Partial Agonists as Probes of Human Platelet Stimulus-Response Coupling in the Response to ADP and Adrenaline

1979 ◽  
Author(s):  
M.C. Scrutton ◽  
J.A. Grant ◽  
C.M. Egan
Keyword(s):  
Cyclic Amp ◽  
Human Platelet ◽  
Shape Change ◽  
Human Platelets ◽  
Stimulus Response ◽  
Partial Agonists ◽  
Aggregation Response ◽  
A 23187 ◽  
Intracellular Ca ◽  
Adp Receptor

Previous studies have identified 2', 3'-dialdehyde-ADF (oADP) and -dialcohol-ADP (or ABI as partial agonists at the ADP receptor, and Clonidine and methoxamine as partial agonists at the n-adrenoceptor.oADP and orADP cause shape change but not aggregation (Euxop, J, Biochem, 88, 543): Clonidine and methoxamine fail to cause aggregation but enhance the response to ADP and other agonists (Nature, in press). Preincubation of platelets with non-aggregating concentrations of the ionophore A-23187 induces a primai aggregation response to o ADP, and a primary aggregation + a secretion response to or ADH This response is specific to the partial agonists; is blocked by an ADP antagonist and by agents which raise cyclic AMP levels (e.g. PGE) or block intracellular Ca++ movemenl (e.g. tetracaine); and is not inhibited by specific chelation of extracellular Ca+. Under similar conditions A-23187 provokes a full aggregation response to Clonidine (which is blocked effectively by yohimbine but not by praiosin) and a primary aggrtion response to methoxamine. Preincubation of platelets with an adenylate cyclase inhibit; (SQ-22536) fails to provoke an aggregation response to oADP or orADP. These data suppt the concept that an increase in oytosolic [Ca2+], rather than a decrease in [cAMP], is key step in initiation of the response of human platelets to ADP and adrenaline.

Inhibition of ADP-Induced Responses in Human Platelets by Agents Elevating the Cyclic AMP Level: Comparison of Aggregation and Shape Change

1984 ◽  
Vol 52 (03) ◽  
pp. 333-335 ◽  
Author(s):  
Vider M Steen ◽  
Holm Holmsen
Keyword(s):  
Inhibitory Action ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Early Step ◽  
Stimulus Response ◽  
Sequential Relationship

SummaryThe inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses.Human platelet-rich plasma was preincubated for 2 min at 37° C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors.These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.

Depressed Responsiveness To Adrenaline In Platelets From Apparently Normal Human Donors – A Familial Trait

1981 ◽  
Author(s):  
M C Scrutton ◽  
K R Bruckdorfer ◽  
R A Hutton
Keyword(s):  
Adenylate Cyclase ◽  
Platelet Function ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
The Other ◽  
Stimulus Response ◽  
A 23187 ◽  
Phospholipid Classes ◽  
Normal Human

Decreased responsiveness to adrenaline has been observed in 5 out of approximately 150 apparently normal unrelated human donors. In 4 donors, familial studies have shown that this trait is inherited. Three of the donors, as well as their affected relatives, exhibit depressed responsiveness to collagen and vasopressin but normal responsiveness to ADP and thrombin in association with the decreased responsiveness to adrenaline. The other two affected donors exhibit normal responsiveness to most other agonists although in one instance depression of responsiveness to vasopressin and absence of a secretory response to ADP may be associated with the decreased adrenaline response.Normal responsiveness can be restored in all instances either by incubating the platelet-rich plasma at 20°C or by addition at 37°C of a low concentration of the divalent cation ionophore, A-23187. No such effect results from addition of an adenylate cyclase inhibitor. All affected platelets have normal ATP and ADP contents, cholesterol to phospholipid ratios, and composition of the phospholipid classes. Mixing experiments demonstrate the absence of a circulating inhibitor of platelet function and suggest that the defect resides in the platelets. We conclude that depressed responsiveness of human platelets to adrenaline may result from a defect in Ca2+ mobilisation to the cytosol. The observed selectivity in the agonists affected may indicate that the stimulus-response coupling pathways converge at the level of an increase in cytosolic Ca2+ concentration.

scholarly journals Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

Blood ◽  
1978 ◽  
Vol 51 (6) ◽  
pp. 1065-1071 ◽  
Author(s):  
S Levy-Toledano ◽  
G Tobelem ◽  
C Legrand ◽  
R Bredoux ◽  
L Degos ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Shape Change ◽  
Antigenic Site ◽  
Human Platelets ◽  
Clot Retraction ◽  
Igg Antibody ◽  
Normal Human ◽  
Induced Aggregation ◽  
Bovine Factor

Abstract In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

Stimulation Of Human Platelets By Lysophosphatidic Acids

1981 ◽  
Author(s):  
D E MacIntyre
Keyword(s):  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Lysosomal Hydrolases ◽  
Stimulus Response ◽  
Ca Antagonist ◽  
Intracellular Ca ◽  
Arachidonate Metabolites ◽  
Irreversible Aggregation ◽  
Subsequent Addition

Production of lysophosphatidic acids (LPA) by “activated” cells may be important in stimulus-response coupling. Using human citrated platelet rich plasma (PRP) we examined the effects of Oleoyl (0), Palmitoyl (P) and Decanoyl (D) LPAS on platelet function. DLPA (≤300 μM) did not affect platelet function, and OLPA and PLPA (≤200 μM) did not inhibit uptake of 3H-5HT (0.3 μM). However OLPA and PLPA (1-30 μM) induced reversable aggregation in the absence of 5HT release (primary aggregation), and higher concentrations (≤300 μM) induced irreversible aggregation and TxB production and release of ≤79% of 5HT, 89% of βTG and ≤73% of β -N-acetyl glucosaminidase that was released by thrombin (1U/ml). LPA-induced release of platelet granule constituents was associated with <5% release of cytoplasmic markers LDH or 3H-adenine. OLPA or PLPA (300 μM) suppressed the elevation of platelet cyclic AMP induced by PGE1. Primary and secondary aggregation induced by LPA were suppressed by PGE1 (1 μ M) and the intracellular Ca antagonist, TMB-8 (200 μM). The Ca transport inhibitor verapamil (100 μM) was ineffective. Indomethacin (30 μM) and the ADP antagonist, β,γ-methylene-ATP (30 μM) inhibited only secondary aggregation. PRP exposed to OLPA or PLPA (30 μM) for greater than two minutes at 37°C failed to respond to subsequent addition of LPA but showed augmented responses to ADP, U 46619, vasopressin and 5HT. OLPA and PLPA resemble the more powerful platelet agonists (e.g., collagen, thrombin) in that they induce secretion of βTG, 5HT and lysosomal hydrolases and cause aggregation that is independent of ADP, 5HT or arachidonate metabolites. Endogenous LPA may function as a mediator of platelet aggregation and degranulation. Alternatively LPA may serve as a mimic of related platelet agonists (acetyl- glyceryl-ether-phosphorylcholine [AGEPC], or PAF-acether).

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

Adenosine 5’-O-(2-Thiodiphosphate) (ADP-β-S) Is A Partial Agonist At The ADP Receptor Of Human Platelets

1981 ◽  
Author(s):  
N J Cusack ◽  
S M O Hourani
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Human Platelet ◽  
Partial Agonist ◽  
Human Platelets ◽  
Simultaneous Addition ◽  
Prostaglandin Receptor ◽  
Human Platelet Aggregation ◽  
Adp Receptor ◽  
Stimulation Of

ADP induces human platelet aggregation and inhibits the stimulation of platelet adenylate cyclase by prostaglandin E1 (PGE1), but analogues of ADP in which the diphosphate group is modified retain only weak aggregating activity. However, ADP-β-S, an ADP analogue in which a terminal phosphate oxygen is replaced by sulphur, is known to be equipotent with ADP as an inhibitor of PGE1-stimulated adenylate cyclase in purified human platelet membranes. We therefore tested ADP-β-S on intact human platelets. ADP-β-S induced human platelet aggregation and inhibited PGE1-stimulated adenylate cyclase, but in botn cases was less potent than ADP and only achieved 75% and 50% respectively of the maximal effects of ADP. Aggregation induced by ADP-β-S was competitively inhibited by ATP (50 μM), a known ADP antagonist.Both these actions of ADP could be inhibited by the simultaneous addition of ADP-β-S (50 μM). Aggregation induced by a stable endoperoxide analogue (11 ,9 -epoxymethano PGH2), which acts at a prostaglandin receptor rather than at an ADP receptor, was not inhibited by the simultaneous addition of ADP-β-S (50 μM). The behaviour of ADP-β-S towards human platelets is therefore tnat of a partial agonist at the ADP receptor.

Arg333 and Arg334 in the C-Terminus of the Human Platelet P2Y1 Receptor Are Crucial for Gq Coupling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1566-1566
Author(s):  
Zhongren Ding ◽  
Florin Tuluc ◽  
Kavita R. Bandivadekar ◽  
Lili Zhang ◽  
Jianguo Jin ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Human Platelet ◽  
Inositol Phosphate ◽  
Shape Change ◽  
P2y Receptors ◽  
Human Platelets ◽  
Carboxyl Terminus ◽  
P2y1 Receptor ◽  
Functional Responses ◽  
C Terminus

Abstract ADP-induced platelet aggregation plays an important role in hemostasis and thrombosis. Human platelets express two ADP receptors: the P2Y1 and P2Y12 receptors. The Gq-activating P2Y1 receptor plays an important role in ADP-induced platelet shape change, aggregation, and thromboxane A2 generation. In this study, we investigated the role of the carboxyl terminus of the human P2Y1 receptor in Gq activation. Human P2Y1 receptors, either wild type (P2Y1-WT) or a mutant in which the C-terminus was truncated (P2Y1-ΔT330-L373), were stably expressed with an HA-tag at the N-terminus in CHO-K1 cells. Stimulation of P2Y1-WT cells with 2-MeSADP caused inositol phosphate production and mobilization of calcium from intracellular stores. In contrast, P2Y1-ΔT330-L373 completely lost its response to 2-MeSADP, indicating that the C-terminus of the human P2Y1 receptor is essential for the activation of Gq. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 carboxyl terminus failed to elicit Gq functional responses, indicating that the P2Y1 carboxyl terminus is essential but not sufficient for Gq activation. Radioligand binding demonstrated that both the P2Y1-WT- and P2Y1-ΔT330-L373- expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. Two additional truncation mutants in the C-terminus, P2Y1-ΔR340-L373 and P2Y1-ΔD356-L373, were expressed in CHO-K1 cells, and responded to 2-MeSADP with functional responses. These results indicate that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y1 receptor are essential for Gq coupling. Finally, the cells expressing a double mutant P2Y1 receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R333R334) in the C-terminus of the human platelet P2Y1 receptor are essential for Gq coupling and subsequent platelet activation.

Effects of Inhibitors of ADP-induced Aggregation on Platelet Nucleo-sidediphosphate Kinase (NDK) Activity

1975 ◽  
Author(s):  
M. A. Packham ◽  
J. F. Mustard ◽  
M. A. Guccione ◽  
D. W. Perry
Keyword(s):  
Sulfonic Acid ◽  
Shape Change ◽  
Human Platelets ◽  
Phosphoryl Group ◽  
Platelet Surface ◽  
Rabbit Platelets ◽  
Adenosine Derivatives ◽  
Adp Receptor ◽  
Presence And Absence ◽  
Induced Aggregation

We showed previously (Am. J. Physiol. 227, 1143, 1974) that ADP reacts with NDK on the surface of platelets; when 14C-ADP is added to suspensions of washed rabbit platelets, it is partially converted to 14C-ATP in the suspending fluid. This is more difficult to show with human platelets because of their greater ATPase activity. With both species, -external ATP (10 μM), and other NTP’s at higher concentrations, enhance formation of “C-ATP form “C-ADP. All the NTP’s inhibit ADP-induced aggregation, possibly by acting as external donors of a phosphoryl group which, in their absence, is normally donated to ADP by platelets. Studies of other inhibitors have been done with suspensions -of washed rabbit platelets. The other NDP’s and AMP inhibit, ADP-induced shape change, aggregation, and conversion of 14C-ADP to 14C-ATP, probably by competition for the same site on NDK. AMP inhibits NDK both in the presence and absence of external ATP. Parachloromercuribenzene sulfonic acid has a similar effect. In contrast, prostaglandin E1 and hydroxylamine inhibit ADP-induced shape change and aggregation, but inhibit NDK only in the absence of external ATP, not in its presence. ADP-induced shape change without aggregation occurs in the presence of EGTA or EDTA but only half as much 14C-ADP is converted to 14C-ATP in the first minute. EGTA does not inhibit NDK in the presence of external ATP but EDTA does. Inhibition of NDK by adenosine was observed in some experiments but the adenosine derivatives 2-n-amylthioadenosino, 2-cyclopentylthioadenosine and 2-cyclophexylthioadenosine (Kohjin Co., Tokyo) inhibited strongly both in the presence and absence of external ATP; these compounds also inhibit ADP-induced aggregation. Thus all compounds tested which inhibit NDK also inhibit ADP-induced aggregation, indicating that NDK may be the ADP receptor on the platelet surface. Donation of a phosphoryl group from the platelets to ADP, catalyzed by this enzyme, may be responsible for initiating ADP-induced platelet aggregation.

scholarly journals Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

Blood ◽  
1978 ◽  
Vol 51 (6) ◽  
pp. 1065-1071
Author(s):  
S Levy-Toledano ◽  
G Tobelem ◽  
C Legrand ◽  
R Bredoux ◽  
L Degos ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Shape Change ◽  
Antigenic Site ◽  
Human Platelets ◽  
Clot Retraction ◽  
Igg Antibody ◽  
Normal Human ◽  
Induced Aggregation ◽  
Bovine Factor

In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

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