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)

By devising and applying quantitative methods for the assay of thrombin and autoprothrombin C and by developing techniques for their purification, it was possible to obtain information about the function and properties of antithrombin. The inhibitor is a protein for which the initial purification steps consist of removing fibrinogen from plasma by heating to 56 degrees for 3 min, removing prothrombin complex by absorption on barium carbonate, absorbing the antithrombin on aluminum hydroxide, and eluting with phosphate buffer. Antithrombin is limited in its capacity to neutralize thrombin activity, and, under some conditions, the rate of inhibition was accelerated, but equivocal results were involved. Heparin cofactor was found to be essential for retarding the formation of thrombin, and, by inference, it is essential for retarding the formation of autoprothrombin C. Heparin cofactor and antithrombin III are the same. Thrombin absorbs on fibrin, and this has been referred to as the "antithrombin I effect." Interference with the thrombin-fibrinogen reaction by mixtures of antithrombin III and heparin is called the "antithrombin II henomenon." The acceleration of thrombin inactivation at the time thrombin forms is called the "antithrombin IV effect." It was discovered that antithrombin III neutralizes thrombin, as well as autoprothrombin C. The inhibitor and the enzyme form a mutual depletion system. To assay for antithrombin III, a standard quantity of thrombin (about 1,100U/ml) was reacted with antithrombin III for 2 hr. The percent thrombin inactivated was then measured. In random samples of human blood, a wide range of antithrombin III concentration was found. The inhibitor is relatively stable in plasma and serum. It is not changed in concentration when Dicumarol therapy is instituted. Ether extraction of plasma reduces antithrombin III activity. Seitz filtration of plasma did not remove activity. Under special conditions, antithrombin III enhances esterase activity of thrombin. Under special conditions, thrombin regenerates from the thrombin-antithrombin III complex. Antithrombin III neutralizes the activity of prethrombin-E and thrombin-E; consequently, an active histidine center found in the B1 chain of thrombin is not essential for the binding of antithrombin. Autoprothrombin II-A activity was neutralized by antithrombin III. Autoprothrombin C was found to be neutralized by antithrombin III; the amounts required varied with the molecular forms of autoprothrombin C. Thrombin and autoprothrombin C apparently occupy the same binding sites on antithrombin III. An equation was developed to account for all the known characteristics of antithrombin III functions. The kinetic aspects of thrombin neutralization were found to correspond exactly with those of autoprothrombin C. Antithrombin III is a high-capacity inhibitor of the two most powerful enzymes in blood coagulation.
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PMID:Antithrombin III: a backward glance o'er travel'd roads. 4 4

1. Cytosolic pH (pHi) and calcium concentration ([Ca2+]i) have been investigated in the presence and absence of physiological HCO3- in human platelets co-loaded with the fluorescent indicators BCECF and Fura-2. Basal pHi and changes evoked by butyrate, thrombin, platelet activating factor (PAF), ADP and phorbol ester were investigated, as were the effects of removing external Na+. 2. In the presence of physiological HCO3- and CO2, basal pHi was 7.02 +/- 0.04 compared with 7.15 +/- 0.05 in the absence of HCO3-. Estimated cytosolic buffering power was reduced from 35.6 +/- 3.0 to 14.5 +/- 0.4 mM/pH unit by the omission of HCO3-. 3. Thrombin evoked an immediate acidification of 0.03 +/- 0.01 pH units in the presence of HCO3- and 0.07 +/- 0.01 pH units in its absence. The acidifications were followed by a slow alkalinization. The final pHi was 0.10 +/- 0.01 units above basal in the presence of HCO3- and 0.08 +/- 0.02 units above basal in the absence of HCO3-. The initial acidification was significantly greater in the absence of HCO3-. The subsequent increase in pHi was similar in the presence and absence of this ion, but the calculated loss of proton equivalents was greater in the presence of HCO3-. 4. Replacement of extracellular Na+ with N-methyl-D-glucamine resulted in a fall in basal pHi and abolished recovery from thrombin-evoked acidification in both the presence and absence of HCO3-. 5. In the presence of HCO3-, PAF and ADP evoked an intracellular acidification similar to that caused by thrombin. However, with PAF and ADP, the subsequent recovery in pHi was slow and did not rise above basal levels. Phorbol dibutyrate, an activator of protein kinase C, evoked a similar elevation in pHi of 0.04 +/- 0.01 units over 3 min in the presence and absence of HCO3-. 6. Stopped-flow fluorimetric measurements were made of both BCECF and Fura-2 fluorescence in the presence of HCO3-. In the presence and absence of external Ca2+, thrombin-evoked rises in [Ca2+]i peaked before any cytoplasmic alkalinization occurred. ADP evoked rapid elevations in [Ca2+]i, but caused no alkalinization.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Agonist-evoked changes in cytosolic pH and calcium concentration in human platelets: studies in physiological bicarbonate. 210 61

We have estimated the changes in cytosolic pH (pHi) that occur when human platelets are stimulated by thrombin. Changes in pHi were estimated (i) from the H+ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H+ efflux that averaged 7.7 +/- 1.6 mumol/10(11) platelets (means +/- SD) leading to an increase in pHi, from 7.05 +/- 0.04 to 7.45 +/- 0.05. Both H+ efflux and pHi changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4'-acetamido 4'-isothiostilbene-2,2'-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO3-/Cl- exchange. Removal of HCO3- or Cl- from the suspending buffer had no effect on the extent of the rise in pHi. After blockade of Na+/H+ exchange by 100 microM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi the rate of which averaged 0.39 unit/min in HCO3(-)-containing medium, and 0.57 unit/min in HCO3(-)-free medium. The cytosolic buffer capacity for H+ was determined by the nigericin/NH4Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO3-, but only 17.2 mmol/(1xpH) in HCO3(-)-free buffer. The total amount of H+ transferred by Na+/H+ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO3- and to 14 mmol/l platelet cytosol in the presence of HCO3-, and is in good agreement with the estimated amount of Na+ uptake by ADP-stimulated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO3-/Cl- exchange. 214 Nov 13

The presence of extracellular bicarbonate potentiated platelet intracellular pH rises induced by thrombin. The effect was most remarkable in sodium-depleted buffers. This effect of bicarbonate was dose-dependent and was inhibited by anion channel blockers. Bicarbonate also potentiated the pHi recovery after acid loading, but had no effect on alkali loading. It was suggested that anion exchange, most probably that of bicarbonate/chloride, contributes to the regulation of intracellular pH in human platelets.
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PMID:Anion channels contribute to the regulation of intracellular pH in human platelets. 247 Jan 62

From 50 to 73% of the lithium contained in platelets of patients receiving oral therapy with lithium carbonate was released by brief thrombin treatment. Similarly, about 50% of the lithium in platelets of normal volunteers incubated with lithium chloride was thrombin-releasable. The data indicate that an amount of lithium approximately equal to 10% of the calcium content was sequestered in the dense bodies (amine storage organelles) of human platelets. Electron microprobe analysis of dense bodies suggests that the addition of lithium did not change the phosphorus content but produced a loss of about 10% of the dense-body calcium. Nevertheless, synthetic solid analogues of the dense-body core incubated with lithium chloride did not sequester lithium preferentially over potassium and failed to exchange calcium for lithium. Thus, the mechanism responsible for the observed changes in platelet dense bodies may be related to selective membrane permeability properties rather than to binding of lithium to nucleotides or pyrophosphate in the dense-body core.
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PMID:Preferential accumulation of lithium in the dense bodies of human platelets. 681 41

Enhanced Na+/H+ exchange has been reported to be increased in patients with essential hypertension. However, early variations of intracellular pH, although influenced by the antiport, are only partially dependent on the exchange. In this study, we measured the initial platelet pH response to agonists in a group of untreated subjects with essential hypertension (EH, n = 24) and in a group of age- and sex-matched normotensive control subjects (CS, n = 24). Intracellular pH was measured with the specific fluorescence indicator 2'7'bis(carboxyethyl)-5,6-carboxyfluorescein. Measurements were performed on platelets in the presence or absence of extracellular calcium, in a carbonate-free medium. Intracellular calcium was measured by the Fura 2 method. Mean pH values were slightly higher in the platelets of EH (7.469 +/- 0.017 U) compared with CS (7.423 +/- 0.012 U, P < .05), although there was a substantial overlap. When stimulated with physiologic agonists ADP and thrombin and with the calcium ionophore ionomycin, a biphasic response consisting of early acidification followed by alkalinization was observed, the second phase not being detectable with ADP. The initial acidification was greater in EH, particularly with ADP (EH, -0.046 +/- 0.002 U; CS, -0.036 +/- 0.002 U, P < .001) and with ionomycin (EH, -0.074 +/- 0.007 U; CS, -0.051 +/- 0.005 U, P < .05). This acidification proved in some way calcium dependent, as it was reduced in the absence of extracellular calcium (EGTA) in both EH and CS. After incubation with amiloride a further decrease in intracellular pH, more marked in EH, was observed. Alkalinization induced by thrombin was increased in EH (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Early agonist-induced intracellular acidification is increased in platelets from patients with essential hypertension. 770 4

The effect of mood stabilizing agents such as lithium, carbamazepine, valproic acid and clonazepam on serotonin(5-HT)- or thrombin-induced intracellular calcium (Ca) mobilization was studied in the platelets of healthy subjects using the fluorescent Ca indicator fura-2. After incubating platelet-rich plasma with these drugs for one or four hours, there was no significant difference in either basal Ca2+ concentration or 5-HT-stimulated Ca response between each agent treatment and control. 5-HT- or thrombin-induced Ca mobilization was not altered by four weeks of lithium carbonate administration in healthy volunteers. These results indicate that these mood stabilizers fail to affect the agonist-stimulated intracellular Ca mobilizing pathway either in vitro or ex vivo in the platelets of healthy subjects.
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PMID:Effect of mood stabilizing agents on agonist-induced calcium mobilization in human platelets. 803 47

Development of a small diameter prosthetic vascular graft with surface based antithrombin properties should aid in maintaining early graft patency in small vessel reconstruction. The purpose of this study was to bind covalently a basecoat protein (canine serum albumin [CSAJ) and a potent antithrombin agent (recombinant hirudin [rHir]) to 4 mm inner diameter poly(carbonate urea) urethane grafts with reactive carboxylic acid groups (cPU). 125I-CSA was covalently bound to 1 cm length segments of cPU grafts using the carbodimide cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). To bind 125I-rHir covalently, CSA was modified with the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) before linkage to the cPU surface with EDC (cPU-CSA-SMCC). 125I-rHir was modified with Traut's reagent and reacted with the cPU-CSA-SMCC surface, covalently linking 125I-rHir to surface bound CSA. 125I-CSA binding to the cPU graft surface (34,235 ng/segment) was ninefold, sevenfold, and 10-fold greater than controls with nonspecifically bound 125I-CSA. Covalent linkage of 125I-rHir to the cPU-CSA-SMCC surface (9,974 ng/segment) was 172, 192, and 142-fold greater than controls with nonspecifically bound 125I-rHir. Surface antithrombin properties were characterized using a chromogenic assay to measure residual thrombin activity. Evaluation of surface antithrombin activity showed significantly greater 131I-thrombin inhibition and binding by the cPU surface with covalently bound 125I-rHir, as compared with controls. Release of 125I-rHir from the cPU surface was minimal as compared with controls. Therefore, rHir can be covalently linked to a novel small diameter polyurethane vascular graft surface while maintaining its potent antithrombin properties.
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PMID:Bioengineering of a novel small diameter polyurethane vascular graft with covalently bound recombinant hirudin. 980 16

A library of compounds were prepared by reacting 2-(bromomethyl)-1, 2-benzisothiazol-3(2H)-one 1,1-dioxide (5) with commercially available carboxylic acids in the presence of potassium carbonate or a tertiary amine base. From this library, (1,1-dioxido-3-oxo-1, 2-benzisothiazol-2(3H)-yl)methyl N-[(phenylmethoxy)carbonyl]-beta-alanate (7b) emerged as a potent inhibitor of human mast cell tryptase (IC50 = 0.85 microM). Extension of the side chain of 7b by two carbons gave (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 5-[[(phenylmethoxy)carbonyl]amino]pentanoate (7d) which was an 8-fold more potent inhibitor (IC50 = 0.1 microM). Further modification of this series produced benzoic acid derivative (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 4-[[(phenylmethoxy)carbonyl]amino]benzoate (7n) which is the most potent inhibitor identified in this series (IC50 = 0.064 microM). These compounds exhibit time-dependent inhibition consistent with mechanism-based inhibition. For 7b, the initial enzyme velocity is not a saturable function of the inhibitor concentration and the initial Ki could not be determined (Ki > 10 microM). The steady-state rate constant, Ki, was determined to be 396 nM. On the other hand, compounds 7d and 7n are time-dependent inhibitors with a saturable initial complex. From these studies, an initial rate constant, Ki, for 7d and 7n was found to be 345 and 465 nM, respectively. The steady-state inhibition constants, Ki, for 7d and 7n were calculated to be 60 and 52 nM, respectively. Compound 7n is a 13-fold more potent inhibitor than 7b, and these kinetic studies indicate that the increase in inhibitory activity is due to an increase in initial affinity toward the enzyme and not an increase in chemical reactivity. These inhibitors generally show high selectivity for tryptase, being 40-fold weaker inhibitors of elastase, being 100-fold weaker against trypsin, and showing no inhibition against thrombin. These compounds are not inhibitors of thrombin, plasmin t-PA, urokinase, and factor Xa (IC50 > 33 microM). In the delayed-type hypersensitivity (DTH) mouse model, a model of skin inflammation, a 5% solution of 7d reduced edema by 69% compared to control animals.
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PMID:1,2-Benzisothiazol-3-one 1,1-dioxide inhibitors of human mast cell tryptase. 982 54

Antenna coupling microwave plasma enables a highly oxidative treatment of the outmost surface of polypropylene (PP) nonwoven fabric within a short time period. Subsequently, grafting copolymerization with acrylic acid (AAc) makes the plasma-treated fabric durably hydrophilic and excellent in water absorbency. With high grafting density and strong water affinity, the pAAc-grafted support greatly becomes feasible as an intensive absorbent and as a support to promote heparin immobilization through amide bonds. For heparin immobilized in acidic condition, the carbonate groups of the molecule tend to dissolve and passive encapsulation of the molecule prevents its functional groups from bonding with the carboxylic acid of pAAc. This effect leads to inhibit the immobilization process and consequently reduces the quantity as well as the bioactivity of the immobilized heparin. In alkaline processing environment, the oxidized uronic acid residues in heparin-related glycans are presumably cleaved and the removal of some oxidized residuals before immobilization process is likely to reduce the chain length of heparin. In the latter case, anticoagulant Factors X and XII, but not thrombin, are unaffected. Anticoagulant activity test using activated partial thromboplastin time (aPTT) is more sensitive in assessing heparin-immobilized surfaces, since it corresponds to Factor X and initiates the inhibition of Factor XII and thrombin. Likewise, platelets adhesion on the surfaces decreases as the process shifted from acidic to alkaline condition, whereas the hydrophilic character of the grafted pAAc markedly contributes to extend physical insertion of platelets. The immobilized heparin has a great part of original bioactivity, depending on the pH of the processing environment and the immobilized quantity. Relative bioactivity based upon aPTT tests is partially held longer than 90 days for the sample prepared in the alkaline or neutral environment.
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PMID:Anticoagulant activity of immobilized heparin on the polypropylene nonwoven fabric surface depending upon the pH of processing environment. 1255


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