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.

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 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 Lyn, PKC-δ, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3056-3063 ◽  
Author(s):  
Ramya Chari ◽  
Soochong Kim ◽  
Swaminathan Murugappan ◽  
Archana Sanjay ◽  
James L. Daniel ◽  
...  
Keyword(s):  
Human Platelets ◽  
Sh2 Domain ◽  
Dense Granule ◽  
Protease Activated Receptors ◽  
Inositol Phosphatase ◽  
Glycoprotein Vi ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Granule Release ◽  
Stimulation Of

Protein kinase C-δ (PKC-δ) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-δ positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-δ in platelets. SH2 domain–containing inositol phosphatase-1 (SHIP-1) is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists SFLLRN and AYPGKF or GPVI agonist convulxin. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-δ on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-δ–null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1 and Y311 of PKC-δ were inhibited. In murine platelets lacking Lyn or SHIP-1, GPVI-mediated dense granule secretions are potentiated, whereas PAR-mediated dense granule secretions are inhibited. Therefore, we conclude that Lyn-mediated phosphorylations of PKC-δ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.

INHIBITION BY NEOMYCIN OF AGONIST-INDUCED POLYPHOSPHOINOSITIDE METABOLISM AND RESPONSES IN HUMAN PLATELETS

1987 ◽  
Author(s):  
Holm Holmsen ◽  
Ole-Bjørn Tysnes ◽  
Adrie J M Verhoeven ◽  
Vidar M Steen ◽  
Lindsey J Moore ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Human Platelets ◽  
Dense Granule ◽  
Stimulus Response ◽  
P Content ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Phosphatidylinositol 4 ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

Signal processing in platelets seems to involve polyphosphoinositide (PPI) metabolism, although direct coupling between PPI metabolism and responses has not been proved. Neomycin binds tightly to PPIs and has been used to probe the involvement of PPI metabolism and responses in platelets. Neomycin(SO4)3 powerfully inhibited ADP- and adrenaline-induced aggregation of platelets in PRP. This was partly due to the sulphate anion; the chloride form was therefore prepared. Platelets were prelabelled in PRP with 32P-Pi. and transferred by gel filtration to a calcium-free Tyrode’s solution (GFP). Increasing concentrations (2-5 mM) of neomycinCl6 caused progressive inhibition of thrombin-induced aggregation, dense granule secretion, acid hydrolase secretion and formation of 32P-phosphatidic acid (PA); the inhibition was immediate, not affected by aspirin and counteracted by increasing thrombin concentrations. Incubation of neomycin (up to 5 mM) with this GFP or with P-Pi. On GFP prepared from unlabelled PRP had no effect on the P content of ATP, phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4,5-bisphosphate (PIP ). Increasing neomycin concentrations caused progressive inhibition of the thrombin-induced init^l (10 sec) decrease, but not of the late (90 sec) incg^ase i-n P-PIP2 while they enhanced the increase in P-PIP. Similar results were obtained ^th collagen and PAF. Both the increase in cytosolic Ca and pH (measured by INDO-I and BCECF, respectively) induced by thrombin were inhibited progressively by increasing concentrations of neomycin. These results are in support for a direct involvement of PPI metabolism in the stimulus-response coupling below the receptor level. However, the failure of neomycin to affect turnover of PIP and PIP2 in nonstimulated platelets suggests that the aminoglycoside does not penetrate the membrane, and only become available to PPI during stimulation.

scholarly journals Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism

1988 ◽  
Vol 253 (2) ◽  
pp. 581-586 ◽  
Author(s):  
V M Steen ◽  
O B Tysnes ◽  
H Holmsen
Keyword(s):  
Narrow Range ◽  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Dense Granule ◽  
Phosphoinositide Metabolism ◽  
Inositol Phospholipids ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Changes ◽  
Induced Aggregation

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.

Protein Kinase Cδ Differentially Regulates Platelet Functional Responses.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1841-1841
Author(s):  
Ramya Chari ◽  
Todd Getz ◽  
Bela Nagy ◽  
Kamala Bhavaraju ◽  
Yingying Mao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Receptor Activation ◽  
Dense Granule ◽  
Small Contribution ◽  
Functional Responses ◽  
Fibrinogen Receptor ◽  
Granule Secretion ◽  
Induced Aggregation ◽  
Protein Kinase Cδ

Abstract Protein Kinase Cδ (PKCδ), a novel PKC isoform is expressed and activated in platelets downstream of PARs and GPVI receptors. In the current study, the role of PKCδ in regulating platelet functional responses was investigated using a pharmacological inhibitor, (δV1-1)TAT (a PKCδ inhibitor) in human platelets. These studies were further confirmed by a knockout approach using PKCδ+/+ and PKCδ−/− mice. In both human and murine platelets, PAR4-mediated dense granule secretions were inhibited, whereas GPVI-mediated dense granule secretions were potentiated. Furthermore, α-granule secretions and thromboxane A2 (TXA2) generation were differentially regulated in murine platelets.. These data suggest a differential role for this isoform in regulating dense granule secretion, α-granule secretion and TXA2 generation. Previous studies have shown that PAR-mediated fibrinogen receptor activation is regulated by a Calcium-dependent and a PKC-dependent pathway. The contribution of PKCδ to PAR-mediated fibrinogen receptor activation was studied by pretreating human and murine platelets with BAPTA. Our results showed a inhibition of AYPGKF-induced aggregation in human and murine platelets in the presence of BAPTA and fibrinogen. These results suggest a small contribution of PKCδ to PAR-4- mediated platelet aggregation and aIIbb3 activation. The in vivo significance of PKCδ was tested using a FeCl3 injury model. While the wildtype mice occluded in 7 minutes, PKCδ −/− mice occluded after 4 minutes of injury with 10 % FeCl3. Therefore, we conclude that PKCδ regulates platelet functional responses such as dense, α-granule secretions, TXA2 generation downstream of both PARs and GPVI receptors, contributes to PAR-4-mediated fibrinogen receptor activation ex vivo and plays a critical role in the thrombus formation in vivo. This study is supported by predoctoral fellowships to Ramya Chari and Swaminathan Murugappan from American Heart Association, Great Rivers affiliate.

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.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document