products and strengthening of local identities in the Peruvian Andes: Experiences from the BioAndes Programme

In the Peruvian Andes, a long history of interaction between the local populations and their natural environment has led to extraordinary levels of agrobiodiversity. However, in sharp contrast with this biological wealth, Andean indigenous populations live under most precarious conditions. Moreover, natural resources are undergoing severe degradation processes and local knowledge about biodiversity management is under serious pressure. Against this background, the BioAndes Programme is developing initiatives based on a biocultural approach that aim at fostering biodiversity through the enhancement of cultural processes. On the basis of intercultural dialogue, joint learning and capacity development, and transdisciplinary action-research, indigenous communities, development practitioners, and researchers strive for the creation of innovative ways to contribute to more sustainable economic, socio-cultural, and political valorization of Andean biodiversity. Project activities are diverse and range from the cultivation, transformation, and commercialization of organic Andean fruits in San Marcos, Cajamarca Department, to the recuperation of natural dying techniques for alpaca wool and traditional weaving in Pitumarca, Cusco Department, and the promotion of responsible ecotourism in both regions. Based on the projects’ first two-years of experience, the following lessons learnt will be presented and discussed: 1. The economic valorization and commercialization of local products can be a powerful tool for the revival and innovation of eroded know-how; at the same time it contributes to the strengthening of local identities, in parallel with the empowerment of marginalized groups such as smallholders and women. 2. Such initiatives are only successful when they are embedded within activities that go beyond the focus on local products and seek the valorization of the entire natural and cultural landscape (e.g. through the promotion of agrotourism and local gastronomy, more sustainable management of local resources including the restoration of ecosystems, and the realization of inventories of local agrobiodiversity and the knowledge related to it). 3. The sustainability of these initiatives, which are often externally induced, is conditioned by the ability of local actors to acquire ownership of projects and access to the knowledge required to carry them out, which also means developing the personal and institutional capacities for handling the whole chain from production to commercialization. 4. The confrontation of different economic rationalities and their underlying worldviews that occur when local or indigenous people integrate into the market economy implies the need for a dialogical co-production of knowledge and collective action by local people, experts from NGOs, and political authorities in order to better control the conditions relating to the market economy. The valorization of local agrobiodiversity shows much potential for enhancing natural and cultural diversity in Southern countries, but only when local communities can participate in the shaping of the conditions under which this happens. Such activities should be designed in the mid- to long-term as part of social learning processes that are carefully embedded in the local context. Supporting institutions play a crucial role in these processes, but should see themselves only as facilitators, while ensuring that control and ownership remain with the local actors.

Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich Fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives.

Platelet concentrates for topical and infiltrative use - commonly termed Platetet-Rich Plasma (PRP) or Platelet-Rich Fibrin (PRF) - are used or tested as surgical adjuvants or regenerative medicine preparations in most medical fields, particularly in sports medicine and orthopaedic surgery. Even if these products offer interesting therapeutic perspectives, their clinical relevance is largely debated, as the literature on the topic is often confused and contradictory. The long history of these products was always associated with confusions, mostly related to the lack of consensual terminology, characterization and classification of the many products that were tested in the last 40 years. The current consensus is based on a simple classification system dividing the many products in 4 main families, based on their fibrin architecture and cell content: Pure Platelet-Rich Plasma (P-PRP), such as the PRGF-Endoret technique; Leukocyte- and Platelet-Rich Plasma (LPRP), such as Biomet GPS system; Pure Platelet-Rich Fibrin (P-PRF), such as Fibrinet; Leukocyte- and Platelet-Rich Fibrin (L-PRF), such as Intra-Spin L-PRF. The 4 main families of products present different biological signatures and mechanisms, and obvious differences for clinical applications. This classification serves as a basis for further investigations of the effects of these products. Perspectives of evolutions of this classification and terminology are also discussed, particularly concerning the impact of the cell content, preservation and activation on these products in sports medicine and orthopaedics.

Prediction of Post-Weaning Fibrinogen Status during Cardiopulmonary Bypass: An Observational Study in 110 Patients.

BACKGROUND After cardiac surgery with cardiopulmonary bypass (CPB), acquired coagulopathy often leads to post-CPB bleeding. Though multifactorial in origin, this coagulopathy is often aggravated by deficient fibrinogen levels. OBJECTIVE To assess whether laboratory and thrombelastometric testing on CPB can predict plasma fibrinogen immediately after CPB weaning. PATIENTS / METHODS This prospective study in 110 patients undergoing major cardiovascular surgery at risk of post-CPB bleeding compares fibrinogen level (Clauss method) and function (fibrin-specific thrombelastometry) in order to study the predictability of their course early after termination of CPB. Linear regression analysis and receiver operating characteristics were used to determine correlations and predictive accuracy. RESULTS Quantitative estimation of post-CPB Clauss fibrinogen from on-CPB fibrinogen was feasible with small bias (+0.19 g/l), but with poor precision and a percentage of error >30%. A clinically useful alternative approach was developed by using on-CPB A10 to predict a Clauss fibrinogen range of interest instead of a discrete level. An on-CPB A10 ≤10 mm identified patients with a post-CPB Clauss fibrinogen of ≤1.5 g/l with a sensitivity of 0.99 and a positive predictive value of 0.60; it also identified those without a post-CPB Clauss fibrinogen <2.0 g/l with a specificity of 0.83. CONCLUSIONS When measured on CPB prior to weaning, a FIBTEM A10 ≤10 mm is an early alert for post-CPB fibrinogen levels below or within the substitution range (1.5-2.0 g/l) recommended in case of post-CPB coagulopathic bleeding. This helps to minimize the delay to data-based hemostatic management after weaning from CPB.