Chelation is the rational treatment modality in metal overload conditions, but chelators are often non-selective and can, hence, cause an imbalance in the homeostasis of physiological metals including calcium and magnesium. The aim of this study was to develop an affordable, rapid but sensitive and precise method for determining the degree of chelation of calcium and magnesium ions and to employ this method for comparison on a panel of known metal chelators. Spectrophotometric method using o-cresolphthalein complexone (o-CC) was developed and its biological relevance was confirmed in human platelets by impedance aggregometry. The lowest detectable concentration of calcium and magnesium ions by o-CC was 2.5 μM and 2 μM, respectively. The indicator was stable for at least 110 days. Four and seven out of twenty-one chelators strongly chelated calcium and magnesium ions, respectively. Importantly, the chelation effect of clinically used chelators was not negligible. Structure-activity relationships for eight quinolin-8-ols showed improvements in chelation particularly in the cases of dihalogen substitution, and a negative linear relationship between pKa and magnesium chelation was observed. Calcium chelation led to inhibition of platelet aggregation in concentrations corresponding to the complex formation. A novel method for screening of efficacy and safety of calcium and magnesium ion chelation was developed and validated.

• MeSH
• agregace trombocytů účinky léků   MeSH
• chelátory * chemie   MeSH
• hořčík * chemie   MeSH
• lidé MeSH
• preklinické hodnocení léčiv metody   MeSH
• trombocyty účinky léků   metabolismus   MeSH
• vápník * analýza   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

BACKGROUND AND PURPOSE: Although the amphiphilic nature of the widely used antithrombotic drug Ticagrelor is well known, it was never considered as a membranotropic agent capable of interacting with the lipid bilayer in a receptor-independent way. In this study, we investigated the influence of Ticagrelor on plasma membrane lipid order in platelets and if this modulates the potency of Ticagrelor at the P2Y12 receptor. EXPERIMENTAL APPROACH: We combined fluorescent in situ, in vitro and in silico approaches to probe the interactions between the plasma membrane of platelets and Ticagrelor. The influence of Ticagrelor on the lipid order of the platelet plasma membrane and large unilamellar vesicles was studied using the advanced fluorescent probe NR12S. Furthermore, the properties of model lipid bilayers in the presence of Ticagrelor were characterized by molecular dynamics simulations. Finally, the influence of an increased lipid order on the dose-response of platelets to Ticagrelor was studied. KEY RESULTS: Ticagrelor incorporates spontaneously into lipid bilayers and affects the lipid order of the membranes of model vesicles and isolated platelets, in a nontrivial composition and concentration-dependent manner. We showed that higher plasma membrane lipid order in platelets leads to a lower IC50 value for Ticagrelor. It is shown that membrane incorporation of Ticagrelor increases its potency at the P2Y12 receptor, by increasing the order of the platelet plasma membrane. CONCLUSION AND IMPLICATIONS: A novel dual mechanism of Ticagrelor action is suggested that combines direct binding to P2Y12 receptor with simultaneous modulation of receptor-lipid microenvironment.

• MeSH
• buněčná membrána * účinky léků   metabolismus   MeSH
• lidé MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• membránové lipidy metabolismus   MeSH
• purinergní receptory P2Y - antagonisté farmakologie   chemie   MeSH
• purinergní receptory P2Y12 * metabolismus   účinky léků   MeSH
• simulace molekulární dynamiky MeSH
• ticagrelor * farmakologie   chemie   MeSH
• trombocyty * účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND AND AIMS: It is well known that elevated cholesterol is associated with enhanced platelet aggregation and patients suffering from familial hypercholesterolemia (FH) have a high risk of thrombotic cardiovascular events. Although decreasing cholesterol level is associated with attenuation of platelet hyperactivity, there are currently no data on the effect of convertase subtilisin/kexin type 9 monoclonal antibodies (PCSK9ab) on platelet reactivity in FH. The aim of the study was to analyse the impact of different therapies including PCSK9ab on platelet aggregation in FH. METHODS: This study enrolled all 15 patients treated in the University Hospital Hradec Králové for FH. PCSK9ab have been administered in 12 of 15 patients while 8 patients were also undergoing lipid apheresis. Blood samples from all patients including pre- and post-apheresis period were tested for platelet aggregation triggered by 7 inducers, and the effect of 3 clinically used drugs (acetylsalicylic acid, ticagrelor and vorapaxar) was compared as well. RESULTS: Although apheresis decreased the reactivity of platelets in general, platelet responses were not different between non-apheresis patients treated with PCSK9ab and apheresis patients (post-apheresis values) with the exception of ristocetin. However, when compared to age-matched healthy population, FH patients had significantly lower platelet aggregation responses to 4 out of 7 used inducers and higher profit from 2 out of 3 used antiplatelet drugs even after exclusion of FH patients regularly receiving conventional antiplatelet treatment. CONCLUSION: This study showed for the first time the suitability of PCSK9ab treatment for reduction of platelet reactivity in FH patients.

• MeSH
• agregace trombocytů * účinky léků   MeSH
• dospělí MeSH
• hyperlipoproteinemie typ II * krev   terapie   farmakoterapie   MeSH
• inhibitory agregace trombocytů * terapeutické užití   farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• monoklonální protilátky terapeutické užití   farmakologie   MeSH
• PCSK9 inhibitory * MeSH
• proproteinkonvertasa subtilisin/kexin typu 9 * imunologie   MeSH
• senioři MeSH
• separace krevních složek * MeSH
• trombocyty účinky léků   metabolismus   imunologie   MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

• MeSH
• alely * MeSH
• Bernardův-Soulierův syndrom krev   diagnóza   genetika   MeSH
• fenotyp * MeSH
• genetická predispozice k nemoci * MeSH
• genetická variace * MeSH
• genetické asociační studie MeSH
• imunofenotypizace MeSH
• lidé MeSH
• mutační analýza DNA MeSH
• počet trombocytů MeSH
• rodokmen MeSH
• trombocytopenie krev   diagnóza   MeSH
• trombocytový glykoproteinový komplex Ib-IX genetika   metabolismus   MeSH
• trombocyty metabolismus   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Geografické názvy
• Česká republika MeSH

The objective of the present study was to evaluate platelet mitochondrial oxygen consumption using high-resolution respirometry (HRR) and metabolic flux analysis (MFA) and to verify the effect of advanced age on these parameters. HRR was used to analyze permeabilized and intact platelets, MFA to measure oxygen consumption rates (OCR), extracellular acidification rates (ECAR) and ATP production rate in intact fixed platelets. Two groups of healthy volunteers were included in the study: YOUNG (20-42 years, n=44) and older adults (OLD; 70-89 years; n=15). Compared to YOUNG donors, platelets from group OLD participants displayed significantly lower values of oxygen consumption in the Complex II-linked phosphorylating and uncoupled states and the Complex IV activity in HRR protocols for permeabilized cells and significantly lower resting and uncoupled respirations in intact cells when analyzed by both methods. In addition, mitochondrial ATP production rate was also significantly lower in platelets isolated from older adults. Variables measured by both methods from the same bloods correlated significantly, nevertheless those acquired by MFA were higher than those measured using HRR. In conclusion, the study verifies compromised mitochondrial respiration and oxidative ATP production in the platelets of aged persons and documents good compatibility of the two most widely used methods for determining the global performance of the electron-transporting system, i.e. HRR and MFA.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• analýza metabolického toku metody   MeSH
• buněčné dýchání MeSH
• dospělí MeSH
• energetický metabolismus * MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• mladý dospělý MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• spotřeba kyslíku MeSH
• stárnutí krev   metabolismus   MeSH
• trombocyty metabolismus   MeSH
• věkové faktory MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Platelet mitochondria can be used in the study of mitochondrial dysfunction in various complex diseases and can help in finding biological markers for diagnosing the disease, monitoring its course and the effects of treatment. The aim of this chapter was to describe in detail the method of measuring mitochondrial respiration in platelets using high-resolution respirometry. The described method was successfully used for the study of mitochondrial dysfunction in neuropsychiatric diseases.

• MeSH
• buněčné dýchání MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• polarografie přístrojové vybavení   metody   MeSH
• trombocyty metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

AIM: To test a novel method of assessment of platelet adhesion to a fibrinogen-coated surface in whole blood under flow conditions. METHODS: We developed a fluidic device that mimics blood flow in vessels. The method of detection of platelet adhesion is based on recording of a scattered laser light signal from a fibrinogen-covered surface. Testing was performed in platelet-rich plasma (PRP) and whole blood of healthy volunteers. Control measurements were performed, followed by tests with inhibition of platelet GPIIa/IIIb and GPIb receptors. Then, the same testing sequence was performed in whole blood of persons with autoimmune thrombocytopenia and type 3 von Willebrand disease. RESULTS: The change in intensity of scattered light was 2.7 (2.4; 4.1) times higher in whole blood (0.2 ± 0.08V, n = 7) than in PRP (0.05 ± 0.02 V, n = 7), p < 0.01. The blocking of GP IIb/IIIa receptors decreased the intensity of scattered light to 8.5 (6.5;12)%; the blocking of GPIb receptors decreased it to 34 (23;58)%, p < 0.01. In the whole blood of a person with autoimmune thrombocytopenia, the inhibition of GPIb receptors decreased platelet adhesion, but no effect was observed in type 3 von Willebrand disease. Inhibition of platelet GPIIb/IIIa receptors alone or combined inhibition of GPIb and GPIIb/IIIa receptors resulted in almost total suppression of adhesion in both cases. CONCLUSION: Our system effectively registers platelet adhesion to a fibrinogen-coated surface under controlled-flow conditions and may successfully be applied to the investigation of platelet adhesion kinetics.

• MeSH
• adhezivita trombocytů * MeSH
• agregace trombocytů MeSH
• fibrinogen metabolismus   MeSH
• inhibitory agregace trombocytů farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• trombocytový glykoproteinový komplex Ib-IX antagonisté a inhibitory   metabolismus   MeSH
• trombocytový glykoproteinový komplex IIb-IIIa antagonisté a inhibitory   metabolismus   MeSH
• trombocyty metabolismus   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Platelet concentrates and especially their further product platelet lysate, are widely used as a replacement for cell culturing. Platelets contain a broad spectrum of growth factors and bioactive molecules that affect cellular fate. However, the cellular response to individual components of the human platelet concentrate is still unclear. The aim of this study was to observe cellular behavior according to the individual components of platelet concentrates. The bioactive molecule content was determined. The cells were supplemented with a medium containing 8% (v/v) of platelet proteins in plasma, pure platelet proteins in deionized water, and pure plasma. The results showed a higher concentration of fibrinogen, albumin, insulin growth factor I (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), in the groups containing plasma. On the other hand, chemokine RANTES and platelet-derived growth factor bb (PDGF-bb), were higher in the groups containing platelet proteins. The groups containing both plasma and plasma proteins showed the most pronounced proliferation and viability of mesenchymal stem cells and fibroblasts. The platelet proteins alone were not sufficient to provide optimal cell growth and viability. A synergic effect of platelet proteins and plasma was observed. The data indicated the importance of plasma in platelet lysate for cell growth.

• MeSH
• albuminy MeSH
• becaplermin metabolismus   MeSH
• buněčné kultury metody   MeSH
• chemokiny metabolismus   MeSH
• fibrinogen metabolismus   MeSH
• fibroblastový růstový faktor 7 MeSH
• fibroblasty metabolismus   MeSH
• hepatocytární růstový faktor MeSH
• insulinu podobný růstový faktor I MeSH
• krevní plazma chemie   MeSH
• kultivační média chemie   MeSH
• lidé MeSH
• mezenchymální kmenové buňky metabolismus   MeSH
• plazma bohatá na destičky metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• protoonkogenní proteiny c-sis metabolismus   MeSH
• trombocyty chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Platelets (PLTs) stored at 20-24 °C have a short shelf life of only 5 days, which can result in their restricted availability. PLT cryopreservation extends the shelf life to 2 years. METHODS: We implemented a method of PLT freezing at -80 °C in 5-6% dimethyl sulfoxide. Buffy-coat-derived leucodepleted fresh PLTs blood group O (FP) were used for cryopreservation. Cryopreserved pooled leucodepleted PLTs (CPP) were thawed at 37 °C, reconstituted in PLT additive solution SSP + and compared to FP regarding PLT content, PLT concentration, pH, volume, PLT loss, anti-A/B antibody titre, total protein, plasma content, and PLT swirling. Clot properties were evaluated via rotational thromboelastometry. PLT microparticle number and surface receptor phenotype were assessed via flow cytometry. RESULTS: CPP met the required quality parameters. The mean freeze-thaw PLT loss was 22.24 %. Anti-A/B antibody titre and plasma content were significantly lower in CPP. CPP were characterised by faster clot initiation and form stable PLT clots. The number of PLT microparticles increased 25 times in CPP and there were more particles positive for the activation marker CD62 P compared to FP. CONCLUSION: Thawing and reconstitution are easy and fast processes if platelet additive solution is used. Low anti-A/B antibody titre and plasma content make possible the use of CPP of blood group O reconstituted in SSP + as universal ABO products, including clinical situations where washed PLTs are required. Clot properties evaluated via rotational thromboelastometry demonstrated that CPP retain a significant part of their activity compare to FP and are haemostatically effective.

• MeSH
• hemostáza MeSH
• kryoprezervace metody   MeSH
• lidé MeSH
• trombocyty metabolismus   MeSH
• vrstva buffy coat metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Pluripotent stem cells of the bone marrow are stimulated by different cytokines to proliferation and differentiation into various types of blood cells. These cytokines are mostly glycoproteins. Erythropoietin stimulates stem cells to the formation of erythrocytes while colony-stimulating factors cause the formation of different types of white blood cells. Stem cell factors play an important role in the maintenance and survival of blood cells of all types. Thrombopoietin stimulates stem cells to proliferation and formation of blood platelets. Granulocyte colony-stimulating factor is probably the most important drug in use. It stimulates stem cells to the formation of neutrophile granulocytes. It is often used in recombinant forms such as filgrastim in the treatment of neutropenia in cancer chemotherapy or AIDS. Its pegylated conjugates such as pegfilgrastim are also available. Its activity can be supported by plerixafor, a small molecule - bicyclam derivative acting as an indirect agonist of stem cells factor. It acts as an antagonist of CXCR4 receptor activation of which brakes hematopoiesis. The treatment of conditions accompanied by thrombocytopenia such as idiopathic thrombocytopenic purpura is currently not performed by thrombopoietin but synthetic agonists of its receptor are preferred. Romiplostim is a peptibody. It consists of a protein part interacting with the thrombopoietin receptor which is, however, different from thrombopoietin, and of Fc fragment of immunoglobulin G1. In contrast, small molecule thrombopoietin receptor agonists represented by eltrombopag can be given orally unlike all of the above.

• MeSH
• benzoáty chemie   farmakologie   MeSH
• buněčná diferenciace účinky léků   MeSH
• faktor růstu kmenových buněk farmakologie   MeSH
• faktory stimulující kolonie farmakologie   MeSH
• hydraziny chemie   farmakologie   MeSH
• knihovny malých molekul chemie   farmakologie   MeSH
• leukocyty mononukleární cytologie   účinky léků   metabolismus   MeSH
• lidé MeSH
• pyrazoly chemie   farmakologie   MeSH
• receptory thrombopoetinu agonisté   metabolismus   MeSH
• trombocyty cytologie   metabolismus   MeSH
• trombopoéza účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH