Development of Impaired Cytochrome C Oxidase Activity In Apheresis Platelets During Storage

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3347-3347
Author(s):  
Yaser A. Diab ◽  
Adia Thomas ◽  
Naomi L.C. Luban ◽  
Edward Wong ◽  
Stephen J. Wagner ◽  
...  
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Conflicts Of Interest ◽  
Atp Depletion ◽  
In Vitro Assays ◽  
P Value ◽  
Soluble Cd40 Ligand

Abstract Abstract 3347 Background: With current storage requirements, the shelf life of platelet (PLT) products is largely limited by the development of deleterious in vitro changes associated with overall reduction in therapeutic efficacy collectively known as the “Platelet Storage Lesion” (PSL). PSL is characterized by a number of biochemical changes including lactate accumulation, bicarbonate depletion and a fall in pH. We hypothesize that these changes reflect a state of impaired oxidative phosphorylation associated with increased reliance on anaerobic glycolysis that evolves during PLT storage. In this study we evaluated the function and expression of “Cytochrome C Oxidase” (COX), a key mitochondrial enzyme in oxidative phosphorylation, with relation to several in vitro markers of PSL. The studies were performed in apheresis PLT stored for up to 7 days under standard blood bank conditions. Methods: Apheresis PLT concentrates were collected in 100% plasma using a Trima Cell Separator (CaridianBCT, Lakewood CO) to provide products with at least 3 × 1011 PLT. All products were collected in Trima® storage bags and were stored in a flatbed reciprocal agitator at 22 ± 2°C for 7 days. Multiple standard in vitro assays were performed on Days 0 (baseline measurement), 2, 4 and 7 of storage including PLT count, mean PLT volume, pH, pO2, pCO2, bicarbonate, lactate & glucose levels, aggregation and ATP release studies (ADP/Collagen), soluble CD40 ligand levels (supernatant) and total intracellular PLT ATP content. In addition, steady state COX kinetics and protein immunoblotting for COX subunits I and IV, were performed using isolated PLT mitochondria from simultaneously collected samples. Data are reported as mean ± 2 SD (n =10 experiments). One-way Analysis of Variance (ANOVA) and post-hoc Tukey's range test were used to compare data obtained at different time points. Differences were considered statistically significant only if the p value was <0.001 taking into the account that performing such large number of tests in the study increased the probability that a significant p value was incorrectly obtained by random chance. Results: PLT COX function declined significantly throughout storage (Table). Steady-state levels of COX I and IV remained essentially unchanged. This decrease in COX function paralleled progressive ATP depletion and time-dependent changes that were consistent with the development of the PSL. Conclusion: During storage of apheresis PLT for 7 days, COX function decreased progressively in association with ATP depletion indicating acquired impairment in oxidative phosphorylation. These findings suggest that bioenergetic failure is associated with PSL. Further studies are required to determine if mitochondrial dysfunction is a cause of PSL, and if it can be prevented or is amenable to therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Sulfide inhibition of and metabolism by cytochrome c oxidase

2013 ◽  
Vol 41 (5) ◽  
pp. 1312-1316 ◽  
Author(s):  
Peter Nicholls ◽  
Doug C. Marshall ◽  
Chris E. Cooper ◽  
Mike T. Wilson
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Sulfide Oxidation ◽  
Oxidase Inhibitor ◽  
Common Features ◽  
Sulfide Inhibition ◽  
Electron Reduction

Hydrogen sulfide (H2S), a classic cytochrome c oxidase inhibitor, is also an in vitro oxidase substrate and an in vivo candidate hormonal (‘gasotransmitter’) species affecting sleep and hibernation. H2S, nitric oxide (NO) and carbon monoxide (CO) share some common features. All are low-molecular-mass physiological effectors and also oxidase inhibitors, capable of binding more than one enzyme site, and each is an oxidizable ‘substrate’. The oxidase oxidizes CO to CO2, NO to nitrite and sulfide to probable persulfide species. Mitochondrial cytochrome c oxidase in an aerobic steady state with ascorbate and cytochrome c is rapidly inhibited by sulfide in a biphasic manner. At least two successive inhibited species are involved, probably partially reduced. The oxidized enzyme, in the absence of turnover, occurs in at least two forms: the ‘pulsed’ and ‘resting’ states. The pulsed form reacts aerobically with sulfide to form two intermediates, ‘P’ and ‘F’, otherwise involved in the reaction of oxygen with reduced enzyme. Sulfide can directly reduce the oxygen-reactive a3CuB binuclear centre in the pulsed state. The resting enzyme does not undergo such a step, but only a very slow one-electron reduction of the electron-transferring haem a. In final reactivation phases, both the steady-state inhibition of catalysis and the accumulation of P and F states are reversed by slow sulfide oxidation. A model for this complex reaction pattern is presented.

Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria

1977 ◽  
Vol 55 (7) ◽  
pp. 706-713 ◽  
Author(s):  
Lars Chr. Petersen ◽  
Hans Degn ◽  
Peter Nicholls
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Concentration ◽  
Respiration Rate ◽  
Redox State ◽  
Rat Liver Mitochondria ◽  
Excess Oxygen ◽  
Succinate Oxidation ◽  
State Reduction

1. Coupled, cytochrome-c-depleted ('stripped') rat liver mitochondria reducing oxygen in the presence of exogenous cytochrome c, with succinate or ascorbate as substrates, show marked declines in the steady-state reduction of cytochrome c in excess oxygen on addition of uncouplers. Calculated ratios of maximal turnover in the uncoupled state and in the energized state for the cytochrome c oxidase (EC 1.9.3.1) reaction lie between 3 and 6, as obtained with reconstituted oxidase-containing vesicles. The succinate-cytochrome c reductase activity in such mitochondria shows a smaller response to uncoupler than that of the oxidase.2. The respiration rates of uncoupled mitochondria oxidizing ascorbate in the presence of added cytochrome c follow a Michaelis–Menten relationship with respect to oxygen concentration, in accordance with the pattern found previously with the solubilized oxidase. But succinate oxidation tends to give nonlinear concave-upward double-reciprocal plots of respiration rate against oxygen concentration, in accordance with the pattern found previously with intact uncoupled mitochondria.3. From simultaneous measurements of cytochrome c steady-state reduction, respiration rate, and oxygen concentration during succinate oxidation under uncoupled conditions it is found that at full reduction of cytochrome c, apparent Km for oxygen is 0.9 μM and the maximal oxidase (aa3) turnover is 400 s−1 (pH 7.4, 30 °C).4. The redox state of cytochrome c in uncoupled systems reflects a simple steady state; the redox state of cytochrome c in energized systems tends towards an equilibrium condition with the terminal cytochrome a3, whose apparent potential under these conditions is more negative than that of cytochrome c.

scholarly journals Culturing embryonic cells from the parthenogenetic clonal marble crayfish (Marmorkrebs) Procambarus virginalis Lyko, 2017 (Decapoda: Astacidea: Cambaridae)

2019 ◽  
Vol 39 (6) ◽  
pp. 758-763
Author(s):  
Heriberto Deleon ◽  
Juan Garcia ◽  
Dionn Carlo Silva ◽  
Oscar Quintanilla ◽  
Zen Faulkes ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Early Stage ◽  
Model Organism ◽  
Coi Gene ◽  
Embryonic Cells ◽  
Oxidase Subunit ◽  
Marbled Crayfish

Abstract The parthenogenetic marbled crayfish, or Marmorkrebs (Procambarus virginalis Lyko 2017), is an emerging model organism. We describe a method to isolate cells from early-stage embryos and culture them in vitro. The identity of the cells was confirmed by sequencing the cytochrome c oxidase subunit I (COI) gene. This technique can be applied for use in the manipulation of embryonic parthenogenetic crayfish cells.

scholarly journals Titration and steady-state behaviour of the 830 nm chromophore in cytochrome c oxidase

1982 ◽  
Vol 203 (3) ◽  
pp. 541-549 ◽  
Author(s):  
P Nicholls ◽  
G A Chanady
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Lag Phase ◽  
State Reduction ◽  
Cytochrome Aa3 ◽  
System A ◽  
Reducing Equivalent ◽  
State Behaviour ◽  
Copper Excess

Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN'N'-tetramethyl-p-phenylenediamine initially introduces two reducing equivalents per mol of cytochrome aa3. The first equivalent reduces the cytochrome a haem iron; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (e.p.r.-detectable copper) but is probably required for reduction of the e.p.r.-undetectable copper. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with ab increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. It is concluded that the e.p.r.-undetectable copper (CuD) is reduced early in the reaction sequence, whereas the detectable copper (CUD) is reduced late. The latter species is probably that responsible for reduction of the cytochrome a3 haem. The magnetic association between undetectable copper and the a3 haem may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.

The Effects of Anti-A and Anti-B On Platelet Function: An in Vitro Model of ABO Non-Identical Transfusion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2120-2120
Author(s):  
Majed A. Refaai ◽  
Neil Blumberg ◽  
Charles W. Francis ◽  
Richard Phipps ◽  
Sherry Spinelli ◽  
...  
Keyword(s):  
Normal Saline ◽  
Conflicts Of Interest ◽  
Platelet Rich Plasma ◽  
Type A ◽  
P Value ◽  
Blood Product Transfusion ◽  
Trauma Patients ◽  
Type B ◽  
Average Percentage

Abstract Abstract 2120 Poster Board II-97 Background: Transfusion of ABO non-identical red blood cells (RBCs) can cause immune mediated hemolytic transfusion reactions. Therefore, only ABO identical RBCs are transfused, except in emergencies, when group O RBCs are transfused. Use of exclusively ABO identical plasma and platelet (PLT) transfusions is not uniformly practiced nor always feasible despite reports of hemolytic reactions. Since PLTs and soluble plasma proteins possess A and B antigens, ABO non-identical PLTs could, theoretically, be activated and/or rendered hypofunctional by anti-A and anti-B antibodies (Abs) in transfused or recipient plasma. Recent findings demonstrate that transfusion of ABO non-identical PLTs is associated with increased bleeding in surgical patients and patients with leukemia. Blunt trauma patients who received at least one ABO non-identical blood product transfusion demonstrated a significantly higher RBC usage (12.3 ± 6.9 SD versus 8.4 ± 9.9 SD, p-value 0.0011) compared to those patients who received only ABO identical transfusions (Transfusion. 2007;47:192A). ABO identical PLT transfusions in leukemia patients were a significant predictor of survival (Leukemia. 2008;22:631-5). In a multi center retrospective analysis of more than one million cancer patients over a period of 9 years, Khorana et al. demonstrated an overall venous thromboembolism (VTE) rate of 4.1%. In multivariate risk factor analysis, the association between blood transfusions and VTE had an odds ratio of 1.35 (1.31-1.39, 95% CI) with a p value of < 0.001 (Arch Intern Med. 2008;168:2377-81). We hypothesized that PLTs activated by ABO Abs might have altered function. Methods and Materials: PLT function was evaluated by testing aggregation in platelet rich plasma (PRP). Aggregation was performed with PRP from 7 type A and 6 type B normal blood donors following a 10 min incubation period at 37°C with either normal saline, group O or AB plasma. PLTs were activated by 20 mM ADP and aggregation quantitated from the maximum change in OD. Similar experiments were repeated utilizing different titration of the commercial anti-A and anti-B anti-sera. Results: Following incubation with O plasma, PLT aggregation was inhibited by a mean of 38% and 18% for group A and B PLTs, respectively (P ≤ 0.005) (Figure). A trend toward inhibition was observed when type A PLTs were incubated with control AB plasma (average of 14%, P = 0.187), whereas type B PLT showed no inhibition when incubated with AB plasma (P = 0.939) (Table 1). PLT aggregation with the anti-sera showed gradual inhibition correlated with the antibody titer (Table 2). Conclusion: Mediators in group O plasma, most likely anti-A and anti-B Abs, cause impaired PLT aggregation in ABO non-identical PLTs. These in vitro findings may explain, at least in part, clinical observations that patients receiving ABO non-identical PLT transfusions experience more bleeding than recipients of ABO identical PLT transfusions. Table 1: PLT aggregation of A and B PRP with saline, O and AB plasma. Blood Donor Type N Average Percentage of Platelet Aggregation (SD) Normal Saline “O” Plasma P value* “AB” Plasma P value A 7 92 (7.4) 54 (9.9) < 0.005 78 (2.9) 0.187 B 6 85 (6.8) 67 (9.8) 0.005 85.3 (7.9) 0.939 P value < 0.05 is considered statistically significant. Figure: PLT function of type A PRP incubated for 10 min at 37°C with O or AB plasma, or normal saline. Figure:. PLT function of type A PRP incubated for 10 min at 37°C with O or AB plasma, or normal saline. Table 2: PLT aggregation of A and B PRP with different titration of the commercial anti-A and anti-B anti-sera. Anti-sera/Plasma Type A PRP P value Type B PRP P value Baseline 93.7 (3.1) — 83.4 (11) — 1:1024 48.7 (8.5) 0.006 36.3 (7.8) 0.0005 1:512 57.3 (2.5) 0.0001 47.7 (7.5) 0.002 1:256 59.5 (3.5) 0.008 59.5 (0.7) 0.002 1:128 55.5 (3.5) 0.006 67 (2.8) 0.027 AB plasma 87.7 (3.2) 0.08 81.2 (16) 0.88 Disclosures: No relevant conflicts of interest to declare.

DNMT3A Mutation Leads to Hematopoietic Dysregulation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2382-2382
Author(s):  
Jie Xu ◽  
Wei-na Zhang ◽  
Tao Zhen ◽  
Yang Li ◽  
Jing-yi Shi ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modification ◽  
Hematopoietic Cells ◽  
In Vitro Assays ◽  
Clinical Samples ◽  
Hematopoietic Stem

Abstract Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

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