THROMBOXANE-A2 MEDIATES THE ACTION OF INOSITOL (1.4.5) TRISPHOSPHATE (IP3) IN SAPONIN-PERMEABILISED PLATELETS

1987 ◽  
Author(s):  
K S Authi ◽  
B J Evenden ◽  
E J Hornby ◽  
N Crawford
Keyword(s):  
Phospholipase A2 ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Thromboxane B2 ◽  
Cyclooxygenase Inhibitors ◽  
Storage Site ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Thromboxane Receptor ◽  
Thromboxane Production

Inositol trisphosphate (IP3) has now been identified as an important intracellular second messenger that can initiate the release of Ca2+ from intracellular stores in a variety of cells, including platelets. We have studied the effects of IP3 on washed platelets permeabilised with saponin (12-14 μg/mi) which allows penetration into the cell of low M.Wt polar molecules. The permeabilised cells show normal responses to the agonists thrombin and collagen. The addition of IP (1-20 μM) after saponin treatment induces shape change, aggregation and secretion of preloaded [14C] 5HT. Concomitant with these responses, thromboxane is produced in a dose related manner. With 20 μM IP3 thromboxane B2 increases from basal levels of 5-4 ± 3-0 ng/ml to 140 ± 23 ng/ml. Both thromboxane production and the platelet responses induced by IP3 are inhibited by pretreatment with the cyclooxygenase inhibitors, indomethacin (EC50 50 μM) and aspirin (EC50 30 μM). Aggregation and secretion responses to IP3 are also inhibited by thromboxane B2 receptor agonists; EPO 92 (R. Jones, Edinburgh) and AH 23848 (Glaxo Ltd.). If Ca2+ EGTA buffers age used with permeabilised platelets to "lock" the cytosolic [Ca2+] at 0.1 μM, thromboxane production is reduced to the basal level. Intact platelets were labelled with Ca2+ (4h incubation) and after washing, resuspension and saponisation, IP3 induced the release of 20% of the cell associated Ca2+. The release was unaffected by pretreatment with antimycin and oligomycin indicating an gndoplasmic reticulum-lige storage site for the sequestered Ca2+. This IP3 -induced Ca2+ release was also not affected by pretreatment with either cyclooxygenase inhibitors or thromboxane receptor antagonists (EPO 92 and AH 23848). We believe these studies indicate that the action of IP3 in sagonised platelets involves release of intracellularly stored Ca2+, activation of phospholipase A2 and cyclooxygenase, and production of thromboxane A2. The release of thromboxane mediates and/or attenuates platelet responses by acting upon platelet surface receptors.

scholarly journals Inhibition of collagen-induced platelet activation by 5'-p- fluorosulfonylbenzoyl adenosine: evidence for an adenosine diphosphate requirement and synergistic influence of prostaglandin endoperoxides

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 565-570 ◽  
Author(s):  
RW Colman ◽  
WR Figures ◽  
LM Scearce ◽  
AM Strimpler ◽  
FX Zhou ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Platelet Surface ◽  
Prostaglandin Endoperoxide ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Absolute Requirement

Abstract The relative roles of platelet autacoids such as adenosine diphosphate (ADP), prostaglandin endoperoxides, and thromboxane A2 (TXA2) in collagen-induced platelet activation are not fully understood. We reexamined this relationship using the ADP affinity analogue, 5'-p- fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies a receptor for ADP on the platelet surface, thereby inhibiting ADP- induced platelet activation. Collagen-induced shape change, aggregation, and fibrinogen binding were each fully inhibited under conditions in which FSBA is covalently incorporated and could not be overcome by raising the collagen used to supramaximal concentrations. In contrast, TXA2 synthesis stimulated by collagen under conditions that produced maximum aggregation was only minimally inhibited by FSBA. Since covalent incorporation of FSBA has been previously shown to specifically inhibit ADP-induced activation of platelets, the present study supports the contention that ADP is required for collagen-induced platelet activation. Under similar conditions, indomethacin, an inhibitor of cyclooxygenase, inhibited collagen-induced shape change, indicating that endoperoxides and/or TXA2 also play a role in this response. Shape change induced by low concentrations (10 nmol/L) of the stable prostaglandin endoperoxide, azo-PGH2, was also inhibited by FSBA. These observations indicate a role for ADP in responses elicited by low concentrations of endoperoxides. However, at higher concentrations of azo-PGH2 (100 nmol/L), inhibition by FSBA could be overcome. Thus, the effect of collagen apparently has an absolute requirement for ADP for aggregation and fibrinogen binding and for both ADP and prostaglandins for shape change. Aggregation and fibrinogen binding induced by prostaglandin endoperoxides also required ADP as a mediator, but ADP is not absolutely required at high endoperoxide concentration to induce shape change.

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.

Thromboxane receptor-mediated bronchial and hemodynamic responses in ovine endotoxemia

1988 ◽  
Vol 254 (2) ◽  
pp. R310-R319 ◽  
Author(s):  
P. G. Kuhl ◽  
J. M. Bolds ◽  
J. E. Loyd ◽  
J. R. Snapper ◽  
G. A. FitzGerald
Keyword(s):  
Respiratory Distress Syndrome ◽  
Respiratory Distress ◽  
Adult Respiratory Distress Syndrome ◽  
Distress Syndrome ◽  
Thromboxane A2 ◽  
Lung Compliance ◽  
Thromboxane B2 ◽  
Simultaneous Increase ◽  
Spectrometric Analysis ◽  
Thromboxane Receptor

The role of thromboxane A2 in sheep endotoxemia, an animal model of the adult respiratory distress syndrome, was investigated by a combined biochemical and pharmacological approach. Endogenous thromboxane biosynthesis was assessed by gas chromatographic-mass spectrometric analysis of urinary (thromboxane B2, 2,3-dinor-thromboxane B2) and plasma (11-dehydrothromboxane B2) metabolites that demonstrated a significant stimulation by endotoxin. The functional relevance of thromboxane A2 was probed with a specific thromboxane-prostaglandin endoperoxide receptor antagonist, SQ 29548. The antagonist significantly blunted the increase in pulmonary arterial pressure, pulmonary vascular resistance, lung lymph flow, and lymph protein clearance induced by endotoxin. Whereas the reduction in lung compliance caused by endotoxin was abolished, the augmented airway resistance was unaffected. From the simultaneous increase in thromboxane biosynthesis and effects of receptor blockade, it was concluded that thromboxane A2 mediates the early pathophysiological changes of sheep endotoxemia. Thromboxane receptor antagonism may offer a potential therapeutic approach to patients at risk of the adult respiratory distress syndrome.

scholarly journals Megakaryocyte interaction with subendothelial extracellular matrix is associated with adhesion, platelet-like shape change, and thromboxane A2 production

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 570-576
Author(s):  
RF Levine ◽  
A Eldor ◽  
E HyAm ◽  
H Gamliel ◽  
Z Fuks ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Guinea Pig ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Functional Capabilities ◽  
Gentle Agitation ◽  
Bovine Endothelial Cells ◽  
The Face ◽  
Thromboxane Production ◽  
Plastic Surfaces

We have examined the morphological and secretory behavior of rat and guinea pig megakaryocytes exposed for up to 24 hours to extracellular matrix produced by cultured bovine endothelial cells. By phase-contrast microscopy of living cells and in more detail by scanning electron microscopy, the megakaryocytes showed a nonreversible adherence, an extensive formation of filopodia around the periphery like the rays of the sun, and a tendency toward flattening. These filopodia were generally linear with attenuated tips and were larger than, but resembled the filopodia of, rat or guinea pig platelets exposed to this extracellular matrix. In contrast, isolated megakaryocytes on glass or on uncoated plastic surfaces did not show these responses; adherence, in the face of gentle agitation before fixation, was minimal, with rare filopodia and no flattening. Megakaryocytes that interacted with the extracellular matrix produced significant amounts of thromboxane A2, but this did not occur on uncoated surfaces and could not be attributed to other contaminating cells in the megakaryocyte suspensions. The appearance in megakaryocytes of these typical platelet responses indicates that megakaryocytes acquire the functional capabilities of platelets by the synthesis and assembly of platelet substances and organelles. Thromboxane production by megakaryocytes stimulated by the extracellular matrix is a readily quantifiable measure of this capacity.

scholarly journals Megakaryocyte interaction with subendothelial extracellular matrix is associated with adhesion, platelet-like shape change, and thromboxane A2 production

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 570-576 ◽  
Author(s):  
RF Levine ◽  
A Eldor ◽  
E HyAm ◽  
H Gamliel ◽  
Z Fuks ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Guinea Pig ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Functional Capabilities ◽  
Gentle Agitation ◽  
Bovine Endothelial Cells ◽  
The Face ◽  
Thromboxane Production ◽  
Plastic Surfaces

Abstract We have examined the morphological and secretory behavior of rat and guinea pig megakaryocytes exposed for up to 24 hours to extracellular matrix produced by cultured bovine endothelial cells. By phase-contrast microscopy of living cells and in more detail by scanning electron microscopy, the megakaryocytes showed a nonreversible adherence, an extensive formation of filopodia around the periphery like the rays of the sun, and a tendency toward flattening. These filopodia were generally linear with attenuated tips and were larger than, but resembled the filopodia of, rat or guinea pig platelets exposed to this extracellular matrix. In contrast, isolated megakaryocytes on glass or on uncoated plastic surfaces did not show these responses; adherence, in the face of gentle agitation before fixation, was minimal, with rare filopodia and no flattening. Megakaryocytes that interacted with the extracellular matrix produced significant amounts of thromboxane A2, but this did not occur on uncoated surfaces and could not be attributed to other contaminating cells in the megakaryocyte suspensions. The appearance in megakaryocytes of these typical platelet responses indicates that megakaryocytes acquire the functional capabilities of platelets by the synthesis and assembly of platelet substances and organelles. Thromboxane production by megakaryocytes stimulated by the extracellular matrix is a readily quantifiable measure of this capacity.

Surface Sialic Acid Prevents Loss of GPIbα during Platelet Storage and Rescues In Vivo Survival of Murine Platelets.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 138-138 ◽  
Author(s):  
Gerard Jansen ◽  
Emma C. Josefsson ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Shape Change ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
Surface Receptors ◽  
Platelet Survival ◽  
Platelet Receptor

Abstract Platelet processing and storage are associated with platelet lesion, e.g. shape change, activation, release reaction and apoptosis, which is partially due to loss of surface receptors. Surface sialic acid is considered to be a key determinant for the survival of circulating blood cells and glycoproteins. However, its role in platelet receptor loss and platelet survival is unclear. In this study, the relationship between surface sialic acid and platelet receptor loss was investigated in vitro and in vivo. Murine platelets stored at room temperature for 6 hours lost surface sialic acid, as evidenced by flow cytometry using FITC conjugated RCA I lectin, which recognizes exposed galactose residues. This loss correlated with a 30–60% loss of surface receptors GPIbα and GPV, but not GPIX and integrin αIIbβ3, as measured by flow cytometry. Treatment of murine platelets with the neuraminidase (NA) substrate fetuin partially decreases the loss of GPIbα and GPV to 10–20%. In vitro, sialic acid was cleaved from the platelet surface by adding NA (α2-3,6,8-NA (V. cholerae) or α2-3,6,-NA (C. perfringens)) to murine platelets. Removal of sialic acid correlated with the removal of 50–60% of surface GPIbα and GPV, but not GPIX and integrin αIIbβ3. Addition of fetuin, or the more specific NA inhibitor 2,3-dehydro-2-deoxy-, sodium salt (DANA), completely prevented this loss, as determined by both flow cytometry and Western blot analysis. Murine platelets treated with α2-3,6,8-NA (V. cholerae) ± the addition of DANA were labeled with the green dye CMFDA and transfused into age-, strain- and sex-matched C57BL/6 mice to measure platelet survival. NA-treated platelets were cleared within minutes after transfusion, whereas the addition of DANA rescued platelet survival to control-count increments. Our study shows that inhibiting the loss of surface sialic acid prevents platelet surface GPIbα and GPV loss during storage in vitro and rescues platelet survival in vivo.

Platelet ADP Receptors Stimulating Shape Change and Inhibiting Adenylate Cyclase

Physiology ◽  
1992 ◽  
Vol 7 (6) ◽  
pp. 274-278
Author(s):  
RW Colman
Keyword(s):  
Platelet Aggregation ◽  
Membrane Protein ◽  
Adenylate Cyclase ◽  
Shape Change ◽  
Surface Membrane ◽  
Thromboxane A2 ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Adp Receptors

Aggregin, a platelet surface membrane protein required for ADP-induced shape change, aggregation, and fibrinogen binding, is distinct from the receptor coupled to adenylate cyclase. Platelet aggregation by epinephrine, thromboxane A2, and collagen requires ADP binding to aggregin. Thrombin and plasmin are independent of ADP and activate platelet calpain, which cleaves aggregin.

Effect of Vitamin E on Platelet Aggregation in Diabetes Mellitus

1984 ◽  
Vol 51 (03) ◽  
pp. 313-316 ◽  
Author(s):  
J Watanabe ◽  
F Umeda ◽  
H Wakasugi ◽  
H Ibayashi
Keyword(s):  
Vitamin E ◽  
Platelet Aggregation ◽  
Vascular Complications ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Thromboxane B2 ◽  
Enhanced Production ◽  
Thromboxane Production ◽  
And Control ◽  
Platelet Thromboxane

SummaryVitamin E is known to be an inhibitor of platelet prostaglandin production and aggregation. The rate of platelet aggregation induced by adenosine diphosphate was significantly increased in diabetics with proliferative retinopathy and the enhanced production of thromboxane B2, a stable metabolite of thromboxane A2, was demonstrated in those patients. On the other hand, vitamin E in platelets was significantly reduced in diabetics compared with age matched controls. In addition, it was shown that vitamin E content in platelets examined in diabetic and control subjects inversely correlated with both the rate of platelet aggregation and thromboxane B2 production during aggregation. It is suggested that the reduced vitamin E levels in diabetic platelets can contribute to the mechanisms of the enhanced platelet thromboxane production and aggregation which relate to the development of vascular complications.

Further Characterization of Wheat Germ Agglutinin Interaction with Human Platelets: Exposure of Fibrinogen Receptors

1986 ◽  
Vol 56 (03) ◽  
pp. 323-327
Author(s):  
Marilyne Lebret ◽  
Francine Rendu
Keyword(s):  
Platelet Activation ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Lectin Binding ◽  
Human Platelets ◽  
Cyclooxygenase Inhibitors ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Fibrinogen Binding ◽  
Exogenous Calcium

SummaryIt was previously shown that (i) Wheat germ agglutinin, (WGA)-induced platelet activation occurred when only 17% of the lectin binding sites were occupied on the platelet surface and (ii) WGA caused the release of a platelet constituent which in turn participates in the observed effect. We now further define the platelet activation induced by WGA: the lectin induces a binding of fibrinogen to specific surface receptors. 125I-fibrinogen binding increases with the WGA concentration from 5 to 15 ug/ ml. Binding occurs without addition of exogenous calcium; its analysis demonstrated 54000 sites with a Ka = 0.8 × 106 M-1, Addition of 1 mM Ca2+ enhances the 125I-fibrinogen binding and reveals a second class of sites with higher affinity (9200 sites, Ka = 0.17 x 108 M-1). This 125I-fibrinogen binding is totally abolished by EDTA, ATP and arginine, and inhibited by 75% by CP/CPK; cyclooxygenase inhibitors and PGE1 also reduce the fibrinogen binding. Thus the WGA-induced fibrinogen binding is (1) release-dependent and (2) responsible for the aggregation process but not for the agglutinating effect of the lectin.

scholarly journals The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin

1984 ◽  
Vol 221 (3) ◽  
pp. 897-901 ◽  
Author(s):  
T J Hallam ◽  
N T Thompson ◽  
M C Scrutton ◽  
T J Rink
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Free Calcium ◽  
Similar Time ◽  
Transient Rise ◽  
Thromboxane Production

Responses to vasopressin were studied in human platelets loaded with the fluorescent Ca2+ indicator, quin2. In the presence of 1 mM external Ca2+, vasopressin caused a transient rise in [Ca2+]i from the basal level near 100nM to about 700 nM; peak [Ca2+]i was reached in a few seconds and the level then declined towards resting over several minutes. In the absence of external Ca2+ there was a much smaller rise of similar time-course, suggesting that vasopressin increases [Ca2+]i mainly by stimulated-influx across the plasma membrane but also by partly releasing internal Ca2+. Inhibition of thromboxane A2 formation somewhat reduced the peak [Ca2+]i in the presence of external Ca2+, but had no effect on the response attributed to release of internal Ca2+. With external Ca2+, vasopressin stimulated shape-change, secretion and aggregation. Secretion and aggregation were decreased by about half following blockage of thromboxane production. The ability of vasopressin to induce shape-change and secretion even at near basal [Ca2+]i suggests that activators other than Ca2+ are involved.

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