Baltetin: a new C-type lectin-like isolated from Bothrops alternatus snake venom which act as a platelet aggregation inhibiting.

C-type lectin-like proteins found in snake venom, known as snaclecs, have important effects on hemostasis through targeting membrane receptors, coagulation factors and other hemostatic proteins. Here, we present the isolation and functional characterization of a snaclec isolated from Bothrops alternatus venom, designated as Baltetin. We purified the protein in three chromatographic steps (anion-exchange, affinity and reversed-phase chromatography). Baltetin is a dimeric snaclec that is approximately 15 and 25 kDa under reducing and non-reducing conditions, respectively, as estimated by SDS-PAGE. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and Edman degradation sequencing revealed that Baltetin is a heterodimer. The first 40 amino acid residues of the N-terminal region of Baltetin subunits share a high degree of sequence identity with other snaclecs. Baltetin had a specific, dose-dependent inhibitory effect on epinephrine-induced platelet aggregation in human platelet-rich plasma, inhibiting up to 69% of platelet aggregation. Analysis of the infrared spectra suggested that the interaction between Baltetin and platelets can be attributed to the formation of hydrogen bonds between the PO32- groups in the protein and PO2- groups in the platelet membrane. This interaction may lead to membrane lipid peroxidation, which prevents epinephrine from binding to its receptor. The present work suggests that Baltetin, a new C-type lectin-like protein isolated from B. alternatus venom, is the first snaclec to inhibit epinephrine-induced platelet aggregation. This could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

Systemic alterations induced by phospholipase A2 , BmooTX-I, isolated from Bothrops moojeni snake venom.

Ophidic accidents are among the problems of public health in Brazil. The components from bothropic venom are responsible for many systemic clinical complications resulting from envenomation. The present work aimed to analyse the systemic changes induced in mice after intraperitoneal administration of BmooTX-I, a myotoxic acidic phospholipase A2 isolated from Bothrops moojeni venom. Urinalysis was performed and the following plasma biochemical markers were documented: urea, creatinine and uric acid (renal function); glucose and amylase (pancreatic function); alanine aminotransferase, alkaline phosphatase and gamma-GT (intra- and extrahepatic function); creatine kinase and enzymatic lactate (muscle function). Our results showed that after the intraperitoneal injection of BmooTX-I the urine of these animals showed glycosuria, proteinuria, haematuria, bacteriuria, bilirubinuria, polyuria and nitrite. The plasma biochemical analysis showed alterations in levels of urea, creatinine and uric acid. Amylase concentration was not altered significantly, but the plasma glucose increased significantly compared to controls. The plasma levels of alanine aminotransferase and alkaline phosphatase decreased and increased, respectively, in these same animals. On the other hand, the plasma γGT concentration did not undergo significant modification compared to the control group. The plasma concentration of CK increased, while the enzymatic lactate concentration decreased after the injection of the BmooTX-I. Therefore, in mice BmooTX-I is capable of causing systemic alterations which manifest as renal, muscular, hepatic and pancreatic impairment.

A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom.

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Abstract Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 groups, present in BaltDC, form hydrogen bonds with the PO 2 groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)

Comparative analysis of local effects caused by Bothrops alternatus and Bothrops moojeni snake venoms: enzymatic contributions and inflammatory modulations.

Bothropic envenomation is characterised by severe local damage caused by the toxic action of venom components and aggravated by induced inflammation. In this comparative study, the local inflammatory effects caused by the venoms of Bothrops alternatus and Bothrops moojeni, two snakes of epidemiological importance in Brazil, were investigated. The toxic action of venom components induced by bothropic venom was also characterised. Herein, the oedema, hyperalgesia and myotoxicity induced by bothropic venom were monitored for various lengths of time after venom injection in experimental animals. The intensity of the local effects caused by B. moojeni venom is considerably more potent than B. alternatus venom. Our results also indicate that metalloproteases and phospholipases A2 have a central role in the local damage induced by bothropic venoms, but serine proteases also contribute to the effects of these venoms. Furthermore, we observed that specific anti-inflammatory drugs were able to considerably reduce the oedema, the pain and the muscle damage caused by both venoms. The inflammatory reaction induced by B. moojeni venom is mediated by eicosanoid action, histamine and nitric oxide, with significant participation of bradykinin on the hyperalgesic and myotoxic effects of this venom. These mediators also participate to inflammation caused by B. alternatus venom. However, the inefficient anti-inflammatory effects of some local modulation suggest that histamine, leukotrienes and nitric oxide have little role in the oedema or myotoxicity caused by B. alternatus venom.