OBJECTIVE: A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. METHODS: HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. RESULTS: This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. CONCLUSIONS: This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.
- MeSH
- antitumorózní látky škodlivé účinky farmakologie MeSH
- chronická myeloidní leukemie * farmakoterapie metabolismus MeSH
- energetický metabolismus účinky léků MeSH
- imatinib mesylát * farmakologie škodlivé účinky MeSH
- inhibitory proteinkinas farmakologie škodlivé účinky MeSH
- karnitin * metabolismus farmakologie MeSH
- lidé MeSH
- mitochondrie metabolismus účinky léků MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- rodina nosičů rozpuštěných látek 22, člen 5 * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Atherosclerosis and thrombosis are underlying causes of acute coronary syndrome and stroke. In clinical practice, several markers (such as LDL, HDL cholesterol, glycaemia, C-reactive protein etc.) of elevated risk are available, however, translation of the population risk to personal risk is rather questionable. For this reason, additional information to explain such condition is of a great interest. Advanced instrumental technique represented by high performance liquid chromatography coupled to high-resolution tandem mass spectrometry will enable, based on chemometric assessment fingerprints of metabolome components, to identify the differences between patients ́ groups that are not evident when employing conventionally measured parameters. Blood samples will be obtained from patients with acute and subacute phase of acute coronary syndrome and stroke. The relationship between the generated data and the degree of oxidative stress will be searched. The possibly detected changes may represent the interface between the atherosclerosis and thrombosis.
Ateroskleróza a trombóza jsou podkladem akutního koronárního syndromu a cévní mozkové příhody. V klinické praxi se ke stratifikaci rizika používají různé markery (LDL, HDL cholesterol, glykémie, C-reaktivní protein atd.), nicméně transformace populačního do individuálního rizika může být diskutabilní. Přes „normální“ hodnoty těchto markerů je stále přítomno riziko příhody. Pokročilé techniky reprezentované ultraúčinnou kapalinovou chromatografií spojenou s tandemovou vysokorozlišovací hmotnostní spektrometrií umožní, na základě chemometricky vyhodnocených záznamů ́fingerprintů ́ složek metabolomu, hledání rozdílů mezi skupinami pacientů, které se v základních, běžně měřených parametrech, významně neliší. Vzorky krve budou odebírány pacientům v akutní a subakutní fází akutního koronárního syndromu a cévní mozkové příhody a budou srovnávány s kontrolní skupinou. Výsledky budou vztaženy ke stupni oxidačního stresu. Provedený předběžný screening ukázal změny především na úrovni lipidomu, které mohou reprezentovat spojnici mezi aterosklerózou a aterotrombózou.
- Klíčová slova
- ateroskleróza, atherosclerosis, oxidační stres, oxidative stress, cévní mozková příhoda, Akutní koronární syndrom, Acute coronary syndrome, Hmotnostní spektrometrie, Mass Spectrometry, aterotrombóza, lipidomika, fibrinogen, atherothrombosis, acute stroke, lipidomics, fibrinogen,
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease-myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.
- MeSH
- akutní myeloidní leukemie * MeSH
- biosenzitivní techniky * MeSH
- glykoproteiny metabolismus MeSH
- glykosylace MeSH
- krevní plazma metabolismus MeSH
- lektiny MeSH
- lidé MeSH
- myelodysplastické syndromy * terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background: Predicting stroke risk in patients with carotid artery stenosis (CS) remains challenging. Circulating biomarkers seem to provide improvements with respect to risk stratification. Methods: Study patients who underwent carotid endarterectomy were categorized into four groups according to symptomatology and compared as follows: symptomatic with asymptomatic patients; and asymptomatic patients including amaurosis fugax (AF) (asymptomatic + AF group) with patients with a transient ischemic attack (TIA) or brain stroke (BS) (hemispheric brain stroke group). Carotid specimens were histologically analyzed and classified based on the American Heart Classification (AHA) standard. As a marker of OS, the plasma levels of malondialdehyde (MDA) were measured. Comparisons of MDA plasma levels between groups were analyzed. Results: In total, 35 patients were included in the study. There were 22 (63%) patients in the asymptomatic group and 13 (37%) in the symptomatic group. Atheromatous plaque (p = 0.03) and old hemorrhage (p = 0.05), fibrous plaque (p = 0.04), myxoid changes (p = 0.02), plaques without hemorrhage (p = 0.04), significant neovascularization (p = 0.04) and AHA classification (p = 0.006) had significant correlations with clinical presentation. There were 26 (74%) patients in the asymptomatic group and 9 (26%) in the hemispheric brain stroke group. Atheromatous plaque (p = 0.02), old hemorrhage (p = 0.05) and plaques without neovascularization (p = 0.02), fibrous plaque (p = 0.03), plaques without hemorrhage (p = 0.02) and AHA classification (p = 0.01) had significant correlations with clinical presentation. There was no significant difference between symptomatic and asymptomatic groups with respect to MDA plasma levels (p = 0.232). A significant difference was observed when MDA plasma levels were compared to asymptomatic + AF and the hemispheric stroke group (p = 0.002). Conclusions: MDA plasma level correlates with the risk of hemispheric stroke (TIA or BS) and is a reliable marker of plaque vulnerability in carotid artery stenosis.
- Publikační typ
- časopisecké články MeSH
We describe the internal structure, spatial organization and dynamic formation of coronary artery thrombi from ST-segment elevation myocardial infarction patients. Scanning electron microscopy (SEM) revealed significant differences among four groups of patients (<2 hours; 2-6 hours; 6-12 hours, and >12 hours) related to the time of ischemia. Coronary artery thrombi from patients presenting less than 2 hours after the infarction were almost entirely composed of platelets, with small amounts of fibrin and red blood cells. In contrast, thrombi from late presenters (>12 hours) consisted of mainly platelets at the distal end, where clotting was initiated, with almost no platelets at the proximal end, while the red blood cell content went from low at the initiating end to more than 90% at the proximal end. Furthermore, fibrin was present mainly on the outside of the thrombi and older thrombi contained thicker fibers. The red blood cells in late thrombi were compressed to a close-packed, tessellated array of polyhedral structures, called polyhedrocytes. Moreover, there was redistribution from the originally homogeneous composition to fibrin and platelets to the outside, with polyhedrocytes on the interior. The presence of polyhedrocytes and the redistribution of components are signs of in vivo clot contraction (or retraction). These results suggest why later thrombi are resistant to fibrinolytic agents and other treatment modalities, since the close-packed polyhedrocytes form a nearly impermeable seal. Furthermore, it is of particular clinical significance that these findings suggest specific disparate therapies that will be most effective at different stages of thrombus development.
- MeSH
- čas zasáhnout při rozvinutí nemoci MeSH
- časové faktory MeSH
- erytrocyty patologie MeSH
- fibrin analýza MeSH
- fibrinolytika * aplikace a dávkování škodlivé účinky MeSH
- hemokoagulace účinky léků fyziologie MeSH
- infarkt myokardu s elevacemi ST úseků * etiologie terapie MeSH
- koronární trombóza * diagnostické zobrazování farmakoterapie metabolismus patologie MeSH
- léková rezistence fyziologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací metody MeSH
- trombektomie metody MeSH
- trombocyty patologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Here, we present the first case of fibrinogen variant FGG c.8G>A. We investigated the behaviour of this mutated fibrinogen in blood coagulation using fibrin polymerization, fibrinolysis, fibrinopeptides release measurement, mass spectrometry (MS), and scanning electron microscopy (SEM). The case was identified by routine coagulation testing of a 34-year-old man diagnosed with thrombosis. Initial genetic analysis revealed a heterozygous mutation in exon 1 of the FGG gene encoding gamma chain signal peptide. Fibrin polymerization by thrombin and reptilase showed the normal formation of the fibrin clot. However, maximal absorbance within polymerization was lower and fibrinolysis had a longer degradation phase than healthy control. SEM revealed a significant difference in clot structure of the patient, and interestingly, MS detected several posttranslational oxidations of fibrinogen. The data suggest that the mutation FGG c.8G>A with the combination of the effect of posttranslational modifications causes a novel case of hypofibrinogenemia associated with thrombosis.
- MeSH
- afibrinogenemie * komplikace genetika MeSH
- dospělí MeSH
- fibrin metabolismus MeSH
- fibrinogen genetika metabolismus MeSH
- fibrinogeny abnormální * genetika metabolismus MeSH
- hemostatika * MeSH
- lidé MeSH
- oxidační stres MeSH
- posttranslační úpravy proteinů MeSH
- trombóza * komplikace genetika MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Congenital fibrinogen disorders are caused by mutations in genes coding for fibrinogen and may lead to various clinical phenotypes. Here, we present a functional and structural analysis of 4 novel variants located in the FGB gene coding for fibrinogen Bβ chain-heterozygous missense BβY416C and BβA68S, homozygous nonsense BβY345*, and heterozygous nonsense BβW403* mutations. The cases were identified by coagulation screening tests and further investigated by various methods. Fibrin polymerization had abnormal development with decreased maximal absorbance in all patients. Plasmin-induced fibrin degradation revealed different lytic phases of BβY416C and BβW403* than those of the control. Fibrinopeptide cleavage measured by reverse phase high pressure liquid chromatography of BβA68S showed impaired release of fibrinopeptide B. Morphological properties, studied through scanning electron microscopy, differed significantly in the fiber thickness of BβY416C, BβA68S, and BβW403*, and in the fiber density of BβY416C and BβW403*. Finally, homology modeling of BβA68S showed that mutation caused negligible alternations in the protein structure. In conclusion, all mutations altered the correct fibrinogen function or structure that led to congenital fibrinogen disorders.
- MeSH
- afibrinogenemie krev diagnóza genetika MeSH
- fenotyp * MeSH
- fibrinogen chemie genetika metabolismus MeSH
- genetická predispozice k nemoci * MeSH
- genetické asociační studie MeSH
- hemokoagulace MeSH
- konformace proteinů MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- molekulární modely MeSH
- mutace * MeSH
- mutační analýza DNA MeSH
- novorozenec MeSH
- senioři MeSH
- vyšetření krevní srážlivosti MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
Fibrinogen, an abundant plasma glycoprotein, is involved in the final stage of blood coagulation. Decreased fibrinogen levels, which may be caused by mutations, are manifested mainly in bleeding and thrombotic disorders. Clinically relevant mutations of fibrinogen are listed in the Human Fibrinogen Database. For the αC-connector (amino acids Aα240-410, nascent chain numbering), we have extended this database, with detailed descriptions of the clinical manifestations among members of reported families. This includes the specification of bleeding and thrombotic events and results of coagulation assays. Where available, the impact of a mutation on clotting and fibrinolysis is reported. The collected data show that the Human Fibrinogen Database reports considerably fewer missense and synonymous mutations than the general COSMIC and dbSNP databases. Homozygous nonsense or frameshift mutations in the αC-connector are responsible for most clinically relevant symptoms, while heterozygous mutations are often asymptomatic. Symptomatic subjects suffer from bleeding and, less frequently, from thrombotic events. Miscarriages within the first trimester and prolonged wound healing were reported in a few subjects. All mutations inducing thrombotic phenotypes are located at the identical positions within the consensus sequence of the tandem repeats.
Alterations in lipid metabolism mediated by oxidative stress play a key role in the process of atherosclerosis and superimposed thrombosis; these can lead to acute coronary syndrome (ACS) and acute ischemic stroke (AIS). Multiple studies have shown that the formation of atheromatous lesions is initiated by oxidation of low-density lipoproteins incorporated into the intima of the vessel wall. Here, we studied lipids in plasma samples from three cohorts: 61 patients with ACS (group A), 49 patients with AIS (group D), and 82 controls (group K). Untargeted lipidomics based on high-performance liquid chromatography coupled to mass spectrometry (UHPLC-HRMS) was employed to obtain comprehensive information on whether relationships exist between these patient categories based on lipid patterns. In addition, malondialdehyde (MDA) as a standard marker of oxidative stress was monitored. The most characteristic lipids in group K were fatty acyls of hydroxyfatty acids (FAHFAs). As expected, MDA concentrations were the lowest in group K. Our findings can better explain ongoing pathologies, both acute and chronic, with the potential for future diagnosis and treatment.
- Publikační typ
- časopisecké články MeSH
During coagulation, the soluble fibrinogen is converted into insoluble fibrin. Fibrinogen is a multifunctional plasma protein, which is essential for hemostasis. Various oxidative posttranslational modifications influence fibrinogen structure as well as interactions between various partners in the coagulation process. The aim was to examine the effects of oxidative stress conditions on fibrin clot formation in arterial atherothrombotic disorders. We studied the changes in in vitro fibrin network formation in three groups of patients-with acute coronary syndrome (ACS), with significant carotid artery stenosis (SCAS), and with acute ischemic stroke (AIS), as well as a control group. The level of oxidative stress marker malondialdehyde measured by LC-MS/MS was higher in SCAS and AIS patients compared with controls. Turbidic methods revealed a higher final optical density and a prolonged lysis time in the clots of these patients. Electron microscopy was used to visualize changes in the in vitro-formed fibrin network. Fibers from patients with AIS were significantly thicker in comparison with control and ACS fibers. The number of fibrin fibers in patients with AIS was significantly lower in comparison with ACS and control groups. Thus, oxidative stress-mediated changes in fibrin clot formation, structure and dissolution may affect the effectiveness of thrombolytic therapy.
- Publikační typ
- časopisecké články MeSH
