Central role of protein kinase Cepsilon in constitutive activation of ERK1/2 and Rac1 in the malignant cells of hairy cell leukemia.

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.

Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara.

In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.

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.

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.

Characterization of a novel protein from Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist.

Many toxins from viperid venoms have been characterised as powerful activators of platelets. Here, the venom from the East African Lowland viper, Proatheris superciliaris, was investigated for its effect on platelets and the coagulation system. Whole P. superciliaris venom stimulated platelet shape change and aggregation; however, the stimulation of platelet activation was unaffected by the structurally distinct Src family kinase inhibitors PP1 and PD0173952, suggesting that platelet activation was mediated by a G protein-coupled receptor. A platelet reactive 34-kDa protein was isolated from P. superciliaris venom which we have designated proatherocytin. This protein induced Src kinase-independent aggregation of both human and mouse platelets that was inhibited by the serine protease inhibitor AEBSF. Proatherocytin did not clot bovine or human fibrinogen, degrade fibrinogen or hydrolyse the serine protease substrate benzoyl-FVR-pNA. It activated human PAR1 on stably transfected rat kidney epithelial cells, whereas no activation of the trypsin receptor PAR2 was shown. Surprisingly, Edman degradation of proatherocytin revealed sequence identity with existing disintegrin-like domains of snake venom metalloproteinases. These results suggest that proatherocytin is a highly selective PAR1 agonist that also causes mouse platelet aggregation, probably through cleavage of PAR4.

Platelets as targets of snake venom metalloproteinases.

For centuries snake venoms have been known to interfere with haemostasis and this is now known basically due either to toxins activating/inhibiting clotting factors, having effects on blood vessels or interfering with platelet function. In this short review, the interaction of one major group of toxins, the snake venom metalloproteinases, with platelets is considered. This is relevant for understanding the mechanism of haemorrhage induced by these toxins.

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 micromol/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.

Bothrops asper metalloproteinase BaP1 is inhibited by alpha(2)-macroglobulin and mouse serum and does not induce systemic hemorrhage or coagulopathy.

The ability of the P-I metalloproteinase BaP1, isolated from the venom of the snake Bothrops asper, to induce systemic bleeding, thrombocytopenia and defibrinogenation was assessed in an experimental mouse model. Intravenous administration of BaP1 caused neither systemic bleeding nor any evidence of pathology in lungs, kidneys, liver, heart and brain. Moreover, there were no alterations in the whole blood clotting time or in platelet numbers. In addition, BaP1 did not inhibit collagen-induced platelet aggregation in vitro. Proteolytic and hemorrhagic activities of BaP1 were readily inhibited by the plasma proteinase inhibitor, alpha(2)-macroglobulin, and normal mouse serum also inhibited hemorrhage. Such inhibition may explain why BaP1 induces multiple local tissue-damaging effects, but is largely devoid of systemic toxicity.

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.

Clinical trial of two antivenoms for the treatmentof Bothrops and Lachesis bites in the northeastern 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.

Characterization of 'basparin A,' a prothrombin-activating metalloproteinase, from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis.

A prothrombin activator, named 'basparin A,' was isolated from the venom of the crotaline snake Bothrops asper, the species responsible for the majority of snakebite cases in Central America. It is an acidic (pI 5.4), 70kDa, single chain P-III metalloproteinase comprising, in addition to the metalloproteinase domain, disintegrin-like, and high-cysteine domains. Basparin A is a glycoprotein displaying immunological cross-reactivity with BaH1, a P-III hemorrhagic metalloproteinase isolated from the same venom. It activates prothrombin through the formation of meizothrombin, without requiring additional cofactors; it is, therefore, a class A snake venom prothrombin activator. In contrast with most venom metalloproteinases, it does not degrade components of the extracellular matrix. Apart from its clotting activity, basparin A inhibits collagen-dependent platelet aggregation in vitro, an effect that does not depend on proteolytic activity. Clotting activity on human plasma is not abrogated by the plasma proteinase inhibitors alpha(2) macroglobulin and murinoglobulin, whereas activity is completely inhibited by Costa Rican polyvalent (Crotalinae) anti-venom. Basparin A does not induce local tissue alterations, such as hemorrhage, myonecrosis, and edema, in mice. Moreover, it does not induce systemic hemorrhage, thrombocytopenia nor prolongation of the bleeding time following intravenous administration. At low doses, the only observed effect induced by basparin A, when injected intravenously or intramuscularly into mice, is defibrin(ogen)ation. At higher doses, intravenous administration resulted in sudden death due to numerous occluding thrombi in pulmonary vessels. Basparin A is likely to play an important role in the coagulopathy associated with B. asper envenoming.

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 H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) 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.

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 alpha2beta1 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 alpha2-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.

Regulation of hairy-cell survival through constitutive activation of mitogen-activated protein kinase pathways.

The hairy cells (HCs) of hairy-cell leukemia are intrinsically activated mature clonal B cells. The aims of this study were to gain further insights into the nature of this activation and to assess its importance for the prolonged HC survival in this chronic disease. We show that HCs contain phosphorylated/activated p38 MAPK, JNK and ERK1/ERK2 (ERK1/2). PKC inhibitors increased the activation of p38 and JNK, but reduced the phosphorylation of ERK1/2. Moreover, PKC inhibition resulted in cell death; cell death was also observed when the activation of ERK1/2 in HCs was abrogated with an inhibitor of MEK1/2 activation. In addition to PKC, active Src kinase was also shown to be involved in the maintenance of Raf-independent ERK activation in HCs. During cell culture on a nonadherent surface, ERK phosphorylation was sustained, while phosphorylation of p38 and JNK decreased. This decrease was not observed in HCs cultured on vitronectin (VN), indicating that p38/JNK activation is probably a consequence of in vivo HC interaction with VN present in abundance in the red pulp of the spleen. Taken together, these results suggest that active p38/JNK make HCs susceptible to apoptosis, but the cells are effectively rescued by ERK activation involving constitutively active PKC and Src. These findings are relevant for the understanding of the prolonged cell survival of HCs and their selective sensitivity to some chemotherapeutic agents.

Use of microarrays for investigating the subtoxic effects of snake venoms: insights into venom-induced apoptosis in human umbilical vein endothelial cells.

The pathological effects of only a small percentage of the total number of protein components of snake venoms are well documented, yet this knowledge has led to a general understanding of the physiological consequences of snake venom poisoning. The aim of this study was to assess the effect of subpathological levels of Crotalus atrox (Western diamondback rattlesnake) and Bothrops jararaca (Jararaca) snake venoms on the gene expression profile of human umbilical vein endothelial cells (HUVEC) in culture. Analysis of the data demonstrated that HUVECs treated with C. atrox venom had 33 genes up-regulated with significant fold changes of 1.5 or greater compared to untreated control cells. Ten genes were down-regulated with 1.5 or greater fold changes. In cells treated with B. jararaca venom, 33 genes were observed to be up-regulated and 11 genes were down-regulated with a fold change of 1.5 or more. More than half of the up-regulated genes and approximately half of the down-regulated genes detected in cells treated with the venoms were found in both data sets underscoring both the similarities and differences between the two venoms. Ontological categorization of the up-regulated genes from endothelial cells treated with either C. atrox or B. jararaca venom gave the cell growth/maintenance and signal transducer groups as having the most members. The ontology of the down-regulated genes from both venom-treated cell samples was more varied but interestingly, the predominant ontology class was also cell growth/maintenance. Many of the up-regulated genes are involved in the Fas ligand/TNF-alpha receptor apoptotic pathway. In summary, these experiments demonstrate the power of gene expression profiling to explore the subtoxic effects of venoms on gene expression and highlight its potential for the discovery of novel insights into a variety of biological processes and signal transduction pathways. Furthermore, these studies illustrate the subtle functional differences between similar venoms that are not always evident from standard analyses.

The reprolysin jararhagin, a snake venom metalloproteinase, functions as a fibrillar collagen agonist involved in fibroblast cell adhesion and signaling.

The integrins alpha(2)beta(1) and alpha(1)beta(1) have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to alpha(2)beta(1) regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the alpha(2)beta(1) integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor alpha(2)beta(1) in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (a disintegrin-like and metalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins.

Cytoprotective antioxidant activity of serum albumin and autocrine catalase in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) cells are long lived in vivo but undergo spontaneous apoptosis when cultured in vitro. Intriguingly, CLL cells also appear to have a specific susceptibility to oxidative stress - a potent inducer of apoptosis. Here, we show that serum albumin can function as a cytoprotective antioxidant of potential relevance to circulating CLL cells, and that autocrine catalase - a hydrogen peroxide-inactivating enzyme that may be released extracellularly - can perform a similar role under the crowded conditions that prevail at sites of tissue involvement. Albumin lowered oxidative stress in cultured CLL cells and inhibited spontaneous and reactive oxidant-induced apoptosis. Maximal effects were observed at a concentration of 10 mg/ml - fourfold lower than that in plasma and twofold higher than that in standard culture medium containing 10% fetal calf serum. Oxidative stress and spontaneous apoptosis were also decreased by cell crowding and by conditioned medium (CM) from crowded CLL cells, indicating that these processes were subject to autocrine regulation. CLL cells were found to express catalase and release enzyme activity into the culture medium. Exogenous catalase decreased oxidative stress and spontaneous apoptosis, and the anti-apoptotic effect of CM from crowded CLL cells was abrogated by the specific catalase inhibitor, 3'-amino-1,2,4-triazole. Together, these data strongly implicate autocrine catalase as a cytoprotective antioxidant. Oxidative stress in CLL cells was greatly diminished by ruthenium red - an inhibitor of mitochondrial reactive oxidant production - and by the glutathione (GSH) precursor N-acetylcysteine, suggesting that the GSH peroxidase antioxidant system may be compromised by lack of available substrate. Our findings highlight the importance of endogenous reactive oxidants in regulating CLL-cell apoptosis, and help to explain why CLL cells survive for prolonged periods in vivo despite their vulnerability to oxidative stress and spontaneous apoptosis when cultured in vitro.