Human Platelet Secretion Research Articles

Inability of divalent cation complexes of A23187 to induce platelet secretion

Extracellular calcium produces a dose-dependent inhibition of the early time course of A23187-induced 14C-5HT secretion in human platelets which is independent of the occurrence of aggregation. Pre-incubation of A23187 with calcium to form the A23187-Ca complex results in markedly prolonged lag periods prior to the onset of secretion at Ca concentrations of 0.25 – 1.0 mM and total abolition of the secretion response at 2.0 mM Ca, indicating that these effects of extracellular Ca are due to specific interactions of Ca and A23187. Under conditions which favor dissociation, the complexes of A23187 with Sr, Ca, and Mg, but not Mn, all induce platelet secretion but produce an inhibition of the early secretion response which generally parallels their relative complex stabilities, i.e., Sr < Ca ≈ Mg < Mn. Under conditions which prevent dissociation, none of these complexes induce secretion. These results indicate that the complexes of A23187 with divalent cations, including Ca, are unable to induce platelet secretion, most likely because of an inability to penetrate the platelet plasma membrane.

Inhibition of aggregation and secretion of human platelets by quercetin and other flavonoids: structure-activity relationships.

Quercetin and 12 other natural flavonoid aglycones inhibit washed human platelet aggregation and secretion of serotonin induced by ADP, collagen or thrombin. The inhibitory effect of flavonoids is of the same order of magnitude as IBMX and dipyridamole. The structural features required for a flavonoid to inhibit human platelet function are similar to those previously reported by us to inhibit cyclic nucleotide phosphodiesterase. The inhibitory effect of flavonoids on human platelet function was diminished by saturation of the C-2, C-3 double bond, lack of the C-4 carbonyl, glycosylation at C-3 and a high number of hydroxyl substituents.

Inhibition of the Platelet Reaction by a High Molecular Weight Phosphoglycoprotein Isolated from Human Platelet Plasma Membranes

The effect of phospho-glycoprotein (HMW-GP), obtained from human platelet plasma membranes, on the aggregation and secretion of human platelets was studied. Incubation of PRP with 4 to 16 micrograms/ml of HMW-GP results in inhibition of ADP-, Epinephrine-, Collagen-, and Thrombin-induced platelet aggregation. The effect is mainly reflected on the secondary wave of aggregation. The inhibitory effect is partially overcome by higher concentrations of inducers, however, even under these conditions, a clear tendency towards disaggregation is observed. 5HT release (Col-induced) is strongly decreased from 50% to 4.5. The inhibitory effect on Thrombin-induced aggregation is markedly dependent on external calcium, being maximal at 5 mM calcium. The HMW-GP does not bind ADP or Thrombin. Membrane conformation is markedly affected, as evidenced by the effect of HMW-GP on the iodination of surface polypeptides of intact platelets. It is suggested that interaction of HMW-GP with the platelet membrane blocks the signal(s) transmission that links stimulus to activation. The inhibition observed might just represent an experimental amplification of the endogenous modulatory function that has been proposed for this high molecular weight phosphoglycoprotein.

The Interrelationship between Thromboxane Biosynthesis, Aggregation and 5-Hydroxytryptamine Secretion in Human Platelets in Vitro

Platelet aggregation, secretion of 5-hydroxy tryptamine and production of thromboxane B2 were monitored simultaneously in human platelet suspensions in the absence and presence of cyclooxygenase or thromboxane synthetase inhibitors. Aggregation, secretion and thromboxane B2 formation in response to either sodium arachidonate or epinephrine were blocked by aspirin or by 1-N-butyl inidazole suggesting that thromboxane biosynthesis was an essential requirement for platelet activation by these agents. In contrast, thrombin and collagen could apparently induce aggregation and secretion via two pathways: at low doses involving thromboxane production, but at higher doses by a direct mechanism independent of thromboxane biosynthesis. In the case of ADP, inhibition of thromboxane production blocked secretion but had little effect on aggregation, indicating that secretion was probably dependent on thromboxane biosynthesis which probably occurred as a result of aggregation. Thus it appears that although the processes of thromboxane production, release of dense granule constituents and aggregation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.

Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides.

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 [(14)C]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 [(14)C]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 (14)C-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, beta-N-acetylglucosaminidase, beta-glucuronidase and beta-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.

Mechanisms of cellular interaction with monosodium urate crystals. IgG-dependent and IgG-independent platelet stimulation by urate crystals.

Monosodium urate crystals (MSU) stimulate suspensions of washed platelets or neutrophils. When MSU crystals are coated with IgG, as occurs in plasma, stimulation is markedly enhanced. These studies which use MSU-induced human platelet serotonin secretion as a model examine the nature of cellular recognition mechanisms for MSU crystals and IgG-coated MSU crystals. F(ab')2 fragments of specific anti-Fc antibody blocked and the lipopolysaccharide of Salmonella minnesota R595 enhanced human platelet secretion induced by IgG-coated urate crystals. These agents had little effect on stimulation by uncoated crystals. This indicated that urate crystals stimulate platelets independently of fluid phase IgG. Urate crystals directly stimulated suspensions of washed rabbit platelets which lack Fc receptors. In contrast to human cells, stimulation was blocked by IgG. This again demonstrated IgG-independent cell stimulation by urate crystals. Calcium pyrophosphate dihyrate crystals could trigger human platelet secretion only when coated with IgG. This suggests that when crystals are coated with IgG, the surface-bound IgG alone may be the stimulus to the cell. This was supported by the finding that polyvinylpyridine-N-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated, but not IgG-coated, urate crystals. These data indicate that urate crystals (and potentially other surface or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of adsorbed IgG or, when coated with IgG, by triggering the cell via immunoglobulin receptors.

Thrombin-induced oxygen consumption, malonyldialdehyde formation and serotonin secretion in human platelets

The relationship of a thrombin-induced burst in O 2 consumption to lipid peroxidation in washed human platelets was investigated by measuring malonyldialdehyde, a by-product of endoperoxide degradation in platelets. The ratio of O 2 consumed to malonyldialdehyde produced was approximately 7:1. Acetylsalicylate blocked the formation of malonyldialdehyde completely and partially inhibited the O 2 burst induced by thrombin. 5,8,11,14-Eicosatetraynoic acid inhibited the O 2 burst as well as the malonyldialdehyde formation completely. The release of [ 14C]serotonin was not affected by either inhibitor.

Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP.

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).