Aura S. Kamiguti

Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases.

Local and systemic haemorrhage are common consequences of crotaline and viperine envenoming. Several studies carried out using purified toxins have indicated that local haemorrhage can be attributed to a distinct class of venom metalloproteinases. Analyses of their cDNAs predict multi-domain enzymes, with an N-terminal metalloproteinase domain, a disintegrin-like domain and a Cys-rich C-terminus. Haemorrhagic metalloproteinases are responsible for degrading proteins of the extracellular matrix and they also have cytotoxic effects on endothelial cells. However, to date very few investigations have been carried out on the effects of venom haemorrhagic metalloproteinases on components of the haemostatic system. We describe here the effects of a high molecular weight haemorrhagic metalloproteinase, jararhagin, from the venom of a South American pit viper Bothrops jararaca, on platelet and plasma components involved in haemostasis. Jararhagin, which is not inhibited in plasma, causes the loss of the platelet collagen receptor alpha 2 beta 1 integrin (gpIa/IIa or VLA-2) and degrades the adhesive plasma protein von Willebrand factor. Alterations of these haemostatic components are known to result in bleeding. This suggests that venom haemorrhagic metalloproteinases, in addition to causing local bleeding, may also contribute to systemic haemorrhage.

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue‐damaging activities

BaP1 is a 22.7‐kD P‐I‐type zinc‐dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue‐damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117–Cys 197, Cys 159–Cys 181, Cys 157–Cys 164). It has the consensus sequence H142E143XXH146XXGXXH152, as well as the sequence C164I165M166, which characterize the “metzincin” superfamily of metalloproteinases. The active‐site cleft separates a major subdomain (residues 1–152), comprising four α‐helices and a five‐stranded β‐sheet, from the minor subdomain, which is formed by a single α‐helix and several loops. The catalytic zinc ion is coordinated by the Nε2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active‐site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P‐I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active‐site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia.

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.

Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites in the north eastern Amazon region of Brazil

The efficacies of specific Bothrops atrox-Lachesis and standard Bothrops-Lachesis antivenoms were compared in the north eastern Amazon region of Brazil. The main aim was to investigate whether a specific antivenom raised against the venom of B. atrox, the most important Amazon snake species from a medical point of view, was necessary for the treatment of patients in this region. Seventy-four patients with local and systemic effects of envenoming by Bothrops or Lachesis snakes were randomly allocated to receive either specific (n = 38) or standard (n = 36) antivenoms. In 46 cases (24 in the standard antivenom group, 22 in the other) the snake was identified either by enzyme immunoassay or by examination of the dead snake, as B. atrox in 45, L. muta in one. Patients were similar in all clinical and epidemiological respects before treatment. Results indicated that both antivenoms were equally effective in reversing all signs of envenoming detected both clinically and in the laboratory. Venom-induced haemostatic abnormalities were resolved within 24 h after the start of antivenom therapy in most patients. The extent of local complications, such as local skin necrosis and secondary infection, was similar in both groups. There were no deaths. The incidence of early anaphylactic reactions was 18% and 19%, respectively for specific and standard antivenoms; none was life-threatening. Measurement of serum venom concentrations by enzyme immunoassay (EIA) confirmed that both antivenoms cleared venom antigenaemia effectively. EIA also revealed that one patient had been bitten by Lachesis muta, although the clinical features in this case were not distinctive.

Snake venom metalloproteinases and disintegrins: interactions with cells

Metalloproteinases and disintegrins are important components of most viperid and crotalid venoms. Large metalloproteinases referred to as MDC enzymes are composed of an N-terminal Metalloproteinase domain, a Disintegrin-like domain and a Cys-rich C-terminus. In contrast, disintegrins are small non-enzymatic RGD-containing cysteine-rich polypeptides. However, the disintegrin region of MDC enzymes bears a high degree of structural homology to that of the disintegrins, although it lacks the RGD motif. Despite these differences, both components share the property of being able to recognize integrin cell surface receptors and thereby to inhibit integrin-dependent cell reactions. Recently, several membrane-bound MDC enzymes, closely related to soluble venom MDC enzymes, have been described in mammalian cells. This group of membrane-anchored mammalian enzymes is also called the ADAM family of proteins due to the structure revealing A Disintegrin And Metalloproteinase domains. ADAMs are involved in the shedding of molecules from the cell surface, a property which is also shared by some venom MDC enzymes.

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function

Atrolysin A and jararhagin are class P‐III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin‐like and a cysteine‐rich. The metalloproteinase and the disintegrin‐like domains of atrolysin A and jararhagin contain peptide sequences that interact with α2β1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine‐rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine‐rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of α2‐expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine‐rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.

The role of matrix metalloproteinase 9 in the pathogenesis of chronic lymphocytic leukaemia

Matrix metalloproteinases (MMPs) are important for the pathogenesis and progression of different tumours. MMPs‐2 and ‐9 are the principal MMPs produced by lymphocytes; these enzymes can degrade a number of matrix proteins but are the two main MMPs that digest type IV collagen, the major component of basement membranes. Therefore, these enzymes are potentially important for tissue invasion and remodelling by malignant lymphocytes. This study showed that chronic lymphocytic leukaemia (CLL) cells produce and secrete variable amounts of pro‐MMP‐9, but no MMP‐2 or tissue inhibitor of metalloproteinase 1 (TIMP‐1). The pro‐enzyme was found in monomeric and dimeric forms and also complexed with lipocalin. Moreover, a small fraction of secreted monomer became associated with the cell surface and activated upon cell adhesion to insolubilized type IV collagen. High levels of intracellular MMP‐9 were associated with advanced (stage C) disease and with poor patient survival. Immunohistochemical studies demonstrated that MMP‐9 was associated with areas of tissue invasion and remodelling. The relatively specific MMP‐9 inhibitors, Ro31‐9790 (3 μmol/l) and TIMP‐1, reduced CLL‐cell migration through type IV collagen and through endothelial monolayers suggesting that the enzyme may also be important in malignant cell entry and egress to and from involved tissue. Our data raise the possibility that MMP‐9 modulation may have therapeutic potential in advanced CLL.

Collagen-induced secretion-dependent phase of platelet aggregation is inhibited by the snake venom metalloproteinase jararhagin

Jararhagin, a 52 kDa metalloproteinase from Bothrops jararaca snake venom, belongs to the family of enzymes with an N-terminal Zn2+-containing enzymatic domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. Both jararhagin and jararhagin C, a 28 kDa-protein from the same venom identical to the disintegrin-like domain of jararhagin, inhibit collagen-induced platelet aggregation. In this study, jararhagin and synthetic linear peptides based on the disintegrin-like domain of jararhagin overlapping with the RGD sequence of venom disintegrins, were shown for the first time to inhibit the release of 5-hydroxytryptamine (5-HT) from platelets preloaded with [14C]5-HT and stimulated with collagen. The normal phosphorylation of the 21-kDa myosin light chain (p21) in response to the stimulation indicated that jararhagin and the peptides did not interfere with platelet shape change. The selective inhibition of the secretion-dependent phase of the platelet response to collagen by the enzyme and its peptides was confirmed by the defective phosphorylation of pleckstrin, a 47-kDa platelet protein (p47) involved in dense granule secretion.

Mass spectrophotometric evidence for P-III/P-IV metalloproteinases in the venom of the Boomslang (Dispholidus typus)

The Boomslang, Dispholidus typus, is a mid- to rear-fanged arboreal colubrid widely distributed throughout much of the African continent. Envenoming by this species is rare although deaths have been recorded. Typical symptoms associated with envenoming include diffuse intravascular coagulation (DIC) caused by fibrinogen consumption and consequent incoagulable blood together with haemorrhage into tissues such as muscle and brain; together, these procoagulant and haemorrhagic effects of the venom result in a very poor prognosis in patients who receive a large dose of venom and who are not treated with antivenom. Renal failure may also result from acute tubular necrosis resulting from pigment nephropathy. Little is known about the toxic components present in the venom; however, proteolytic activity has been reported although the proteinases involved have not been identified. In this study we provide LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) data supporting the presence of class P-III/P-IV snake venom metalloproteinases (SVMPs) in Boomslang venom. Using a polyclonal antibody raised against the P-III haemorrhagic toxin (Jararhagin) obtained from the venom of the Brazilian pit viper, Bothrops jararaca, we identified by western blot a 65 kDa protein from Boomslang venom which cross-reacted with the jararhagin antibody. A corresponding band from SDS-PAGE was subjected to tryptic digestion followed by LC/MS/MS sequence analysis of the digestion mixture. A variety of peptide sequences were identified in the digest, one of which was clearly homologous with a highly conserved region of the disintegrin-like domains of P-III/P-IV SVMPs. These data provide the first structural evidence for the presence of SVMPs in Boomslang venom; it is possible that SVMPs may also be present in the venoms of other colubrids, which cause similar symptoms in envenomed humans. In other snake venoms, most notably those of the Viperinae and Crotalinae subfamilies, many of the coagulopathic and haemorrhagic syndromes associated with systemic and local envenoming are attributed to SVMPs. The identification of a P-III/P-IV SVMP sequence in D. typus venom suggests that many of the pathological signs resulting from envenoming by this species may also be due to the presence of SVMPs in the venom. It is hoped that these results may accelerate research into colubrid venoms and may provide new insights into novel and more efficacious treatments for colubrid envenoming.

B-Cell Receptor Translocation to Lipid Rafts and Associated Signaling Differ between Prognostically Important Subgroups of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which interaction of the malignant cells with antigen is thought to play a key role. Individual CLL-cell clones markedly differ in their ability to respond to B-cell receptor ligation, but the mechanism underlying the frequent hyporesponsiveness is incompletely understood. Our aim was to further clarify the extent and cause of the B-cell receptor signaling abnormality in CLL and to assign pathophysiologic relevance to the presence or absence of B-cell receptor responsiveness. We show that extracellular signal-regulated kinase-2 phosphorylation, intracellular Ca2+ increases, CD79a phosphorylation, and translocation of the B-cell receptor to lipid rafts in response to ligation with anti-immunoglobulin M (as a surrogate for antigen) are features of CLL cells with relatively unmutated VH genes (<5% deviation from germ line) and a poor prognosis. B-cell receptor stimulation in these cases also promoted cell survival. In clones with mutated VH genes (>5% deviation from germ line), surface immunoglobulin M ligation failed to induce receptor translocation to rafts or to prolong cell survival. This failure of receptor translocation observed in mutated CLL cells was associated with the constitutive exclusion of the B-cell receptor from rafts by a mechanism involving src-dependent interactions between the B-cell receptor and the actin cytoskeleton. We conclude that exposure to antigen promotes the survival of unmutated CLL clones, contributing to the poor prognosis of this group. In contrast, hyporesponsive mutated CLL clones may have developed into a stage where continuous exposure to antigen results in relative tolerance to antigenic stimulation mediated by the exclusion of the B-cell receptor from lipid rafts.