scholarly journals Metabolic and functional consequences of introducing inositol 1,4,5-trisphosphate into saponin-permeabilized human platelets

1986 ◽  
Vol 233 (3) ◽  
pp. 707-718 ◽  
Author(s):  
K S Authi ◽  
B J Evenden ◽  
N Crawford
Keyword(s):  
Arachidonic Acid ◽  
Shape Change ◽  
Human Platelets ◽  
Dense Granule ◽  
Water Soluble ◽  
Marker Enzyme ◽  
Intact Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Functional Consequences ◽  
Granule Release

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.

Conditions Affecting the Responses of Human Platelets to Epinephrine

1988 ◽  
Vol 60 (02) ◽  
pp. 209-216 ◽  
Author(s):  
Chantal Lalau Keraly ◽  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Hidenori Suzuki ◽  
J Fraser Mustard
Keyword(s):  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Dense Granule ◽  
Primary Phase ◽  
Lag Phase ◽  
Acid Citrate Dextrose ◽  
Two Phases ◽  
Citrate Dextrose ◽  
Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

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.

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.

Activation of G12/13 Pathway Is Essential for the Dense Granule Release in Platelets.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1564-1564
Author(s):  
Jianguo Jin ◽  
Soochong Kim ◽  
Satya Kunapuli
Keyword(s):  
Knockout Mouse ◽  
Partial Agonist ◽  
Shape Change ◽  
P2y Receptors ◽  
Dense Granule ◽  
Null Mouse ◽  
Granule Release ◽  
Platelet Shape ◽  
Stimulation Of ◽  
Platelet Secretion

Abstract Platelet secretion is an important physiological event in hemostasis. A number of agonists such as thrombin and thromboxane A2 induce platelet secretion. ADP does not cause dense granule release in aspirin-treated platelets, although ADP induce platelet shape change, aggregation and alpha granule release through Gq and Gi pathways. The protease activated receptors 1 and 4, and the thromboxane receptor activate the G12/13 pathways in addition to the Gq pathways. We postulated that the platelet dense granule release reaction depends on both Gq and G12/13, and in the absence of signaling through either G protein abolishes secretion. In other words, co-stimulation of Gq and G12/13 pathways is a major requirement for dense granule release in platelets. We rationalize that because U46619 and thrombin can activate both Gq and G12/13, they cause platelet dense granule release, whereas ADP fails to cause platelet secretion from dense granules because it does not activate G12/13. As a first step towards testing this hypothesis, we supplemented ADP signaling in aspirin-treated platelets with selective activation of G12/13 pathways using YFLLRNP, a partial agonist of PAR-1. YFLLRNP selectively active G12/13 signaling pathway without activating Gq or Gi pathways at low concentrations. YFLLRNP (60 μM) or 2MeSADP (100 nM) failed to cause dense granule release. However, addition of YFLLRNP (60 μM) and 2MeSADP (100 nM) together caused dense granule release. We proceeded to confirm these results in Gq null mice by selectively activating phospholipase C (PLC), a downstream signaling molecule from Gq. In aspirin-treated Gαq knockout mouse platelets 80 μM m-3M3FBS, a direct PLC activator, causes calcium mobilization from intracellular stores and platelet shape change, but does not cause dense granule secretion. We have previously shown that Gq/PLC pathways downstream of ADP are not sufficient for aggregation and require concomitant signaling from Gi for platelet aggregation. Thus, lack of aggregation by PLC activation alone in G?q knockout mouse platelets is consistent with our previous observations. In G?q null mouse platelets, m-3M3FBS (80 μM) or AYPGKF (500 μM) alone failed to cause dense granule release. However, addition of m-3M3FBS (80 μM) and AYPGKF (500 μM) together caused dense granule release. In addition, consistent with our previous findings, co-stimulation of G12/13 pathways and Gi (AYPGKF + 2MeSADP) in G?q knockout mouse platelets caused aggregation, but failed to cause dense granule release. We conclude that supplemental signaling from G12/13 is required for Gq-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G12/13 pathways.

scholarly journals Platelet activation by immobilized monoclonal antibody: evidence for a CD9 proximal signal

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1753-1759
Author(s):  
L Griffith ◽  
J Slupsky ◽  
J Seehafer ◽  
L Boshkov ◽  
AR Shaw
Keyword(s):  
Platelet Aggregation ◽  
Hla Class I ◽  
Human Platelets ◽  
Dense Granule ◽  
Polystyrene Latex ◽  
Cross Linking ◽  
Latex Beads ◽  
Dose Dependent ◽  
Aggregation Of Platelets ◽  
Granule Release

Anti-CD9 monoclonal antibodies (MoAbs) are reported to activate human platelets through stimulation of the Fc gamma II receptor. We show here that nonstimulatory F(ab')2 fragments of the anti-CD9 MoAb 50H.19 induce dense-granule release and dose-dependent platelet aggregation when attached to polystyrene latex beads. Cross-linking F(ab')2 fragments of MoAb 50H.19 by F(ab')2 fragments of goat anti-mouse IgG does not result in platelet aggregation unless the second antibody is bound to latex beads, indicating that immobilization, and not cross- linking of the stimulus, is critical to the initiation of the CD9 signal. In contrast, F(ab')2 fragments of the second antibody readily induce the aggregation of platelets treated with the anti-Fc gamma II receptor MoAb IV.3. Immobilization of MoAb per se is insufficient to induce an activation signal because intact and F(ab')2 fragments of nonstimulatory MoAb directed to glycoprotein Ib and HLA class I do not become stimulatory when attached to beads. CD9-induced activation requires cytoskeletal rearrangement because it is inhibited by cytochalasin B. Aggregation is blocked by inhibitors of the thromboxane pathway, indicating that CD9 activates phospholipase C indirectly through prior activation of phospholipase A2.\

scholarly journals Characteristics of the synergistic actions of platelet agonists

Blood ◽  
1981 ◽  
Vol 57 (4) ◽  
pp. 685-691
Author(s):  
EM Huang ◽  
TC Detwiler
Keyword(s):  
Arachidonic Acid ◽  
Shape Change ◽  
Human Platelets ◽  
Threshold Concentration ◽  
Synergistic Action ◽  
Characteristic Pattern ◽  
Clear Distinction

Platelets are activated by many different agonists that act synergistically. Since there is a characteristic pattern of responses to each agonist, and since there is a clear distinction between weak and strong agonists, understanding the nature of the synergism and its physiologic significance requires characterization of the pattern of responses to the synergistic action of the various agonists. Shape change, aggregation, and secretion of ATP by human platelets in citrated plasma were analyzed after activation by ADP, epinephrine, arachidonic acid, gamma-thrombin, or collagen, either singly or in pairs. The patterns of responses were characteristic of the agonist in higher concentration relative to its threshold concentration; if neither was clearly higher, the pattern of responses was intermediate between the responses characteristic of each agonist. No combination of weak agonists had the characteristics of a strong agonist. These results help define the extent to which platelet responses can be attributed to the synergistic actions of weak agonists.

THE ROLE OF GPIIb-IIIa IN MODULATION OF ADHESION REACTIONS

1987 ◽  
Author(s):  
J C Mattson ◽  
D W Estry ◽  
D Peterson ◽  
R LaFevre ◽  
J Chirco
Keyword(s):  
Shape Change ◽  
Atp Release ◽  
Dense Granule ◽  
Clot Retraction ◽  
Virtual Absence ◽  
Fibrinogen Binding ◽  
Related Defect ◽  
Rice University ◽  
Granule Release

We have previously reported that patients with Glanzmann’s Thrombasthenia (GT) fail to adhere to a carbon-formvar surface and undergo contact-induced shape change in a non-flow system. The ability of ADP to reverse this adhesion defect suggested that it may be secondary to defective dense granule release rather that a direct requirement for GPIIb-IIIa. To further assess the role of GPIIb-IIIa in adhesion, we examined the effect of two mouse monoclonal antibodies to the GPIIb-IIIa complex, AP2 (IgG, kappa) from T. Kunicki, Milwaukee Blood Center and MAb36 (IgM, lambda) from D. Peterson, Rice University. AP2 (1:50 dil) and MAb36 (1:200 dil) both completely abolished aggregation by ADP, collagen and epinephrine and prevented clot retraction. In a transmission EM (TEM) whole mount assay of adhesion and contact-induced shape change, both antibodies inhibited platelet attachment to the substrate and impaired spreading in those few platelets that did attach. This antibody-induced adhesion defect was reversed by the addition of 2×10−6 m ADP just prior to exposure of platelets to the activating surface. In parallel studies, antibody treated platelets demonstrated a dose-related defect in ATP release as measured in a Lumiaggregometer with total absence of release at antibody dilutions that abolished aggregation. Using a colloidal gold-fibrinogen probe, virtual absence of binding of exogenous fibrinogen was demonstrated in antibody treated platelets induced to. spread by ADP stimulation. These studies suggest that while GPIIb-IIIa may play a role in adhesion in non-flow systems, as suggested by the altered adhesion seen in GT platelets, adhesion and adhesion-induced shape change can be supported by ADP stimulation in the absence of fibrinogen binding to GPIIb-IIIa.

scholarly journals Platelet activation by immobilized monoclonal antibody: evidence for a CD9 proximal signal

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1753-1759 ◽  
Author(s):  
L Griffith ◽  
J Slupsky ◽  
J Seehafer ◽  
L Boshkov ◽  
AR Shaw
Keyword(s):  
Platelet Aggregation ◽  
Hla Class I ◽  
Human Platelets ◽  
Dense Granule ◽  
Polystyrene Latex ◽  
Cross Linking ◽  
Latex Beads ◽  
Dose Dependent ◽  
Aggregation Of Platelets ◽  
Granule Release

Abstract Anti-CD9 monoclonal antibodies (MoAbs) are reported to activate human platelets through stimulation of the Fc gamma II receptor. We show here that nonstimulatory F(ab')2 fragments of the anti-CD9 MoAb 50H.19 induce dense-granule release and dose-dependent platelet aggregation when attached to polystyrene latex beads. Cross-linking F(ab')2 fragments of MoAb 50H.19 by F(ab')2 fragments of goat anti-mouse IgG does not result in platelet aggregation unless the second antibody is bound to latex beads, indicating that immobilization, and not cross- linking of the stimulus, is critical to the initiation of the CD9 signal. In contrast, F(ab')2 fragments of the second antibody readily induce the aggregation of platelets treated with the anti-Fc gamma II receptor MoAb IV.3. Immobilization of MoAb per se is insufficient to induce an activation signal because intact and F(ab')2 fragments of nonstimulatory MoAb directed to glycoprotein Ib and HLA class I do not become stimulatory when attached to beads. CD9-induced activation requires cytoskeletal rearrangement because it is inhibited by cytochalasin B. Aggregation is blocked by inhibitors of the thromboxane pathway, indicating that CD9 activates phospholipase C indirectly through prior activation of phospholipase A2.\

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.

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