fibrin and 4-(2-aminoethyl)benzenesulfonylfluoride

The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500 μL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na

Topics: Animals; Blood Coagulation; Bothrops; Crotalid Venoms; Female; Fibrin; Hepatocyte Growth Factor; Male; Metalloproteases; Protein Precursors; Rats, Wistar; Serine Endopeptidases; Serine Proteinase Inhibitors; Sulfones

The role of red blood cells (RBCs) in coagulation is not well understood. Overt exposure of phosphatidylserine on surfaces of RBCs provide docking sites for formation of the prothrombinase complex, which further aids in amplification of coagulation leading to subsequent thrombosis. No studies to date have evaluated heparin inhibition of the RBC-prothrombinase system. Therefore, this study examines the ability of heparin and a covalent antithrombin-heparin complex (ATH) to inhibit the RBC-prothrombinase system. Discontinuous inhibition assays were performed to obtain k₂ values for inhibition of free or prothrombinase-bound Xa by antithrombin and unfractionated heparin (AT + UFH) versus ATH. In addition, components of the complex (prothrombin, RBCs or Va) were excluded prior to reaction with inhibitors to investigate potential mechanisms involved. Inhibition of thrombin generation, fibrinogen conversion and plasma clotting by the RBC-prothrombinase system was also examined. Protection of Xa was observed for AT + UFH and not for ATH reactions. Inhibition rates for ATH were significantly faster when compared with AT + UFH results. The greatest impact on Xa inhibition was observed from factor Va omission for both inhibitors. ATH inhibited thrombin generation, fibrinogen conversion and plasma clotting better compared with AT + UFH. This study determined potential control of coagulation contributed by RBCs. Moreover, greater control of coagulation is achieved by covalently linking heparin to AT.

Topics: Adult; Anticoagulants; Antithrombins; Blood Coagulation; Erythrocytes; Factor V; Factor Xa; Factor Xa Inhibitors; Fibrin; Fibrinogen; Heparin; Humans; Kinetics; Serine Proteinase Inhibitors; Sulfones

Thromboelastography analysis providing a global assessment of coagulation is gaining new interest in clinical practice. MinimalTF triggered whole blood thromboelastography provides a valuable tool for studying the kinetics of clot formation (expressed by the parameters R, K and alpha-angle) and the physical characteristics of the clot, such as its firmness and the elastic modulus shear (expressed by the parameters maximal amplitude MA and G). We studied the influence of fibrin polymerization and platelet functional status on each parameter of thromboelastographic trace obtained by minimalTF activation inWB by employing increasing concentrations of a fibrin polymerization inhibitors (the tetrapeptide Gly-Pro-Arg-Pro-OH.AcOH; Pefabloc-FG) and an inhibitor of actin polymerization (Cytochalasin D). Pefabloc-FG at concentrations higher than 5 mg/ml prolonged the R and K times and decreased the alpha-angle in a concentration-dependent manner but it did not modify MA and G parameters. At the concentration of 5 mg/ml, Pefabloc-FG completely inhibited clot formation. Cytochalasin D had no effect on R time but decreased the alpha-angle, MA and G parameters by reaching a plateau at the concentration of 5 microM. The effect of cytochalasin D was more pronounced on MA and G than on the alpha-angle. A combination of both Pefabloc-FG (0.5 mg/ml) and cytochalasin D (50 microM) significantly decreased alpha-angle compared to control as well as their single effect. However, G value was dramatically reduced in the presence of cytochalasin D exposure, without any additional effect when both inhibitors were combined. This study confirms the importance of fibrin polymerisation on the kinetics of thrombus formation and demonstrates the close association between the quality of the thrombus and the functional status of platelets. Normal platelet contractile forces are of major importance for the maximum amplitude of TEG which is related to the strength and elastic modulus of the thrombus.

Topics: Biomechanical Phenomena; Blood Coagulation; Blood Platelets; Citric Acid; Cytochalasin D; Dose-Response Relationship, Drug; Fibrin; Humans; Oligopeptides; Sulfones; Thrombelastography; Thrombosis

Digestive gland protease pH optima and specific activities determined in Penaeus indicus with casein, azocasein, Azocoll, and Congo red fibrin as substrates were pH 7.7-9.2, 210-371 micromol of tyrosine/mg of homogenate protein/min; pH 7.8, 36; pH 6.0-7.0, 7; and pH 8.9-9.2, 7A delta0.001 U/mg of homogenate protein/min, respectively. Activity in the shrimp was stable during frozen storage but relatively labile and very low (1.043 azocasein units) in the Norwegian lobster, Nephrops norvegicus. The high activity in shrimp is significant in aquaculture and may be a source of proteolytic enzymes for industrial use. The rapid deterioration after landing may be a consequence of the high and stable activity. The low activity in the lobster may present a problem in culture and requires a more critical choice of feed as well as further investigation. 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride was a very convenient, fast-acting, and effective inhibitor of shrimp trypsin and chymotrypsin but did not completely inhibit general protease activity in shrimp and had a negligible effect on the lobster. A significant component of that activity may be from nonserine proteases (such as the exoproteases carboxypeptidase A and B and the leucine aminopeptidases), whose proportion relative to the serine proteases may be greater in the lobster.

Topics: Animal Feed; Animals; Azo Compounds; Buffers; Caseins; Collagen; Digestive System; Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Exopeptidases; Fibrin; Hydrogen-Ion Concentration; Nephropidae; Penaeidae; Peptide Hydrolases; Serine Endopeptidases; Substrate Specificity; Sulfones; Tromethamine