cytochrome-c-t and Thrombocytopenia

Thrombocytopenia (TP) Cargeeg is a unique autosomal dominant disorder, affecting a seven-generation family, caused by cytochrome C (CYCS) mutation that dysregulates platelet formation. The CYCS mutation in this disorder is a glycine 41 replacement by serine, which yields a cytochrome C variant with enhanced apoptotic pathway activity in vitro. The deregulated apoptosis in this disorder affects megakaryocytes (MK) during platelet formation, leading to early and ectopic platelet release in the bone marrow (BM). Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome C activity is not a critical regulator of physiological apoptosis in most cells. The pathophysiology of this unique inherited TP, with unaltered platelet survival and normal MK content in the BM, has implications for physiological and pathological mechanisms altering MK apoptosis, with implications for other unexplained thrombocytopenic disorders.

Topics: Apoptosis; Base Sequence; Cytochromes c; Female; Humans; Male; Megakaryocytes; Mutation, Missense; Pedigree; Thrombocytopenia

Platelet concentrate (PC) transfusion is administrated to reduce the hemostatic complications in patients with thrombocytopenia. Strength platelet against oxidative stress conditions lead to decrease in platelet storage lesion (PSL). This study was aimed to evaluate L-carnitine (LC) effects on platelet oxidative stress and platelet apoptosis during storage time. PC bags were randomly selected and each bag was divided into two equal parts. L-carnitine was added to test groups. Normal saline was added to control groups. Platelets count, mean platelet volume (MPV), pH, Platelet aggregation, nitric oxide metabolism (nitric/nitrate), total antioxidant capacity (TAC), malondealdehyde concentration (MDA), lactate dehydrogenase (LDH) enzyme activity, mitochondrial reactive oxygen species (ROS) and cytochrome C releasing were assayed by standard methods in 1, 3, 5 and 7 days of platelet storage. LDH enzyme activity was increased during storage but it had lower level in L-carnitine-treated platelets. LC treatment led to reduction in MDA concentration (3.35 ± 0.98 vs 5.3 ± 1.32, p = 0.003 and 6.52 ± 1.88 vs 5.67 ± 1.25, p = 0.005 for day 5 and day 7 respectively). Increased level of TAC was detected in LC-treated platelets in comparison to control (0.29 ± 0.06 vs 0.21 ± 0.05, p = 0.008 and 0.22 ± 0.03 vs 0.16 ± 0.03, p = 0.003 for day 5 and day 7 respectively). Interestingly, mitochondrial ROS and cytochrome C releasing was significantly lower in LC-treated versus control group during platelet storage. L-carnitine not only decreases mitochondrial ROS but also reduces cytochrome C releasing in PCs during storage. It might be considered as safe additive to decrease PSL in the future.

Topics: Apoptosis; Blood Platelets; Blood Preservation; Carnitine; Cytochromes c; Humans; Oxidative Stress; Platelet Transfusion; Protective Agents; Reactive Oxygen Species; Thrombocytopenia

Mitochondrial cytochrome c is associated with electron transfer in the respiratory chain and in apoptosis. Four cytochrome c variants have been identified in families that suffer from mild autosomal dominant thrombocytopenia, a platelet disorder associated with increased apoptosis. Three out of the four substitutions, G41S, Y48H and A51V are located on the 40-57 Ω-loop. The G41S and Y48H variants perturb key physicochemical and dynamic properties that result in enhanced functional features associated with apoptotic activity. Herein we characterise the ferric A51V variant. We show by chemical denaturation that this variant causes the native state to be destabilized. Through azide binding kinetics, the population of a pentacoordinate heme form, whereby the Met80 axial ligand is dissociated, is estimated to be of equal magnitude to that found in the Y48H variant. This pentacoordinate form gives rise to peroxidase activity, which despite the similar pentacoordinate population of the A51V variant to that of the Y48H variant, the peroxidase activity of the A51V variant is suppressed. Far-UV circular dichroism spectroscopy and pH jump studies, suggest that a combination of structural and dynamic features in addition to the population of the pentacoordinate form regulate peroxidase activity in these disease variants. Additionally, the steady-state ratio of ferric/ferrous cytochrome c when in turnover with cytochrome c oxidase has been investigated for all 40-57 Ω-loop variants. These studies show that the lower pK

Topics: Animals; Apoptosis; Cattle; Cell Respiration; Cytochromes c; Electrons; Heme; Humans; Iron; Molecular Dynamics Simulation; Mutation, Missense; Peroxidase; Thrombocytopenia

Topics: Alleles; Amino Acid Substitution; Cytochromes c; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Variation; Genotype; Humans; Models, Molecular; Mutation; Phenotype; Platelet Count; Protein Conformation; Structure-Activity Relationship; Thrombocytopenia

Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71-85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (p K

Topics: Alkalies; Cytochromes c; Humans; Hydrogen-Ion Concentration; Isomerism; Kinetics; Models, Molecular; Peroxidase; Point Mutation; Protein Conformation; Protein Denaturation; Protein Stability; Thrombocytopenia

We report a patient with thrombocytopenia from a Japanese family with hemophilia A spanning four generations. Various etiologies of thrombocytopenia, including genetic, immunological, and hematopoietic abnormalities, determine the prognosis for this disease. In this study, we identified a novel heterozygous mutation in a gene encoding cytochrome c, somatic (CYCS, MIM123970) using whole exome sequencing. This variant (c.301_303del:p.Lys101del) is located in the α-helix of the cytochrome c (CYCS) C-terminal domain. In silico structural analysis suggested that this mutation results in protein folding instability. CYCS is one of the key factors regulating the intrinsic apoptotic pathway and the mitochondrial respiratory chain. Using the yeast model system, we clearly demonstrated that this one amino acid deletion (in-frame) resulted in significantly reduced cytochrome c protein expression and functional defects in the mitochondrial respiratory chain, indicating that the loss of function of cytochrome c underlies thrombocytopenia. The clinical features of known CYCS variants have been reported to be confined to mild or asymptomatic thrombocytopenia, as was observed for the patient in our study. This study clearly demonstrates that thrombocytopenia can result from CYCS loss-of-function variants.

Topics: Amino Acid Substitution; Biomarkers; Cytochromes c; Cytokines; DNA Mutational Analysis; Female; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Japan; Male; Middle Aged; Mutation; Pedigree; Protein Conformation, alpha-Helical; Protein Domains; Structure-Activity Relationship; Thrombocytopenia

Naturally occurring mutations to cytochrome c (cyt-c) have been identified recently in patients with mild autosomal dominant thrombocytopenia (low platelet levels), which yield cyt-c mutants with enhanced apoptotic activity. However, the molecular mechanism underlying this low platelet production and enhanced apoptosis remain unclear. Therefore, an attempt is made herein for the first time to investigate the effects of mutations of glycine 41 by serine (G41S) and tyrosine 48 by histidine (Y48H) on the conformational and dynamic changes of apoptotic (Fe

Topics: Binding Sites; Catalytic Domain; Cytochromes c; Humans; Hydrogen Bonding; Molecular Dynamics Simulation; Mutation; Protein Structure, Secondary; Solvents; Thrombocytopenia

Thrombocytopenia Cargeeg is a rare autosomal dominant disorder and one of three thrombocytopenias caused by mutation of cytochrome c (Online Mendelian Inheritance in Man entry THC4). Our previous observations of platelet-like structures in the marrow space and early platelet production in vitro suggested that the low platelet phenotype in Thrombocytopenia Cargeeg subjects is caused by premature release of platelets into non-vascular regions of the bone marrow. We now show that two processes of platelet release occur in Thrombocytopenia Cargeeg subjects. Circulating platelets have a normal marginal microtubule coil, and cultured megakaryocytes derived from peripheral blood cells of Thrombocytopenia Cargeeg subjects form proplatelets normally and release platelets containing a marginal microtubule coil, consistent with effective platelet release via the proplatelet mechanism. In contrast, platelet-like structures within the extravascular bone marrow space have the dimensions of platelets but lack the marginal microtubule coil, suggesting abnormal proplatelet-independent platelet release. The mechanism of extravascular platelet release remains unclear. The failure to recapitulate this mechanism in vitro implies that the phenotype is not simply an intrinsic property of CYCS mutation-carrying megakaryocytes, but is dependent on the interaction between these cells and their environment.

Topics: Blood Platelets; Bone Marrow; Cells, Cultured; Cytochromes c; Humans; Megakaryocytes; Microtubules; Mutation; Thrombocytopenia

It has been shown, both experimentally and clinically, that water-pipe smoke (WPS) exposure adversely affects the cardiovascular system (CVS) through the generation of oxidative stress and inflammation. Betaine, a naturally occurring compound in common foods, has antioxidant and anti-inflammatory actions. However, its potential to mitigate the adverse effect of WPS on the CVS has never been reported before. This is the subject of this study in mice.. Mice were exposed daily for 30 min to either normal air (control), or to WPS for two consecutive weeks. Betaine was administered daily by gavage at a dose of 10mg/kg, 1h before either air or WPS exposure.. Betaine mitigated the in vivo prothrombotic effect of WPS in pial arterioles and venules. Moreover, it reversed the WPS-induced decrease in circulating platelets. Likewise, betaine alleviated platelet aggregation in vitro, and the shortening of activated partial thromboplastin time and prothrombin time induced by WPS. Betaine reduced the increase of plasminogen activator inhibitor-1 and fibrinogen concentrations in plasma induced by WPS. Betaine also diminished the WPS-induced increase of plasma concentrations of interleukin 6 and tumor necrosis factor α, and attenuated the increase of lipid peroxidation and superoxide dismutase. Immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome C by cardiomyocytes of the WPS-exposed mice. These effects were averted by betaine.. Our findings suggest that betaine treatment significantly mitigated WPS-induced hypercoagulability, and inflammation, as well as systemic and cardiac oxidative stress.

Topics: Administration, Oral; Animals; Antioxidants; Betaine; Blood Platelets; Cytochromes c; Fibrinogen; Gene Expression; Interleukin-6; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Nitric Oxide Synthase Type II; Oxidative Stress; Partial Thromboplastin Time; Plasminogen Activator Inhibitor 1; Platelet Aggregation; Primary Cell Culture; Prothrombin Time; Smoking; Superoxide Dismutase; Thrombocytopenia; Thrombosis; Tumor Necrosis Factor-alpha

Inherited thrombocytopenias are heterogeneous diseases caused by at least 20 genes playing different role in the processes of megakaryopoiesis and platelet production. Some forms, such as thrombocytopenia 4 (THC4), are very rare and not well characterized. THC4 is an autosomal dominant mild thrombocytopenia described in only one large family from New Zealand and due to a mutation (G41S) of the somatic isoform of the cytochrome c (CYCS) gene. We report a novel CYCS mutation (Y48H) in patients from an Italian family. Similar to individuals carrying G41S, they have platelets of normal size and morphology, which are only partially reduced in number, but no prolonged bleeding episodes. In order to determine the pathogenetic consequences of Y48H, we studied the effects of the two CYCS mutations in yeast and mouse cellular models. In both cases, we found reduction of respiratory level and increased apoptotic rate, supporting the pathogenetic role of CYCS in thrombocytopenia.

Topics: Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Cells, Cultured; Child, Preschool; Cytochromes c; DNA Mutational Analysis; Embryo, Mammalian; Energy Metabolism; Family Health; Female; Fibroblasts; Humans; Lung; Male; Mice; Molecular Sequence Data; Mutation, Missense; Oxygen Consumption; Pedigree; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Thrombocytopenia

Topics: Adult; Blood Platelets; Cell Size; Cohort Studies; Cytochromes c; Female; Genetic Testing; Humans; Italy; Male; Mutation; Platelet Count; Thrombocytopenia

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Cytochromes c; Female; Genetic Linkage; Humans; Male; Megakaryocytes; Mutation; Oxidation-Reduction; Pedigree; Platelet Count; Serine; Signal Transduction; Thrombocytopenia