Platelet Aggregation Caused by Dithiothreitol

1984 ◽  
Vol 51 (01) ◽  
pp. 119-124 ◽  
Author(s):  
M B Zucker ◽  
N C Masiello
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Electrophoretic Patterns ◽  
Discernible Effect ◽  
Variable Extent ◽  
Triton X ◽  
Induced Aggregation ◽  
Platelet Shape

SummaryMacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DTT- induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0° C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to “expose” the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I- labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

scholarly journals Effects of Lectins on Blood Platelets from Various Species

1977 ◽  
Author(s):  
O. Tangen ◽  
B. Karlstam ◽  
S. Bygdeman
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Human Platelets ◽  
Serotonin Release ◽  
Variable Degree ◽  
Con A ◽  
Platelet Release ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Platelet Shape

Earlier it has been shown that different lectins induce a variable degree of aggregation of platelets. The present study confirmed previous data and demonstrated that wheat germ agglutinin (WGA) was very active, 1eucoagglutinin had about a tenth of the activity of WGA on a concentration basis, and Con A had a weak aggregating effect on human gel filtered platelets (GFP). Soy bean lectin did not aggregate human GFP.The fact that adenosine inhibited WGA- and leucoagglutinin-induced aggregation that WGA and Con A caused serotonin release, and that the aggregation- curves indicated platelet shape change are indications that the lectins influenced glycosyl moieties involving one or more molecules relevant to release and aggregation reaction.GFP were markedly more responsive to the lectins than platelets in plasma, probably due to interfering glycosyl groups amongst the plasma constituents.Platelets from man, rabbit, rat, cow and pig reacted differently towards the lectins, human platelets being the most reactive and bovine and porcine platelets being almost unreactive. These results pose intriguing questions regarding the glycosyl content of platelet membranes in different species and their relation to platelet release and aggregation.

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.

LUMI-AGGREGOMETER STUDIES OF THE INITIAL ATP-SECRETION FROM COLLAGEN-ADHERENT PLATELETS

1987 ◽  
Author(s):  
R Malmgren
Keyword(s):  
Platelet Adhesion ◽  
Rank Order ◽  
Shape Change ◽  
Dense Granule ◽  
Dependent Manner ◽  
Equal Degree ◽  
Atp Secretion ◽  
Ic50 Values ◽  
Dose Dependent ◽  
Platelet Shape

We have earlier, with the use of a lumi-aggregometer and sub-aggregating doses of collagen (0.2-0.8 ug/ml PRP), been able to detect the initial, aspirin-insensitive secretion of ATP from the collagen-adherent platelets, and to correlate this secretion to the doses of collagen, and onset and degree of subsequent shape change of non-adherent platelets (Malmgren, Thromb Res 4:445, 1986). The present study shows, that 200 ATU of hirudin,which reduced near-maximal aggregation and ATP-secretion induced by high collagen doses (2.5 ug/ml PRP) from 3.35 ± 0.2 uM to 2.85 ± 0.1 uM, did neither reduce the secreted amount of ATP that were 82.5 ± 15 nM in control samples and 90 ± 27.5 nM in hirudin-treated samples, nor reduce platelet shape change when platelets were challenged with 0.31 ug collagen /ml PRP. (200 ATU hirudin completely abolished an equal degree of platelet shape change induced by 0.01 U thrombin). Assuming that 3 % of the platelets in PRP were actually adhering to the collagen fibrils, the secreted amount corresponds to 14.6 ±0.04 pmoles ATP/106adheringplatelets, amounts which closely represented 100 % of their dense granule content. The finding confirms that hirudin does not inhibit platelet adhesion and also indicates, that thrombin-mediated activation of secretory pathways appears not to be involved during the initial phase of platelet-collagen interactions.Dipyridamole (DPA) and dibutyryl cAMP (DBcAMP) inhibited ATP-secretion and platelet aggregation in a dose-dependent manner at high collagen concentrations, but only DBcAMP caused a dose-dependent reduction of ATP secretion (IC50 =10-4 M) induced by sub-aggregating doses of collagen. DPA was devoid of effect in this respect and thus did not inhibit platelet adhesion.Yohimbine, dihydroergotamine and phentolamine reduced ATP-secretion induced by sub-aggregating collagen doses in the mentioned rank order of potency, and with IC50 values in the micromolar range. Ketanserin, ritanserin and propranolol were devoid of effect. The findings suggest that the initial collagen-plate-let interaction involve alfareceptor-mediated mechanisms that may encompass adhesion, while DBcAMP probably interacts with secretory mechanisms connected to phosphatidylinositol turnover.

Possible Differences In The Mode Of Action Of Ditazole And Non-Steroidal Anti-Inflammatory Drugs As Potential Antithrombotic Agents

1981 ◽  
Author(s):  
A K Sim ◽  
A P McCraw ◽  
L Caprino ◽  
F Antonetti ◽  
L Morelli
Keyword(s):  
Platelet Aggregation ◽  
Mode Of Action ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Dose Range ◽  
Oral Drug ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
Induced Aggregation ◽  
Platelet Shape

Ditazole (4,5-diphenyl-2-diethanolamino-oxazole), a weak anti-inflammatory drug, has been shown to be a potent inhibitor of platelet aggregation, adhesiveness and bleeding time. Acetylsalicylic acid (ASA), dipyridamole and a combination of these two drugs induced a platelet shape change which was much shorter lasting than their effect on platelet aggregation. Conversely, similar doses of ditazole induced a potent shape change but no effect on aggregation. Ditazole has now been shown to reversibly antagonise thromboxane A2 (TXA2)-induced contraction of rabbit aortic strips at an optimal concentration of 25 μm in the perfusate. Separately, over a dose range of 50-400 mg/kg/p.o., TXA2 production was inhibited between 39% and 85% in spontaneously clotted rabbit blood. In addition, we have shown that TXA2 formation following arachidonic acid-induced aggregation of platelet-rich plasma (PRP) is similarly inhibited. Ditazole however did not inhibit prostacyclin (PGI2) production in rabbit aortic rings following oral drug administration over a dose range of 50-400 mg/kg. At 1000 and 2000 mg/kg PGI2 production was inhibited by 23% and 41% respectively. TXA2 and PGI2 levels were measured by radioimmunoassay of their stable derivatives TXB2 and 6-keto-PGF1α. It is suggested that the mode of action of ditazole may be more specific than the cyclo-oxygenase/PG-synthetase blocking activity of most other non-steroidal anti-inflammatory drugs.

scholarly journals The effect of aggregation and release on platelet prothrombin- converting activity

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 659-672 ◽  
Author(s):  
AC Cox ◽  
P Inyangetor ◽  
CT Esmon ◽  
BN White
Keyword(s):  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Dense Body ◽  
Shape Change ◽  
Factor Xa ◽  
Dense Bodies ◽  
Activated Platelets ◽  
Induced Aggregation ◽  
Body Secretion ◽  
Platelet Shape

Abstract Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel- filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.

Regulation of canine platelet function II. Catecholamines

1983 ◽  
Vol 245 (1) ◽  
pp. R100-R109 ◽  
Author(s):  
K. M. Meyers ◽  
L. Y. Huston ◽  
R. M. Clemmons
Keyword(s):  
Arachidonic Acid ◽  
Adrenergic Receptor ◽  
Dense Granule ◽  
Platelet Response ◽  
Receptor Agonists ◽  
Alpha Adrenergic Receptor ◽  
Acid Metabolites ◽  
Induced Aggregation ◽  
The Relationship ◽  
Platelet Shape

The action of epinephrine (E) on canine platelet aggregation is described. Although E did not induce a change in platelet shape or aggregation, potentiation of aggregation induced by the following agents was observed at physiological E concentrations (that is, less than 10 nM/1): arachidonic acid; the dense granule agonists, ADP and serotonin (5-HT); and collagen. Epinephrine-induced potentiation was in part independent of formation of arachidonic acid metabolites, and E potentiated the aggregating action of the bivalent cationophore A23187. Potentiation was inhibited by alpha-adrenergic receptor antagonists phenoxybenzamine, phentolamine, and ergotamine, and mimicked by alpha-adrenergic receptor agonists norepinephrine, clonidine, and in some cases, phenylephrine. The beta-adrenergic receptor agonists isoproterenol and dobutamine inhibited ADP-induced aggregation, and this action was presented by pretreating the platelets with propranolol and dichloroisoproterenol. An augmentation of the aggregation response of platelets to arachidonic acid was observed in blood samples withdrawn when circulating catecholamines were elevated. The physiological implication of epinephrine acting as a gain controller that alters the relationship between actuating signal and the platelet response to an agonist is discussed.

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 Changes in phosphatidylinositol-4,5-bisphosphate 10 seconds after stimulation of washed rabbit platelets with ADP

Blood ◽  
1982 ◽  
Vol 60 (6) ◽  
pp. 1247-1250
Author(s):  
JD Vickers ◽  
RL Kinlough-Rathbone ◽  
JF Mustard
Keyword(s):  
Shape Change ◽  
Adenosine Diphosphate ◽  
Cellular Responses ◽  
Release Reaction ◽  
Rabbit Platelets ◽  
Induced Aggregation ◽  
Platelet Shape ◽  
Stimulation Of

Adenosine diphosphate (ADP) induced aggregation of rabbit platelets, without the release reaction, causes a significant decrease (7%) in the amount of phosphatidylinositol-4,5-bisphosphate (PIP2) at 10 sec and at 60 sec (11%). In platelets prelabeled with 32P-phosphate, this decrease in PIP2 is associated with a decrease in PIP2 radioactivity, which is significant at 50 sec. The decrease in PIP2 is sufficient to mobilize about 0.18 nmole Ca2+/10(9) platelets. In view of the key role played by Ca2+ in ADP-induced platelet shape change and aggregation, this evidence is compatible with the hypothesis that changes in PIP2 can be a source of calcium for cellular responses to agonists.

New Evidence For An Adp-Independent Mechanism Of Thrombin Induced Platelet Activation

1981 ◽  
Author(s):  
W R Figures ◽  
R F Colman ◽  
S Niewiarowski ◽  
T Morinelli ◽  
Y Wachtfogel ◽  
...  
Keyword(s):  
Shape Change ◽  
Fibrinogen Binding ◽  
Sds Page ◽  
Independent Mechanism ◽  
Human Thrombin ◽  
Induced Aggregation ◽  
New Evidence ◽  
Adenosine Nucleotide ◽  
Platelet Shape

The adenosine nucleotide analog, 5’fluorosulfonylbenzoyl adenosine (5’FSBA) is a potent inhibitor of ADP induced platelet shape change, aggregation, and fibrinogen binding and covalently incorporates into a single membrane polypeptide (Mr=100,000). To evaluate the role of released intracellular ADP in thrombin induced aggregation and fibrinogen binding, the effects of 5’FSBA on these events were studied. Following incubation with 5’FSBA, the platelets became unresponsive to ADP but were aggregated by purified human thrombin (0.2 U/ml). However, the aggregation was slower and less extensive than that of untreated control platelets. Binding of 125I-fibrinogen to control platelets occured after exposure to ADP or after exposure to thrombin in the presence of hirudin to inhibit clotting. Binding of 125I-fibrinogen was absent in platelets treated with 5’FSBA after exposure to ADP and diminished after incubation with thrombin (0.2 U/ml, 5 min.).The effect of thrombin on the single platelet membrane polypeptide modified by 5’FSBA was examined by SDS-PAGE. Thrombin treatment (0.5 U/ml, 30 min.) of cells labeled with (H)-5’FSBA resulted in partial cleavage of this polypeptide as evident by a decrease in the (H) FSBA-modified polypeptide in the thrombin treated samples as compared to controls. Unlike chymotrypsin activation of platelets, no fragment of the protein at 75,000 daltons was associated with the membranes of thrombin treated cells. The results of these experiments indicate that thrombin induced platelet aggregation and fibrinogen binding depend on both ADP binding which is blocked by 5’FSBA and the proteolytic cleavage of the same 100,000 dalton membrane polypeptide cleaved by chymotrypsin.

Proteolysis of the platelet surface: dissociation of shape change from aggregation

1986 ◽  
Vol 250 (4) ◽  
pp. H550-H557
Author(s):  
E. Kornecki ◽  
Y. H. Ehrlich ◽  
D. H. Hardwick ◽  
R. H. Lenox
Keyword(s):  
Adenylate Cyclase ◽  
Binding Sites ◽  
Prostaglandin E1 ◽  
Direct Evidence ◽  
Shape Change ◽  
Adenylate Cyclase System ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Induced Aggregation ◽  
Platelet Shape

Stimulation of intact platelets by ADP results in a shape change followed by aggregation in the presence of fibrinogen. ADP was found to induce a shape change in chymotrypsin-treated platelets that was similar in extent and initial velocity to that of intact (untreated) platelets. Scanning-electron microscopy verified an ADP-induced shape change in chymotrypsin-treated platelets. This shape change could be completely blocked by stimulators of platelet adenylate cyclase (forskolin, prostaglandin E1, and prostacyclin). On the other hand, the aggregation of chymotrypsin-treated platelets by fibrinogen was not dependent on the presence of ADP and could not be blocked by forskolin, prostaglandin E1, or prostacyclin, even though the levels of cyclic AMP (cAMP) formed in chymotrypsin-treated platelets were comparable to levels that completely inhibited the ADP-induced aggregation of intact platelets. This lack of inhibition of platelet aggregation was not due to degradation of the adenylate cyclase or prostaglandin receptors, since chymotrypsin-treated platelets were found to have a functional adenylate cyclase system that could be stimulated by forskolin, prostaglandin E1, and prostacyclin and inhibited by ADP and epinephrine, similar to that of intact platelets. These results provide direct evidence that cAMP does not interact with fibrinogen binding sites once they have become permanently exposed on the surface of platelets. Pretreatment of platelets with chymotrypsin therefore appears to be a useful tool that allows for the dissociation of platelet shape change from aggregation, without inhibiting either response.

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