Museum DNA reveals a new, potentially extinct species of rinkhals (Serpentes: Elapidae: Hemachatus) from the Eastern Highlands of Zimbabwe.

Genetic information plays a pivotal role in species recognition and delimitation, but rare or extinct animals can be difficult to obtain genetic samples from. While natural history wet collections have proven invaluable in the description of novel species, the use of these historical samples in genetic studies has been greatly impeded by DNA degradation, especially because of formalin-fixation prior to preservation. Here, we use recently developed museum genomics approaches to determine the status of an isolated population of the elapid snake genus Hemachatus from Zimbabwe. We used multiple digestion phases followed by single strand sequencing library construction and hybridisation capture to obtain 12S and 16S rDNA sequences from a poorly preserved tissue sample of this population. Phylogenetic and morphological analyses in an integrated taxonomic framework demonstrate that the Zimbabwean rinkhals population represents an old and highly distinct lineage, which we describe as a new species, Hemachatus nyangensis sp. nov. Our phylogenetic dating analysis is compatible with venom spitting having evolved in response to the threat posed by early hominins, although more data are required for a robust test of this hypothesis. This description demonstrates the power of museum genomics in revealing rare or even extinct species: Hemachatus from Zimbabwe are only known from a small area of the Eastern Highlands known for high endemism. No living specimens have been seen since the 1980s, most likely due to dramatic land-use changes in the Eastern Highlands, suggesting that the species could be extinct. In view of its recognition as a highly distinct lineage, urgent action is required to determine whether any populations survive, and to safeguard remaining habitat.

Comparative venomics and preclinical efficacy evaluation of a monospecific Hemachatus antivenom towards sub-Saharan Africa cobra venoms.

Cobras are the most medically important elapid snakes in Africa. The African genera Naja and Hemachatus include snakes with neurotoxic and cytotoxic venoms, with shared biochemical, toxinological and antigenic characteristics. We have studied the antigenic cross-reactivity of four sub-Saharan Africa cobra venoms against an experimental monospecific Hemachatus haemachatus antivenom through comparative proteomics, preclinical assessment of neutralization, and third generation antivenomics. The venoms of H. haemachatus, N. annulifera, N. mossambica and N. nigricollis share an overall qualitative family toxin composition but depart in their proportions of three-finger toxin (3FTxs) classes, phospholipases A2 (PLA2s), snake venom metalloproteinases (SVMPs), and cysteine-rich secretory proteins (CRISPs). A monospecific anti-Hemachatus antivenom produced by Costa Rican Instituto Clodomiro Picado neutralized the lethal activity of the homologous and heterologous neuro/cytotoxic (H. haemachatus) and cyto/cardiotoxic (N. mossambica and N. nigricollis) venoms of the three spitting cobras sampled, while it was ineffective against the lethal and toxic activities of the neurotoxic venom of the non-spitting snouted cobra N. annulifera. The ability of the anti-Hemachatus-ICP antivenom to neutralize toxic (dermonecrotic and anticoagulant) and enzymatic (PLA2) activities of spitting cobra venoms suggested a closer kinship of H. haemachatus and Naja subgenus Afrocobra spitting cobras than to Naja subgenus Uraeus neurotoxic taxa. These results were confirmed by third generation antivenomics. BIOLOGICAL SIGNIFICANCE: African Naja species represent the most widespread medically important elapid snakes across Africa. To gain deeper insight into the spectrum of medically relevant toxins, we compared the proteome of three spitting cobras (Hemachatus haemachatus, Naja mossambica and N. nigricollis) and one non-spitting cobra (N. annulifera). Three finger toxins and phospholipases A2 are the two major protein families among the venoms analyzed. The development of antivenoms of broad species coverage is an urgent need in sub-Saharan Africa. An equine antivenom raised against H. haemachatus venom showed cross-reactivity with the venoms of H. haemachatus, N. mossambica and N. nigricollis, while having poor recognition of the venom of N. annulifera. This immunological information provides clues for the design of optimum venom mixtures for the preparation of broad spectrum antivenoms.

Proteomic and toxinological characterization of the venom of the South African Ringhals cobra Hemachatus haemachatus.

The protein composition and toxinological profile of the venom of the African spitting elapid Hemachatus haemachatus (Ringhals) were characterized by bottom-up proteomics and functional in vitro and in vivo assays. Venom is composed of abundant three-finger toxins (3FTxs; 63.3%), followed by phospholipases A2 (PLA2s; 22.8%), snake venom metalloproteinases (SVMPs; 7.1%), cysteine-rich secretory proteins (CRISPs; 4.1%) and Kunitz type protease inhibitors (KTPIs; 1.5%). 3FTxs are the main responsible for lethality and myotoxicity in mice and in vitro anticoagulant activity. In contrast to closely related spitting species, whose venom 3FTxs induces dermonecrosis, the 3FTxs of H. haemachatus did not induce dermonecrotic activity. The venom showed in vitro PLA2 activity, and most likely PLA2s contribute to some extent in venom lethality, as judged by partial reduction in toxicity after inhibition of their catalytic activity. Despite its relatively high content of SVMPs, compared to most elapids, the venom of H. haemachatus did not exert hemorrhagic effect, proteolytic activity on azocasein or defibrinogenating activity. Toxicovenomic characterization of H. haemachatus venom revealed that RP-HPLC fractions with higher abundance of 3FTxs presented lethal activity, while fractions with high content of PLA2s did not, underscoring the role of 3FTxs in the pathophysiology caused by this venom. BIOLOGICAL SIGNIFICANCE: The proteomic composition and toxinological profile of the venom of Ringhals snake, Hemachatus haemachatus, a cobra-like spitting snake endemic to southern Africa, were investigated. In vitro, Ringhals venom showed anticoagulant and phospholipase A2 activities, but was devoid of proteolytic activity on azocasein. In mice, venom induced lethality and myotoxicity, but no local hemorrhage or dermonecrosis. The lack of dermonecrotic activity is in sharp contrast to venoms of closely related spitting cobras which present a similar relative abundance of 3FTxs but are potently dermonecrotic. 3FTxs, the most abundant protein family in the venom, are predominantly responsible for toxic effects. PLA2 enzyme inactivation experiments suggest that H. haemachatus venom lethality is not dependent on PLA2s, but instead is more related to neurotoxic or cardiotoxic 3FTxs. The characterization of this venom, based on proteomic and toxicovenomic approaches, is useful for more in depth studies associated with biogeography, phylogeny, toxinology and antivenom efficacy towards the venom of this species, and its association with related elapids.

Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus.

Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki' and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 µM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex.

Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex.

Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa. Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki' of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively. Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.

Caracterização da pancreatite aguda induzida por fosfolipases A2 secretorias / Characterization of the acute pancreatitis induced by secretory A2

A pancreatite aguda é uma doença inflamatória do pâncreas caracterizada por intensa necrose pancreática e efeitos sistêmicos secundários como lesão pulmonar, os quais são a principal causa da mortalidade observada nessa doença. Há evidências de que as fosfolipases A2 (PLA2) têm um importante papel na fisiopatologia da pancreatite aguda. O objetivo deste trabalho foi investigar a capacidade de PLA2s de induzir pancreatite em ratos e os mecanismos envolvidos nesse fenômeno. As seguintes PLA2s foram utilizadas: piratoxina-I (homólogo Lys-49 de PLA2 desprovido de atividade catalítica), bothropstoxina-II (homólogo Asp-49 de PLA2 com baixa atividade catalítica) e a PLA2 proveniente do veneno de Naja mocambique mocambique (que possui alta atividade catalítica). Ratos Wistar machos (200-250 g) provenientes do CEMIB/UNICAMP foram utilizados. As PLA2s foram injetadas no ducto biliopancreático de animais anestesiados e após diferentes tempos experimentais foram avaliados o extravasamento de proteínas plasmáticas no pâncreas, infiltrado de neutrófilos no pâncreas e pulmão e amilase sérica. A análise histológica do pâncreas e pulmão também foi realizada em alguns grupos experimentais. Piratoxina-I foi capaz de causar extravasamento de proteínas plasmáticas no pâncreas, infiltrado de neutrófilos no pulmão e os níveis séricos de amilase. Além disso, a piratoxina-I causou alterações histológicas nos tecidos pancreático (infiltrado de neutrófilos, necrose de células acinares e edema intersticial) e pulmonar (edema intersticial e diminuição do espaço alveolar), que foram mais evidentes nos tempos iniciais da pancreatite (4-12h). Bothropstoxina-II e a PLA2 do veneno de Naja mocambique moçambique, à semelhança da Piratoxina-I, também foram capazes de aumentar o extravasamento de proteínas plasmáticas e o influxo de neutrófilos no tecido pancreático por mecanismos não relacionados à sua atividade catalítica. Entretanto, o influxo de neutrófilos para o pulmão e o aumento dos níveis séricos de amilase causados por essas PLA2s foi dependente de sua atividade catalítica. As PLA2s também causaram secreção de amilase de ácinos pancreáticos isolados, que foi dependente da atividade catalítica dessas xxii enzimas. Adicionalmente, com o objetivo de entender o mecanismo envolvido na pancreatite induzida pela PLA2 de Naja mocambique mocambique animais foram tratados com os seguintes agentes farmacológicos: pentoxifilina (inibidor da síntese de TNF-α), SR140333 (antagonista de receptor NK1), icatibant (antagonista de receptor B2), L-NAME (inibidor não seletivo das NOS), aminoguanidina (inibidor preferencial da NOS induzível), indometacina (inibidor não seletivo de COX), celecoxib (inibidor seletivo de COX-2), PCA4248 (antagonista dos receptores de PAF) e AA861 (inibidor da 5-lipoxigenase). Em conjunto, nossos dados mostraram que os efeitos locais e secundários são multimediados, envolvendo a participação de bradicinina, substância P, NO, TNF-α, PAF e metabólitos das COXs. Em conclusão, demonstramos que as PLA2s secretórias são capazes de induzir pancreatite aguda em ratos quando injetadas no ducto biliopancreático, um quadro caracterizado por efeitos inflamatórios locais e secundários cuja mediação farmacológica envolve vários fatores. Além disso, a pancreatite aguda induzida pelas PLA2s reproduz algumas alterações observadas na pancreatite aguda em humanos, representando uma nova estratégia de estudo da fisiopatologia da pancreatite aguda.

Acute pancreatitis is an inflammatory disease of the pancreas that is characterized by intense pancreatic necrosis and remote systemic effects such as the lung injury that is the main cause of death during acute pancreatitis. There are evidences that phospholipases A2 (PLA2) have an important role in the acute pancreatitis pathophysiology. The aim of this work was to investigate the ability of PLA2 to induce acute pancreatitis in rats, and the mechanisms underlying this phenomenon. The following PLA2s were used: piratoxin-I (a Lys-49 PLA2 homologue devoid of catalytic activity), bothropstoxin-II (an Asp-49 PLA2 homologue with low catalytic activity) and PLA2 from Naja mocambique mocambique venom (high catalytic activity). Male Wistar rats (200-250g) provided by CEMIBUNICAMP have been used. The PLA2s were injected into the common bile duct of anesthetized rats, after which pancreatic plasma protein extravasation, pancreatic and lung neutrophil infiltration and serum levels of amylase were measured. Histology of the pancreatic and lung tissue has also been carried out in some experimental group. Piratoxin-I was able to increase the pancreatic plasma protein extravasation, lung neutrophil infiltration and serum amylase levels. In addition, Piratoxin-I caused histological changes in the pancreatic (neutrophil infiltration, areas of acinar cell necrosis and interstitial edema) and lung (interstitial edema and diminuition of alveolar space) tissues, which were more evident in the early periods (4-12h) after the injection. Bothropstoxin-II and PLA2 from Naja mocambique mocambique venom were also able to increase the plasma protein extravasation and neutrophil influx in the pancreatic tissue by mechanisms unrelated to their catalytic activity. However, the remote lung neutrophil influx caused by these PLA2s was shown to depend on their catalytic activity. The enhancement of serum amylase levels was also dependent on the catalytic activity of these enzymes. The PLA2s also caused amylase secretion from isolated pancreatic acini, which was dependent on their catalytic activity. Next, in order to further understand the mechanisms involved in pancreatitis induced by PLA2 Naja mocambique mocambique, animals were treated with the following xxiv pharmacological agents: pentoxifylline (TNF-α synthesis inhibitior), SR140333 (NK1 receptor antagonist), icatibant (B2 receptor antagonist), L-NAME (non-selective NOS inhibitor), aminoguanidine (preferential inducible NOS inhibitor), indomethacin (nonselective COX inhibitor), celecoxib (selective COX-2 inhibitor), PCA4248 (PAF receptor antagonist) and AA861 (5-lipoxygenase inhibitor). Taken together our data showed that local and remote effects are multimediated, involving the participation of bradykinin, substance P, NO, TNF-α, PAF and COXs metabolites. In conclusion, we have shown that secretory PLA2s are able to induce acute pancreatitis in rats when injected into the common bile duct. Therefore, PLA2-induce acute pancreatitis reproduces some aspects of the human disorder representing a new strategy to study the pathophysiology of acute pancreatitis.