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.

Species Variability in Platelet and other Cellular Responsiveness to Thrombin Receptor-derived Peptides

1994 ◽  
Vol 72 (04) ◽  
pp. 627-633 ◽  
Author(s):  
Thomas M Connolly ◽  
Cindra Condra ◽  
Dong-Mei Feng ◽  
Jacquelynn J Cook ◽  
Maria T Stranieri ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Intracellular Signaling ◽  
Shape Change ◽  
Fibroblast Cell ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Human Thrombin ◽  
Cellular Responsiveness ◽  
Aggregation Of Platelets

SummaryThe aggregation of platelets from a variety of animal species in response to thrombin receptor-derived activating peptides was evaluated. A series of 14-(SFLLRNPNDKYEPF), 7-(SFLLRNP-NH2), 6-(SFLLRN-HN2) or 5-(SFLLR-NH2) residue peptides, the structures of which were based on the deduced amino acid sequence of the human thrombin receptor, promoted full aggregation of platelets in plasma from humans, African Green and Rhesus monkeys, baboons and guinea pigs at 4-50 μM depending on the peptide used. Platelets in plasma from rabbit, dog, pig, and hamster underwent a shape change but failed to aggregate in response to these peptides over 3 log units of peptide up to 800 μM, despite being fully responsive to human thrombin. However, because the receptor peptides induced shape change in the platelets from these non-aggregating species, they apparently can activate some of the intracellular signaling system(s) usually initiated by thrombin in these platelets. In contrast, platelets from rats did not undergo shape change or aggregate in response to the peptides. A 7-residue receptor-derived peptide based on the deduced amino acid sequence of the clone of the hamster thrombin receptor (SFFLRNP-N2) was nearly as efficacious as the corresponding human receptor-derived 7-residue peptide to promote aggregation of human platelets. However, the hamster peptide could not promote aggregation of hamster platelets in plasma at up to 800 μM peptide, while a shape change response was elicited. Platelets from rats, rabbits and pigs also did not aggregate in response to this peptide derived from the hamster thrombin receptor, but all species except the rat underwent a shape change. Longer 17-residue peptides derived from the sequences of the hamster or mouse thrombin receptors elicited aggregation of human platelets but no aggregation of the hamster platelets. In contrast, the human 14- and 5-residue and the hamster 7-residue thrombin receptor-derived peptides promoted mitogenesis of CCL39 cells, a hamster fibroblast cell line. Finally, the human 6-residue thrombin receptor-derived peptide promoted contraction of normal and de-endothelialized canine coronary artery rings, despite having no pro-aggregatory effect on canine platelets. Taken together, these results demonstrate that the thrombin receptor-derived peptides are able to mimic many, but not all, of the activating effect of thrombin in different tissues from multiple species. The heterogeneity of responsiveness to these peptides should be taken into account in future experiments designed to elucidate the mechanism of thrombin stimulation of platelets and other cells.

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.

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.

Tachyphylaxis in Human Blood Platelets Induced by Various Agonists and the Effect on Response To other Agonists

1979 ◽  
Author(s):  
P.A. Ruggles ◽  
M.C. Scrutton
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Common Pathway ◽  
Stimulus Response ◽  
Reversible Aggregation ◽  
Agonist Concentration ◽  
Subsequent Addition ◽  
Human Blood Platelets

Tachyphylaxis of human platelets to ADP, adrenaline, thrombin, 5-HT and vasopressin (VP) was induced by preincubation of stirred citrated PRP with an agonist concentration which induced primary reversible aggregation. The effect was demonstrable within 2 mins, after addition of some of the agonists and persisted for at least 30 mins. The extent of tachyphylaxis showed a positive correlation with agonist concentration used to induce the initial reversible response. Partial aganists at the ADP (2’, 3’-dialcohol ADP) or α-adreno-(clonidine) receptors did not induce significant tachyphylaxis to subsequent addition of the full agonist. In most instances platelets iaade tachyphylactic to a given agonist showed an unchanged or enhanced response to all other agonists including arachidonate. However tachyphylaxis to ADP, 5HT or thrombin was associated with an attenuated response to collagen. Furthermore tachyphylaxis to thrombin also caused attenuation of the response to VP and arachidonate. Induction of tachyphylaxis to VP, or addition of oxytocin (an inhibitor of aggregation induced by VP) had no effect on the response to thrombin. Thus the region of the thrombin receptor responsible for induction of tachyphylaxis to this agonist is not identical with that at which VP interacts. If stimulus-response coupling involves a common pathway for most agonists these data suggest that development of tachyphylaxis depends on events which preceed the effect of the agonists en this common pathway.

Mechanism of platelet plug formation and role of adenosine diphosphate

1964 ◽  
Vol 206 (6) ◽  
pp. 1267-1274 ◽  
Author(s):  
Theodore H. Spaet ◽  
Marjorie B. Zucker
Keyword(s):  
Connective Tissue ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Suitable Model ◽  
Plug Formation ◽  
Rat Platelets

Traumatized rat omentum was used to demonstrate the development of "platelet plugs" following agitation in platelet-rich plasma. In the absence of divalent cation there was only platelet adhesion to connective tissue fibers; in the presence of divalent cation masses of platelets formed (cohesion) even in plasma adequately anticoagulated with heparin. Exposure of these platelet masses to thrombin produced greater compactness and stability. Human and rat platelets behaved alike with the traumatized rat omentum; platelets from two patients with von Willebrand's disease gave normal reactions whereas platelets from a patient with thrombasthenia showed adhesion only. Exposure of human platelets to washed connective-tissue fragments or to thrombin elicited clumping accompanied by release of serotonin and of adenine nucleotides (AN) of which about one-third was adenosine diphosphate. Intermediate concentrations of connective tissue and thrombin also caused clumping but no liberation of AN or serotonin. ADP caused intense clumping but failed to liberate serotonin or additional ADP. It is suggested that cohesion reaction is mediated by release of ADP. The traumatized omentum appears to be a suitable model for studying the hemostatic process.

scholarly journals Release of human platelet factor V activity is induced by both collagen and ADP and is inhibited by aspirin

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 448-455
Author(s):  
WJ Vicic ◽  
B Lages ◽  
HJ Weiss
Keyword(s):  
Human Platelet ◽  
Initial Response ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Dense Granule ◽  
Antimycin A ◽  
Factor V ◽  
Platelet Factor ◽  
Gradient Separation ◽  
Density Gradient Separation

Factor V activity in suspensions of human platelets washed by albumin density gradient separation increased in response to stimulation by both collagen and adenosine diphosphate (ADP). The appearance of factor V activity extracellularly had the characteristics of platelet secretion and was partially inhibited by aspirin and by the antimetabolites 2-deoxyglucose and antimycin A. Some increase in factor V activity was also observed in platelet suspensions during the initial response to ADP; this activity was not detected extracellularly, but remained associated with the platelets. Patients with storage pool deficiency (SPD) whose platelets are deficient only in dense granule substances released normal amounts of factor V activity, whereas decreased amounts were released in a patient whose platelets have both dense and alpha granule deficiencies. These findings suggest that a portion of platelet factor V is associated with, and released from, alpha granules.

Dichotomous Regulation of Myosin Phosphorylation and Shape Change by Rho-Kinase and Calcium in Intact Human Platelets

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1665-1672 ◽  
Author(s):  
Markus Bauer ◽  
Michaela Retzer ◽  
Jonathan I. Wilde ◽  
Petra Maschberger ◽  
Markus Essler ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Shape Change ◽  
Rho Kinase ◽  
Adenosine Diphosphate ◽  
Receptor Activation ◽  
Human Platelets ◽  
Peptide Ligand ◽  
Thromboxane Receptor ◽  
Mlc Phosphorylation ◽  
Stimulation Of

Both Rho-kinase and the Ca2+/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca2+. Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca2+ and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein–coupled platelet receptors, independent of an increase in cytosolic Ca2+. Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca2+/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).

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.

Platelet Aggregation and Fibrinogen Binding in Human, Rhesus Monkey, Guinea-Pig, Hamster and Rat Blood: Activation by ADP and a Thrombin Receptor Peptide and Inhibition by Glycoprotein IIb/IIIa Antagonists

1993 ◽  
Vol 70 (03) ◽  
pp. 531-539 ◽  
Author(s):  
Nigel S Cook ◽  
Hans-Günter Zerwes ◽  
Carlo Tapparelli ◽  
Max Powling ◽  
Jagjit Singh ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Guinea Pig ◽  
Rhesus Monkey ◽  
Whole Blood ◽  
Flow Cytometric Analysis ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Important Species ◽  
Rat Blood ◽  
Fibrinogen Binding

SummaryPlatelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 μM) and TRP (1-10 μM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation.Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50’s of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50’s (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.These results highlight important species differences in the response of platelets to activation by two different agonists and also in their inhibition by GP-IIb/IIIa antagonists. In particular, platelets from the rat and hamster were insensitive to agonists and antagonists, whereas guinea-pig and rhesus monkey platelets responded with an affinity similar to human platelets. Since these studies were performed in whole blood, the results should be representative of those expected in animal experiments. These recently developed methods for studying platelet responses in small aliquots of whole blood are simple to perform and provide important information concerning the optimal choice of species for subsequent in vivo studies with these compounds.

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