scholarly journals Thrombopoietin potentiates activation of human platelets in association with JAK2 and TYK2 phosphorylation

1996 ◽  
Vol 316 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Belén RODRÍGUEZ-LIÑARES ◽  
Steve P. WATSON
Keyword(s):  
Shape Change ◽  
Physiological Role ◽  
Human Platelets ◽  
Dense Granule ◽  
Functional Responses ◽  
Dramatic Effect ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Dense Granule Secretion ◽  
Granule Secretion

Thrombopoietin (TPO), also known as the c-mpl ligand, stimulates rapid tyrosine phosphorylation of multiple proteins in human platelets including the Janus family kinases JAK2 and TYK2. On its own, TPO has no effect on platelet aggregation and dense-granule secretion but induces a general potentiation of these responses by other stimuli. The most dramatic effect is observed against threshold concentrations of agonists for aggregation. Shape change or weak reversible aggregation induced by low concentrations of thrombin, collagen and the thromboxane mimetic, U46619, are converted into irrreversible aggregation in the presence of TPO. A similar result is obtained in the presence of the ADP scavenger apyrase and cyclo-oxygenase inhibitor indomethacin. TPO also induces potentiation of dense-granule secretion measured through release of 5-hydroxy[3H]tryptamine. This effect is most striking against low concentrations of stimuli and is independent of aggregation as it is observed in the presence of chelation of extracellular Ca2+ with EGTA. TPO potentiates activation of phospholipase C and elevation of intracellular Ca2+, providing a molecular explanation for potentiation of functional responses. TPO may have an important physiological role in priming platelet activation in thrombocytopenia, an action that may help to compensate for the reduced platelet density.

scholarly journals Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

1979 ◽  
Vol 182 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Holm Holmsen ◽  
Linda Robkin ◽  
H. James Day
Keyword(s):  
Shape Change ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Antimycin A ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Dense Granule Secretion ◽  
Granule Secretion

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [14C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [14C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected 14C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, β-N-acetylglucosaminidase, β-glucuronidase and β-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)–response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

2011 ◽  
Vol 436 (2) ◽  
pp. 469-480 ◽  
Author(s):  
Knut Fälker ◽  
Linda Haglund ◽  
Peter Gunnarsson ◽  
Martina Nylander ◽  
Tomas L. Lindahl ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Human Platelets ◽  
Dense Granule ◽  
Receptor Internalization ◽  
Functional Responses ◽  
Protease Activated Receptors ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Aggregation

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both Gα12/13 and Gαq signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2+ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y12 receptor-induced Gαi signalling accounted for the loss of the aggregation response, as mimicking Gαi/z signalling with 2-MeS-ADP (2-methylthioadenosine-5′-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

scholarly journals Evidence for a role for Gαi1 in mediating weak agonist-induced platelet aggregation in human platelets: reduced Gαi1 expression and defective Gi signaling in the platelets of a patient with a chronic bleeding disorder

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4828-4835 ◽  
Author(s):  
Yatin M. Patel ◽  
Kirti Patel ◽  
Salman Rahman ◽  
Mark P. Smith ◽  
Gillian Spooner ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Immunoblot Analysis ◽  
Human Platelets ◽  
Dense Granule ◽  
Bleeding Disorder ◽  
Functional Responses ◽  
Dense Granule Secretion ◽  
High Doses ◽  
Granule Secretion

AbstractWe have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Gαi signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Gα subunits indicated normal levels of Gαi2,Gαi3,Gαz, and Gαq in patient platelets. However, the Gαi1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Gαi1 or Gαi2 gene sequences. Our studies implicate the minor expressed Gαi subtype Gαi1 as having an important role in regulating signaling pathways associated with the activation of αIIbβ3 and subsequent platelet aggregation by weak agonists.

THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION

1987 ◽  
Author(s):  
C T Poll ◽  
J Westwick
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Free Calcium ◽  
Fluorescent Properties ◽  
Fura 2 ◽  
Stimulus Response ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

scholarly journals Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 103-106 ◽  
Author(s):  
RL Kinlough-Rathbone ◽  
ML Rand ◽  
MA Packham
Keyword(s):  
Guinea Pig ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Dense Granule ◽  
Thrombin Receptor ◽  
Amino Acid Residues ◽  
Reversible Aggregation ◽  
Human Thrombin ◽  
Rat Platelets

Human platelets are aggregated and induced to release their granule contents and form thromboxane by peptides as short as 6-amino acid residues (SFLLRN) corresponding to the newly released N-terminus of the thrombin receptor that is cleaved by thrombin. Using washed platelets, we found that these responses to SFLLRN (2 to 6 mumol/L) were enhanced by fibrinogen. However, neither SFLLRN nor SFLLRNPNDKYEPF had any effect on washed rabbit or rat platelets, although they were fully responsive to human thrombin. Concentrations of the peptides as high as 100 mumol/L did not cause the platelets of rabbits or rats to change shape, aggregate, release granule contents, or form thromboxane. SFLLRN did not affect the extent of aggregation induced by adenosine diphosphate (ADP) or a low concentration of thrombin. Pig platelets responded to 50 mumol/L SFLLRN with reversible aggregation, which was enhanced by fibrinogen, but not accompanied by the release of dense granule contents. Guinea pig platelets aggregated and released granule contents in response to 25 or 50 mumol/L of SFLLRN, but responded with only shape change to lower concentrations. Thus, these experiments indicate that rabbit and rat platelets lack a functional response to human thrombin receptor peptides that fully activate the previously described human thrombin receptor, despite a full response of both rabbit and rat platelets to human thrombin, and that pig and guinea pig platelets have incomplete responses to these human thrombin receptor peptides. The results suggest that platelets of rabbits and rats, and perhaps guinea pigs and pigs, respond to thrombin through an alternative receptor that has also been suggested to be present on human platelets.

nPKC Epsilon Negatively Regulates Platelet Functional Responses

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2855-2855
Author(s):  
Yamini Saraswathy Bynagari ◽  
Bela Nagy ◽  
Kamala Bhavaraju ◽  
Donna Woulfe ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Dense Granule ◽  
Dependent Manner ◽  
P2y1 Receptor ◽  
Wild Type ◽  
Functional Responses ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Protein Kinase C (PKC) are family of serine threonine kinases, known to regulate various platelet functional responses. Among them novel class of PKC isoforms (nPKC) including delta(δ), theta(𝛉), eta(η), and epsilon(ε) are expressed in platelets. Although, the role of nPKC ε and η in platelets is fairly understood, not much is known about nPKC ε and η in platelets. In this study, we investigated the role of nPKC ε in platelet functional responses using ADP-induced signaling as our stereotype. ADP causes platelet activation via Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor. Thus, we primarily studied the role of P2Y1 receptor in nPKC ε activation. ADP activated nPKC ε in time- and concentration- dependent manner. In the presence of P2Y1 receptor antagonist MRS-2179 and in P2Y1 knockout (KO) murine platelets ADP failed to activate nPKC ε, suggesting that ADP activates nPKC ε via P2Y1 receptor. We further investigated the functional role of nPKC ε using specific nPKC ε inhibitory RACK peptide (ε V1-2). ε V1-2 is a peptide designed to compete with native nPKC ε to bind ε-Receptors for activated C Kinase (ε-RACK) and thereby inhibits nPKC ε catalytic activity due to decreased substrate accessibility. ADP-induced thromboxane generation in human platelets pretreated with ε V1-2 peptide was more compared to the platelets pretreated with control peptide. Similarly, ADP-induced thromboxane generation in platelets derived from nPKC ε KO mouse was more compared to the wild type (WT) littermates. However, ADP- induced alpha granule secretion and aggregation in aspirin treated platelets derived from PKC ε KO mice was not significantly different from platelets derived from wild type littermates. These data suggest that nPKC e regulates an unknown pathway, which primarily regulates thromboxane generation with minimal effects on aggregation and alpha granule secretion. Furthermore, we also investigated the role of nPKC ε in PAR- and GPVI- mediated platelet aggregation and dense granule secretion. Interestingly, in both aspirin-treated and non-aspirin-treated platelets PAR- and GPVI- mediated platelet aggregation and dense granule secretion were potentiated. Consistent with ex vivo studies, FeCl3-induced arterial thrombosis was enhanced in nPKC ε KO mice compared to WT littermates.

The actin cytoskeleton differentially regulates platelet α-granule and dense-granule secretion

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3879-3887 ◽  
Author(s):  
Robert Flaumenhaft ◽  
James R. Dilks ◽  
Nataliya Rozenvayn ◽  
Rita A. Monahan-Earley ◽  
Dian Feng ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Fusion ◽  
Dense Granule ◽  
Protein Receptor ◽  
Low Concentrations ◽  
Latrunculin A ◽  
High Concentrations ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Exocytosis

AbstractStimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced α-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced α-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca2+- or guanosine triphosphate (GTP)-γ-S-induced α-granule secretion. Latrunculin A-dependent α-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates α-granule and dense granule secretion.

Requirement for Thrombin Receptor Occupancy During Platelet Secretion Under Aggregating and Non-Aggregating Conditions

1987 ◽  
Vol 58 (04) ◽  
pp. 1053-1059 ◽  
Author(s):  
Kazuo Koike ◽  
Holm Holmsen
Keyword(s):  
Receptor Occupancy ◽  
Human Platelets ◽  
Dense Granule ◽  
Secretory Process ◽  
Cell Contact ◽  
Acid Hydrolase ◽  
High Concentration ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Fold Excess

SummaryThe requirement for receptor occupancy in thrombin-induced secretion in human platelets has been studied. When increasing concentrations of thrombin were added to gel-filtered platelets containing a constant, high concentration of hirudin, dense granule secretion was initiated at lower thrombin concentrations than those required for α-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 62-fold excess of hirudin produced abrupt stop of dense granule secretion and a-granule secretion when added to non-aggregating (no stirring) platelets shortly after thrombin; it had no affect after these secretory process had reached about 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, the three secretory processes increased their rates and were abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule and α-granule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum.It is concluded that a-granule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for acid hydrolase secretion. α-Granule secretion might, however, require longer occupancy than dense granule secretion. Aggregation is believed to potentiate secretion through close cell contact and the secretion processes were inhibited by hirudin through hirudin’s effect on aggregation.

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