Thromboxane A2/prostaglandin H2 mobilizes calcium in human blood platelets

1985 ◽  
Vol 249 (1) ◽  
pp. H1-H7 ◽  
Author(s):  
L. D. Brace ◽  
D. L. Venton ◽  
G. C. Le Breton
Keyword(s):  
Calcium Release ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Blood Platelets ◽  
Photon Counting ◽  
Thromboxane A2 ◽  
Receptor Interaction ◽  
Significant Release ◽  
Human Blood Platelets ◽  
Calcium Redistribution

The present study investigated the mechanism by which thromboxane A2/prostaglandin H2 (TXA2/PGH2) stimulates platelet activation. Previous studies in isolated platelet vesicles have suggested that TXA2/PGH2 functions to release calcium from intraplatelet stores. On this basis, we investigated whether TXA2/PGH2 causes mobilization of calcium in intact platelets. Calcium redistribution was measured using the fluorescent probe, chlortetracycline (CTC), and a photon-counting microspectrofluorometer. Human platelet-rich plasma was incubated with CTC (50 microM) for 40 min at 25 degrees C. Shape change was induced with arachidonic acid (AA, 100 microM) or ADP (0.75-1.0 microM). It was found that AA addition resulted in a significant release of intraplatelet calcium. This release was blocked by inhibition of the cyclooxygenase with indomethacin (20 microM) or the specific TXA2/PGH2 antagonist, 13-azaprostanoic acid (13-APA, 100 microM). On the other hand, neither indomethacin nor 13-APA had any effect on calcium release stimulated by ADP. However, prostacyclin (13 nM) inhibited both AA- and ADP-induced calcium release. These findings provide evidence that cyclooxygenase products of AA, i.e., TXA2 and/or PGH2 directly caused the mobilization of intraplatelet calcium. Furthermore, this calcium mobilization appears to be mediated through a specific TXA2/PGH2 receptor interaction.

Reversal of thromboxane A2/prostaglandin H2 and ADP-induced calcium release in intact platelets

1985 ◽  
Vol 249 (1) ◽  
pp. H8-H13
Author(s):  
L. D. Brace ◽  
D. L. Venton ◽  
G. C. Le Breton
Keyword(s):  
Receptor Antagonist ◽  
Calcium Release ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Calcium Mobilization ◽  
Receptor Interaction ◽  
Calcium Sequestration ◽  
Platelet Shape

We previously demonstrated that thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2), ADP, and A23187 cause calcium mobilization in intact human platelets. Other studies have also shown that platelet shape change and aggregation induced by a variety of platelet agonists can be reversed by specific antagonists. In the present study, we used the fluorescent calcium probe chlortetracycline to evaluate whether the reversal of platelet activation involves a resequestration of intraplatelet calcium. It was found that the TXA2/PGH2 receptor antagonist 13-azaprostanoic acid (13-APA) reversed calcium mobilization and shape change induced by AA but not that induced by ADP. A similar specificity of action was observed using the specific ADP receptor antagonist, ATP, in that ATP only reversed ADP-induced calcium release and shape change. In contrast, prostacyclin reversed both AA and ADP-induced calcium redistribution and shape change. In the latter experiments, a net calcium sequestration was actually observed on prostacyclin addition. These findings indicate that the resequestration of released calcium leads to platelet deactivation. Furthermore, there appear to be at least two mechanisms by which a reduction in cytosolic calcium can be produced: specific interruption of the agonist-receptor interaction, for example, 13-APA antagonism of TXA2/PGH2; and stimulation of platelet adenosine 3',5'-cyclic monophosphate production by prostacyclin and consequent calcium sequestration.

Ca2+ mobilization in blood platelets as visualized by chlortetracycline fluorescence

1981 ◽  
Vol 241 (4) ◽  
pp. H613-H619
Author(s):  
N. E. Owen ◽  
G. C. Le Breton
Keyword(s):  
Prostaglandin E1 ◽  
Ethylenediaminetetraacetic Acid ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Photon Counting ◽  
Low Doses ◽  
Oxygenase Activity ◽  
Membrane Bound ◽  
The Relationship ◽  
Human Blood Platelets

Binding of intracellular Ca2+ was measured in intact human blood platelets using the fluorescent Ca2+ probe, chlortetracycline, and a photon-counting microspectrofluorometer. Low doses of epinephrine, A23187, or prostaglandin endoperoxide analog U46619 induced a release of intraplatelet membrane-bound Ca2+. When platelet transmembrane Ca2+ flux was blocked by verapamil or ethylenediaminetetraacetic acid (EDTA), Ca2+ mobilization in response to epinephrine was inhibited, whereas A23187- or U46619-induced Ca2+ release was unchanged. When indomethacin was used to inhibit cyclo-oxygenase activity, Ca2+ mobilization in response to epinephrine or U46619 was partially blocked, whereas Ca2+ release in response to A23187 was unaltered. The relationship between platelet cyclic adenosine 3',5'-monophosphate (cAMP) and intraplatelet Ca2+ binding was also investigated. Prostaglandin E1 or prostacyclin was found to markedly elevate cAMP as well as enhance platelet Ca2+ binding. These effects were augmented by inhibition of phosphodiesterase activity using RO201724. The relationship between cAMP and Ca2+ binding was linear in the range of 10-60 pmoles cAmP/ml platelet-rich plasma. In addition the increase in cAMP stimulated by prostaglandin E1 or prostacyclin reduced the ability of epinephrine, A23187, or U46619 to induce intraplatelet Ca2+ mobilization.

Cinemicroscopic Visualization of Shape Change and Motility in Normal and Abnormal Living Human Platelets

1979 ◽  
Author(s):  
L. R. Zacharski ◽  
R. D. Allen ◽  
R. Rosenstein ◽  
S. Widirtsky
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Rapid Test ◽  
Human Platelets ◽  
Epifluorescence Microscopy ◽  
Optical Sectioning ◽  
Dense Bodies ◽  
Cine Film ◽  
20 Nm ◽  
Human Blood Platelets

Living human blood platelets (P) have been examined with a high extinction differential interference contrast (DIC) microscope capable of detecting structures as small as 20 nm in diameter. During the quarter hour required for complete transformation, the entire shape change sequence is clearly visible, including disk to sphere transformation, extension and retraction of pseudopodia, and spreading and ruffling of the hyalomere. The exocytosis of intact 5-hydroxy tryptamine (serotonin)-containing dense bodies has been observed both by DIC microscopy and by epifluorescence microscopy in P stained with mepacrine. The release of dense bodies is associated with the formation of one or more “craters” in the upper surface of the granulomere. With optical sectioning, it is evident that certain “craters” represent internal chambers of the open canalicular system. Using these techniques, abnormalities in P motility have been observed in hereditary P disorders. In summary, the ability to observe and record permanently on cine film the motility of living P provides a rapid test of P function which allows quantitation of normal vs. abnormal motile behavior.

Epinephrine induces Ca2+ uptake in human blood platelets

1980 ◽  
Vol 239 (4) ◽  
pp. H483-H483
Author(s):  
N. E. Owen ◽  
H. Feinberg ◽  
G. C. Le Breton
Keyword(s):  
Human Blood ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Receptor Interaction ◽  
Induced Aggregation ◽  
Selective Increase ◽  
Dose Dependent ◽  
Primary Platelet ◽  
Human Blood Platelets

Human blood platelets isolated by albumin density gradient centrifugation take up Ca2+ during 10-6M epinephrine-induced primary aggregation but not during 10-6 M ADP-induced primary aggregation. Platelet uptake of Ca2+ is dose-dependent over a range of 10-7) to 10-5 M epinephrine. Antagonism of the platelet α-receptor by phentolamine (10-6 M) results in inhibition of both epinephrine-stimulated Ca2+ uptake and aggregation. The Ca2+ antagonist verapamil (50 μM) blocks Ca2+ uptake and epinephrine-induced aggregation, but not ADP-induced aggregation. The verapamil inhibition of aggregation is reduced on Ca2+ addition. These results suggest that epinephrine acts to stimulate primary platelet aggregation through a specific receptor interaction that results in a selective increase in platelet membrane permeability to Ca2+.

Freeze-Fracture Studies of Human Blood Platelets Activated by Thrombin Using Rapid Freezing

1985 ◽  
Vol 54 (03) ◽  
pp. 574-578 ◽  
Author(s):  
Hans Hols ◽  
Jan J Sixma ◽  
J Leunissen-Bijvelt ◽  
Arie Verkley
Keyword(s):  
Human Blood ◽  
Shape Change ◽  
Blood Platelets ◽  
Secretory Granules ◽  
Freeze Fracture ◽  
Rapid Freezing ◽  
Freeze Fracturing ◽  
Morphological Alterations ◽  
Thrombin Activation ◽  
Human Blood Platelets

SummaryIn this study the influence of thrombin activation on human blood platelets has been followed by freeze-fracturing electron microscopy using rapid freezing in order to catch the initial changes in shape and the morphological alterations during the process of exocytosis of secretory granules. We found that isolation of the platelets by itself leads to some degree of shape change, which made it impossible to study the resting discoid platelet by rapid freezing.Activation of the platelets by thrombin induced dilation of the “surface connecting system (SCS)” with formation of large vacuoles as a result of fusion of the secretory granules with SCS. No intermediary fusion stages or structures were observed even using rapid freezing. Volcano-like protrusions and the corresponding complementary pits were seen at the SCS. These structures were interpreted by us as fractures through protoplasmic channels crossing the SCS. These channels originate during the swelling of the SCS as a result of the fusion of secretory granules with the SCS.

Effects of Aggregating Agents, Cytochalasin B (CB) and Neuraminidase (N) on the Ultrastructure of Freeze Dried (FD) Human blood Platelets

1975 ◽  
Author(s):  
R. B. Davis
Keyword(s):  
Human Blood ◽  
Plasma Membranes ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Adenosine Diphosphate ◽  
Surface Complexes ◽  
Release Reaction ◽  
Cryoprotective Agents ◽  
Freeze Dried ◽  
Human Blood Platelets

Frozen and freeze dried human blood platelets remain intact morphologically when preserved by cryoprotective agents. These studies have investigated effects of 1) the release reaction, 2) discoid stabilization by CB, and 3) N on platelet morphology in FD specimens. Platelets were collected in 1/10 volume of acid citrate and platelet rich plasma (PRP) obtained by centrifugation. Aggregating agents (adenosine diphosphate, 2 × 10-6 M, epinephrine, 5 × 10-5 M, collagen, 30 μg/ml, thrombin 0.2 U/ml) CB (25 μg/ml), trypsin (3 mgs/ml), and N (20 U/ml), then cryoprotective agents, were added. Platelets were FD and the ultrastructure examined. Aggregating agents were associated with 1 ) the appearance of amorphous electron dense material within platelets extending via channels to the exterior; 2) membranous complexes contiguous to plasma membranes; 3) numerous organelles adjacent to the plasma membrane. Platelets in artificial thrombi also showed homogeneous electron opaque areas and membrane rich surface complexes. CB caused vacuolization and previously reported concentric membranous structures were noted in trypsinized platelets. N did not prevent interplatelet bridging. In conclusion, aggregated FD platelets differ from platelets fixed by traditional means, providing morphologic support that platelet organelles and membrane systems relate structurally to the platelet exterior as well as the canalicular system to provide a catalytic lipoprotein surface during the release reaction.

scholarly journals Incorporation and turnover of eicosapentaenoic and docosahexaenoic acids in human blood platelets in vitro

1992 ◽  
Vol 281 (2) ◽  
pp. 309-316 ◽  
Author(s):  
M Croset ◽  
Y Bayon ◽  
M Lagarde
Keyword(s):  
Fatty Acids ◽  
Human Blood ◽  
Molecular Species ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Total Radioactivity ◽  
Significant Release ◽  
Subsequent Activation ◽  
Human Blood Platelets

Mass changes in the incorporation of linoleic (C18:2), eicosapentaenoic (C20:5) and docosahexaenoic (C22:6) acids in human blood platelet phospholipids were induced by incubating the cells and these fatty acids complexed to albumin. The remodelling of [14C]C18:2, [14C]C20:5 and [14C]C22:6 in classes, subclasses and molecular species of platelet phospholipids was studied in resting and thrombin-stimulated cells. More than 85% of the incorporation was located in phospholipids, representing 5-fold and 2.5-fold increases in the phospholipid C20:5 and C22:6 endogenous content respectively. Thrombin stimulation induced a 30% degradation of 1-acyl-2-C20:5-glycerophosphocholine (GPC) and 1-acyl-2-C22:6-GPC, but did not induce significant release of C18:2 from 1-acyl-2-C18:2-GPC. There was no change in the [14C]fatty acid composition of 1-alkyl-2-acyl-GPC. Thrombin-dependent increases in 1-alkenyl-2-C20:5-glycerophosphoethanolamine (GPE) and 1-alkenyl-2-C22:6-GPE of 2.1-fold and 2.5-fold respectively accounted for the rise in GPE radioactivity and partly compensated for the loss of these fatty acids from 1,2-diacyl-GPC: transfer to 1-alkenyl-2-acyl-GPE was 0.4 and 1.5 nmol/10(9) platelets for C20:5 and C22:6 respectively. [14C]C20:5 and [14C]C22:6 were incorporated into six different species of 1,2-diacyl-GPC, with acylation in the major endogenous forms (C18:1 +C16:0 and C18:0 species) representing 76% and 66% respectively of the total radioactivity present in 1,2-diacyl-GPC. Stimulation by thrombin induced significant release of these fatty acids from the main molecular species of 1,2-diacyl-GPC, but significantly stimulated the synthesis of alkenyl forms of GPE containing C18:1/C22:6 +C16:0/C22:6, C18:0/C22:6 and C18:0/C20:5. C18:0/C18:2, the major endogenous C18:2 molecular species, represented only 10.5% of the incorporation; none of the [14C]C18:2 molecular species was a substrate for transfer towards 1-alkenyl-2-acyl-GPE. It is concluded that when C20:5 and C22:6, but not C18:2, are acylated in 1,2-diacyl-GPC, they participate in thrombin-dependent phospholipid remodelling, and might compete with the turnover and release of arachidonic acid from platelet phospholipids and the subsequent activation of the cells.

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