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.

Genetic and Pharmacological Analyses of Involvement of Src-family, Syk and Btk Tyrosine Kinases in Platelet Shape Change

2001 ◽  
Vol 85 (02) ◽  
pp. 331-340 ◽  
Author(s):  
Markus Bauer ◽  
Petra Maschberger ◽  
Lynn Quek ◽  
Stephen Briddon ◽  
Debabrata Dash ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Src Family Kinases ◽  
G Protein Coupled Receptors ◽  
Protein Tyrosine Phosphorylation ◽  
G Protein Coupled ◽  
Platelet Shape ◽  
Stimulation Of

SummaryPlatelet shape change was found to be associated with an increase in protein tyrosine phosphorylation upon stimulation of thrombin-, ADPand thromboxane A2-G-protein coupled receptors in human platelets and thromboxane A2 receptors in mouse platelets. By using PP1 and PD173956, two structurally unrelated specific inhibitors of Src-family tyrosine kinases, and mouse platelets deficient in the Src-kinase Fyn or Lyn, we show that Src-family kinases cause the increase in protein tyrosine phosphorylation. We further detected that the non-Src tyrosine kinase Syk was activated during shape change in a manner dependent on Src-family kinaseactivation. The pharmacological experiments and the studies on Fyn-, Lyn- and Syk-deficient mouse platelets showed that neither Src-family kinases nor Syk are functionally involved in shape change. Also human platelets deficient of the tyrosine kinase Btk showed a normal shape change. Binding of PAC-1 that recognizes activated integrin αIIb β3 complexes on the platelet surface was enhanced during shape change and blocked by inhibition of Src-kinases. We conclude that the activation of Src-kinases and the subsequent Syk stimulation upon activation of G-protein coupled receptors are not involved in the cytoskeletal changes underlying shape change of human and mouse platelets, but that the stimulation of this evolutionary conserved pathway leads to integrin αIIb β3 exposure during shape change.

Fibrinogen Binding Is Independent of an Increase in Intracellular Calcium Concentration in Thrombin Degranulated Platelets

1995 ◽  
Vol 73 (02) ◽  
pp. 304-308 ◽  
Author(s):  
Fabio M Pulcinelli ◽  
James L Daniel ◽  
Silvia Riondino ◽  
Pier Paolo Gazzaniga ◽  
Leon Salganicoff
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Calcium ◽  
Binding Sites ◽  
Calcium Concentration ◽  
Calcium Release ◽  
Shape Change ◽  
Cytosolic Calcium ◽  
Calcium Mobilization ◽  
Induce Platelet Aggregation ◽  
Fibrinogen Binding

SummaryIn a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C(PLC) activation and calcium mobilization in TDP activated by ADP, epinephrine and U46619.ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.

IRREVERSIBLE BLOCKADE OF THE THROMBOXANE A2/PROSTAGLANDIN H2 RECEPTOR OF HUMAN PLATELETS BY AZIDO-BSP

1987 ◽  
Author(s):  
H Zehender ◽  
E C Witte ◽  
K Stegemeier ◽  
A Patscheke
Keyword(s):  
Shape Change ◽  
Thromboxane A2 ◽  
High Specificity ◽  
Short Wave ◽  
Human Platelets ◽  
Serotonin Release ◽  
Irreversible Inhibition ◽  
High Concentrations ◽  
Very High ◽  
Platelet Shape

Azido-BSP (4-[2-(4-azido-benzenesulphonylamino)-ethyl]phen-oxyacetic acid) is a photolabile derivative of the competitive thromboxane A2 /prostaglandin H2 (TXA2/PGH2) receptor antagonist sulotroban (=BM 13.177). If protected from short wave light, azido-BSP reversibly inhibited the platelet shape change induced by the PGH2 analogue U 46619 but notthe shape change induced by ADP or PAF. Schild analysis revealed an apparent KD=0.2 μM with washed platelets. The irreversible inhibition requiredirradiation of the platelet suspensionwith UVlight (254 nm) for 5 minutes in the presenceof azido-BSP. After this treatment,the platelets were washed twice and used forplatelet function tests. Treatment with 0.5 μM of azido-BSP suppressed the U 46619(10 μM)-induced (3H)serotonin release and 1 μM of azido-BSP was necessary to block the U 46619(2 μM)-inducedaggregation.The platelet shape change induced by U 46619 (0.01μM) was only partially inhibited, even at very high concentrations (50μM) of the antagonist.This suggests that a small portion of the TXA2/PGH2 receptors could not be blocked bythe photoaffinity treatment with azido-BSP. After treatment with 1 μM azido-BSP, the shape change stimulated by ADP or PAF was not reduced. This indicates a high specificity of thephotoaffinity ligand for the TXA2/PGH2 receptor. It is concluded that UV irradiation of azido-BSP generates anitrene intermediate that covalently links to the majority of the TXA2/PGH2 receptors. Azido-BSP provides a specific tool for tagging and subsequent purification of the TXA2/PGH2 receptor of platelets.(Supported by the Deutsche Forschungsgemeinschaft, Grant Pa263).

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.

Hypersensitivity to Thromboxane A2 in Cholesterol-Rich Human Platelets

1990 ◽  
Vol 64 (04) ◽  
pp. 594-599 ◽  
Author(s):  
Takuya Tomizuka ◽  
Kyohei Yamamoto ◽  
Aizan Hirai ◽  
Yasushi Tamura ◽  
Sho Yoshida
Keyword(s):  
Calcium Concentration ◽  
Binding Capacity ◽  
Thromboxane A2 ◽  
Cytosolic Calcium ◽  
Cholesterol Content ◽  
Human Platelets ◽  
Dissociation Rate ◽  
Platelet Membrane ◽  
Maximal Concentration ◽  
Equilibrium Dissociation

SummaryThe effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,ll-epithio-ll,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.

Evidence for Ca2+-independent phosphorylation of human platelet myosin

1987 ◽  
Author(s):  
J L Daniel ◽  
M Rigmaiden
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Human Platelets ◽  
Myosin Phosphorylation ◽  
Kinase C ◽  
Synthetic Inhibitor ◽  
Measurable Change ◽  
Platelet Shape

Phosphorylation of platelet myosin is thought to be required for activation of the contractile events occurring during platelet activation. At present the only known mechanism for Onitiating myosin phosphorylation is through a Ca2+-calmodulin-dependent activation of myosin light chain kinase. However, our previous studies using the fluorescent Ca2+-indicator quin2 indicated that both platelet shape change and myosin phosphorylation could be induced in an EGTA-containing media in the absence of a measurable change in cytosolic free Ca2+ concentration (Hallam, Daniel, Kendrick-Jones & Rink. Biochem. J. 232 (1985) 373). In order to confirm this finding, we fyave investigated the regulation of myosin phosphorylation usin^+a preparation of electrically-permeabilized platelets and Ca2+ buffers to control the internal Ca2+ concentration. Fifty percent myosin phosphorylation was obtained at 700 nM Ca2+. When thrombin (5 U/ml) was added to this system, this curve shifted both to the left and upward; 50% myosin phosphorylation was obtained at 400 nM Ca2+.A synthetic inhibitor of protein kinase C, H7, had no effect on myosin phosphorylation in the absence of agonist but did inhibit the thrombin-induced shift to left suggesting that protein kinase C may modulate myosin phosphorylation. We also compared the effects of H7 agonist-induced myosin phosphorylation and shape change in control and an quin2 loaded platelets. Comparable inhibition of both phosphorylation and the rate of shape change was observed with both quin2 and H7. Addition of H7 to quin2-loaded platelets resulted in complete inhibition of both agonist-induced shape change and myosin phosphorylation. These results indicate that both protein kinase C and Ca2+-dependent reactions are involved in complete expression of myosin phosphorylation in human platelets.

scholarly journals The Potent Inhibition of Vapiprost, a Novel Thromboxane A2 Receptor Antagonist, on the Secondary Aggregation and ATP Release of Human Platelets.

1997 ◽  
Vol 20 (6) ◽  
pp. 625-631 ◽  
Author(s):  
Shuichi HORIE ◽  
Mayumi YAMADA ◽  
Megumi SATOH ◽  
Shinobu NORITAKE ◽  
Sayuri HIRAISHI ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Atp Release ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Thromboxane A2 Receptor ◽  
Potent Inhibition

Antivasoconstrictor and Antiaggregatory Activities of Picotamide Unrelated to Thromboxane A2 Antagonism

1997 ◽  
Vol 78 (05) ◽  
pp. 1385-1391 ◽  
Author(s):  
Roberta Vezza ◽  
Domenico Spina ◽  
Ronald J Tallarida ◽  
Malevika Nathan ◽  
Clive P Page ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Inhibitory Effects ◽  
Experimental Conditions ◽  
Vasoactive Substances ◽  
Vasoconstrictor Effect ◽  
Synthase Inhibitor

SummaryPicotamide is a dual thromboxane (Tx) A2 receptor antagonist/Tx synthase inhibitor although some observations suggest an anti-vasoconstrictor effect independent of TxA2 inhibition/antagonism. The aim of our study was to assess whether picotamide antagonises vascular contractions induced by different vasoactive substances in vitro. Picotamide inhibited competitively the contraction of rabbit aortic rings induced by the TxA2 mimetic U46619 (pA2 = 3.59) but also the contractions induced by phenylephrine (pA2 = 3.93) and serotonin (5-HT) (pA2 = 5.81) although in a not competitive way. Picotamide did not inhibit potassium-induced contractions, thus excluding aspecific effects on vascular smooth muscle. Picotamide inhibited 5-HT-induced platelet aggregation in vitro with an IC50 (212 μM) similar to that found when other aggregating stimuli are used, but it did not affect shape change (IC50> 1 mM) suggesting that the effects of picotamide can not be ascribed to 5-HT2-receptor antagonism; in the same experimental conditions neither a Tx-receptor antagonist (BM13.177) nor a dual Tx-receptor antagonist/synthase inhibitor (ridogrel) affected 5-HT-induced platelet responses.Our studies demonstrate that picotamide exerts antivasoconstrictor and platelet inhibitory effects unrelated to TxA2 antagonism. This activity may contribute to the anti-thrombotic/anti-ischaemic effects of the drug in vivo.

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.

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