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.

scholarly journals Synthetic lipopeptide Pam3CysSer(Lys)4 is an effective activator of human platelets

1994 ◽  
Vol 266 (6) ◽  
pp. C1684-C1691 ◽  
Author(s):  
M. Berg ◽  
S. Offermanns ◽  
R. Seifert ◽  
G. Schultz
Keyword(s):  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Cellular Effects ◽  
Prostacyclin Receptor ◽  
Serotonin Secretion ◽  
Peptide Moiety ◽  
Lipid Moiety ◽  
Molecular Masses ◽  
Aggregation Of Platelets

Lipopeptide analogues of the NH2-terminus of bacterial lipoprotein are known to induce activation of macrophages, neutrophils, and lymphocytes. We studied the effect of the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-( S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine [Pam3CysSer(Lys)4] on several functions of human platelets. Pam3CysSer(Lys)4 led to the aggregation of platelets and induced the secretion of serotonin with an effectiveness similar to thrombin. These cellular effects of Pam3CysSer(Lys)4 were concentration dependent, being half maximal at 2-3 microM and maximal at 10-30 microM. Another lipopeptide also induced platelet aggregation and serotonin secretion but was less potent and less effective than Pam3CysSer(Lys)4. The lipid moiety and the peptide moiety of Pam3CysSer(Lys)4 alone were without any effect. Lipopeptides also stimulated tyrosine phosphorylation of several proteins with molecular masses similar to those found to be tyrosine phosphorylated in response to thrombin, and Pam3CysSer(Lys)4 led to an increase in the cytosolic calcium concentration. All studied responses of platelets to lipopeptides were inhibited by the prostacyclin receptor agonist cicaprost. Taken together, our data show that lipopeptides are effective activators of human platelets and that this activation is susceptible to the action of physiological platelet inhibitors.

scholarly journals Defective signal transduction induced by thromboxane A2 in a patient with a mild bleeding disorder: impaired phospholipase C activation despite normal phospholipase A2 activation

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 994-1000 ◽  
Author(s):  
I Fuse ◽  
M Mito ◽  
A Hattori ◽  
W Higuchi ◽  
A Shibata ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Phospholipase C ◽  
Binding Capacity ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Dissociation Rate ◽  
Bleeding Disorder ◽  
Normal Ranges ◽  
Txa2 Receptor ◽  
Dissociation Rate Constants

Abstract A patient with a mild bleeding disorder whose platelets responded defectively to thromboxane A2 (TXA2) was identified, and the mechanism of this dysfunction was analyzed. The platelets were defective in shape change, aggregation, and release reaction in response to synthetic TXA2 mimetic (STA2). When the platelet TXA2 receptor was examined with both a 125I-labeled derivative of a TXA2 receptor antagonist ([125I]-PTAOH) and [3H]-labeled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (kd) and the maximal concentrations of binding sites (Bmax) of the platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 could not induce IP3 formation and intracellular Ca2+ mobilization, whereas these responses to thrombin were within normal ranges. GTPase activity was also decreased when the patient's platelet membrane was challenged with STA2. On the other hand, lysophosphatidylinositol formation, which is a direct indicator of phospholipase A2 (PLA2) activation, was found to be normal when the [3H]-inositol-labeled platelets were challenged with STA2. Thromboxane B2 (TXB2) was also produced in response to STA2. These results suggested that the abnormality in these platelets was impaired coupling between TXA2 receptor and phospholipase C (PLC) activation. Furthermore, it is also suggested that the activation of PLA2 and PLC are separable events in thromboxane-induced platelet activation.

scholarly journals Spiking in cytosolic calcium concentration in single fibrinogen-bound fura-2-loaded human platelets

1992 ◽  
Vol 283 (2) ◽  
pp. 379-383 ◽  
Author(s):  
J W Heemskerk ◽  
J Hoyland ◽  
W T Mason ◽  
S O Sage
Keyword(s):  
Calcium Concentration ◽  
Calcium Signalling ◽  
Peak Frequency ◽  
Cytosolic Calcium ◽  
Light Level ◽  
Optical Probe ◽  
Human Platelets ◽  
Free Calcium ◽  
Fura 2 ◽  
Subsequent Activation

Fura-2-loaded human platelets were immobilized on a fibrinogen-coated surface and the cytosolic free calcium concentration ([Ca2+]i) was measured in single platelets by low-light-level video-ratio image-processing of the optical probe signal. Some fibrinogen-bound platelets showed repetitive spiking in [Ca2+]i with a mean frequency of about 2/min, which increased to 5/min in the presence of ADP. Other cells showed no activity until the addition of agonist. When immobilized in the presence of prostaglandin I2 and the fibrinogen antagonist Arg-Gly-Asp-Ser, the platelets adhered less firmly to fibrinogen, and in many [Ca2+]i remained low and constant. Subsequent activation of such platelets with ADP evoked oscillations in [Ca2+]i with a peak frequency of about 5/min and which persisted for at least 5 min. These results indicate that human platelets, like many other non-excitable cells, have an elaborate system of calcium signalling involving spiking.

Defective Signal Transduction through the Thromboxane A2 Receptor in a Patient with a Miid Bleeding Disorder: Deficiency of the Inositol 1,4,5-Triphosphate Formation despite Normal G-protein Activation

1997 ◽  
Vol 77 (05) ◽  
pp. 0991-0995 ◽  
Author(s):  
Tetsuo Mitsui ◽  
Shinkichi Yokoyama ◽  
Yukitoshi Shimizu ◽  
Michihiko Katsuura ◽  
Kaori Akiba ◽  
...  
Keyword(s):  
Thromboxane A2 ◽  
Dissociation Rate ◽  
Bleeding Disorder ◽  
Ionophore A23187 ◽  
Protein Activation ◽  
Prolonged Bleeding Time ◽  
Normal Ranges ◽  
G Protein Activation ◽  
Dissociation Rate Constants ◽  
Equilibrium Dissociation

SummaryWe describe an 11-year-old girl with a mild bleeding disorder since early childhood. The disorder was characterized by a prolonged bleeding time, and the patient’s platelets showed defective aggregation responses to thromboxane A2 (TXA2) mimetic U46619 and arachidonic acid. In contrast, the platelets showed normal responses to thrombin and Ca ionophore A23187. When the platelet TXA2 receptor was examined with the [3H]-labeled TXA2 agonist U46619, the equilibrium dissociation rate constants (kd) and the maximal concentration of binding sites (Bmax) of the patient's platelets were within normal ranges. Normal GTPase activity was also induced in the patient's platelets by stimulation with U46619, however, inositol 1,4,5-triphosphate (IP3) formation was not induced by U46619. These results suggested that the patient’s platelets had a defect in phospholipase C activation beyond TXA2 receptors.

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.

Lack of effect of ouabain on calcium homeostasis in rat platelets: comparative study with human platelets

1994 ◽  
Vol 266 (3) ◽  
pp. R651-R657
Author(s):  
T. Oshima ◽  
T. Ishida ◽  
H. Matsuura ◽  
M. Ishida ◽  
K. Ishibashi ◽  
...  
Keyword(s):  
Wistar Rats ◽  
Time Course ◽  
Calcium Concentration ◽  
Sodium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Time Dependent ◽  
Maximal Dose ◽  
Peak Response ◽  
Rat Platelets

The precise mechanisms of Ca2+ handling in rat platelets are not fully understood. We sought to determine whether rat platelets possess a Na(+)-Ca2+ exchanger. First, we investigated the time course of the effect of ouabain (10(-4) M) on cytosolic sodium concentration ([Na+]i) and Ca2+ homeostasis in platelets from Wistar rats in comparison with those from humans. Ouabain increased platelet [Na+]i in both rat and human platelets. Whereas ouabain induced a time-dependent increase in basal and thrombin-stimulated (0.3 units/ml) cytosolic calcium concentration ([Ca2+]i) in human platelets, no change was found in rat platelets. Furthermore, 90-min pretreatment of rat platelets with ouabain did not affect the [Ca2+]i response to thrombin (0.1-1.0 units/ml) or a maximal dose of ionomycin (5 microM). Also the decline in [Ca2+]i after the peak response evoked by these agonists in the absence of extracellular Ca2+ was not changed by ouabain pretreatment. Similarly, replacement of extracellular Na+ had no influence on any of these determinations. Thus decreasing the plasma membrane Na+ gradient did not affect basal [Ca2+]i, thrombin-induced mobilization of Ca2+ from intracellular stores, internal Ca2+ discharge capacity, or Ca2+ extrusion from cytosol of rat platelets. In contrast to human platelets, a Na(+)-Ca2+ exchange mechanism does not appear to play a significant role in Ca2+ homeostasis of rat platelets.

Elevation of cytoplasmic free calcium concentration by stable thromboxane A2 analogue in human platelets

1983 ◽  
Vol 117 (3) ◽  
pp. 663-669 ◽  
Author(s):  
Yasuhiro Kawahara ◽  
Junji Yamanishi ◽  
Yutaka Furuta ◽  
Kozo Kaibuchi ◽  
Yoshimi Takai ◽  
...  
Keyword(s):  
Calcium Concentration ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Free Calcium ◽  
Free Calcium Concentration ◽  
Thromboxane A2 Analogue ◽  
Cytoplasmic Free Calcium

scholarly journals ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store

1990 ◽  
Vol 265 (3) ◽  
pp. 675-680 ◽  
Author(s):  
S O Sage ◽  
R Reast ◽  
T J Rink
Keyword(s):  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Second Phase ◽  
Stopped Flow ◽  
Bivalent Cation ◽  
Fura 2 ◽  
Fast Phase ◽  
Lower Temperature ◽  
Delayed Phase

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.

Calcium Entry in Nonexcitable Cells: Lessons from Human Platelets

Physiology ◽  
1992 ◽  
Vol 7 (3) ◽  
pp. 108-113
Author(s):  
SO Sage ◽  
MP Mahaut-Smith ◽  
TJ Rink
Keyword(s):  
Calcium Signaling ◽  
Platelet Activation ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Calcium Entry ◽  
Cytosolic Calcium Concentration ◽  
Stimulus Response ◽  
Signaling Mechanisms

A rise in cytosolic calcium concentration plays an important role in platelet activation. As well as helping define stimulus-response coupling in these intriguing and clinically important cells, the study of calcium mobilisation in platelets serves as an important model for elucidating calcium signaling mechanisms in general.

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