Ana M. Salazar

Cloning, expression, and hemostatic activities of a disintegrin, r-mojastin 1, from the mohave rattlesnake (Crotalus scutulatus scutulatus).

Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216 bp disintegrin cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet adhesion to fibronectin with an IC50 of 58.3 nM and ADP-induced platelet aggregation in whole blood with an IC50 of 46 nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting with an IC50 of 95.6 nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 7.95676 kDa.

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.

Comparative hemostatic parameters in BALB/c, C57BL/6 and C3H/He mice.

This study describes micro-methods to determine biological parameters in plasma of three strains of mice. Platelet count was significantly different among strains. C57BL/6 mice showed the highest values (988 x 10(3)/microL) and BALB/c the lowest (782 x 10(3)/microL). Fibrinogen levels were 2.55 (C57BL/6), 2.37 (BALB/c) and 2.28 g/L (C3H/He). Some inter-strain differences were observed in factor XIII (94, 118 and 114%) and plasminogen levels (142, 80 and 135%) in C57BL/6, BALB/c and C3H/He, respectively. Additionally, we observed individual mice factor XIII and plasminogen levels between 80 to 200% and 65 to 180%, respectively, in relation to pooled human plasma; and between 70 to 185% and 70 to 155%, respectively, against pooled mice plasma. To our knowledge, this is first report in the literature in diverse mice strains regarding hemostasis, mainly on factor XIII, plasminogen levels, and a very simple test that allows measurement of endogenous fibrinolytic activity present in the plasma. The different results are discussed in relationship with existing literature regarding if the animals in some studies were maintained under strict pathogen-free conditions, the collection of blood was from the heart or eye and if the analysis method was tested by counting manually or automatically. This work could contribute useful knowledge to the field of investigations regarding hemostatic disorders using mouse models, especially for laboratories that are not well equipped.

Venom variation in hemostasis of the southern Pacific rattlesnake (Crotalus oreganus helleri): Isolation of hellerase

Envenomations by the southern Pacific rattlesnake (Crotalus oreganus helleri) are the most common snakebite accidents in southern California. Intraspecies venom variation may lead to unresponsiveness to antivenom therapy. Even in a known species, venom toxins are recognized as diverse in conformity with interpopulational, seasonal, ontogenetic and individual factors. Five venoms of individual C. oreganus helleri located in Riverside and San Bernardino counties of southern California were studied for their variation in their hemostatic activity. The results demonstrated that Riverside 2 and San Bernardino 1 venoms presented the highest lethal activity without hemorrhagic activity. In contrast, San Bernardino 2 and 3 venoms had the highest hemorrhagic and fibrinolytic activities with low lethal and coagulant activities. Riverside 1, Riverside 2 and San Bernardino 1 venoms presented a significant thrombin-like activity. San Bernardino 2 and 3 venoms presented an insignificant thrombin-like activity. In relation to the fibrinolytic activity, San Bernardino 3 venom was the most active on fibrin plates, which was in turn neutralized by metal chelating inhibitors. These results demonstrate the differences amongst C. oreganus helleri venoms from close localities. A metalloproteinase, hellerase, was purified by anionic and cationic exchange chromatographies from San Bernardino 3 venom. Hellerase exhibited the ability to break fibrin clots in vitro, which can be of biomedically importance in the treatment of heart attacks and strokes.

Hemostatic and toxinological diversities in venom of Micrurus tener tener, Micrurus fulvius fulvius and Micrurus isozonus coral snakes.

The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A₂s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD₅₀s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD₅₀ of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD₅₀s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bβ chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders.

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of A alpha and B beta chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of A alpha and B beta chains at 5 min and of gamma chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm(2)/microg, respectively using 25 microg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD(50) of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 microg protein, showed that B. isabelae venom contained higher specific activity (50 mm(2)/microg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.

Activities against hemostatic proteins and adrenal gland ultrastructural changes caused by the brown widow spider Latrodectus geometricus (Araneae: Theridiidae) venom

Brown widow spider (BrWS) (Latrodectus geometricus) venom produces intense systemic reactions such as cramps, harsh muscle nociceptive, nauseas, vomiting and hypertension. The proposed pathogenic mechanisms resulting in these accidents have principally been damages occurring at the nervous system. However, it is suspected that there is also damage of the adrenal glands, as a result of the experimental animals clinical manifestations, which developed symptoms compatible with acute adrenal insufficiency. We have currently found that the adrenal gland is damaged by this venom gland homogenates (VGH) producing severe alterations on cortex cells resulting in death by acute adrenal insufficiency. In general, the ultrastructural study on the glands of mice under transmission electronic microscopy observations showed alterations in the majority of the intracellular membranes within 3 to 24h. BrWSVGH also showed specific actions on extracellular matrix proteins such as fibronectin, laminin and fibrinogen. In addition, zymogram experiments using gelatin as substrates detected gelatinolytic activity. The molecular exclusion fractionation of crude BrWSVGH resulted in 15 fractions, of which F1 and F2 presented alpha/beta-fibrinogenase and fibronectinolytic activities. Fractions F6, F14 and F15 showed only alpha-fibrinogenase activity; in contrast, the gelatinolytic action was only observed in fraction F11. Only metalloproteinase inhibitors abolished all these proteolytic activities. Our results suggest that adrenal cortex lesions may be relevant in the etiopathogenesis of severe brown widow spider envenoming. To our knowledge, this is the first report on adrenal gland damages, fibrinogenolytic activity and interrelations with cell-matrix adhesion proteins caused by L.geometricus VGH. The venom of this spider could be inducing hemostatic system damages on envenomed patients.

Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph

Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are ≥ 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.

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.

The effects of Lonomin V, a toxin from the caterpillar (Lonomia achelous), on hemostasis parameters as measured by platelet function

Platelets play a central role in hemostasis during vascular injury. Patients affected with the hemorrhagic syndrome caused by contact with Lonomia achelous caterpillars (Lac) Lepidoptera distributed in various South American countries, show digestive, pulmonary and intraperitoneal bleeding in combination with hematomas and echymosis. In the present study, we have evaluated the effects of Lonomin V (serine protease isolated from Lac hemolymph) on some functional properties of platelets, evaluating its importance in primary hemostasis. Platelet adhesion to fibrinogen was reduced by 19, 20, 36, and 37% after pre-treated with 0.2, 2, 20 and 40 nM of Lonomin V, respectively. Pre-incubation of the platelets with 408 nM of Lonomin V, for 4 min at 37 °C, resulted in complete inhibition of the collagen-induced platelet aggregation, in contrast to 56% inhibition of the ADP - induced platelet aggregation. Lonomin V also inhibited anti-α(IIb)β(3) integrin binding to platelets by 56, 57, 52 and 54% at concentrations of 0.2, 2, 20 and 40 nM respectively. Additionally, Lonomin V inhibited anti-P-selectin binding to platelets by 28, 37, 33 and 33% at the same concentrations. The platelets tested with Lonomin V did not modify their viability. In summary, Lonomin V inhibited platelet aggregation, probably caused by the degradation of collagen. The anti-platelet activity of Lonomin V has been shown to be unique and a potentially useful tool for investigating cell-matrix and cell-cell interactions and for the development of antithrombotic agents in terms of their anti-adhesive activities.