The characterization of trans-pecos copperhead (Agkistrodon contortrix pictigaster) venom and isolation of two new dimeric disintegrins.

The vast amounts of toxins within the venom of snakes, while known to cause medical emergencies, display various biological functions. Trans-pecos copperhead (Agkistrodon contortrix pictigaster) crude venom separated by cation-exchange chromatography showed several fractions with fibrinolytic, hemorrhagic, gelatinase and platelet activities. Venom fractions 1, 2, 4, 5, and 12-17 contained fibrinolytic activity. Venom fractions 1, 2, 5 and 12-14 had hemorrhagic activity. Fractions 1, 2, 12, 13 and 17 contained gelatinase activity. Reverse-Phase C18 High Performance Liquid Chromatography was also used to purify and isolated disintegrins from this venom. Anti-platelet aggregation activity of the C18 fractions collected and performed on whole human blood showed that they inhibited platelet aggregation in presence of several agonists. Results from both SDS-PAGE and N-terminal sequencing determined that pictistatin 1 obtained from the Trans-Pecos copperhead venom was a dimeric disintegrin, and pictistatin 2 was a heterodimeric disintegrin. The molecules with anti-platelet activity could be considered in the development of more effective drugs, for numerous blood-related diseases such as stroke, heart attacks, thrombosis, and other medical conditions. In this study, we are presenting the first report of the purification, isolation, and partial characterization of two new dimeric disintegrins isolated from the venom of trans-pecos copperhead.

Overwiew of the Coagulation System and laboratory tests for its study. / Generalidades del sistema de la coagulación y pruebas para su estudio.

In the sixties, the clotting cascade was proposed, which describes the coagulation process as a sequence of enzymatic events initiated by two different pathways, the intrinsic and the extrinsic pathways, converging on a common pathway, to generate a multifunctional enzyme, thrombin, whose main function is to convert fibrinogen into fibrin, a protein that polymerizes spontaneously to form the building block of a hemostatic clot. Later, it was proposed a cell-based model of the hemostasis according to that coagulation does not occur as a consequence of linear sequential enzyme activation pathways, but rather via a network of simultaneous interactions between plasmatic and transmembrane proteins, as well as several cellular types, that allow the formation of highly efficient enzymatic complexes that lead to thrombin generation. In this review, we summarize these two approaches highlighting the functions of thrombin within the hemostasis and the inhibition mechanisms that regulate the blood coagulation. Moreover, we described different tests that are used to assess the function of the coagulation system, such as: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, ecarin clotting time, and the use of chromogenic substrates to evaluate individual coagulation factors. Finally, because of thrombin generation is a fundamental part of the blood coagulation and, an estimation of how well a particular individual can generate thrombin may correlate with either a risk of bleeding or thrombosis, we also include the existing methods to evaluate the potential of thrombin generation in an individual.

[Hemostasis alterations in sickle cell syndrome]. / Alteraciones de la hemostasia en el síndrome drepanocítico.

Sickle cell syndrome (SCS) includes a group of congenital hemolytic anemias associated to the presence of hemoglobin S, which is characterized by acute pain episodes and progressive damage of different organs. Some patients with sickle cell syndrome have shown, when compared with healthy individuals, an increased risk of presenting stroke, pulmonary hypertension, avascular necrosis of joints, acute chest syndrome and pregnancy complications, associated to a hypercoagulable state induced by alterations in different components of hemostasis, such as changes that include activation of the endothelium, platelet activity, coagulation and fibrinolytic systems. This paper compiles hemostasis disorders, associated with thrombotic manifestations, reported until now in sickle cell syndrom. These patients have an increase in activation markers of the coagulation system, such as prothrombin fragment 1.2, thrombin-antithrombin complex, etc., depletion of natural anticoagulant proteins, abnormal activation of the fibrinolytic system and increased tissue factor expression. Similarly, abnormal expression of glycoproteins and increased adhesion and platelet aggregation have been reported. All these alterations produce a hypercoagulable state, which induces, among other things, the appearance of thrombotic complications. In view of the importance of controlling the different complications that can occur in patients with sickle cell syndrome, we recommend the implementation, in diagnosis and monitoring studies, of the evaluation of the different components of the hemostatic system, identifying alterations at an early stage and applying effective treatments to prevent thrombotic complications.

Isolation and characterization of two new non-hemorrhagic metalloproteinases with fibrinogenolytic activity from the mapanare (Bothrops colombiensis) venom.

Colombienases are acidic, low molecular weight metalloproteinases (Mr of 23,074.31 Da colombienase-1 and 23,078.80 Da colombienase-2; pI of 6.0 and 6.2, respectively) isolated from Bothrops colombiensis snake venom. The chromatographic profile in RP-HPLC and its partial sequence confirmed its high homogeneity. Both colombienases present fibrino(geno)lytic activity, but did not show any hemorrhagic, amidolytic, plasminogen activator or coagulant activities, and no effect on platelet aggregation induced by collagen or ADP. Both enzymes were strongly active on fibrinogen Aα chains followed by the Bß chains, and colombienases-2, at high doses, also degraded the γ chains. This activity was stable at temperatures ranging between 4 and 37 °C, with a maximum activity at 25 °C, and at pHs between 7 and 9. The homology demonstrated by the comparison of sequences, with zinc-dependent metalloproteinases, as well as the metal chelant effects on, confirmed that the colombienases were metalloproteinases, particularly to α-fibrinogenases belonging to the P-I class of SVPMs (20-30 kDa), which contain only the single-domain proteins. The biological characteristics of the colombienases confer a therapeutic potential, since they contain a high fibrino(geno)lytic activity, devoid of hemorrhagic activity. These metalloproteinases might be explored as thrombolytic agents given that they dissolve fibrin clots or prevent their formation.

[Epidemiology of ophidism in Venezuela (1996-2004)]. / Epidemiologia del ofidismo en Venezuela (1996-2004).

The data of accidents caused by snakebites in Venezuela, registered at the morbidity statistics of the Direction of Epidemiology and Strategic Analysis of the Ministry of Health and Social Development were analyzed. During the years of 1996-2004, 53,792 snakebites were registered in Venezuela (5,976 cases average per year), with a higher incidence during the year 2004 (7,486 incidents). Zulia reported the highest frequency of all the states (5,975 cases); meanwhile the Midwestern region, constituted by Lara, Portuguesa, Falc6n and Yaracuy states, had a higher morbidity for snake bites. The highest incidence, distributed per states was registered in Cojedes, during the year 2001, with 228.72 cases per 100,000 inhabitants. When it was determined by regions, the highest incidence occurred during the year 2004 at los Llanos with 63.81 per 100,000 inhabitants. The median of the incidence rate for Venezuela during the period was of 21.46 accidents per 100,000 inhabitants. The classification of the endemic areas for ophidism, according to the percentiles 23, 50, 75 and 90, organized the country in: (a) states and regions of very high endemicity, (b) high endemicity, (c) middle, (d) low and (e) very low endemicity. These epidemiological data indicated that the accidents caused by snakes constitute a collective health problem in Venezuela.

Characterization of toxins from the broad-banded water snake Helicops angulatus (Linnaeus, 1758): isolation of a cysteine-rich secretory protein, Helicopsin.

Helicops angulatus (broad-banded water snake) according to recent proposals is presently cited in the family Dipsadidae, subfamily Xenodontinae, forming the tribe Hydropsini along with the genera Hydrops and Pseudoeryx. The current work characterizes the proteolytic and neurotoxic activities of H. angulatus crude toxins from salivary excretion (SE) and describes the isolation and identification of a cysteine-rich secretory protein (CRISP) called helicopsin. The SE lethal dose (LD50) was 5.3 mg/kg; however, the SE did not contain hemorrhagic activity. Helicopsin was purified using activity-guided, Superose 12 10/300 GL molecular exclusion, Mono Q10 ion exchange, and Protein Pak 60 molecular exclusion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a highly purified band of approximately 20 kDa. The minimal lethal dose for helicopsin was 0.4 mg/kg. Liquid chromatography mass spectrometry (LC-MS/MS) analysis identified 2 unique peptides MEWYPEAAANAER and YTQIVWYK, representing a protein sequence (deleted homology) belonging to cysteine-rich secretory proteins, which are conserved in snake venoms (CRISPs). CRISPs are a large family of cysteine-rich secretory proteins found in various organisms and participate in diverse biological processes. Helicopsin exhibited robust neurotoxic activity as evidenced by immediate death (~8 min) due to respiratory paralysis in NIH mice. These observations for helicopsin purified from H. angulatus provide further evidence of the extensive distribution of highly potent neurotoxins in the Colubroidea superfamily of snakes than previously described.

Description of envenomation by the "gusano-pollo" caterpillar (Megalopyge opercularis) in Venezuela.

Lepidoptera is a large order of insects, with more than 180,000 species word-wide, showing larval stages of butterflies and moths known as wormlike caterpillars. Almost 12 families of butterflies around the world are capable of causing severe human injuries, varying from dermatitis, renal failure, hemostatic alterations, respiratory failure and neurotoxic symptoms. These caterpillars are coated in long, hair-like setae containing venom to protect themselves against aggressive predators. The setae cause a painful reaction, upon contact, due to presence of neurotoxins. These caterpillars are extensively dispersed all through North America and often, during the dry and wet seasons in tropical regions, being able to sustain two annual larval generations. There exist several species of Megalopyge caterpillars; however, Megalopyge opercularis is the most widely distributed species in Latin America and the United States. This work reports, to our knowledge, the first case of envenomation by the "gusano-pollo" (Megalopyge opercularis), a stinging caterpillar, described in Venezuela. The patient in this report presented severe symptoms, including systemic reactions such as intense hand pain irradiated to the upper arm, restricted swelling, headache, dizziness, serious chest distress and shock-like symptoms that required hospitalization. Symptoms improved upon treatment with opiaceous analgesic drugs.

Anti-platelet effect of cumanastatin 1, a disintegrin isolated from venom of South American Crotalus rattlesnake.

Disintegrins have been previously described in the venom of several snake families inhibiting signal transduction, cell-cell interactions, and cell-matrix interactions and may have therapeutic potential in heart attacks, thrombotic diseases, and cancers. This investigation describes the first disintegrin isolated from South American Crotalus venom (Venezuelan rattlesnake Crotalus durissus cumanensis), which inhibits platelet adhesion to matrix proteins. C. d. cumanensis crude venom was first separated on a Sephadex G-100 column into 4 fractions (SI to SIV). Crude venom and SIII fraction significantly diminished platelet adhesion to fibrinogen (Fg) and to fibronectin (Fn). Anti-adhesive SIII fraction was further separated by DEAE-Sephacel followed by C-18 reverse phase high performance liquid chromatography (HPLC). The platelet anti-adhesive fraction obtained was designated as cumanastatin-1. This disintegrin has a mass of 7.442 kDa as determined by mass spectrometry (MALDI-TOF/TOF) and pI of 8.5. Cumanastatin-1 also inhibited ADP-induced platelet aggregation with an IC(50) of 158 nM. However, it did not significantly inhibit collagen and thrombin-induced platelet aggregation. Cumanastatin-1 considerably inhibited anti-alpha(IIb)beta(3) integrin binding to platelets in a dose-dependent manner; however, it did not present any effect on the alpha(5)beta(1) integrin or on P-selectin.

Discreplasminin, a plasmin inhibitor isolated from Tityus discrepans scorpion venom.

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.

Contribution of endothelial cell and macrophage activation in the alterations induced by the venom of Micrurus tener tener in C57BL/6 mice.

An acute inflammatory response, cellular infiltrates, anemia, hemorrhage and endogenous fibrinolysis activation were previously described in C57BL/6 mice injected with M. tener tener venom (Mtt). As the endothelium and innate immunity may participate in these disturbances and due to our poor understanding of the alterations produced by these venoms when the neurotoxic component is not predominant, we evaluated the effects in an in vitro model. At 24 h, the release of pro-inflammatory mediators was detected in peritoneal macrophages. At different times, the release of pro-inflammatory (TNF-α, IL-6, NO and E-Selectin), pro-coagulant (vWF and TF) and pro-fibrinolytic (uPA) mediators were seen in liver sinusoidal endothelial cells (LSECs). These results suggest that Mtt venom activates macrophages and endothelium, thus inducing the release of mediators, such as TNF-α, that orchestrate the acute inflammatory response and the later infiltration of mononuclear cells into liver in C57BL/6 mice. In addition, endothelium activation promotes TF expression, which may in turn modulate the inflammatory and hemostatic response. These findings suggest crosstalk between inflammation and hemostasis in the alterations observed in Micrurus envenomation, where the neurotoxic manifestations do not predominate.

Mutacytin-1, a New C-Type Lectin-Like Protein from the Venezuelan Cuaima (Lachesis muta muta Linnaeus, 1766) (Serpentes: Viperidae) Snake Venom Inducing Cardiotoxicity in Developing Zebrafish (Danio rerio) Embryos.

Envenomation by the Venezuelan bushmaster snake (Lachesis muta muta) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys, which is a Ca2+-dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 µg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.

The action of Lonomin V (Lonomia achelous) on fibronectin functional properties.

Lonomia achelous caterpillar, Lepidoptera distributed along some South American countries, induces a hemorrhagic syndrome in people who come into contact with its bristles. A clinical characteristic in these patients is that fresh healed wounds are re-opened and bleed. In order to explain this symptomatology, we evaluated the effect of Lonomin V (a protein isolated from L. achelous hemolymph), on some functional properties of fibronectin, which in turn plays an important role in the hemostasis. The effect of Lonomin V on fibronectin was studied by SDS-PAGE in reduced condition, binding to gelatin and heparin, crosslinking to fibrin and platelet adhesion. Formation of degradation products of 120, 66, 50, 40 and 29 kDa, some of which retain affinity to heparin and gelatin were observed; however, the fibronectin degradation fragments presented a significant decrease of crosslinking capacity to fibrin and platelet adhesion, suggesting that the proteolysis of fibronectin by Lonomin V induces changes in its crosslinking sites and on platelet receptors. These findings might partially explain the wound dehiscence observed in the patients. Due to its effect on adhesive proteins with concomitant impairment of some functional properties, Lonomin V might be useful for cellular adhesion studies involved in hemostasis such as platelet adhesion.

Intraspecies differences in hemostatic venom activities of the South American rattlesnakes, Crotalus durissus cumanensis, as revealed by a range of protease inhibitors.

Crotalus durissus cumanensis is an endemic rattlesnake found in Venezuela and Colombia. In this study, a comparative analysis of hemorrhagic, coagulation and fibrino(geno)lytic activities in the presence or absence of protease inhibitors was performed with venoms of the same species Crotalus durissus cumanensis, from seven geographical regions of Venezuela (Lagunetica, Santa Teresa, Carrizales, Guarenas, Anzoátegui, Margarita and Maracay). Lagunetica, Carrizales and Anzoátegui venoms induced hemorrhagic activity. All venoms, except that of snakes from the Carrizales region presented thrombin-like activity, which was inhibited completely by phenylmethanesulfonyl fluoride and ethylene glycol-bis-N, N,N',N'-tetraacetic acid. This effect of the latter could be explained by the high chelant calcium effect, which is a cofactor for the fibrin polymerization process. Soybean trypsin inhibitor was effective on Santa Teresa venom. Antithrombin III/Hep complex and phenantroline partially inhibited this activity in all venoms except Margarita and Anzoátegui, respectively, which were not inhibited. Serine protease inhibitors were more effective against thrombin, kallikrein and plasmin-like amidolytic activities. Additionally, metalloprotease inhibitors significantly inhibited the t-PA-like amidolytic activity and completely the hemorrhagic and fibrino(geno)lytic activities. In conclusion, the thrombin-like activity observed in these venoms was partially reduced by serine protease inhibitors, indicating the possible presence of catalytic domains in those enzymes that do not interact with these inhibitors. Using protease inhibitors on venom hemostatic activities could contribute to our understanding of the active components of snake venom on the hemostatic system, and further reveal the intraspecies variation of venoms, which is important in the treatment of envenomation, and in addition represents an interesting model for the study of venom in hemostasis.

[Effect of Tityus discrepans scorpion venom on global coagulation test. Preliminary studies]. / Efecto del veneno del escorpión Tityus discrepans sobre las pruebas globales de la coagulación. Estudios preliminares.

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.

Pro-inflammatory response and hemostatic disorder induced by venom of the coral snake Micrurus tener tener IN C57BL/6 mice.

Micrurus venoms are known to induce mainly neurotoxicity in victims. However, other manifestations, including hemorrhage, edema, myotoxicity, complement activation, and hemostatic activity have been reported. In order to develop a more complete pharmacological profile of these venoms, inflammatory responses and hemostasis were evaluated in C57BL/6 mice treated with a sub-lethal dose of M. t. tener (Mtt) venom (8 µg/mouse), inoculated intraperitoneally. The venom induced moderate bleeding into the abdominal cavity and lungs, as well as infiltration of leukocytes into the liver. After 30 min, the release of pro-inflammatory mediators (TNF-α, IL-6, and NO) were observed, being most evident at 4 h. There was a decrease in hemoglobin and hematocrit levels at 72 h, a prolongation in coagulation times (PT and aPTT), a decrease in the fibrinogen concentration and an increase in fibrinolytic activity. In this animal model, it was proposed that Mtt venom induces inflammation with the release of mediators such as TNF-α, in response to the toxins. These mediators may activate hemostatic mechanisms, producing systemic fibrinolysis and hemorrhage. These findings suggest alternative treatments in Micrurus envenomations in which neurotoxic manifestations do not predominate.

Intraspecies geographical variability in the South American tigra mariposa (Bothrops venezuelensis Sandner 1952) snake venom activities.

Bothrops venezuelensis snake venoms, from five localities in the North-Central Venezuelan regions, showed biochemical and haemostatic differences. In this study, bioactivities of B. venezuelensis venoms from different regions (Aragua state; Waraira Repano (Capital District); Baruta, La Boyera and Lagunetica (Miranda state)) were compared using both natural and synthetic substrates. The protein contents of these venoms were Lagunetica 89%, La Boyera 79%, Baruta 71%, Waraira Repano 68% and Aragua 64%. Toxic activities effects were: Intraperitoneal LD50s: Aragua-14 mg/kg; Waraira Repano-6.4 mg/kg; Baruta: 8.3 mg/kg; La Boyera-4.4 mg/kg; Lagunetica-16.2 mg/kg. The MHD results: Aragua-21.4 µg/mouse; Waraira Repano-2.5 µg/mouse; Baruta-1.2 µg/mouse; La Boyera-1.4 µg/mouse and Lagunetica-12 µg/mouse. The hide powder azure results: Aragua-1.24 U/mg; La Boyera-2.26 U/mg; Baruta-2.83 U/mg; Lagunetica-3.28 U/mg and Waraira Repano-5.77 U/mg. Esterase specific activity on BAEE results: Waraira Repano-666.66 U/mg; La Boyera-805.5 U/mg; Baruta-900.00 U/mg; Lagunetica-922.19 U/mg and Aragua-1960.67 U/mg. Casein zymography showed digestion bands in the molecular weight above 100 and at 66.2 and 21.5 kDa. Analysis of casein degradation by SDS-PAGE showed two different degradation patterns. Fibrinolytic activity (mm2/µg) on fibrin plates results: Aragua-6.07; Lagunetica-27.6; Waraira Repano-35.7; La Boyera-44.27 and Baruta-45.63. In the fibrinogenolytic assay, the five venoms completely degraded the α chain after 1 min of incubation. None of the venoms completely degraded the ß and γ chains after 24 h incubation. The research indicated that venoms of B. venezuelensis of different geographic areas in Venezuela exhibit variances in composition and component concentrations; except the Aragua venom, all of them had high proteolytic activities.

A comparative analysis of the clotting and fibrinolytic activities of the snake venom (Bothrops atrox) from different geographical areas in Venezuela.

Venom constitution within the same snake species can present considerable geographical variations. Bothrops atrox venoms were obtained from adult snakes captured at different geographical locations: Parguasa (Bolívar state); Puerto Ayacucho 1, Serranía del Cuao and Puerto Ayacucho 2 (Amazon state). The coagulant and fibrinolytic activities of these venoms were compared. Amidolytic activity of crude snake venom was measured by a micromethod designed in our laboratory. Coagulant activity on plasma and fibrinogen due to thrombin-like activity in venoms was also determined. Crude snake venom fibrinolytic activity by the fibrin plate method was assayed. Chromatographic studies were developed on Protein-Pack 300 column. Polyacrylamide gel electrophoresis was carried out under reduced conditions. After SDS-PAGE of samples, the fibrin-zymography was tested on agarose-fibrin plates. The results demonstrated several differences among B. atrox venoms from different geographical areas. Chromatograms and SDS-PAGE profiles indicated that venoms from the same species presented differences in the molecular mass of their components. The procoagulant activity depended on the utilized method (amidolytic versus clotting). Parguasa and Puerto Ayacucho 2 venoms presented procoagulant activity for both methods. Furthermore, Parguasa venom had also the highest hemorrhagic activity and the lowest LD50. In relation to the fibrinolytic activity, Puerto Ayacucho 1 venom was the most active, equally for fibrin plates as for the amidolytic method (t-PA like). This venom had the lowest coagulant activity, which induced us to think that probably its procoagulant activity was interfered by its fibrinolytic activity.

Individual venom variability in the South American rattlesnake Crotalus durissus cumanensis.

Crotalus durissus cumanensis snake venoms from different Venezuelan regions, showed biochemical and hemostatic variations. Fibrino(geno)lytic, hemorrhagic and procoagulant activities and gel-filtration chromatography and SDS-PAGE profiles were analyzed. Differences were observed in fibrinolytic activity: kallikrein-like amidolytic activity was highest in venoms of Santa Teresa, and Margarita. Lagunetica and Carrizales venoms showed the maximum fibrin lysis. The highest hemorrhagic activity was seen in Lagunetica venom. Margarita had the lowest LD(50) of 0.18. Lagunetica, Carrizales and Anzoátegui induced a rapid degradation of fibrinogen alpha chains and slower degradation on beta chains, which could possibly due to a higher content of alpha fibrinogenases in these venoms. This fibrinogenolytic activity is decreased by metalloprotease inhibitors. All venoms, except Carrizales, presented thrombin-like activity. Anzoátegui, Carrizales and Lagunetica, in which fibrinolytic activity was present, showed the largest concentration of high molecular mass components. These results represent a new finding, not previously described, of fibrinolytic activity in South American C. durissus venoms. Santa Teresa and Margarita had fibrinolytic activity, and lack of hemorrhagic activity, representing an important finding in Venezuelan venoms since the description of a fibrinolytic molecule without hemorrhagic activity can have valuable potential in thrombolytic therapy.

[Fibronectin. Structure and functions associated to hemostasis. Review]. / Fibronectina. Estructura y funciones asociadas a la hemostasia. Revisión.

Fibronectin is an adhesive glycoprotein present in a soluble form in plasma and in an insoluble form in the extracellular matrix of many tissues. The human plasma level of this protein is about 300 +/- 100 microg/mL. It is synthesized and secreted by a wide variety of cells, consequently is one of the components of greater distribution in the body that participates in the biochemical reactions of diverse physiological and pathological processes. Due to the presence of multifunctional domains in its structure, fibronectin interacts with diverse components of the coagulation and fibrinolysis. It may bind to collagen, fibrinogen, fibrin, heparin, factor XIII and platelets, among others, regulating processes of importance in hemostasis such as: platelet adhesion and aggregation, tissue remodelling during wound healing, and activation of fibrinolysis by the plasminogen activators.

Hemostatic disorders induced by skin contact with Lonomia obliqua (Lepidoptera, Saturniidae) caterpillars.

Patients envenomed by Lonomia sp caterpillars initially experience a mild burning pain, headache, nausea, vomiting, and skin and mucosal hemorrhages. Some patients can rapidly progress to a severe coagulopathy that presents as visceral or intracerebral hemorrhaging. We studied the hemostatic alterations that occurred in 14 patients who were envenomed by Lonomia obliqua in Southern Brazil and presented at the Hospital São Vicente de Paulo (Passo Fundo, RS), Brazil during the summers of 1993 and 1994 when Lonomia antivenom was not yet available for treatment. The patients were classified into to 4 clinical groups: 0 (two patients), I (eight patients), II (two patients), and III (two patients). The patients were admitted to the hospital between 4 hours and five days after contact with the caterpillars. In this study, the coagulation parameters of the patients were followed up for up to 172 hours after the accidents. The patients received no treatment with the exceptions of two patients who received blood transfusions and antifibrinolytic treatment. The observed abnormalities related to blood coagulation and fibrinolytic factors were similar regardless of the severity of the bleeding symptoms. These findings suggest that alterations in hemostatic parameters without thrombocytopenia are not predictors of the seriousness of such accidents. Thus, consumptive disorder and reactive fibrinolysis are not proportional to mild coagulopathy. Furthermore, these patients recovered. The hemostatic parameters of most of the patients normalized between 96 and 120 h after the accident.