Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.
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PMID:Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom. 642 17

Effects of seven purified phospholipases A2 from the venoms of snakes (Naja naja atra, Trimeresurus mucrosquamatus and T. gramineus) and honey bee (Apis mellifera) on rabbit washed platelet suspension in the absence of bovine serum albumin have been studied. Only phospholipases A2 from N. n. atra, T. mucrosquamatus and A. mellifera venoms induced platelet aggregation with small amounts of 14C-serotonin release. They showed tachyphylaxis and also cross-tachyphylaxis in inducing platelet aggregation. The former two phospholipases A2 exhibited biphasic responses in which irreversible aggregations appeared at concentrations of 1-10 micrograms/ml. At higher concentrations, they elicited the reversible aggregation. Exogenous Ca2+ was essential to their activity. Indomethacin and EDTA completely abolished both phospholipase A2 induced platelet shape change and aggregation, while mepacrine, prostaglandin E1, verapamil and nitroprusside inhibited only the aggregation response. p-Bromophenacyl bromide-modified phospholipases A2, which almost completely lost enzymatic activity, failed to induce platelet aggregation. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol inhibited the phospholipase A2-induced platelet aggregation. These phospholipases A2 induced thromboxane B2 formation which was inhibited by EDTA and indomethacin, but not by prostaglandin E1. Pre-treatment of platelet suspension with phospholipase A2 from N. n. atra or A. mellifera venom (50 micrograms/ml) inhibited platelet aggregation induced by sodium arachidonate or collagen, but not that induced by thrombin or ionophore A-23187. Exogenous sodium arachidonate or lysophosphatidylcholine also showed unaltered inhibitory spectrum on platelet aggregation. It is concluded that phospholipases A2 induce platelet aggregation by virtue of their enzymatic activity, cleaving the membrane phospholipids resulting in arachidonic acid release and formation of thromboxane A2. On the other hand, the cleaved products, lysophosphatidylcholine, arachidonic acid or arachidonate metabolites (via lipoxygenase pathway) may be responsible for anti-platelet activity.
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PMID:Effect of the purified phospholipases A2 from snake and bee venoms on rabbit platelet function. 644 10

Plasma soluble fibrin monomer complexes (SFMC) in 10 patients with nephrotic syndrome were measured to demonstrate the contribution of hypoalbuminemia to the SFMC formation. The levels of SFMC as well as plasma fibrinogen (Fbg) levels were significantly higher than those in control subjects. There was a negative correlation between the levels of SFMC and serum albumin, and also between Fbg and serum albumin. The increase in SFMC levels which indicates the intravascular generation of thrombin might be correlated to hypoalbuminemia in nephrotic syndrome with hypercoagulability.
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PMID:Plasma soluble fibrin monomer complexes in nephrotic syndrome--with reference to hypoalbuminemia. 646 12

We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.
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PMID:Detection of protein C activation in humans. 654 15

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.
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PMID:Production of nerve growth-stimulating factor(s) from chick embryo heart cells. Use of Cytodex 3 microcarriers and serum-free media. 672 97

The biosynthetic mechanism of prostaglandin D2 in human platelet-rich plasma has been investigated. Platelet-rich plasma was separated into washed platelets and platelet-poor plasma, and [1-14C]prostaglandin H2 was incubated with each fraction. The enzymatic conversion of the endoperoxide to prostaglandin D2 was found only in platelet-poor plasma and not in washed platelets or platelet lysate. This prostaglandin D synthetase activity was purified to homogeneity and identified as serum albumin by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and immunoelectrophoresis. The optimal pH and Km value for prostaglandin H2 were 9.0 and 6 microM, respectively. Glutathione was not required for the activity. Although prostaglandin H2 ws converted to prostaglandin D2 and E2 in the reaction, only the prostaglandin D2 formation was dependent on the protein amount and abolished by prior boiling. The action of this activity under physiological conditions was examined in a model system constituted of serum albumin and washed platelets. Prostaglandin D2 formation was observed in association with thrombin-evoked platelet aggregation in this system and was proportional to the number of platelets and the concentration of serum albumin, suggesting that thrombin-stimulated platelets released prostaglandin H2, and the latter compound was then converted to prostaglandin D2 by the action of serum albumin. Consistent with this interpretation, prostaglandin H2 added to platelet-rich plasma was converted in part to prostaglandin D2, and the aggregation caused by this endoperoxide was greatly enhanced by neutralizing the action of prostaglandin D2 with anti-prostaglandin D2 antiserum.
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PMID:Characterization of the biosynthetic pathway of prostaglandin D2 in human platelet-rich plasma. 681 98

Covalently bound conjugates of human serum albumin and heparin were prepared as compounds which could improve the blood-compatibility of polymer surfaces either by preadsorption or by covalent coupling of the conjugates onto blood contacting surfaces. The conjugates (10-16 weight % of heparin) were obtained by a condensation reaction between albumin and heparin using 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Unreacted albumin and heparin were removed by diethyl-aminoethyl (DEAE)-cellulose and Cibacron Blue Sepharose chromatography respectively. The activity of the heparin component incorporated in the albumin-heparin conjugates (Ac) was compared with that of the heparin used for the synthesis of the conjugates (Anat) by thrombin time, inhibition of Factor Xa and the activated partial thromboplastin time (APTT) assays. The Ac/Anat ratio for the above assays was as follows: Thrombin time 1.25, Factor Xa inhibition 0.5. and APTT 0.5. Gel filtration chromatography showed broad-molecular weight distributions. The conjugates were fractionated using immobilized antithrombin III (ATIII). High ATIII and low ATIII affinity conjugate fractions showed the same behavior as ATIII fractionated heparin with respect to thrombin times and Factor Xa inhibition.
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PMID:Covalently bound conjugates of albumin and heparin: synthesis, fractionation and characterization. 683 42

Addition of several arginine-specific serine proteases to culture medium conditioned by fibroblasts results in the proteases being taken into sodium dodecyl sulfate-stable complexes with a secreted factor termed protease nexin (PN) (Baker, J. B., Low, D. A., Simmer, R. L., and Cunningham, D. D. (1980) Cell 21, 37-45). PN not only inhibits these degradative enzymes but also mediates their binding, internalization, and degradation by the cells (Low, D. A., Baker, J. B., Koonce, W. C., and Cunningham, D. D. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2340-2344). Here we describe a simple procedure for purifying milligram quantities of PN from serum-free medium conditioned by human foreskin cells. Accumulation of PN in the medium is increased by using high density microcarrier cultures supplemented with epidermal growth factor and bovine serum albumin. Application of ultrafiltration-concentrated medium to a heparin-Sepharose column followed by extensive washing of the column with buffer containing 0.2 M NaCl and elution with buffer containing 1.0 M NaCl results in the recovery of 60-90% of the input PN in a form that is 90-97% pure. This preparation can be further purified by hydrophobic chromatography on octyl-agarose. Purified PN has a molecular mass of approximately 51 kilodaltons. On nonequilibrium pH gradient electrophoresis it migrates as five bands with isoelectric points between 7.5 and 7.8. Purified PN exhibits all the properties attributed to PN in culture medium. These include: 1) formation of sodium dodecyl sulfate-stable complexes with thrombin, urokinase, and plasmin; 2) inhibition of protease activity; 3) heparin-enhanced inhibition of thrombin; and 4) cellular binding of protease-PN complexes in a heparin-sensitive reaction. When thrombin-PN complexes are dissociated with 1 M hydroxylamine a smaller form of PN (approximately 46 kilodaltons) is detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the complexed PN is proteolytically modified.
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PMID:Purification of human protease nexin. 688 87

In vitro exper iments showed that polymeric hydrogels containing covalently immobilized heparin affect the parameters of blood clotting system. This is not accounted for by the elution of heparin into the surrounding medium and this property of heparin-containing polymers is caused by specific interaction of immobilized heparin with plasma proteins. By the methods of coagulograms and thromboelastography it was revealed that the decrease of total blood anticoagulant activity takes place due to the decrease in blood of fibrinogen, prothrombin and thrombin content and the suppression of fibrin stabilizing factor activity. Free heparin content in blood was left unaffected. It was found that plasma proteins permeate the hydrophilic polymer matrix and interact with immobilized heparin. Binding strength for immobilized heparin is decreased in the array fibrinogen congruent to thrombin much greater than plasmin greater than serum albumin. Complexes of immobilized heparin with fibrinogen, thrombin and plasmin display lytic action towards unstabilized fibrin.
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PMID:On the interaction of heparin-containing polymers with plasma proteins and blood. 713 50

Experiments were performed to determine if the sulfhydryl protease, papain (EC 3.4.22.2), reacts with the plasma protease inhibitor antithrombin (antithrombin III, heparin cofactor) on a specific manner analogous to the reaction of thrombin (EC 3.4.21.5) and other serine proteases with this inhibitor. The esterolytic activity of papain is blocked by the addition of antithrombin, but not by antithrombin-thrombin complex or by protein substrates such as bovine serum albumin. Likewise, in the presence of papain, antithrombin was unable to displace the active site dye proflavine from thrombin, or to inhibit thrombin-catalysed hydrolysis of an anilide substrate. The reaction of antithrombin and papain was not accelerated by low concentrations of heparin. Approximately stoichiometric amounts of heparin completely inhibited the reaction of papain with antithrombin. The mutual inhibition indicates that plasma antithrombin does react with papain but the reaction differs from the interaction with coagulation factors, particularly in the heparin effect.
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PMID:Reaction of antithrombin with proteases. Evidence for a specific reaction with papain. 740


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