Generation of hyperfunctional recombinant human factor IX variants expressed in human cell line SK-Hep-1.

OBJECTIVE: To develop recombinant factor IX (FIX) variants with augmented clotting activity. RESULTS: We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 µg-mL), followed by FIX-LLW (1.5 µg-mL) and FIX-ALL (0.9 µg-mL). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 µg-mL). However, the biological activities of FIX variants were 15-31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection. CONCLUSION: We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.

De Novo Assessment and Review of Pan-American Pit Viper Anticoagulant and Procoagulant Venom Activities via Kinetomic Analyses.

Snakebite with hemotoxic venom continues to be a major source of morbidity and mortality worldwide. Our laboratory has characterized the coagulopathy that occurs in vitro in human plasma via specialized thrombelastographic methods to determine if venoms are predominantly anticoagulant or procoagulant in nature. Further, the exposure of venoms to carbon monoxide (CO) or O-phenylhydroxylamine (PHA) modulate putative heme groups attached to key enzymes has also provided mechanistic insight into the multiple different activities contained in one venom. The present investigation used these techniques to characterize fourteen different venoms obtained from snakes from North, Central, and South America. Further, we review and present previous thrombelastographic-based analyses of eighteen other species from the Americas. Venoms were found to be anticoagulant and procoagulant (thrombin-like activity, thrombin-generating activity). All prospectively assessed venom activities were determined to be heme-modulated except two, wherein both CO and its carrier molecule were found to inhibit activity, while PHA did not affect activity (Bothriechis schlegelii and Crotalus organus abyssus). When divided by continent, North and Central America contained venoms with mostly anticoagulant activities, several thrombin-like activities, with only two thrombin-generating activity containing venoms. In contrast, most venoms with thrombin-generating activity were located in South America, derived from Bothrops species. In conclusion, the kinetomic profiles of venoms obtained from thirty-two Pan-American Pit Viper species are presented. It is anticipated that this approach will be utilized to identify clinically relevant hemotoxic venom enzymatic activity and assess the efficacy of locally delivered CO or systemically administered antivenoms.

Flocculation kinetics of low-turbidity raw water and the irreversible floc breakup process.

The main objective of this study was to propose an improvement to the flocculation kinetics model presented by Argaman and Kaufman, by including a new term that accounts for the irreversible floc breakup process. Both models were fitted to the experimental results obtained with flocculation kinetics assays of low turbidity raw water containing Microcystis aeruginosa cells. Aluminum sulfate and ferric chloride were used as coagulants, and three distinct average velocity gradient (G) values were applied in the flocculation stage (20, 40 and 60 s-1). Experimental results suggest that the equilibrium between the aggregation and breakup process, as depicted by Argaman and Kaufman's original model, might not be constant over time, since the residual turbidity increased in various assays (phenomenon that was attributed to the irreversible floc breakup process). In the aluminum sulfate assays, the residual turbidity increase was visible when G = 20 s-1 (dosages of 60 and 80 mg L-1). For the ferric chloride assays, the phenomenon was noticed when G = 60 s-1 (dosages of 60 and 80 mg L-1). The proposed model presented a better fit to the experimental results, especially at higher coagulant dosages and/or higher values of average velocity gradient (G).

Development of a magnetic coagulant based on Moringa oleifera seed extract for water treatment.

In this work, to evaluate the effectiveness of the coagulation/flocculation using a natural coagulant, using Moringa oleifera Lam functionalized with magnetic iron oxide nanoparticles, producing flakes that are attracted by an external magnetic field, thereby allowing a fast settling and separation of the clarified liquid, is proposed. The removal efficiency of the parameters, apparent color, turbidity, and compounds with UV254nm absorption, was evaluated. The magnetic functionalized M. oleifera Lam coagulant could effectively remove 90 % of turbidity, 85 % of apparent color, and 50 % for the compounds with absorption at UV254nm, in surface waters under the influence of an external magnetic field within 30 min. It was found that the coagulation/flocculation treatment using magnetic functionalized M. oleifera Lam coagulant was able to reduce the values of the physico-chemical parameters evaluated with reduced settling time.

The use of a natural coagulant (Opuntia ficus-indica) in the removal for organic materials of textile effluents.

The goal of this study was to investigate the activity of the coagulant extracted from the cactus Opuntia ficus-indica (OFI) in the process of coagulation/flocculation of textile effluents. Preliminary tests of a kaolinite suspension achieved maximum turbidity removal of 95 % using an NaCl extraction solution. Optimization assays were conducted with actual effluents using the response surface methodology (RSM) based on the Box-Behnken experimental design. The responses of the variables FeCl3, dosage, cactus dosage, and pH in the removal of COD and turbidity from both effluents were investigated. The optimum conditions determined for jeans washing laundry effluent were the following: FeCl3 160 mg L(-1), cactus dosage 2.60 mg L(-1), and pH 5.0. For the fabric dyeing effluent, the optimum conditions were the following: FeCl3 640 mg L(-1), cactus dosage 160 mg L(-1), and pH 6.0. Investigation of the effects of the storage time and temperature of the cactus O. ficus-indica showed that coagulation efficiency was not significantly affected for storage at room temperature for up to 4 days.

Protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves: enzymatic characterization and procoagulant and fibrinogenolytic activities.

Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors.

Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. The determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects.

Individual variability in the venom proteome of juvenile Bothrops jararaca specimens.

Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of Bothrops jararaca of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities; however, their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC-MS analysis showed significant differences between venoms; however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most B. jararaca toxin classes. Profiles of venoms analyzed under nonreducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and l-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in B. jararaca exists from a very early animal age and is not a result of ontogenetic and diet changes.

Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: molecular sequence analysis of its cDNA and biochemical properties.

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.

New insights into the complex mixture of latex cysteine peptidases in Calotropis procera.

The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and ß(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.

Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters.

Lectins are carbohydrate recognition proteins. cMoL, a coagulant Moringa oleifera Lectin, was isolated from seeds of the plant. Structural studies revealed a heat-stable and pH resistant protein with 101 amino acids, 11.67 theoretical pI and 81% similarity with a M. oleifera flocculent protein. Secondary structure content was estimated as 46% α-helix, 12% ß-sheets, 17% ß-turns and 25% unordered structures belonging to the α/ß tertiary structure class. cMoL significantly prolonged the time required for blood coagulation, activated partial thromboplastin (aPTT) and prothrombin times (PT), but was not so effective in prolonging aPTT in asialofetuin presence. cMoL acted as an anticoagulant protein on in vitro blood coagulation parameters and at least on aPTT, the lectin interacted through the carbohydrate recognition domain.

Systemic effects induced by the venom of the snake Bothrops caribbaeus in a murine model.

Snakebite envenoming by Bothrops caribbaeus, an endemic viperid from the Lesser Antillean island of Saint Lucia, is clinically characterized by local tissue damage and systemic thrombosis that can lead to cerebral, myocardial or pulmonary infarctions and venous thromboses. Systemic effects (lethality, pulmonary hemorrhage, thrombocytopenia and coagulopathy) induced by intravenous (i.v.) administration of B. caribbaeus venom were studied in mice. The role of snake venom metalloproteinases (SVMPs) in these systemic alterations was assessed by inhibition with the chelating agent calcium disodium ethylenediaminetetraacetic acid (CaNa(2)EDTA). A snake C-type lectin-like (snaclec) and a type P-III hemorrhagic SVMP were isolated and characterized from this venom, and the effect of venom and the isolated snaclec on human platelet aggregation was studied in vitro. Results indicate that SVMPs play an important role in the overall toxicity of B. caribbaeus venom, being responsible for systemic hemorrhage and lethality, but not thrombocytopenia, whereas the isolated snaclec is involved in the thrombocytopenic effect. Both venom and snaclec induce platelet aggregation/agglutination. Moreover, the snaclec binds directly to glycoprotein Ib (GPIb) and induces agglutination in washed fixed platelets. On the other hand, B. caribbaeus venom hydrolyzed fibrinogen in vitro and induced a partial drop of fibrinogen levels with an increase in fibrin/fibrinogen degradation products (FDP) levels in vivo. The negative result for D-dimer (DD) in plasma is consistent with the lack of microscopic evidence of pulmonary thrombosis and endothelial cell damage. Likewise, no increments in plasma sE-selectin levels were detected. The absence of thrombosis in this murine model suggests that this effect may be species-specific.

Biochemical and biological characterization of two serine proteinases from Colombian Crotalus durissus cumanensis snake venom.

Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 µg and 0.571 µg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.

Comparison of coagulants and coagulation aids for treatment of meat processing wastewater by column flotation.

The physicochemical treatment of the wastewater from a meat processing industry was studied using three ferric salts as coagulants in conjunction with four different polymers as coagulation aids by batch column flotation. The effluent was characterized in terms of pH (6.5-6.7), turbidity (1000-12000 NTU), total solids (TS) (2300-7000mgl(-1)), oils and greases (OG) (820-1050mgl(-1)), and biochemical and chemical oxygen demands (BOD(5) and COD) (1200-1760 and 2800-3230mgl(-1)), respectively. The treatments achieved typical organic load reductions of oils and greases, and total solids (up to 85%), as well as biochemical and chemical oxygen demands (between 62.0-78.8% and 74.6-79.5%, respectively). The research also found that the utilization of a column flotation achieved high efficiency of organic matter removal and its operation as a primary treatment showed no significant dependence of pollutant removal and air flow rate.

Endothelial dysfunction in patients with antiphospholipid syndrome assessed with positron emission tomography.

BACKGROUND: There is limited knowledge about endothelial dysfunction in patients with primary antiphospholipid syndrome (PAPS). The purpose of this study was to evaluate endothelial function in patients with PAPS assessed by positron emission tomography. METHODS AND RESULTS: A 3-phase protocol--rest, cold pressor test (CPT), and adenosine positron emission tomography with nitrogen 13 ammonia--was used in 18 patients with PAPS and 18 healthy volunteers (HVs). Myocardial blood flow (MBF) was measured in each phase, with calculation of the endothelial-dependent vasodilation index, the increase in the MBF in response to CPT, and the myocardial flow reserve. An important trend was found in the myocardial flow reserve (2.76 +/- 1.04 in PAPS group vs 3.27 +/- 0.72 in HV group, P > .05), in the endothelial-dependent vasodilation index (1.19 +/- 0.31 in PAPS group vs 1.55 +/- 0.37 in HV group, P < .05), and in the percent change in the MBF in response to CPT (from rest) (19% +/- 31% in PAPS group vs 55% +/- 37% in HV group, P < .05). CONCLUSION: The CPT results obtained in this study showed that the PAPS patients studied have endothelial dysfunction.