Hemorrhagic Thrombocytopathy with Platelet Thromboxane A2 Receptor Abnormality: Defective Signal Transduction with Normal Binding Activity

1987 ◽  
Vol 57 (02) ◽  
pp. 158-164 ◽  
Author(s):  
Fumitaka Ushikubi ◽  
Minoru Okuma ◽  
Kenji Kanaji ◽  
Tateo Sugiyama ◽  
Toshiya Ogorochi ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Binding Capacity ◽  
Thromboxane A2 ◽  
Binding Activity ◽  
Dissociation Rate ◽  
Ion Concentration ◽  
Free Calcium ◽  
Normal Ranges ◽  
Txa2 Receptor ◽  
Dissociation Rate Constants

SummarySubnormal platelet responses to thromboxane A2 (TXA2) were found in a patient with polycythemia vera, and the mechanism of this dysfunction was analyzed. The patient’s platelets showed defective aggregation and release reaction to arachidonic acid, enzymatically generated TXA2 and synthetic TXA2 mimetics (STA2, U-46619). In contrast, they showed normal responses to thrombin. When the platelet TXA2 receptor was examined with both a 125I-labelled derivative of a TXA2 receptor antagonist ([125I]-PTA-OH) and a 3H-labelled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (Kd) and the maximal concentrations of binding sites (Bmax) of the patient’s platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 failed to induce normal elevation in the. cytoplasmic free calcium ion concentration, phosphatidic acid formation and 40 kD protein phosphorylation in the patient’s platelets, whereas these responses to thrombin were within normal ranges. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) also evoked normal response in the 40 kD protein phosphorylation in the patient’s platelets. These results suggested that the patient’s platelets had TXA2 receptor abnormalities which were characterized by defective transduction of the binding signal to postreceptor reactions after normal TXA2 binding.

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 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

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.

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.

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.

Mutations of the Platelet Thromboxane A2 (TXA2) Receptor in Patients Characterized by the Absence of TXA2–induced Platelet Aggregation despite Normal TXA2 Binding Activity

1999 ◽  
Vol 82 (11) ◽  
pp. 1528-1531 ◽  
Author(s):  
Wataru Higuchi ◽  
Akira Hattori ◽  
Yoshifusa Aizawa ◽  
Ichiro Fuse
Keyword(s):  
Platelet Aggregation ◽  
Mutational Analysis ◽  
Thromboxane A2 ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Mutant Type ◽  
Platelet Disorder ◽  
Group A ◽  
Txa2 Receptor ◽  
Group B

SummaryPreviously, we reported five cases of platelet dysfunction characterized by the absence of thromboxane A2 (TXA2) – induced platelet aggregation despite normal TXA2 binding activity. In this platelet disorder, patients were divided into two groups; i.e. those whose platelets lacked or did not lack phospholipase C (PLC) activation (Group A and Group B, respectively) (Thromb Haemost 1996; 76: 1080). Furthermore, in one of the patients, we showed that a single amino acid substitution (Arg60 to Leu) in the first cytoplasmic loop of the TXA2 receptor (TXR) was responsible for this platelet disorder. However, mutational analysis of the TXR in the remaining patients has not been performed. Based on this background, we investigated the mutations of the TXR in these patients, and found that all of the patients have the same abnormality of the TXR (Arg60 → Leu), although the Group A patients were homozygous and the Group B patients were heterozygous for this mutation.This mutation is the only abnormality which has been found in this platelet disorder, and in patients heterozygous for this mutation, the mutant type TXR suppresses wild-type receptor-mediated platelet aggregation by a mechanism independent of PLC activation.

Pathogenetic Analysis of Five Cases with a Platelet Disorder Characterized by the Absence of Thromboxane A2(TXA2)-Induced Platelet Aggregation in Spite of Normal TXA2 Binding Activity

1996 ◽  
Vol 76 (06) ◽  
pp. 1080-1085 ◽  
Author(s):  
Ichiro Fuse ◽  
Akira Hattori ◽  
Masao Mito ◽  
Wataru Higuchi ◽  
Kazuaki Yahata ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Bleeding Time ◽  
Thromboxane A2 ◽  
Binding Activity ◽  
Normal Limits ◽  
Signaling Mechanisms ◽  
Platelet Disorder ◽  
Txa2 Receptor

SummaryFive patients with mild bleeding tendencies characterized by defective thromboxane A2 (TXA2)-induced platelet aggregation are reported. The platelets of all the patients had the ability to bind exogenous TXA2. Bleeding time was markedly prolonged in one patient. In three of the five patients, synthetic TXA2 mimetic (STA2)-induced platelet responses, including IP3 formation, Ca2+ mobilization, phosphatidic acid formation and GTPase activities were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. However, in the remaining two patients, these responses were all within normal limits. This suggests that the defective site of this type of platelet disorder is heterogenous and that signaling mechanisms other than the TXA2 receptor-phospholipase C pathway are also involved in TXA2-induced platelet aggregation.

scholarly journals Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver

1981 ◽  
Vol 194 (2) ◽  
pp. 385-394 ◽  
Author(s):  
J S Bonifacino ◽  
S H Sánchez ◽  
A C Paladini
Keyword(s):  
Rat Liver ◽  
Rate Constant ◽  
Binding Capacity ◽  
Inhibitory Effect ◽  
Binding Activity ◽  
Dissociation Rate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Solubilized Form ◽  
Triton X

Lactogenic receptors from rat liver microsomal fraction (‘microsomes’) were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 × 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 × 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.

High-affinity Cortisol receptor activity in the liver of the brook trout, Salvelinus fontinalis (Mitchill)

1987 ◽  
Vol 65 (10) ◽  
pp. 2498-2503 ◽  
Author(s):  
P. K. Chakraborti ◽  
M. Weisbart
Keyword(s):  
Brook Trout ◽  
Association Constant ◽  
Glucocorticoid Receptors ◽  
Binding Capacity ◽  
Gel Filtration ◽  
Nuclear Extract ◽  
Binding Activity ◽  
Dissociation Rate ◽  
High Affinity

In vitro binding of [3H]cortisol to brook trout liver cytosol and salt-soluble nuclear extracts demonstrated a high affinity (cytosol association constant, Ka = (0.18 ± 0.03) × 109M−1, n = 4; nuclear extract association constant, Ka = (0.033 ± 0.01) × 109M−1, n = 4) and low maximum binding capacity (cytosol, Nmax = 167 ± 18 fmol∙mg−1 protein, n = 4; nuclear extract, Nmax = 858 ± 81 fmol∙mg−1 protein, n = 4). Kinetics of binding of [3H]cortisol to cytosol at 2 °C revealed association and dissociation rate constants of 6.95 × 104∙M−1∙s−1 and 2.38 × 10−4 s−1, respectively. Gel filtration chromatography using Sephacryl S-300 as the matrix showed a molecular mass for the cytosol binding activity of 319 000 ± 4486 Da and Stokes' radius of 6.23 ± 0.03 nm (n = 3). Competition studies with different steroids indicated considerable specificity for the cytosol binding. The competitive hierarchy was as follows: dexamethasone > triamcinolone acetonide > Cortisol > corticosterone > cortisone > testosterone > estrone > 17β-estradiol > 11-deoxycortisol > progesterone > 11β, 17α, 21-trihydroxy-4-pregnen-3,20-dione-21-phosphate > 17α,20β-dihydroxy-4-pregnen-3-one > pregnenolone. These results support the concept of cytosolic and nuclear forms of glucocorticoid receptors in the liver of brook trout.

Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

1996 ◽  
Vol 54 ◽  
pp. 738-739
Author(s):  
W.G. Wier
Keyword(s):  
Local Control ◽  
Cardiac Tissue ◽  
Cell Function ◽  
Isolated Heart ◽  
Cardiac Cells ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Heart Cell ◽  
Free Calcium ◽  
Heart Cells

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

Sign in / Sign up

Export Citation Format

Share Document