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Gutiérrez, José María - One of the best experts on this subject based on the ideXlab platform.
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Intravascular Hemolysis induced by phospholipases A2 from the venom of the Eastern coral snake, Micrurus fulvius: Functional profiles of hemolytic and non-hemolytic isoforms
'Elsevier BV', 2018Co-Authors: Fernández, María Laura, Arce Bejarano Ruth, Gutiérrez, José María, Yunes Quartino, Pablo Javier, Fernández Julián, Camacho, Luis F., Kuemmel Daniel, Fidelio, Gerardo Daniel, Lomonte BrunoAbstract:A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe Intravascular Hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A2 (PLA2) isoforms which cause direct Hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA2-17, a direct hemolytic enzyme, and PLA2-12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause Intravascular Hemolysis: PLA2-17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA2-12 lacked these effects. PLA2-12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA2-17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA2-17 could hydrolyze substrate at a pressure of 20 mN m−1, in contrast to PLA2-12 or the non-toxic pancreatic PLA2. This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA2-12 and PLA2-17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA2-17 presents a less basic interfacial surface than PLA2-12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA2s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge.Fil: Fernández, María Laura. Universidad de Costa Rica; Costa RicaFil: Yunes Quartino, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Arce Bejarano, Ruth. Universidad de Costa Rica; Costa RicaFil: Fernández, Julián. Universidad de Costa Rica; Costa RicaFil: Camacho, Luis F.. Universidad de Costa Rica; Costa RicaFil: Gutiérrez, José María. Universidad de Costa Rica; Costa RicaFil: Kuemmel, Daniel. Universität Osnabrück; AlemaniaFil: Fidelio, Gerardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Lomonte, Bruno. Universidad de Costa Rica; Costa Ric
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Intravascular Hemolysis induced by phospholipases A2 from the venom of the Eastern coral snake, Micrurus fulvius: functional profile of hemolytic and non-hemolytic isoforms
'Elsevier BV', 2018Co-Authors: Fernández Montes De Oca, María Laura, Arce Bejarano Ruth, Lomonte Bruno, Gutiérrez, José María, Camacho, Luis F., Kuemmel Daniel, Yunes Quartino, Pablo J., Fernández Ulate Julián, Fidelio, Gerardo DanielAbstract:A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe Intravascular Hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A2 (PLA2) isoforms which cause direct Hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA2-17, a direct hemolytic enzyme, and PLA2-12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause Intravascular Hemolysis: PLA2-17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA2-12 lacked these effects. PLA2-12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA2-17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA2-17 could hydrolyze substrate at a pressure of 20 mNm−1, in contrast to PLA2-12 or the non-toxic pancreatic PLA2. This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA2-12 and PLA2-17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA2-17 presents a less basic interfacial surface than PLA2-12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA2s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge.Universidad de Costa Rica/[741-B4-100]/UCR/Costa RicaInternational Centre for Genetic Engineering and Biotechnology/[grant CRP/13/006]/ICGEB/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP
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Intravascular Hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2
Toxicon 90, 2014Co-Authors: Arce Bejarano Ruth, Lomonte Bruno, Gutiérrez, José MaríaAbstract:artículo -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2014. Este documento es privado debido a limitaciones de derechos de autor.Intravascular Hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of Intravascular Hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent Hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, Hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing Intravascular Hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of Intravascular Hemolysis.ICGEB-Italia. Universidad de Costa Rica.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP
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Intravascular Hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2
Toxicon 90, 2014Co-Authors: Arce Bejarano Ruth, Lomonte Bruno, Gutiérrez, José MaríaAbstract:artículo -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2014. Este documento es privado debido a limitaciones de derechos de autor.Intravascular Hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of Intravascular Hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent Hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, Hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing Intravascular Hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of Intravascular Hemolysis.ICGEB-Italia. Universidad de Costa Rica.UCR::Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP
Peter Hillmen - One of the best experts on this subject based on the ideXlab platform.
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eculizumab prevents Intravascular Hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low level extravascular Hemolysis occurring through c3 opsonization
Haematologica, 2010Co-Authors: Anita Hill, Richard Kelly, Louise Arnold, Stephen J. Richards, Matthew J. Cullen, Russell P. Rother, Peter HillmenAbstract:Background Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by Intravascular Hemolysis which has been demonstrated to be effectively controlled with eculizumab. However, lactate dehydrogenase levels remain slightly elevated and haptoglobin levels remain low in some patients suggesting residual low-level Hemolysis. This may be due to C3-mediated clearance of paroxysmal nocturnal hemoglobinuria red blood cells through the reticuloendothelial system. Design and Methods Thirty-nine samples from patients not treated with eculizumab and 31 samples from patients treated with eculizumab were obtained (for 17 of these 31 samples there were also samples taken prior to eculizumab treatment). Membrane bound complement was assessed by flow cytometry. Direct antiglobulin testing was carried out using two methods. Lactate dehydrogenase was assayed to assess the degree of Hemolysis. Results Three of 39 patients (8%) with paroxysmal nocturnal hemoglobinuria not on eculizumab had a positive direct antiglobulin test, while the test was positive in 21 of 31 (68%) during eculizumab treatment. Of these 21 patients who had a positive direct antiglobulin test during eculizumab treatment, 17 had been tested prior to treatment; only one was positive. Flow cytometry using anti-C3 monoclonal antibodies was performed on the 21 direct antiglobulin test-positive, eculizumab-treated patients; the median proportion of C3-positive total red blood cells was 26%. Among the eculizumab-treated patients, 16 of the 21 (76.2%) with a positive direct antiglobulin test received at least one transfusion compared with one of ten (10.0%) of those with a negative test ( P <0.01). Among the eculizumab-treated patients, the mean hemoglobin value for the 21 with a positive direct antiglobulin test was 9.6±0.3 g/dL, whereas that in the ten patients with a negative test was 11.0±0.4 g/dL ( P =0.02). Conclusions These data demonstrate a previously masked mechanism of red cell clearance in paroxysmal nocturnal hemoglobinuria and suggests that blockade of complement at C5 allows C3 fragment accumulation on some paroxysmal nocturnal hemoglobinuria red cells, explaining the residual low-level Hemolysis occurring in some eculizumab-treated patients.
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eculizumab prevents Intravascular Hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low level extravascular Hemolysis occurring through c3 opsonization
Haematologica, 2010Co-Authors: Anita Hill, Richard Kelly, Louise Arnold, Stephen J. Richards, Matthew J. Cullen, Russell P. Rother, Peter HillmenAbstract:Background Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by Intravascular Hemolysis which has been demonstrated to be effectively controlled with eculizumab. However, lactate dehydrogenase levels remain slightly elevated and haptoglobin levels remain low in some patients suggesting residual low-level Hemolysis. This may be due to C3-mediated clearance of paroxysmal nocturnal hemoglobinuria red blood cells through the reticuloendothelial system.Design and Methods Thirty-nine samples from patients not treated with eculizumab and 31 samples from patients treated with eculizumab were obtained (for 17 of these 31 samples there were also samples taken prior to eculizumab treatment). Membrane bound complement was assessed by flow cytometry. Direct antiglobulin testing was carried out using two methods. Lactate dehydrogenase was assayed to assess the degree of Hemolysis.Results Three of 39 patients (8%) with paroxysmal nocturnal hemoglobinuria not on eculizumab had a positive direct antiglobulin test, while the test was positive in 21 of 31 (68%) during eculizumab treatment. Of these 21 patients who had a positive direct antiglobulin test during eculizumab treatment, 17 had been tested prior to treatment; only one was positive. Flow cytometry using anti-C3 monoclonal antibodies was performed on the 21 direct antiglobulin test-positive, eculizumab-treated patients; the median proportion of C3-positive total red blood cells was 26%. Among the eculizumab-treated patients, 16 of the 21 (76.2%) with a positive direct antiglobulin test received at least one transfusion compared with one of ten (10.0%) of those with a negative test (P
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the clinical sequelae of Intravascular Hemolysis and extracellular plasma hemoglobin a novel mechanism of human disease
JAMA, 2005Co-Authors: Russell P. Rother, Peter Hillmen, Leonard Bell, Mark T GladwinAbstract:ContextThe efficient sequestration of hemoglobin by the red blood cell membrane and the presence of multiple hemoglobin clearance mechanisms suggest a critical need to prevent the buildup of this molecule in the plasma. A growing list of clinical manifestations attributed to hemoglobin release in a variety of acquired and iatrogenic hemolytic disorders suggests that Hemolysis and hemoglobinemia should be considered as a novel mechanism of human disease.Evidence AcquisitionPertinent scientific literature databases and references were searched through October 2004 using terms that encompassed various aspects of Hemolysis, hemoglobin preparations, clinical symptoms associated with plasma hemoglobin, nitric oxide in Hemolysis, anemia, pulmonary hypertension, paroxysmal nocturnal hemoglobinuria, and sickle-cell disease.Evidence SynthesisHemoglobin is released into the plasma from the erythrocyte during Intravascular Hemolysis in hereditary, acquired, and iatrogenic hemolytic conditions. When the capacity of protective hemoglobin-scavenging mechanisms has been saturated, levels of cell-free hemoglobin increase in the plasma, resulting in the consumption of nitric oxide and clinical sequelae. Nitric oxide plays a major role in vascular homeostasis and has been shown to be a critical regulator of basal and stress-mediated smooth muscle relaxation and vasomotor tone, endothelial adhesion molecule expression, and platelet activation and aggregation. Thus, clinical consequences of excessive cell-free plasma hemoglobin levels during Intravascular Hemolysis or the administration of hemoglobin preparations include dystonias involving the gastrointestinal, cardiovascular, pulmonary, and urogenital systems, as well as clotting disorders. Many of the clinical sequelae of Intravascular Hemolysis in a prototypic hemolytic disease, paroxysmal nocturnal hemoglobinuria, are readily explained by hemoglobin-mediated nitric oxide scavenging.ConclusionA growing body of evidence supports the existence of a novel mechanism of human disease, namely, Hemolysis-associated smooth muscle dystonia, vasculopathy, and endothelial dysfunction.
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the clinical sequelae of Intravascular Hemolysis and extracellular plasma hemoglobin a novel mechanism of human disease
JAMA, 2005Co-Authors: Russell P. Rother, Peter Hillmen, Leonard Bell, Mark T GladwinAbstract:ContextThe efficient sequestration of hemoglobin by the red blood cell membrane and the presence of multiple hemoglobin clearance mechanisms suggest a critical need to prevent the buildup of this molecule in the plasma. A growing list of clinical manifestations attributed to hemoglobin release in a variety of acquired and iatrogenic hemolytic disorders suggests that Hemolysis and hemoglobinemia should be considered as a novel mechanism of human disease.Evidence AcquisitionPertinent scientific literature databases and references were searched through October 2004 using terms that encompassed various aspects of Hemolysis, hemoglobin preparations, clinical symptoms associated with plasma hemoglobin, nitric oxide in Hemolysis, anemia, pulmonary hypertension, paroxysmal nocturnal hemoglobinuria, and sickle-cell disease.Evidence SynthesisHemoglobin is released into the plasma from the erythrocyte during Intravascular Hemolysis in hereditary, acquired, and iatrogenic hemolytic conditions. When the capacity of protective hemoglobin-scavenging mechanisms has been saturated, levels of cell-free hemoglobin increase in the plasma, resulting in the consumption of nitric oxide and clinical sequelae. Nitric oxide plays a major role in vascular homeostasis and has been shown to be a critical regulator of basal and stress-mediated smooth muscle relaxation and vasomotor tone, endothelial adhesion molecule expression, and platelet activation and aggregation. Thus, clinical consequences of excessive cell-free plasma hemoglobin levels during Intravascular Hemolysis or the administration of hemoglobin preparations include dystonias involving the gastrointestinal, cardiovascular, pulmonary, and urogenital systems, as well as clotting disorders. Many of the clinical sequelae of Intravascular Hemolysis in a prototypic hemolytic disease, paroxysmal nocturnal hemoglobinuria, are readily explained by hemoglobin-mediated nitric oxide scavenging.ConclusionA growing body of evidence supports the existence of a novel mechanism of human disease, namely, Hemolysis-associated smooth muscle dystonia, vasculopathy, and endothelial dysfunction.
Anita Hill - One of the best experts on this subject based on the ideXlab platform.
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eculizumab prevents Intravascular Hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low level extravascular Hemolysis occurring through c3 opsonization
Haematologica, 2010Co-Authors: Anita Hill, Richard Kelly, Louise Arnold, Stephen J. Richards, Matthew J. Cullen, Russell P. Rother, Peter HillmenAbstract:Background Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by Intravascular Hemolysis which has been demonstrated to be effectively controlled with eculizumab. However, lactate dehydrogenase levels remain slightly elevated and haptoglobin levels remain low in some patients suggesting residual low-level Hemolysis. This may be due to C3-mediated clearance of paroxysmal nocturnal hemoglobinuria red blood cells through the reticuloendothelial system.Design and Methods Thirty-nine samples from patients not treated with eculizumab and 31 samples from patients treated with eculizumab were obtained (for 17 of these 31 samples there were also samples taken prior to eculizumab treatment). Membrane bound complement was assessed by flow cytometry. Direct antiglobulin testing was carried out using two methods. Lactate dehydrogenase was assayed to assess the degree of Hemolysis.Results Three of 39 patients (8%) with paroxysmal nocturnal hemoglobinuria not on eculizumab had a positive direct antiglobulin test, while the test was positive in 21 of 31 (68%) during eculizumab treatment. Of these 21 patients who had a positive direct antiglobulin test during eculizumab treatment, 17 had been tested prior to treatment; only one was positive. Flow cytometry using anti-C3 monoclonal antibodies was performed on the 21 direct antiglobulin test-positive, eculizumab-treated patients; the median proportion of C3-positive total red blood cells was 26%. Among the eculizumab-treated patients, 16 of the 21 (76.2%) with a positive direct antiglobulin test received at least one transfusion compared with one of ten (10.0%) of those with a negative test (P
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eculizumab prevents Intravascular Hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low level extravascular Hemolysis occurring through c3 opsonization
Haematologica, 2010Co-Authors: Anita Hill, Richard Kelly, Louise Arnold, Stephen J. Richards, Matthew J. Cullen, Russell P. Rother, Peter HillmenAbstract:Background Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by Intravascular Hemolysis which has been demonstrated to be effectively controlled with eculizumab. However, lactate dehydrogenase levels remain slightly elevated and haptoglobin levels remain low in some patients suggesting residual low-level Hemolysis. This may be due to C3-mediated clearance of paroxysmal nocturnal hemoglobinuria red blood cells through the reticuloendothelial system. Design and Methods Thirty-nine samples from patients not treated with eculizumab and 31 samples from patients treated with eculizumab were obtained (for 17 of these 31 samples there were also samples taken prior to eculizumab treatment). Membrane bound complement was assessed by flow cytometry. Direct antiglobulin testing was carried out using two methods. Lactate dehydrogenase was assayed to assess the degree of Hemolysis. Results Three of 39 patients (8%) with paroxysmal nocturnal hemoglobinuria not on eculizumab had a positive direct antiglobulin test, while the test was positive in 21 of 31 (68%) during eculizumab treatment. Of these 21 patients who had a positive direct antiglobulin test during eculizumab treatment, 17 had been tested prior to treatment; only one was positive. Flow cytometry using anti-C3 monoclonal antibodies was performed on the 21 direct antiglobulin test-positive, eculizumab-treated patients; the median proportion of C3-positive total red blood cells was 26%. Among the eculizumab-treated patients, 16 of the 21 (76.2%) with a positive direct antiglobulin test received at least one transfusion compared with one of ten (10.0%) of those with a negative test ( P <0.01). Among the eculizumab-treated patients, the mean hemoglobin value for the 21 with a positive direct antiglobulin test was 9.6±0.3 g/dL, whereas that in the ten patients with a negative test was 11.0±0.4 g/dL ( P =0.02). Conclusions These data demonstrate a previously masked mechanism of red cell clearance in paroxysmal nocturnal hemoglobinuria and suggests that blockade of complement at C5 allows C3 fragment accumulation on some paroxysmal nocturnal hemoglobinuria red cells, explaining the residual low-level Hemolysis occurring in some eculizumab-treated patients.
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blockade of Intravascular Hemolysis in pnh with the terminal complement inhibitor eculizumab unmasks low level Hemolysis potentially occurring through c3 opsonization
Blood, 2006Co-Authors: Anita Hill, Stephen J. Richards, Matthew J. Cullen, Russell P. Rother, Antonio M. Risitano, Duncan Cole, Carmine Selleri, Patrizia Ricci, Bruno Rotoli, Lucio LuzzattoAbstract:Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by Intravascular Hemolysis, often resulting in the need for red blood cell (RBC) transfusions. PNH RBCs lack two complement regulatory molecules - CD59, a terminal complement inhibitor, and CD55, a C3 convertase inhibitor. Eculizumab, a humanized monoclonal antibody that inhibits terminal complement by binding to C5, effectively controls Intravascular Hemolysis as determined by a dramatic reduction in lactate dehydrogenase (LDH) to levels in or just above the normal range. Control of Intravascular Hemolysis in these patients led to a reduction in, or cessation of, RBC transfusions. During eculizumab treatment, a majority of patients demonstrate evidence of residual, low-level Hemolysis; LDH levels remain slightly elevated, haptoglobin levels are low or undetectable, and bilirubin levels are above normal. We hypothesized that this low-level residual Hemolysis may be due to clearance of PNH RBCs through a C3b-mediated mechanism. Therefore we investigated C3 deposition on RBC in PNH patients before and on eculizumab. A direct antiglobulin test (DAT) using monoclonal anti-C3d was positive in 29 out of 39 PNH patients on eculizumab. Of these 29 DAT-positive patients, who were all receiving transfusions, 25 had DAT testing prior to eculizumab therapy and only one of these was positive. DAT was negative in all of 8 normal volunteers. By two-color flow cytometric analysis with anti-CD59 and anti-C3, the majority of patients on eculizumab demonstrated three distinct RBC populations: CD59+/C3− (normal RBCs); CD59-/C3− (PNH RBCs without C3 coating); and CD59-/C3+ (PNH RBCs coated by C3). No CD59+/C3+ RBCs were observed. Of 21 DAT positive eculizumab treated patients tested, the median proportion of total RBCs that were C3b positive was 17.6%. 18 of 29 [62%] eculizumab patients with a positive DAT received at least one transfusion during eculizumab therapy compared with 1 of 10 [10%] for DAT negative patients (p=0.01), although even patients who did not become transfusion independent during eculizumab treatment showed a marked reduction in transfusion requirement. The median hemoglobin value for the 29 DAT positive eculizumab patients was 9.8 g/dL compared with 11.3 g/dL in the 10 DAT negative eculizumab patients (p= 0.08). No apparent relationship between LDH and DAT positivity was observed. It is proposed that resolution of Intravascular Hemolysis in PNH patients on eculizumab results in deposition of C3b on the surface of PNH RBCs which may explain, at least in part, the residual low level Hemolysis occurring in some patients. This appears to be a previously undescribed mechanism of RBC clearance in PNH, most likely obscured by the rapidity of Intravascular Hemolysis in the absence of eculizumab therapy. Despite the low-level residual Hemolysis, patients continue to receive significant benefit from eculizumab treatment.
José María Gutiérrez - One of the best experts on this subject based on the ideXlab platform.
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Intravascular Hemolysis induced by phospholipases a2 from the venom of the eastern coral snake micrurus fulvius functional profiles of hemolytic and non hemolytic isoforms
Toxicology Letters, 2017Co-Authors: Maria Laura Fernandez, José María Gutiérrez, Ruth Arcebejarano, Pablo Javier Yunes Quartino, Julian Fernandez, Luis F Camacho, Daniel Kuemmel, Gerardo D Fidelio, Bruno LomonteAbstract:Abstract A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe Intravascular Hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A2 (PLA2) isoforms which cause direct Hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA2-17, a direct hemolytic enzyme, and PLA2-12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause Intravascular Hemolysis: PLA2-17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA2-12 lacked these effects. PLA2-12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA2-17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA2-17 could hydrolyze substrate at a pressure of 20 mN m−1, in contrast to PLA2-12 or the non-toxic pancreatic PLA2. This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA2-12 and PLA2-17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA2-17 presents a less basic interfacial surface than PLA2-12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA2s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge.
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Intravascular Hemolysis induced by the venom of the eastern coral snake micrurus fulvius in a mouse model identification of directly hemolytic phospholipases a2
Toxicon, 2014Co-Authors: Ruth Arcebejarano, Bruno Lomonte, José María GutiérrezAbstract:Intravascular Hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of Intravascular Hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent Hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, Hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing Intravascular Hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of Intravascular Hemolysis.
Arce Bejarano Ruth - One of the best experts on this subject based on the ideXlab platform.
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Intravascular Hemolysis induced by phospholipases A2 from the venom of the Eastern coral snake, Micrurus fulvius: Functional profiles of hemolytic and non-hemolytic isoforms
'Elsevier BV', 2018Co-Authors: Fernández, María Laura, Arce Bejarano Ruth, Gutiérrez, José María, Yunes Quartino, Pablo Javier, Fernández Julián, Camacho, Luis F., Kuemmel Daniel, Fidelio, Gerardo Daniel, Lomonte BrunoAbstract:A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe Intravascular Hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A2 (PLA2) isoforms which cause direct Hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA2-17, a direct hemolytic enzyme, and PLA2-12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause Intravascular Hemolysis: PLA2-17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA2-12 lacked these effects. PLA2-12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA2-17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA2-17 could hydrolyze substrate at a pressure of 20 mN m−1, in contrast to PLA2-12 or the non-toxic pancreatic PLA2. This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA2-12 and PLA2-17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA2-17 presents a less basic interfacial surface than PLA2-12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA2s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge.Fil: Fernández, María Laura. Universidad de Costa Rica; Costa RicaFil: Yunes Quartino, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Arce Bejarano, Ruth. Universidad de Costa Rica; Costa RicaFil: Fernández, Julián. Universidad de Costa Rica; Costa RicaFil: Camacho, Luis F.. Universidad de Costa Rica; Costa RicaFil: Gutiérrez, José María. Universidad de Costa Rica; Costa RicaFil: Kuemmel, Daniel. Universität Osnabrück; AlemaniaFil: Fidelio, Gerardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Lomonte, Bruno. Universidad de Costa Rica; Costa Ric
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Intravascular Hemolysis induced by phospholipases A2 from the venom of the Eastern coral snake, Micrurus fulvius: functional profile of hemolytic and non-hemolytic isoforms
'Elsevier BV', 2018Co-Authors: Fernández Montes De Oca, María Laura, Arce Bejarano Ruth, Lomonte Bruno, Gutiérrez, José María, Camacho, Luis F., Kuemmel Daniel, Yunes Quartino, Pablo J., Fernández Ulate Julián, Fidelio, Gerardo DanielAbstract:A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe Intravascular Hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A2 (PLA2) isoforms which cause direct Hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA2-17, a direct hemolytic enzyme, and PLA2-12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause Intravascular Hemolysis: PLA2-17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA2-12 lacked these effects. PLA2-12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA2-17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA2-17 could hydrolyze substrate at a pressure of 20 mNm−1, in contrast to PLA2-12 or the non-toxic pancreatic PLA2. This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA2-12 and PLA2-17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA2-17 presents a less basic interfacial surface than PLA2-12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA2s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge.Universidad de Costa Rica/[741-B4-100]/UCR/Costa RicaInternational Centre for Genetic Engineering and Biotechnology/[grant CRP/13/006]/ICGEB/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP
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Intravascular Hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2
Toxicon 90, 2014Co-Authors: Arce Bejarano Ruth, Lomonte Bruno, Gutiérrez, José MaríaAbstract:artículo -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2014. Este documento es privado debido a limitaciones de derechos de autor.Intravascular Hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of Intravascular Hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent Hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, Hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing Intravascular Hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of Intravascular Hemolysis.ICGEB-Italia. Universidad de Costa Rica.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP
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Intravascular Hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: Identification of directly hemolytic phospholipases A2
Toxicon 90, 2014Co-Authors: Arce Bejarano Ruth, Lomonte Bruno, Gutiérrez, José MaríaAbstract:artículo -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2014. Este documento es privado debido a limitaciones de derechos de autor.Intravascular Hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of Intravascular Hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent Hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, Hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing Intravascular Hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of Intravascular Hemolysis.ICGEB-Italia. Universidad de Costa Rica.UCR::Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP