ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Following Venezuelan traditional medicine, females with heavy menstrual blood loss (menorrhagia) drink Brownea grandiceps Jacq. flowers (BG) decoctions to reduce the bleeding. In a previous study, we demonstrated that BG aqueous extract (E) possesses a potent anti-fibrinolytic activity capable of inhibiting plasmin, the main serine-protease that degrades fibrin. It is widely known that plasmin inhibitors are often used as anti-fibrinolytics to reduce bleeding during surgeries with high risk of blood loss such as cardiac, liver, vascular, tooth extraction and large orthopedic procedures, as well as for menorrhagia treatments. The aim of this work was to isolate and characterize from BGE the compound responsible for the reported anti-fibrinolytic activity. MATERIALS AND METHODS: A decoction of BG was prepared; then it was homogenized, centrifuged and lyophilized to obtain BGE. Subsequently the extract was fractionated by gel filtration and reverse phase using HPLC and the active compound was characterized by MALDI-ToF MS. The kinetic parameters of anti-plasmin activity were evaluated by an amidolytic assay using a chromogenic substrate; also the anti-plasmin activity was estimated by fibrin plate method. Data were analyzed by nonparametric statistics. RESULTS: The active compound was a condensed tannin denominated Browplasminin, which is capable of inhibiting the plasmin activity in a dose-dependent manner when measured in fibrin plates or by the amidolytic activity method; it also has a minor effect on the FXa activity. However, it does not affect the activity of other serine-proteases such as trypsin, t-PA or u-PA. Browplasminin consists predominately of heteroflavan-3-ols of catechin with B-type linkages, and extents up to heptadecamers (~ 5000Da), with hexose residues attached to the polymer that presents a high degree of galloylation. Its IC50 for plasmin was 47.80µg/mL and for FXa was 237.08µg/mL, while the Ki were 0.76 and 61.61µg/mL for plasmin and FXa, respectively. CONCLUSIONS: The overall outcome of this study suggests that Browplasminin could be responsible for reducing heavy menstrual bleeding in women because its kinetic parameters showed that is a good plasmin inhibitor.
Subject(s)
Antifibrinolytic Agents/pharmacology , Fabaceae/chemistry , Fibrinolysin/antagonists & inhibitors , Plant Extracts/pharmacology , Antifibrinolytic Agents/administration & dosage , Antifibrinolytic Agents/isolation & purification , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Flowers , Hydrolyzable Tannins , Inhibitory Concentration 50 , Medicine, Traditional , Plant Extracts/administration & dosage , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , VenezuelaABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Brownea grandiceps flowers are used in Venezuelan folk medicine as anti-hemorrhagic in women with heavy menstrual blood loss (menorrhagia). However, prior to this study, there were no scientific investigations to support this fact, because the aqueous extract from Brownea grandiceps flowers had not been previously evaluated neither phytochemically nor biologically. The objective of this work was to evaluate in vitro the effects of aqueous extract from Brownea grandiceps flowers on the coagulation system and fibrinolysis. MATERIALS AND METHODS: An infusion of Brownea grandiceps flowers (160g) was performed; then, it was homogenized, centrifuged and lyophilized to obtain the aqueous extract, and this was called BGE. Subsequently, the extract was characterized on the one hand, phytochemically and on the other hand, biologically, employing prothrombin time (PT), partial thromboplastin time (PTT) and thrombin time (TT) to determine the effects on extrinsic, intrinsic and common coagulation pathways, respectively. In addition to that, the fibrinogenolytic and fibronectinase activity was evaluated by SDS-PAGE using Tris-Tricine system and analyzed by densitometric study utilizing ImageJ program. Also, by using specific chromogenic substrates for Factor Xa (FXa), thrombin, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasmin, it was assessed whether BGE exhibited some enzyme-like activity, and inhibitory activity of the afore mentioned enzymes. Fibrinolytic and antifibrinolytic activities were determined by a fibrin plate method. Data were analyzed by an nonparametric method. RESULTS: BGE presented tannins, saponins, glycosides, alkaloids, flavonoids, coumarins, and did not contain triterpenoids and steroids. Also, BGE at low concentrations (250-1250µg/mL) reduced the PT, while higher concentrations (15000-25000µg/mL) prolonged this time. However, BGE concentrations between 1250 and 25000µg/mL prolonged the PTT. Prolongation of PT and PTT was observed at high concentrations and was due to FXa inhibitor found in BGE and this effect could be strengthened by degradation of fibrinogen and fibronectin, which were also produced by BGE. Moreover, BGE did not clot fibrinogen or human plasma, and neither did it cleave the chromogenic substrates specific to FXa nor thrombin. These results suggest the pro-coagulant components could be acting on some factor of the extrinsic pathway, since only PT was shortened. Furthermore, BGE did not hydrolyze the chromogenic substrate specific to plasmin, t-PA and u-PA nor did it produce fibrin degradation. However, all BGE concentrations tested inhibited the plasmin activity in a dose-dependent manner. CONCLUSIONS: The outcomes of this study reveal the presence of fibrinogenolytic, fibronectinase and anti-FXa components in BGE, plus anti-plasmin compounds that could be acting as antifibrinolytic, thus delaying the fibrin degradation in pathophysiological processes, as it has been observed in women presenting with menorrhagia due to a high plasmin concentration. Where this anti-plasmin compound, along with pro-coagulant components also present in BGE, could be made responsible for reducing heavy menstrual bleeding in women, since a deficiency in one or more blood coagulation factors such as factor VII, V or X, is a potential cause of menorrhagia.
Subject(s)
Blood Coagulation/drug effects , Fabaceae/chemistry , Fibrinolysis/drug effects , Flowers/chemistry , Plant Extracts/pharmacology , Blood Coagulation Factors/metabolism , Flowers/enzymology , Humans , Partial Thromboplastin Time , Plant Extracts/chemistry , Prothrombin Time , Serine Endopeptidases/metabolism , Thrombin TimeABSTRACT
Several fibrin(ogen)olytic enzymes from Tityus discrepans (Buthidae, Buthoidea) venom (TdV) were partially purified on a Sephadex G-50 column, by affinity and molecular exclusion high-performance chromatography. Fractions SB1-I and SB1-II had fibrinolytic, fibrinogenolytic (Aα-chains degradation) and tissue plasminogen activator (t-PA)-like activities. SB1-III was only fibrinogenolytic (fast degradation of Aα-chains and slower degradation of fibrinogen Bß-chains). These results showed the presence of α-fibrinogenases in TdV. The fibrino(geno)lytic activity in these fractions was abolished by metalloprotease inhibitors (MPI). Fractions SB3-I and SB3-II contain fibrinogenolytic (Aα-chains degradation) and fibronectinolytic activities. Also fraction SB3-I had a t-PA-like activity. Activities in SB3-I and SB3-II were abolished by serine protease inhibitors (SPI). None of the fractions degraded fibrinogen γ-chains. Fibrinogen degradation by active fractions is associated with an anticoagulant effect supported by a reduced coagulant activity. The overall outcome suggests that metalloproteases and serine proteases in TdV are responsible for fibrin(ogen)olytic activity because MPI and SPI inhibited these activities.
Subject(s)
Fibrin/metabolism , Scorpion Venoms/enzymology , Scorpions/enzymology , Animals , Metalloproteases/metabolism , Serine Proteases/metabolismABSTRACT
Tityus discrepans venom (TdV) produces a variety of haemostatic manifestations including alveoli fbrin deposition and/ or prothrombin and partial thromboplastin time (PT, PTT) alterations in mammals. In vitro studies have demonstrated that TdV contains tissue plasminogen activator-like (t-PA), fbrinolytic and plasmin inhibitory compounds and produces platelets activation through GPVI and a novel Src-dependent signalling pathway. The aim of this study is to describe the initial characterization of procoagulant and anticoagulant components from TdV. This venom was fractionated by exclusion molecular chromatography on a Sephadex G-50 column. The eluted material was collected as fve fractions called S1 to S5. These fractions and the whole venom were used to evaluate factor Xa- and thrombin-like activities, fbrinogen degradation, furthermore thrombin- and factor Xa-inhibitory activities. The results demonstrated that TdV contain components with factor Xa-like activity (procoagulants) as well fbrinogenolytic compounds present in the fraction S1 and components with factor Xa inhibitory activity in the fractions S4 and S5 (anticoagulants).
El veneno de Tityus discrepans (TdV) produce en mamíferos una variedad de manifestaciones hemostáticas tales como depósitos de fbrina en alveolos y/o alteración en los tiempos de protrombina y tromboplastina parcial (PT, PTT). Estudios in vitro han demostrado que el TdV contiene componentes semejantes al activador del plasminógeno tipo tisular (t-PA), fbrino-líticos, compuestos que inhiben la actividad de plasmina y además componentes que promueven la activación de plaquetas a través del receptor GPVI y por una nueva vía de señalización dependiente de las Src kinasas. El objetivo de este estudio es describir la caracterización inicial de componentes procoagulantes y anticoagulantes a partir del TdV. Este veneno fue fraccionado por cromatografía de exclusión molecular sobre una columna Sephadex G-50. El material eluido fue colectado en cinco fracciones denominadas S1 a S5. Estas fracciones y el veneno completo fueron usados para evaluar actividades semejantes a factor Xa y trombina, degradación de fbrinógeno, como también la inhibición de la actividad del factor Xa y de la trombina. Los resultados demostraron que TdV contiene componentes con actividad semejante al factor Xa (procoagulantes) y compuestos fbrinogenolíticos presentes en la fracción S1, además de componentes con actividad inhibitoria del factor Xa presentes en la fracción S4 y S5 (anticoagulantes).
Subject(s)
Blood Coagulation , Factor Xa , Fibrinolysis , Scorpion Venoms/analysis , Scorpion Venoms/enzymology , Anticoagulants , Coagulants , Scorpion Venoms/chemical synthesisABSTRACT
Tityus discrepans venom (TdV) produces a variety of haemostatic manifestations including alveoli fbrin deposition and/ or prothrombin and partial thromboplastin time (PT, PTT) alterations in mammals. In vitro studies have demonstrated that TdV contains tissue plasminogen activator-like (t-PA), fbrinolytic and plasmin inhibitory compounds and produces platelets activation through GPVI and a novel Src-dependent signalling pathway. The aim of this study is to describe the initial characterization of procoagulant and anticoagulant components from TdV. This venom was fractionated by exclusion molecular chromatography on a Sephadex G-50 column. The eluted material was collected as fve fractions called S1 to S5. These fractions and the whole venom were used to evaluate factor Xa- and thrombin-like activities, fbrinogen degradation, furthermore thrombin- and factor Xa-inhibitory activities. The results demonstrated that TdV contain components with factor Xa-like activity (procoagulants) as well fbrinogenolytic compounds present in the fraction S1 and components with factor Xa inhibitory activity in the fractions S4 and S5 (anticoagulants).(AU)
El veneno de Tityus discrepans (TdV) produce en mamíferos una variedad de manifestaciones hemostáticas tales como depósitos de fbrina en alveolos y/o alteración en los tiempos de protrombina y tromboplastina parcial (PT, PTT). Estudios in vitro han demostrado que el TdV contiene componentes semejantes al activador del plasminógeno tipo tisular (t-PA), fbrino-líticos, compuestos que inhiben la actividad de plasmina y además componentes que promueven la activación de plaquetas a través del receptor GPVI y por una nueva vía de señalización dependiente de las Src kinasas. El objetivo de este estudio es describir la caracterización inicial de componentes procoagulantes y anticoagulantes a partir del TdV. Este veneno fue fraccionado por cromatografía de exclusión molecular sobre una columna Sephadex G-50. El material eluido fue colectado en cinco fracciones denominadas S1 a S5. Estas fracciones y el veneno completo fueron usados para evaluar actividades semejantes a factor Xa y trombina, degradación de fbrinógeno, como también la inhibición de la actividad del factor Xa y de la trombina. Los resultados demostraron que TdV contiene componentes con actividad semejante al factor Xa (procoagulantes) y compuestos fbrinogenolíticos presentes en la fracción S1, además de componentes con actividad inhibitoria del factor Xa presentes en la fracción S4 y S5 (anticoagulantes).(AU)
ABSTRACT
In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbß3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.
Subject(s)
Blood Platelets/drug effects , Platelet Activation/drug effects , Platelet Membrane Glycoproteins/metabolism , Scorpion Venoms/pharmacology , src-Family Kinases/blood , Animals , Blood Platelets/metabolism , Cells, Cultured , Chickens , Humans , Mice , Mice, Inbred C57BL , Platelet Membrane Glycoproteins/deficiency , Platelet Membrane Glycoproteins/genetics , Signal Transduction , TransfectionABSTRACT
Tityus discrepans venom (TdV) produces digestive hemorrhages, disseminated intravascular coagulation, alveoli fibrin deposition and/or prothrombin and partial thromboplastin time alterations in humans. T. discrepans venom presents an in vitro tissue plasminogen activator-like (tPA-like), fibrino(geno)lytic and plasmin inhibitory activities. The plasmin inhibitor, called discreplasminin, was isolated from TdV. Discreplasminin has a pI of 8.0 and a relative molecular weight of <6,000 Da. Discreplasminin and aprotinin strongly inhibited plasmin activity and moderately tPA activity, while epsilon amino caproic acid (EACA) moderately inhibited both enzymes. In presence and absence of fibrin, the plasmin generation by tPA was completely inhibited by aprotinin and discreplasminin. EACA in the absence of fibrin partially inhibited plasmin generation (37%); however, it produced a total inhibition of plasmin generation on a fibrin surface. The tPA-clot lysis assay showed that discreplasminin acts like aprotinin inducing a slight delay in lysis time and lysis rate; in contrast, EACA presented a total inhibitory effect on fibrin lysis. These results suggest that discreplasminin presents an anti-fibrinolytic mechanism similar to aprotinin. Discreplasminin probably interacts with the active sites of plasmin and tPA. The presence of discreplasminin and other similar components in scorpion venom could partially explain the generalized fibrin deposition which was found previously in rams.
Subject(s)
Antifibrinolytic Agents/isolation & purification , Antifibrinolytic Agents/pharmacology , Fibrinolysin/antagonists & inhibitors , Scorpion Venoms/chemistry , Scorpions/chemistry , Animals , Antifibrinolytic Agents/chemistry , Fibrin/metabolism , Hemostasis , Isoelectric Point , Molecular Weight , Plasminogen Activators/metabolism , Tissue Plasminogen Activator/metabolismABSTRACT
Tityus discrepans (Td) scorpion venom modifies clotting times in humans. We studied the in vitro venom effect on partial thromboplastin time (PTT), prothrombin time (PT) and its direct clotting activity, using fresh human plasma and purified fibrinogen as substrates. Whole venom (WV) was fractioned with a Protein Pak 125 molecular exclusion column (0.5 mL/min, CH3COONH4 20 mM, pH 4.7). Six fractions (F1 through F6) with retention times ranging from 12.8 to 31 min were obtained. WV (78 to 625 microg/mL) and fraction F1 (10 to 42.5 microg/mL), shortened PTT; WV (700 to 1000 microg/mL) and fraction F6 (16.5 to 700 microg/mL), prolonged PTT. WV (40 to 240 microg/mL) and fraction F2 (5 to 40 microg/mL), prolonged PT. No thrombin-like activity was found with this venom on human plasma or purified fibrinogen. Td venom contains procoagulant components, able to shorten PTT. In addition, the venom contains anticoagulant components which prolong PT and PTT.
Subject(s)
Blood Coagulation/drug effects , Partial Thromboplastin Time , Prothrombin Time , Scorpion Venoms/pharmacology , Animals , Blood Coagulation Tests , HumansABSTRACT
El veneno del escorpión Tityus discrepans (Td) altera los tiempos de coagulación en humanos. En este trabajo se estudió el efecto in vitro de este veneno sobre el tiempo de tromboplastina parcial (TTP), el tiempo de protrombina (TP) y su actividad coagulante directa, utilizando como substrato plasma humano fresco y/o fibrinógeno purificado. El veneno completo (VC) fue fraccionado con una columna de exclusión molecular Protein Pak 125 (0,5 mL/min, CH3COONH4 20 mM, pH 4,7). Seis fracciones (F1 a F6) eluyeron con tiempos de retención entre 12,8 y 31 minutos. El VC (78-625 µg/mL) y la fracción F1 (10-42,5 µg/mL), acortaron el TTP; el VC (700-1000 µg/mL) y la fracción F6 (16,5-700 µg/mL), alargaron esta prueba. El VC (40-240 µg/mL) y la fracción F2 (5-40 µg/mL), prolongaron el TP. No se detectó actividad coagulante parecida a trombina sobre plasma humano o fibrinógeno purificado. Estos resultados evidencian que en el veneno de Td existen componentes con acción procoagulante, que acortan el TTP. Además, presenta componentes anticoagulantes que inducen un alargamiento del TP y TTP.
Subject(s)
Animals , Anticoagulants , Factor VIII , In Vitro Techniques , Scorpions , Scorpion Venoms/adverse effects , Neuropharmacology , VenezuelaABSTRACT
We studied the effects of benzydamine (BZ) and cyclophosphamide (CP) on acute lung injury caused by Tityus discrepans venom. Mice (male, IVIC strain, approximately 25g) were pretreated with either BZ (20microg/g) or CP (100microg/g) i.p. or saline. Envenoming (2microg/g mouse) was induced sc. Lung fraction area occupied by fibrin (FF), nuclei (NF), alveolar space (AS) and parenchyma (PF) were determined. Venom increased FF, NF and PF, significantly, and decreased AS. BZ antagonised venom's effect on FF sharply, antagonised partially effects on PF and AS, but was not able to antagonise effect on NF. CP abolished venom effects on NF, AS and PF, but was not able to antagonise the effect on FF. CP was slightly less effective than BZ in reducing FF. Fibrin deposition was associated to leukocyte sequestration. Blocking pro-inflammatory leukocyte metabolic pathways with BZ diminished FF, suggesting that neutrophil activation, inflammation and coagulation are correlated in the genesis of scorpionism acute lung injury.
