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Semiconductor Nanoparticles Exhibit Cytotoxicity and Mitochondrial Impairment through Ros Stimulation in A459 Cells
The Role of KIR6.2 in Mediating NOX2 Induced Revascularization with Obstruction of Blood Flow
chemistry, and other reference thiols in C. elegans. We have found that 12-SH caused significant toxicity to adult C. elegans in liquid culture beginning at 100 μM concentration. the toxic effect was mildly exacerbated by food restriction and cultivation of the worms without fluorodeoxyuridine, an inhibitor of DNA synthesis and reproduction. Notably, we observed a rather unusual, gradual built-up of toxicity over several days, as would be expected for a mechanism involving chain-transfer activity. in contrast, dodecanol (12-OH) was fully lethal to the worms within just a few hours. Consistently with a thiyl-dependent mechanism in membranes, methylated dodecylthiol (12-S-Me) and less lipophilic thiols like butylthiol (4-SH) were significantly less toxic than 12SH. Our results indicate a toxic effect of thiol groups in biological membranes in vivo, and they are compatible with a thiyl radicaldependent mechanism.
Shampa Chatterjee1, Aron B. Fisher1, Hui Wang1, Sheldon I. Feinstein1, Nan Kang Hong1, and Elizabeth A. Browning1 1 University of Pennsylvania, United States
doi: 10.1016/j.freeradbiomed.2013.10.750
contribute to lung inflammation after sepsis while the switch to non-canonical autophagy and selective removal of damaged mitochondria favors tissue repair and cell survival. Thus, early enhancement or restoration of mitophagy after sepsis may be a potential adjunct for the treatment of sepsis.
doi: 10.1016/j.freeradbiomed.2013.10.748
We reported earlier that stop of blood flow or ischemia results in the generation of reactive oxygen species (ROS) via the closure of a KATP (KIR6.2) channel that causes endothelial membrane depolarization and NADPH oxidase (NOX2) activation. This study was undertaken to understand the physiological role of obstruction of blood flow in signaling. Investigations into angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) in vitro and in the hind limb in vivo revealed that flow adapted PMVEC (72 h flow adaptation, 1 h ischemia and injected into a Matrigel matrix) showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of KATP channels or the NOX2 subunit of NADPH oxidase. in the in vivo model of ischemia (femoral artery ligation), revascularization, as ascertained by microscopic evaluation, was high in wild type mice and was significantly decreased in mice with knockout of KATP channel and in mice orally fed with a KATP channel agonist. in transgenic mice with endothelial specific NOX2 expression, the revascularization observed was intermediate between that of wild type and knockout of KATP channel or NOX2. Increased HIF- Į DFWLYDWLRQ and VEGF expression was observed in ischemic tissue of wild type mice but not in KATP channel and NOX2 null mice. This is a first report of a mechanosensitive ion channel (KATP channel) initiating endothelial signaling that drives revascularization.
doi: 10.1016/j.freeradbiomed.2013.10.749
Toxicity of Intramembrane Thiols in Vivo Antonio J. De Giacomo1, Mario Schindeldecker1, Christian Behl1, Parvana Hajieva1, and Bernd Moosmann1 1 University of Mainz, Germany Bioinformatic analyses have indicated a significant underrepresentation of cysteine residues in relation to oxidative stress, such as in aerobic versus anaerobic bacteria, archaea, eukaryotes, and animals. in particular, the membrane protein complexes of the mitochondrial respiratory chain in animals seem to avoid cysteine in inverse correlation with respiratory rate and longevity. We hypothesized that the reason for the evolutionary elimination of cysteine from highly oxidation-prone membranes might be found in the specific redox chemistry of thiol groups in hydrophobic phases, which has been well established in polymer chemistry: intramembrane thiols might exhibit toxicity in cells due to a thiyl radical-dependent chain-transfer activity in free radical chain reactions. Thus, we have investigated the effects of 12dodecylthiol (12-SH), a prototypic chain-transfer agent in polymer
Semiconductor Nanoparticles Exhibit Cytotoxicity and Mitochondrial Impairment through Ros Stimulation in A459 Cells Vicente Escamilla-Rivera1, Marisela Uribe-Ramirez1, and Andrea De Vizcaya-Ruiz1 1 CINVESTAV-IPN, Mexico Copper indium gallium diselenide (CIGS) and cadmium sulfide (CdS) nanoparticles (NP), are specifically designed to improve the efficiency of photovoltaic solar cells (PV), by increasing the capture of photons from the sunlight and converting them into free electrons, these NP are considered as the most promising materials for PV devices for its low manufacture cost and high efficiency. Due to the growing concern about the risks associated to the use of engineered NP, and the need of information for safe handling and disposal, we assessed the redox ability of CIGS and CdS and the induction of cytotoxicity mediated by reactive oxygen species (ROS) in human alveolar epithelial A549 cells. in order to determine the intrinsic oxidative properties of the NP, we performed the acellular dithiothreitol (DTT) oxidation assay, results showed a greater oxidant activity of CIGS vs. CdS, 41.21 and 1.2 pmol '77 ȝJ-1*min-1 respectively. to guarantee the stability of NP dispersion, we suspended NP in 2 mg/mL of BSA solution before the addition to culture media, this led to a reduction in the size and number of agglomerates compared with the NP-water dispersions. Average NP size and zeta potential were 357 nm with -14.7mV for CIGS and 533 nm with -17.2 mV for CdS in a stable dispersion. Induction of cytotoxicity was evaluated using the crystal violet and MTT reduction assays, CIGS induced more cytotoxicity than CdS in all the concentrations and time points evaluated. the evaluation of intracellular ROS levels using DCFH-DA, showed that CIGS NP induce a significant dose-dependent increase in intracellular ROS after 6h, with a maximum of 1.92-fold versus control after exposure to ȝJP/ while CdS NP induced a moderate ROS increase of 1.5-fold, which was not concentration-dependent. with the purpose to determine if CIGS and CdS NP have mitochondria as a potential target, we evaluated the changes of mitochondrial membrane potential (ǻȥP , using the ratio between tetramethylrhodamine ethyl ester perchlorate (TMRE) and Mitotracker Green fluorescence. After 6h, both CIGS and CdS NP caused a decrease of ǻȥP in a similar extent (28 and ǻȥP LQGLFDWLQJ depolarization of the mitochondrial inner membrane. Antioxidant pre-treatment with Trolox prevented the loss of ǻȥP and cytotoxicity induced by both NP, suggesting that the induction of ROS is the main mechanism by which these NP are cytotoxic. Our results support the hypothesis that intrinsic properties of NP
6)5%0
6
are underlying factors that determine cellular toxicity of engineered NP. for semiconductors engineered NP such as CIGS and CdS bandgap energy is the property of electrons transfer from an energy level to another, that in an intracellular environment may favor the formation of oxidizing molecules leading to cellular toxicity, such as organelle impairment and cell death. Acknowledgment: ICyT-DF 396/10 for partial financial support.
doi: 10.1016/j.freeradbiomed.2013.10.751
Metabolic Fate of Endogenous and ExogenouslyAdministered Electrophilic Fatty Acid Nitroalkenes: the Formation and Actions of Nitro-Fatty AcidContaining Complex Lipids
Marco Fazzari1,2, Nicholas Khoo1, Steven Woodcock1, Franca GolinBisello1, Francisco J. Schopfer1, and Bruce A. Freeman1 1 University of Pittsburgh, United States, 2Rimed Foundation, Italy Electrophilic nitrated fatty acids (NO2-FA) are endogenous signaling mediators generated by reaction of unsaturated fatty acids with nitric oxide (NO) and nitrite (NO2)-derived species. They act to modulate transcription factor and enzyme function via post-translational protein modifications, yielding beneficial metabolic and anti-inflammatory responses. Some metabolic pathways involving NO2-FA (e.g. saturation, desaturation, Eoxidation, and thiol Michael addition) has been recently described, but there remains a large gap in knowledge regarding the detection and characterization of electrophilic fatty acid-containing complex lipids. the standards 1,2-dipalmitoyl-3-conjugatedlineoylglycerol (TGcLA) and 1,2-dipalmitoyl-3-(14and15)NO2conjugated-lineoylglycerol (TGNO2cLA) were synthesized and characterized by HPLC-MSMS. the treatment of TGcLA with gastric acid in the presence of NO2 induced nitration, confirmed by comparison with 15N-labeled standards and the analysis of nitrated free fatty acids upon lipase hydrolysis. the supplementation of 3T3-L1 adipocytes with nitro-oleic acid (NO2OA) showed incorporation of nitroalkane and shorter chain length metabolites into triglycerides. HPLC-high resolution mass spectrometry and kinetic analysis affirmed that NO2-OA was rapidly metabolized by nitroalkene reductases and mitochondrial E-oxidation enzymes prior to esterification to triglycerides. Electrophilic NO2-FA incorporation into triglycerides was also detected after lipase hydrolysis of 3T3-L1 cell lipids and the analysis of adipose tissue obtained from NO2-OA-treated mice. These observations reveal that sources of unsaturated fatty acidcontaining triglycerides in the diet (e.g. plant or animal lipids) support the formation of NO2-FA upon digestion. the direct nitration of complex lipids or the incorporation of free NO2-FA into complex lipids thus forms a reservoir of electrophilic lipids that can react directly with nucleophilic targets or mediate signaling actions upon hydrolysis to free NO2-FA by cellular lipases. Overall, this metabolic profile contributes to the highly favorable pharmacokinetics and low toxicity of endogenously-generated and exogenously-administered NO2-FA.
doi: 10.1016/j.freeradbiomed.2013.10.752
6
Post-Translational Modification and Inhibition of Glutamate Cysteine Ligase Activity by Acrolein and Cigarette Smoke Extract
Kristofer S Fritz1, Donald S Backos1, Robyn L Poerschke1, Jason M Fritz1, Dennis R Petersen1, and Christopher C Franklin1 1 University of Colorado Anschutz Medical Campus, United States Acrolein is a KLJKO\ UHDFWLYH Įȕ-unsaturated aldehyde that is present at extremely high levels in cigarette smoke (CS) and is capable of altering a wide variety of normal cellular functions due to its ability to form covalent adducts on cellular DNA and proteins. Acrolein is metabolized via conjugation with glutathione (GSH), which results in the depletion of cellular GSH stores and necessitates de novo GSH biosynthesis to reestablish GSH homeostasis. the enzyme catalyzing the rate-limiting step of GSH biosynthesis is glutamate cysteine ligase (GCL), which is a heterodimeric holoenzyme consisting of catalytic (GCLC) and modulatory (GCLM) subunits. We recently reported that the a,bunsaturated aldehyde 4-HNE directly modifies the GCL subunits, resulting in altered GCL enzymatic activity. in this study, we demonstrate that GCLC and GCLM are also targets for posttranslational modification by acrolein and CS extract (CSE). Acrolein and CSE had biphasic effects on GCLC enzymatic activity, inhibiting both GCLC and GCL holoenzyme activity at higher concentrations. Acrolein- and CSE-mediated modification of the GCL subunits also prevented GCL holoenzyme formation. Mass spectrometry analysis revealed that multiple cysteine and lysine residues on both GCL subunits were targets for acroleinmediated modification. in silico homology modeling indicates that many of these residues are near the putative interaction interface of GCLC and GCLM, which may provide the molecular basis for the inhibitory effects of acrolein- and CSE-mediated carbonylation on GCL subunit heterodimerization and holoenzyme formation. in aggregate, these findings suggest that acrolein and CSE can directly modify and inhibit GCLC activity and GCL holoenzyme formation and activity, which likely contribute to the loss of cellular GSH homeostasis in CS-induced emphysema.
doi: 10.1016/j.freeradbiomed.2013.10.753
Standard Preservation of Platelets Rich Plasma Induces Oxidative Damage and Fibrinolytic Enzymes Activation in Thrombocytes
Jaime González-Cuevas1, Jesus Javier Garcia-Bañuelos1, José Navarro-Partida2, and Juan Armendariz-Borunda1,3 1 Universidad de Guadalajara, Mexico, 2Universidad Autónoma de Nayarit, Mexico, 3O.P.D. Hospital Civil de Guadalajara, Guadalajara, Mexico Background: Therapeutic effectiveness of platelet rich plasma (PRP) is related to technical procedures for procurement, storage conditions and aging. the maintenance of PRP until use involves avoiding platelet activation. It has been demonstrated that platelet activation and aggregation are regulated by metalloproteases (MMPs) and plasmin. This work focuses on elucidating activation and of oxidative damage of platelets during storage. Methods: Platelet rich plasmas were obtained from 110 healthy donors from a transfusable medical service and kept under stirring at a temperature of 20-Û& 3URWHLQ H[WUDFWLRQ ZDV performed from platelets homogenates at different times of
6)5%0
chemistry, and other reference thiols in C. elegans. We have found that 12-SH caused significant toxicity to adult C. elegans in liquid culture beginning at 100 μM concentration. the toxic effect was mildly exacerbated by food restriction and cultivation of the worms without fluorodeoxyuridine, an inhibitor of DNA synthesis and reproduction. Notably, we observed a rather unusual, gradual built-up of toxicity over several days, as would be expected for a mechanism involving chain-transfer activity. in contrast, dodecanol (12-OH) was fully lethal to the worms within just a few hours. Consistently with a thiyl-dependent mechanism in membranes, methylated dodecylthiol (12-S-Me) and less lipophilic thiols like butylthiol (4-SH) were significantly less toxic than 12SH. Our results indicate a toxic effect of thiol groups in biological membranes in vivo, and they are compatible with a thiyl radicaldependent mechanism.
Shampa Chatterjee1, Aron B. Fisher1, Hui Wang1, Sheldon I. Feinstein1, Nan Kang Hong1, and Elizabeth A. Browning1 1 University of Pennsylvania, United States
doi: 10.1016/j.freeradbiomed.2013.10.750
contribute to lung inflammation after sepsis while the switch to non-canonical autophagy and selective removal of damaged mitochondria favors tissue repair and cell survival. Thus, early enhancement or restoration of mitophagy after sepsis may be a potential adjunct for the treatment of sepsis.
doi: 10.1016/j.freeradbiomed.2013.10.748
We reported earlier that stop of blood flow or ischemia results in the generation of reactive oxygen species (ROS) via the closure of a KATP (KIR6.2) channel that causes endothelial membrane depolarization and NADPH oxidase (NOX2) activation. This study was undertaken to understand the physiological role of obstruction of blood flow in signaling. Investigations into angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) in vitro and in the hind limb in vivo revealed that flow adapted PMVEC (72 h flow adaptation, 1 h ischemia and injected into a Matrigel matrix) showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of KATP channels or the NOX2 subunit of NADPH oxidase. in the in vivo model of ischemia (femoral artery ligation), revascularization, as ascertained by microscopic evaluation, was high in wild type mice and was significantly decreased in mice with knockout of KATP channel and in mice orally fed with a KATP channel agonist. in transgenic mice with endothelial specific NOX2 expression, the revascularization observed was intermediate between that of wild type and knockout of KATP channel or NOX2. Increased HIF- Į DFWLYDWLRQ and VEGF expression was observed in ischemic tissue of wild type mice but not in KATP channel and NOX2 null mice. This is a first report of a mechanosensitive ion channel (KATP channel) initiating endothelial signaling that drives revascularization.
doi: 10.1016/j.freeradbiomed.2013.10.749
Toxicity of Intramembrane Thiols in Vivo Antonio J. De Giacomo1, Mario Schindeldecker1, Christian Behl1, Parvana Hajieva1, and Bernd Moosmann1 1 University of Mainz, Germany Bioinformatic analyses have indicated a significant underrepresentation of cysteine residues in relation to oxidative stress, such as in aerobic versus anaerobic bacteria, archaea, eukaryotes, and animals. in particular, the membrane protein complexes of the mitochondrial respiratory chain in animals seem to avoid cysteine in inverse correlation with respiratory rate and longevity. We hypothesized that the reason for the evolutionary elimination of cysteine from highly oxidation-prone membranes might be found in the specific redox chemistry of thiol groups in hydrophobic phases, which has been well established in polymer chemistry: intramembrane thiols might exhibit toxicity in cells due to a thiyl radical-dependent chain-transfer activity in free radical chain reactions. Thus, we have investigated the effects of 12dodecylthiol (12-SH), a prototypic chain-transfer agent in polymer
Semiconductor Nanoparticles Exhibit Cytotoxicity and Mitochondrial Impairment through Ros Stimulation in A459 Cells Vicente Escamilla-Rivera1, Marisela Uribe-Ramirez1, and Andrea De Vizcaya-Ruiz1 1 CINVESTAV-IPN, Mexico Copper indium gallium diselenide (CIGS) and cadmium sulfide (CdS) nanoparticles (NP), are specifically designed to improve the efficiency of photovoltaic solar cells (PV), by increasing the capture of photons from the sunlight and converting them into free electrons, these NP are considered as the most promising materials for PV devices for its low manufacture cost and high efficiency. Due to the growing concern about the risks associated to the use of engineered NP, and the need of information for safe handling and disposal, we assessed the redox ability of CIGS and CdS and the induction of cytotoxicity mediated by reactive oxygen species (ROS) in human alveolar epithelial A549 cells. in order to determine the intrinsic oxidative properties of the NP, we performed the acellular dithiothreitol (DTT) oxidation assay, results showed a greater oxidant activity of CIGS vs. CdS, 41.21 and 1.2 pmol '77 ȝJ-1*min-1 respectively. to guarantee the stability of NP dispersion, we suspended NP in 2 mg/mL of BSA solution before the addition to culture media, this led to a reduction in the size and number of agglomerates compared with the NP-water dispersions. Average NP size and zeta potential were 357 nm with -14.7mV for CIGS and 533 nm with -17.2 mV for CdS in a stable dispersion. Induction of cytotoxicity was evaluated using the crystal violet and MTT reduction assays, CIGS induced more cytotoxicity than CdS in all the concentrations and time points evaluated. the evaluation of intracellular ROS levels using DCFH-DA, showed that CIGS NP induce a significant dose-dependent increase in intracellular ROS after 6h, with a maximum of 1.92-fold versus control after exposure to ȝJP/ while CdS NP induced a moderate ROS increase of 1.5-fold, which was not concentration-dependent. with the purpose to determine if CIGS and CdS NP have mitochondria as a potential target, we evaluated the changes of mitochondrial membrane potential (ǻȥP , using the ratio between tetramethylrhodamine ethyl ester perchlorate (TMRE) and Mitotracker Green fluorescence. After 6h, both CIGS and CdS NP caused a decrease of ǻȥP in a similar extent (28 and ǻȥP LQGLFDWLQJ depolarization of the mitochondrial inner membrane. Antioxidant pre-treatment with Trolox prevented the loss of ǻȥP and cytotoxicity induced by both NP, suggesting that the induction of ROS is the main mechanism by which these NP are cytotoxic. Our results support the hypothesis that intrinsic properties of NP
6)5%0
6
are underlying factors that determine cellular toxicity of engineered NP. for semiconductors engineered NP such as CIGS and CdS bandgap energy is the property of electrons transfer from an energy level to another, that in an intracellular environment may favor the formation of oxidizing molecules leading to cellular toxicity, such as organelle impairment and cell death. Acknowledgment: ICyT-DF 396/10 for partial financial support.
doi: 10.1016/j.freeradbiomed.2013.10.751
Metabolic Fate of Endogenous and ExogenouslyAdministered Electrophilic Fatty Acid Nitroalkenes: the Formation and Actions of Nitro-Fatty AcidContaining Complex Lipids
Marco Fazzari1,2, Nicholas Khoo1, Steven Woodcock1, Franca GolinBisello1, Francisco J. Schopfer1, and Bruce A. Freeman1 1 University of Pittsburgh, United States, 2Rimed Foundation, Italy Electrophilic nitrated fatty acids (NO2-FA) are endogenous signaling mediators generated by reaction of unsaturated fatty acids with nitric oxide (NO) and nitrite (NO2)-derived species. They act to modulate transcription factor and enzyme function via post-translational protein modifications, yielding beneficial metabolic and anti-inflammatory responses. Some metabolic pathways involving NO2-FA (e.g. saturation, desaturation, Eoxidation, and thiol Michael addition) has been recently described, but there remains a large gap in knowledge regarding the detection and characterization of electrophilic fatty acid-containing complex lipids. the standards 1,2-dipalmitoyl-3-conjugatedlineoylglycerol (TGcLA) and 1,2-dipalmitoyl-3-(14and15)NO2conjugated-lineoylglycerol (TGNO2cLA) were synthesized and characterized by HPLC-MSMS. the treatment of TGcLA with gastric acid in the presence of NO2 induced nitration, confirmed by comparison with 15N-labeled standards and the analysis of nitrated free fatty acids upon lipase hydrolysis. the supplementation of 3T3-L1 adipocytes with nitro-oleic acid (NO2OA) showed incorporation of nitroalkane and shorter chain length metabolites into triglycerides. HPLC-high resolution mass spectrometry and kinetic analysis affirmed that NO2-OA was rapidly metabolized by nitroalkene reductases and mitochondrial E-oxidation enzymes prior to esterification to triglycerides. Electrophilic NO2-FA incorporation into triglycerides was also detected after lipase hydrolysis of 3T3-L1 cell lipids and the analysis of adipose tissue obtained from NO2-OA-treated mice. These observations reveal that sources of unsaturated fatty acidcontaining triglycerides in the diet (e.g. plant or animal lipids) support the formation of NO2-FA upon digestion. the direct nitration of complex lipids or the incorporation of free NO2-FA into complex lipids thus forms a reservoir of electrophilic lipids that can react directly with nucleophilic targets or mediate signaling actions upon hydrolysis to free NO2-FA by cellular lipases. Overall, this metabolic profile contributes to the highly favorable pharmacokinetics and low toxicity of endogenously-generated and exogenously-administered NO2-FA.
doi: 10.1016/j.freeradbiomed.2013.10.752
6
Post-Translational Modification and Inhibition of Glutamate Cysteine Ligase Activity by Acrolein and Cigarette Smoke Extract
Kristofer S Fritz1, Donald S Backos1, Robyn L Poerschke1, Jason M Fritz1, Dennis R Petersen1, and Christopher C Franklin1 1 University of Colorado Anschutz Medical Campus, United States Acrolein is a KLJKO\ UHDFWLYH Įȕ-unsaturated aldehyde that is present at extremely high levels in cigarette smoke (CS) and is capable of altering a wide variety of normal cellular functions due to its ability to form covalent adducts on cellular DNA and proteins. Acrolein is metabolized via conjugation with glutathione (GSH), which results in the depletion of cellular GSH stores and necessitates de novo GSH biosynthesis to reestablish GSH homeostasis. the enzyme catalyzing the rate-limiting step of GSH biosynthesis is glutamate cysteine ligase (GCL), which is a heterodimeric holoenzyme consisting of catalytic (GCLC) and modulatory (GCLM) subunits. We recently reported that the a,bunsaturated aldehyde 4-HNE directly modifies the GCL subunits, resulting in altered GCL enzymatic activity. in this study, we demonstrate that GCLC and GCLM are also targets for posttranslational modification by acrolein and CS extract (CSE). Acrolein and CSE had biphasic effects on GCLC enzymatic activity, inhibiting both GCLC and GCL holoenzyme activity at higher concentrations. Acrolein- and CSE-mediated modification of the GCL subunits also prevented GCL holoenzyme formation. Mass spectrometry analysis revealed that multiple cysteine and lysine residues on both GCL subunits were targets for acroleinmediated modification. in silico homology modeling indicates that many of these residues are near the putative interaction interface of GCLC and GCLM, which may provide the molecular basis for the inhibitory effects of acrolein- and CSE-mediated carbonylation on GCL subunit heterodimerization and holoenzyme formation. in aggregate, these findings suggest that acrolein and CSE can directly modify and inhibit GCLC activity and GCL holoenzyme formation and activity, which likely contribute to the loss of cellular GSH homeostasis in CS-induced emphysema.
doi: 10.1016/j.freeradbiomed.2013.10.753
Standard Preservation of Platelets Rich Plasma Induces Oxidative Damage and Fibrinolytic Enzymes Activation in Thrombocytes
Jaime González-Cuevas1, Jesus Javier Garcia-Bañuelos1, José Navarro-Partida2, and Juan Armendariz-Borunda1,3 1 Universidad de Guadalajara, Mexico, 2Universidad Autónoma de Nayarit, Mexico, 3O.P.D. Hospital Civil de Guadalajara, Guadalajara, Mexico Background: Therapeutic effectiveness of platelet rich plasma (PRP) is related to technical procedures for procurement, storage conditions and aging. the maintenance of PRP until use involves avoiding platelet activation. It has been demonstrated that platelet activation and aggregation are regulated by metalloproteases (MMPs) and plasmin. This work focuses on elucidating activation and of oxidative damage of platelets during storage. Methods: Platelet rich plasmas were obtained from 110 healthy donors from a transfusable medical service and kept under stirring at a temperature of 20-Û& 3URWHLQ H[WUDFWLRQ ZDV performed from platelets homogenates at different times of
6)5%0