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Haematologica Apr 2014Platelet release by megakaryocytes is regulated by a concert of environmental and autocrine factors. We previously showed that constitutively released adenosine...
Platelet release by megakaryocytes is regulated by a concert of environmental and autocrine factors. We previously showed that constitutively released adenosine diphosphate by human megakaryocytes leads to platelet production. Here we show that adenosine diphosphate elicits, in human megakaryocytes, an increase in cytosolic calcium concentration, followed by a plateau, which is lowered in the absence of extracellular calcium, suggesting the involvement of Store-Operated Calcium Entry. Indeed, we demonstrate that megakaryocytes express the major candidates to mediate Store-Operated Calcium Entry, stromal interaction molecule 1, Orai1 and canonical transient receptor potential 1, which are activated upon either pharmacological or physiological depletion of the intracellular calcium pool. This mechanism is inhibited by phospholipase C or inositol-3-phosphate receptor inhibitors and by a specific calcium entry blocker. Studies on megakaryocyte behavior, on extracellular matrix proteins that support proplatelet extension, show that calcium mobilization from intracellular stores activates signaling cascades that trigger megakaryocyte adhesion and proplatelet formation, and promotes extracellular calcium entry which is primarily involved in the regulation of the contractile force responsible for megakaryocyte motility. These findings provide the first evidence that both calcium mobilization from intracellular stores and extracellular calcium entry specifically regulate human megakaryocyte functions.
Topics: Adenosine Diphosphate; Adult; Calcium; Calcium Signaling; Cell Adhesion; Cell Movement; Cells, Cultured; Collagen Type I; Extracellular Space; Female; Humans; Megakaryocytes; Pregnancy; RNA, Messenger; Thrombopoiesis
PubMed: 24463213
DOI: 10.3324/haematol.2013.096859 -
PloS One 2013Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our...
Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg(2+)-ion dependencies of sliding velocities of a high duty ratio class-5 myosin motor, myosin-5b from D. discoideum using in vitro motility assays. Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg(2+)-ions. Free Mg(2+)-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg(2+)-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg(2+)-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg(2+)-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (ΔH(Mg) and ΔS(Mg)) of a fast equilibrium between magnesium free (AM·D) and magnesium bound acto-myosin-ADP (AM· Mg(2+)D) states and the corresponding enthalpic barriers associated with ADP release (ΔH1(‡) and ΔH2(‡)). The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions.
Topics: Adenosine Diphosphate; Dictyostelium; Magnesium; Myosin Type V; Thermodynamics
PubMed: 23738001
DOI: 10.1371/journal.pone.0064797 -
Scientific Reports Oct 2022Storage of platelet concentrates (PC) at cold temperature (CT) is discussed as an alternative to the current standard of storage at room temperature (RT). Recently, we...
Storage of platelet concentrates (PC) at cold temperature (CT) is discussed as an alternative to the current standard of storage at room temperature (RT). Recently, we could show that cold-induced attenuation of inhibitory signaling is an important mechanism promoting platelet reactivity. For developing strategies in blood banking, it is required to elucidate the time-dependent onset of facilitated platelet activation. Thus, freshly prepared platelet-rich-plasma (PRP) was stored for 1 and 2 h at CT (2-6 °C) or at RT (20-24 °C), followed by subsequent comparative analysis. Compared to RT, basal and induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation levels were decreased under CT within 1 h by approximately 20%, determined by Western blot analysis and flow cytometry. Concomitantly, ADP- and collagen-induced threshold aggregation values were enhanced by up to 30-40%. Furthermore, platelet-covered areas on collagen-coated slides and aggregate formation under flow conditions were increased after storage at CT, in addition to induced activation markers. In conclusion, a time period of 1-2 h for refrigeration is sufficient to induce an attenuation of inhibitory signaling, accompanied with an enhancement of platelet responsiveness. Short-term refrigeration may be considered as a rational approach to obtain PC with higher functional reactivity for the treatment of hemorrhage.
Topics: Adenosine Diphosphate; Blood Platelets; Blood Preservation; Collagen; Platelet Aggregation; Refrigeration
PubMed: 36207457
DOI: 10.1038/s41598-022-21124-4 -
Frontiers in Bioscience (Landmark... May 2022Platelet-derived extracellular vesicles (PEVs) are small vesicles released by activated platelets that are gaining growing interest in the field of vascular biology. The...
BACKGROUND
Platelet-derived extracellular vesicles (PEVs) are small vesicles released by activated platelets that are gaining growing interest in the field of vascular biology. The mode of platelet activation is a critical determinant of PEVs release, phenotype and function. However, only very limited information is available concerning the impact of the platelet purification procedure on PEVs release.
METHODS
Washed or isolated platelets were separated by differential centrifugations. For washed platelets, the platelet pellet was washed by resuspension in PIPES buffer and finally resuspended in HEPES buffer. Isolated platelets were obtained by directly resuspending the platelet pellet in HEPES, skipping the washing steps in PIPES buffer. PEVs release was induced in washed or isolated platelets by stimulation with different agonist and analysed by Nanoparticle Tracking Analysis.
RESULTS
Isolated platelets showed a higher release of PEVs upon adenosine diphosphate (ADP) stimulation compared to washed platelets, whereas PEVs released upon stimulation with strong agonists (thrombin, collagen, A23187, U46619) were similar in the two groups. This different responsiveness to ADP was also observed as a higher α-granules release and protein kinase C activation in isolated platelets compared to washed ones. Residual plasma contamination appeared to be essential for the ability of platelets to release PEVs in response to ADP.
CONCLUSIONS
In conclusion, our study strongly suggests that procedure adopted for platelets preparation is a critical determinant of PEVs release upon ADP stimulation.
Topics: Adenosine Diphosphate; Blood Platelets; Extracellular Vesicles; HEPES; Platelet Activation
PubMed: 35638428
DOI: 10.31083/j.fbl2705161 -
Indian Journal of Cancer Mar 2022Human epidermal growth factor receptor 2 (HER2)-negative subset is the most heterogeneous group of metastatic breast cancers (MBCs) as it includes both hormone receptor... (Review)
Review
Human epidermal growth factor receptor 2 (HER2)-negative subset is the most heterogeneous group of metastatic breast cancers (MBCs) as it includes both hormone receptor (HR)-positive and HR-negative breast cancer (or TNBC), which have different therapies and treatment challenges. Though endocrine therapy (ET) remains the treatment backbone in HR-positive HER2-negative cases, about 40% of the patients show intrinsic or acquired resistance to ET due to multiple mechanisms. Combining different therapies such as ET and other targeted therapies with or without chemotherapy fails to give continued benefit, unlike cyclin-dependent kinase (CDK) 4/6 inhibitors that have shown a great benefit. TNBC has conventionally been treated ineffectively with systemic chemotherapy. Recently, poly (ADP-ribose) polymerase inhibitors (PARPi) have emerged for HER2-negative breast cancer (BC) patients, including TNBC. Olaparib and talazoparib have recently been approved in germline BRCA-mutated (gBRCAm) HER2-negative MBC. Additionally, ongoing trials of PARPi in combination with various therapies are expected to provide more and better treatment options for gBRCAm HER2-negative breast cancer.
Topics: Adenosine Diphosphate; Adenosine Diphosphate Ribose; Breast Neoplasms; Female; Humans; Receptor, ErbB-2; Ribose
PubMed: 35343197
DOI: 10.4103/ijc.IJC_30_21 -
Scientific Reports Sep 2021The GroEL-GroES chaperonin complex is a bacterial protein folding system, functioning in an ATP-dependent manner. Upon ATP binding and hydrolysis, it undergoes multiple...
The GroEL-GroES chaperonin complex is a bacterial protein folding system, functioning in an ATP-dependent manner. Upon ATP binding and hydrolysis, it undergoes multiple stages linked to substrate protein binding, folding and release. Structural methods helped to reveal several conformational states and provide more information about the chaperonin functional cycle. Here, using cryo-EM we resolved two nucleotide-bound structures of the bullet-shaped GroEL-GroES complex at 3.4 Å resolution. The main difference between them is the relative orientation of their apical domains. Both structures contain nucleotides in cis and trans GroEL rings; in contrast to previously reported bullet-shaped complexes where nucleotides were only present in the cis ring. Our results suggest that the bound nucleotides correspond to ADP, and that such a state appears at low ATP:ADP ratios.
Topics: Adenosine Diphosphate; Binding Sites; Chaperonin 10; Chaperonin 60; Cryoelectron Microscopy; Escherichia coli Proteins; Protein Binding
PubMed: 34521893
DOI: 10.1038/s41598-021-97657-x -
Acta Crystallographica. Section D,... Jul 2018The DEAH-box ATPase Prp2 plays a key role in the activation of the spliceosome as it promotes the transition from the B to the catalytically active B* spliceosome. Here,...
The DEAH-box ATPase Prp2 plays a key role in the activation of the spliceosome as it promotes the transition from the B to the catalytically active B* spliceosome. Here, four crystal structures of Prp2 are reported: one of the nucleotide-free state and three different structures of the ADP-bound state. The overall conformation of the helicase core, formed by two RecA-like domains, does not differ significantly between the ADP-bound and the nucleotide-free states. However, intrinsic flexibility of Prp2 is observed, varying the position of the C-terminal domains with respect to the RecA domains. Additionally, in one of the structures a unique ADP conformation is found which has not been observed in any other DEAH-box, DEAD-box or NS3/NPH-II helicase.
Topics: Adenosine Diphosphate; Chaetomium; Crystallization; Crystallography, X-Ray; DEAD-box RNA Helicases; Fungal Proteins; Molecular Conformation; Protein Binding; Protein Conformation
PubMed: 29968674
DOI: 10.1107/S2059798318006356 -
The Indian Journal of Medical Research Feb 2011Cholera toxin (CT) was discovered exactly half a century ago by S.N. De. We have come a long way since this epoch-making discovery. Retrospectively, science had to wait... (Review)
Review
Cholera toxin (CT) was discovered exactly half a century ago by S.N. De. We have come a long way since this epoch-making discovery. Retrospectively, science had to wait a long time since Koch's prediction of the existence of a toxin, and its actual discovery by De. CT is not just another enterotoxin that causes the signs and symptoms of the dreaded disease, cholera. It is unique in many respects, starting from its structure to its functions. CT is a multifunctional protein that is capable of influencing the immune system in many ways. It not only has remarkable adjuvant properties, but also acts as an anti-inflammatory agent, by modulating specific signal transduction pathways. Its immunomodulatory properties can be harnessed for treatment of various autoimmune disorders, and have shown great promise in the area of immunotherapeutics. CT can truly be considered as a paradigm of a multifunctional protein.
Topics: Adenosine Diphosphate; Animals; Autoimmune Diseases; Cholera Toxin; Cholera Vaccines; Humans; Immunologic Factors; Immunotherapy
PubMed: 21415492
DOI: No ID Found -
Proceedings of the National Academy of... Aug 2009The influence of the state of the bound nucleotide (ATP, ADP-Pi, or ADP) on the conformational free-energy landscape of actin is investigated. Nucleotide-dependent...
The influence of the state of the bound nucleotide (ATP, ADP-Pi, or ADP) on the conformational free-energy landscape of actin is investigated. Nucleotide-dependent folding of the DNase-I binding (DB) loop in monomeric actin and the actin trimer is carried out using all-atom molecular dynamics (MD) calculations accelerated with a multiscale implementation of the metadynamics algorithm. Additionally, an investigation of the opening and closing of the actin nucleotide binding cleft is performed. Nucleotide-dependent free-energy profiles for all of these conformational changes are calculated within the framework of metadynamics. We find that in ADP-bound monomer, the folded and unfolded states of the DB loop have similar relative free-energy. This result helps explain the experimental difficulty in obtaining an ordered crystal structure for this region of monomeric actin. However, we find that in the ADP-bound actin trimer, the folded DB loop is stable and in a free-energy minimum. It is also demonstrated that the nucleotide binding cleft favors a closed conformation for the bound nucleotide in the ATP and ADP-Pi states, whereas the ADP state favors an open confirmation, both in the monomer and trimer. These results suggest a mechanism of allosteric interactions between the nucleotide binding cleft and the DB loop. This behavior is confirmed by an additional simulation that shows the folding free-energy as a function of the nucleotide cleft width, which demonstrates that the barrier for folding changes significantly depending on the value of the cleft width.
Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Binding Sites; Deoxyribonuclease I; Hydrolysis; Protein Conformation; Protein Folding
PubMed: 19620726
DOI: 10.1073/pnas.0902092106 -
Journal of Veterinary Science Jan 2019Platelet activation has a major role in hemostasis and thrombosis. Various agonists including adenosine diphosphate (ADP) and thrombin interact with G protein-coupled...
Platelet activation has a major role in hemostasis and thrombosis. Various agonists including adenosine diphosphate (ADP) and thrombin interact with G protein-coupled receptors (GPCRs) which transduce signals through various G proteins. Recent studies have elucidated the role of GPCRs and their corresponding G proteins in the regulation of events involved in platelet activation. However, agonist-induced platelet activation in companion animals has not been elucidated. This study was designed to characterize the platelet response to various agonists in dog platelets. We found that 2-methylthio-ADP-induced dog platelet aggregation was blocked in the presence of either P2Y₁ receptor antagonist MRS2179 or P2Y₁₂ receptor antagonist AR-C69931MX, suggesting that co-activation of both the P2Y₁ and P2Y₁₂ receptors is required for ADP-induced platelet aggregation. Thrombin-induced dog platelet aggregation was inhibited in the presence of either AR-C69931MX or the PKC inhibitor GF109203X, suggesting that thrombin requires secreted ADP to induce platelet aggregation in dog platelets. In addition, thrombin-mediated Akt phosphorylation was inhibited in the presence of GF109203X or AR-C69931MX, indicating that thrombin causes G stimulation through the P2Y₁₂ receptor by secreted ADP in dog platelets. Unlike human and murine platelets, protease-activated receptor 4 (PAR4)-activating peptide AYPGKF failed to cause dog platelet aggregation. Moreover, PAR1-activating peptide SFLLRN or co-stimulation of SFLLRN and AYPGKF failed to induce dog platelet aggregation. We conclude that ADP induces platelet aggregation through the P2Y₁ and P2Y₁₂ receptors in dogs. Unlike human and murine platelets, selective activation of the PAR4 receptor may be insufficient to cause platelet aggregation in dog platelets.
Topics: Adenosine Diphosphate; Animals; Blood Platelets; Dogs; Female; Hemostatics; Male; Oligopeptides; Platelet Aggregation; Receptors, Purinergic P2Y; Thionucleotides; Thrombin
PubMed: 30541187
DOI: 10.4142/jvs.2019.20.1.10