Evidence for tight metabolic control of the receptor-activated polyphosphoinositide cycle in human platelets.
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ABSTRACT:
SUBMITTER: Steen VM
PROVIDER: S-EPMC1133474 | biostudies-other | 1989 Oct
REPOSITORIES: biostudies-other
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Project description:Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.
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Project description:Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subsequent structure activity relationship analysis yielded several selective PAR4 antagonists as well as possible new scaffolds for future antagonist development.
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Project description:With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)-stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP-stimulated platelets. PAR4-AP-mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP-mediated FV secretion and microparticle generation to PAR1-AP-mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP-stimulated platelets, compared with PAR1-AP-stimulated platelets. Rho-kinase inhibition reduced PAR4-AP-mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP-mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.
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Project description:Several members of the kinesin superfamily are known to play a prominent role in the motor-driven transport processes that occur in mitotic cells. Here we describe a new mitotic human kinesin-like protein, RB6K (Rabkinesin 6), distantly related to MKLP-1. Expression of RB6K is regulated during the cell cycle at both the mRNA and protein level and, similar to cyclin B, shows a maximum during M phase. Isolation of the RB6K promoter allowed identification of a CDE-CHR element and promoter activity was shown to be maximal during M phase. Immunofluorescence microscopy using antibodies raised against RB6K showed a weak signal in interphase Golgi but a 10-fold higher signal in prophase nuclei. During M phase, the newly synthesized RB6K does not colocalise with Rab6. In later stages of mitosis RB6K localized to the spindle midzone and appeared on the midbodies during cytokinesis. The functional significance of this localization during M phase was revealed by antibody microinjection studies which resulted exclusively in binucleate cells, showing a complete failure of cytokinesis. These results substantiate a crucial role for RB6K in late anaphase B and/or cytokinesis, clearly distinct from the role of MKLP-1.
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Project description:PurposeTo understand the molecular pathways that control early human embryo development.MethodsImproved methods of linear amplification of mRNAs and whole human genome microarray analyses were utilized to characterize gene expression in normal appearing 8-Cell human embryos, in comparison with published microarrays of human fibroblasts and pluripotent stem cells.ResultsMany genes involved in circadian rhythm and cell division were over-expressed in the 8-Cells. The cell cycle checkpoints, RB and WEE1, were silent on the 8-Cell arrays, whereas the recently described tumor suppressor, UHRF2, was up-regulated >10-fold, and the proto-oncogene, MYC, and the core element of circadian rhythm, CLOCK, were elevated up to >50-fold on the 8-Cell arrays.ConclusionsThe canonical G1 and G2 cell cycle checkpoints are not active in totipotent human blastomeres, perhaps replaced by UHRF2, MYC, and intracellular circadian pathways, which may play important roles in early human development.
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Project description:Thrombin inhibits adenylate cyclase and stimulates GTP hydrolysis by high-affinity GTPase(s) in membranes of human platelets at almost identical concentrations. Both of these thrombin actions are similar to those observed with agonist-activated alpha 2-adrenoceptors coupling to the inhibitory guanine nucleotide-binding protein N1. However, stimulation of GTP hydrolysis caused by adrenaline (alpha 2-adrenoceptor agonist) and by thrombin at maximally effective concentrations was partially additive, whereas with regard to adenylate cyclase inhibition no additive response was observed. Furthermore, treatment of platelet membranes with pertussis toxin, which inactivates Ni and largely abolishes thrombin- and adrenaline-induced adenylate cyclase inhibition and adrenaline-induced GTPase stimulation, decreased the thrombin-induced stimulation of GTP hydrolysis by only about 30%. Additionally, the thiol reagent N-ethylmalemide (NEM) at rather low concentrations abolished thrombin- and adrenaline-induced stimulation of GTP hydrolysis was decreased by only 30-40% by treatment of platelet membranes with even high concentrations of NEM. Treatment with cholera toxin, which inhibits GTPase activity of the Ns (stimulatory guanine nucleotide-binding) protein, has no effect on thrombin-stimulated GTP hydrolysis. The data suggest that thrombin interaction with its receptor sites in platelet membranes leads to stimulation of two GTP-hydrolysing enzymes. One of these enzymes is apparently Ni and is also activated by agonist-activated alpha 2-adrenoceptors and is inactivated by pertussis toxin and NEM treatment. The other GTP-hydrolysing enzyme activated by thrombin may represent a guanine nucleotide-binding protein apparently involved in the coupling of thrombin receptors to the phosphoinositide phosphodiesterase.
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Project description:Essentials Platelets employ proteins/signaling pathways traditionally thought reserved for nuclear niche. We determined retinoic-acid-receptor alpha (RAR?) expression and function in human platelets. RAR?/actin-related protein-2/3 complex (Arp2/3) interact via non-genomic signaling in platelets. RAR? regulates Arp2/3-mediated actin cytoskeletal dynamics and platelet spreading.Background Platelets utilize proteins and pathways classically reserved for the nuclear niche. Methods We determined whether human platelets express retinoic-acid-receptor family members, traditionally thought of as nuclear transcription factors, and deciphered the function of RAR?. Results We found that RAR? is robustly expressed in human platelets and megakaryocytes and interacts directly with actin-related protein-2/3 complex (Arp2/3) subunit 5 (Arp2/3s5). Arp2/3s5 co-localized with RAR? in situ and regulated platelet cytoskeletal processes. The RAR? ligand all-trans retinoic acid (atRA) disrupted RAR?-Arp2/3 interactions. When isolated human platelets were treated with atRA, rapid cytoskeletal events (e.g. platelet spreading) were inhibited. In addition, when platelets were cultured for 18 h in the presence of atRA, actin-dependent morphological changes (e.g. extended cell body formation) were similarly inhibited. Using in vitro actin branching assays, RAR? and Arp2/3-regulated complex actin branch formation was demonstrated. Consistent with inhibition of cytoskeletal processes in platelets, atRA, when added to this branching assay, resulted in dysregulated actin branching. Conclusion Our findings identify a previously unknown mechanism by which RAR? regulates Arp2/3-mediated actin cytoskeletal dynamics through a non-genomic signaling pathway. These findings have broad implications in both nucleated and anucleate cells, where actin cytoskeletal events regulate cell morphology, movement and division.
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Project description:Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the ?IIb?3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, ?IIb?3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.
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Project description:Human platelets incubated with [32P]Pi and [3H]arachidonate were transferred to a Pi-free Tyrode's solution by gel filtration. The labile phosphoryl groups of ATP and ADP as well as Pi in the metabolic pool of these platelets had equal specific radioactivity which was identical to that of[32P]phosphatidate formed during treatment of the cells with thrombin for 5 min. Therefore, the 32P radioactivity of phosphatidate was a true, relative measure for its mass. The thrombin-induced formation of[32P]-phosphatidate had the same time course and dose-response relationships as the concurrent secretion of acid hydrolases. 125I-alpha-Thrombin bound maximally to the platelets within 13s and was rapidly dissociated from the cells by hirudin; readdition of excess 125I-alpha-thrombin caused rapid rebinding of radioligand. This binding-dissociation-rebinding sequence was paralleled by a concerted start-stop-restart of phosphatidate formation and acid hydrolase secretion. [3H]Phosphatidylinositol disappearance was initiated upon binding but little affected by thrombin dissociation and rebinding. ATP deprivation caused similar changes in the time courses for [32P]-phosphatidate formation and acid hydrolase secretion which were different from those of [3H]phosphatidylinositol disappearance. The metabolic stress did not alter the magnitude (15%) of the initial decrease in phosphatidylinositol-4,5-bis[32P]phosphate, but did abolish the subsequent increase of phosphatidylinositol-4,5-bis[32P]-phosphate in the thrombin-treated platelets. It is concluded that in thrombin-treated platelets (1) phosphatidate synthesis, but not phosphatidylinositol disappearance, is tightly coupled to receptor occupancy and acid hydrolase secretion in platelets, (2) successive phosphorylations to phosphatidylinositol-4,5-bisphosphate is unlikely to be the main mechanism for phosphatidylinositol disappearance, and (3) only a small fraction (15%) of phosphatidylinositol-4,5-bisphosphate is susceptible to hydrolysis.
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Project description:We have shown previously that purified human neutrophil cathepsin G is a strong platelet agonist. We now demonstrate that cathepsin G exhibits saturable, reversible binding to human platelets which is characteristic of binding to a specific receptor. At room temperature, cathepsin G displayed apparent positive co-operativity of binding, as indicated by sigmoidal binding curves and a Hill coefficient greater than unity. By contrast, binding curves conducted with native enzyme at 0.5 degrees C displayed a much smaller degree of sigmoidicity, and binding studies performed with phenylmethanesulphonyl fluoride-treated enzyme at 22-25 degrees C exhibited hyperbolic binding curves. The concentrations of cathepsin G required to give half-saturation (S0.5) with inhibitor-treated enzyme or with native enzyme at either room temperature or 0.5 degrees C were all similar, suggesting that sigmoidal binding curves did not result from an alteration in the affinity of the binding sites for cathepsin G. However, platelets bound approximately twice as many molecules of native enzyme as molecules of phenylmethanesulphonyl fluoride-treated cathepsin G per cell. From these observations it can be inferred that the apparent positive co-operativity may in part reflect the exposure of binding sites due to the proteolytic activity of cathepsin G. However, this conclusion is not supported by experiments conducted with subsaturating cathepsin G concentrations, which demonstrated that ligand binding did not show an expected increase at longer time intervals. Measurement of Ca2+ mobilization and cathepsin G binding in the same platelet suspensions demonstrated that elevations in cytosolic free Ca2+ concentration had achieved near-maximal levels in the presence of 15 micrograms of cathepsin G/ml, whereas maximal binding was observed at approx. 35 micrograms/ml, indicating that only a fraction of the total binding sites need be occupied to elicit platelet activation. Pretreatment of platelets with forskolin or phorbol 12-myristate 13-acetate (PMA) decreased cathepsin G binding by approx. 60% and 40% respectively, indicating that the receptor may be desensitized or down-regulated by phosphorylation due to protein kinases. Since forskolin and PMA could diminish receptor availability by activating negative feedback mechanisms, inhibition of negative signal-transduction pathways could conversely play a role in the up-regulation of cathepsin G binding. In any event, these results show that cathepsin G is an agonist that must bind to platelets to initiate processes associated with cell activation, and suggest a role for cathepsin G in platelet function.
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