Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human platelets were loaded with the fluorescent Na(+)-sensitive dye sodium-binding benzofuran isophtalate (SBFI), and changes in the fluorescence excited at 345 and 385 nm were analyzed after manipulations that evoked predictable changes in the cytosolic Na+ concentration ([Na+]i). Raising [Na+]i by either gramicidin D or monensin specifically increased the fluorescence excited at 345 nm and decreased that excited at 385 nm. Hence, calculation of changes in the 345/385 nm excitation ratio yields an estimate of actual changes in [Na+]i. A transient activation of Na+/H+ exchange evoked by addition of acidified platelets to buffer, pH 7.4, evoked a transient rise in [Na+]i. The re-establishment of basal [Na+]i could be prevented by ouabain, indicating an involvement of the Na+,K(+)-ATPase. Upon stimulation by 0.5 unit/ml of thrombin, [Na+]i immediately increased by 16 +/- 4 mM and this rise continued for at least 60 min after addition of agonist, albeit at a lower rate. This latter sustained rise could not be curtailed by scavenging thrombin by means of hirudin. Addition of ouabain or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced a comparable slow rise in the 345/385 excitation ratio. This may indicate a protein kinase C-mediated inhibition by thrombin of the Na+,K(+)-ATPase. In the absence of extracellular Ca2+ (Ca2+o), the [Na+]i gain was augmented to 38 +/- 9 mM. This additional uptake of Na+ was prevented by (i) Mn2+ ions, (ii) La3+ ions, (iii) the blocker of receptor-mediated Ca2+ entry (1-[beta[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-im ida zole hydrochloride), and (iv) by hirudin which reversed receptor occupancy by thrombin. These findings suggest that the additional thrombin-induced [Na+]i gain in the absence of Ca2+o is due to Na+ influx through a Ca2+ entry pathway. The increase in [Na+]i in the presence of Ca2+o results from Na+ influx via Na+/H+ exchange.
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PMID:Further characterization of the mechanisms mediating the rise in cytosolic free Na+ in thrombin-stimulated platelets. Evidence for inhibition of the Na+,K(+)-ATPase and for Na+ entry via a Ca2+ influx pathway. 164 80

Depletion of intracellular calcium stores appears to increase plasma membrane permeability for calcium by an as yet obscure mechanism. We found that the Ca2+ ionophore, A23187, and thrombin elevate cytosolic calcium ([Ca2+]i) equally and cause tyrosine phosphorylation of a 130-kDa protein and to a lesser extent 80- and 60-kDa proteins. Chelation of [Ca2+]i by 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid/acetomethoxy ester decreased thrombin-induced tyrosine phosphorylation responses. These results suggested that [Ca2+]i elevation promotes tyrosine phosphorylation. Tyrosine phosphorylation persisted in the presence or absence of extracellular calcium after thrombin stimulation but subsided rapidly after A23187 addition if extracellular calcium was present. When Ca2+/ATPase activity, which is apparently required to maintain calcium stores, is inhibited by low temperature, tyrosine phosphorylation of the 130-kDa protein occurs. Rewarming platelets reverses tyrosine phosphorylation only if extracellular calcium is present. Thapsigargin, a calcium ATPase inhibitor, also induces tyrosine phosphorylation of the 130-kDa protein and prevents dephosphorylation of this protein when added prior to rewarming. These observations suggest that homeostatic levels of calcium in storage compartments favor tyrosine dephosphorylation of specific proteins. Thus the levels of [Ca2+]i and stored calcium appear to control tyrosine phosphorylation antagonistically. Tyrosine phosphorylation may play a role in regulating calcium channel function.
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PMID:Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins. 183 59

Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.
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PMID:Different calcium pools in human platelets and their role in thromboxane A2 formation. 183 1

Endothelin has been shown to affect a broad range of renal functions, including rat inner medullary collecting duct Na/K ATPase activity, renin release, renal blood flow, and glomerular filtration rate. The source of endothelin in the kidney has been assumed to be endothelial cells. However, the inner medulla contains the highest concentration of immunoreactive endothelin in the kidney. Additionally, MDCK cells, a distal tubule-like cell line, synthesize endothelin. In order to determine if primary renal tubule cells release endothelin, supernatants collected from rat inner medullary collecting duct cells in culture were tested for endothelin-1 detected by specific radioimmunoassay. Inner medullary collecting duct cells produced endothelin-1 in a time-dependent manner, releasing 1,016.7 +/- 60.1 pg of endothelin-1 per mg/cell protein/24 h. Inner medullary collecting duct cells expressed a 2.2-kilobase mRNA on blot hybridization with rat prepro endothelin-1 cDNA. Vasopressin, thrombin, bradykinin, and epinephrine did not affect endothelin-1 release. These data demonstrate endothelin-1 production by inner medullary collecting duct cells and suggest a possible autocrine role for the peptide.
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PMID:Endothelin synthesis by rat inner medullary collecting duct cells. 195 27

The head of the myosin molecule (i.e., subfragment 1 with a heavy chain of 95 kDa) is usually obtained by chymotryptic cleavage in the presence of a divalent cation chelator. In the present work, we used another specific proteolytic enzyme, thrombin, to produce a limited cut within the myosin molecule, resulting in a new species of N-terminal fragment. Treatment of skeletal muscle myosin yielded a 97-kDa split heavy chain associated with intact light chains, corresponding to a single cut. The ATPase activities of this new S-1 derivative were slightly affected by the breakdown. It recognized actin in an ATP-dependent manner, as expected, with an affinity 2-5 times higher than that of the usual chymotryptic S-1 preparation but with a very different electron microscopic pattern. Functional differences are noted, and we involve them more precisely in relation to possible structural aspects of the additional C-terminal segment extending the usual S-1 heavy chain from 95 to 97 kDa.
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PMID:New subfragment 1 of skeletal muscle myosin obtained by thrombin cleavage. 213 79

Previous reports have suggested that the physical properties of cell membranes and calcium homeostasis in both the central and peripheral nervous system are changed in Alzheimer's disease (AD). This study has examined the biophysical properties of erythrocyte and platelet membranes by measuring the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and possible related changes in lipid peroxidation. In addition, we have studied calcium homeostasis by measuring thrombin-stimulated changes in intraplatelet free calcium and Ca2(+)-ATPase activity in AD and healthy age and sex-matched controls. Our results show that there was no significant difference in the fluorescence anisotropy of DPH in erythrocyte membranes isolated from the three groups. There was also no significant difference in lipid peroxidation levels in erythrocytes and plasma of AD patients compared to controls. However, there was a significant reduction in the fluorescence anisotropy of DPH in platelet membranes from AD patients, compared with healthy controls. Recent evident suggests that the increase in platelet membrane fluidity results from alterations in internal membranes. We measured the specific activities of enzyme markers associated with intracellular and plasma membranes in platelets from AD patients and healthy controls. There was a significant reduction in the specific activity of antimycin A-insensitive NADH-cytochrome-c reductase (a specific marker for smooth endoplasmic reticulum (SER)), in AD patients compared to controls, but no change in the specific activity of bis(p-nitrophenyl)phosphate phosphodiesterase (a specific marker for plasma membrane). We have also shown that SER mediated [Ca2+] homeostasis is possibly impaired in AD platelets, i.e., the percentage of thrombin-stimulated increase in intraplatelet [Ca2+] above basal levels was significantly higher in AD compared to matched controls and there were significant reductions in the specific activities of Ca2+/Mg2(+)-ATPase and Ca2(+)-ATPase (but not Mg2(+)-ATPase) in AD platelets. Finally electron microscopic analysis of platelets showed that there was a significant increase in the incidence of abnormal membranes in AD patients compared to controls. The ultrastructural abnormalities seem to consist of proliferation of a system of trabeculated cisternae bounded by SER. These results suggest that both SER structure and function might be defected in AD platelets, which could explain the fluidity changes observed here.
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PMID:Platelet and erythrocyte membrane changes in Alzheimer's disease. 214

Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction.
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PMID:Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes. 214 51

Platelet intracellular Ca2+ concentration ([Ca2+]i) and its response to stimuli (ADP and thrombin) were studied in 15 insulin-dependent and 22 non-insulin-dependent diabetes mellitus patients with the fluorescent probe Fura 2. The activity of Ca2(+)-ATPase and Na(+)-K(+)-ATPase, membrane fluidity, and cholesterol and phospholipid content were also determined in platelet membranes. Compared with control subjects, diabetic patients showed 1) increased platelet [Ca2+]i in the resting state, 2) higher Ca2+ levels after stimulation with thrombin and ADP, due entirely to increased resting concentrations, 3) reduced activity of Na(+)-K(+)-ATPase, 4) increased activity of Ca2(+)-ATPase, 5) higher fluidity of the platelet membrane, and 6) increased membrane concentration of total phospholipids. Na(+)-K(+)-ATPase activity was inversely related to platelet [Ca2+]i in each group studied, whereas Ca2(+)-ATPase activity was positively correlated with intracellular Ca2+ levels. The data obtained in diabetic subjects suggest an abnormality in Ca2+ and Na+ transport across the platelet membrane that might be responsible for the reported platelet hyperreactivity to stimuli in diabetes.
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PMID:Altered cellular Ca2+ and Na+ transport in diabetes mellitus. 216 3

The new fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) was used for determination of the cytosolic free Na+ concentration, [Na+]i, in human platelets. The dye could be loaded into platelets in the form of its acetoxymethyl ester (SBFI-AM). Calibration of the fluorescence in terms of [Na+]i was done by measuring the 345/385 nm excitation ratio (emission 490 nm) at various extracellular Na+ concentrations, [Na+]o, in the presence of gramicidin D. The 345/385 intensity ratio increased almost linearly when [Na+]i was stepwise raised from 20 to 60 mM. The basal value for [Na+]i was found to be 26.0 +/- 4.5 mM (n = 15). Incubation of platelets in Na(+)-free buffer decreased [Na+]i, whereas inhibition of the (Na+ + K+)-ATPase by 0.5 mM ouabain increased [Na+]i to 56 +/- 4 mM (n = 4) within 60 min. Activation of Na+/H+ exchange by exposing platelets to propionic acid also raised [Na+]i, and a comparable effect was produced by the Na+/H+ ionophore monensin. Activation of platelets with thrombin (0.1-0.5 unit/ml) also increased the 345/385 nm intensity ratio, an effect that was not seen in Na(+)-free buffer or after raising intracellular cAMP by treatment of platelets with prostaglandin E1. On the average, [Na+]i was raised to 59.5 +/- 5.3 mM (n = 15) at 10 min after addition of thrombin without a significant decrease for further 10 min. An increase in [Na+]i was also seen when platelets were challenged with the Ca2+ ionophore ionomycin, an effect that did not occur in the absence of Na+o. Our findings confirm earlier reports which demonstrated a rise in [Na+]i in stimulated platelets and show that SBFI is a useful tool for determination of [Na+]i in resting and stimulated platelets.
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PMID:Stimulation by thrombin increases the cytosolic free Na+ concentration in human platelets. Studies with the novel fluorescent cytosolic Na+ indicator sodium-binding benzofuran isophthalate. 217 43

Recent findings on endothelin, an endothelium-derived vasoconstrictor substance and endogenous digitalislike Na+, K+-ATPase inhibitor (s) obtained from animal and clinical experiments are reviewed. Endothelin is one of the most potent vasoconstrictive substances ever found; the pressor responses last for more than one hour after the bolus injection in rats. Because the pressor responses have not been attenuated by any known receptor-antagonists, the vasoconstriction is mediated by the endothelin receptors. Since messenger RNA for endothelin increases with thrombin, it may be involved in the damages of blood vessels. Radioimmunoassay for endothelin revealed that immunoreactive endothelin is increased in plasma of patients with chronic renal failure. Therefore, the plasma level of endothelin can be an index for some circulatory disorders. Since the Na+, K+-ATPase inhibitor cross-reacts with antidigoxin and antiouabain antibody, it is called a digitalis-like substance. We have demonstrated that cells containing the immunoreactive-substance to antidigoxin and antiouabain antibodies are restricted in the paraventricular and supraoptic nucleus of the hypothalamus, and that the plasma digoxinlike immunoreactivity increases with intracerebroventricular and intravenous infusions of hypertonic saline in rats. Because plasma concentrations of the immunoreactive substance significantly correlate with blood pressure, the substance seems to be involved in hypertension associated with excess intake of sodium salt.
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PMID:[Endothelin and endogenous digitalis-like substance in cardiovascular regulation: a review]. 268 97


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