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)

Platelets lose their ability to aggregate when deprived of divalent cations. This usually was studied by incubating human citrated platelet-rich plasma with EDTA or EGTA and then adding enough CaCl2 to combine with the chelating agent. Incubation for 5-7 min at 37 degrees C caused irreversible loss of the platelets' ability to adhere to glass and to aggregate with ADP, epinephrine, A23187, vasopressin, or serotonin or upon rewarming after chilling and markedly reduced aggregation with collagen or thrombin. Control samples incubated with saline, CaEDTA, or CaEGTA were not inhibited. Untreated platelets washed and incubated in solutions treated with resins that remove divalent cations lost their ability to aggregate in 30 min. More than about 0.26 mM Mg2+ partially protected the platelets. Unlike aggregation, ADP-induced shape change, clot retraction caused by thrombin or ADP plus reptilase, and thrombin-induced 14C-serotonin release were not inhibited after incubation. Aggregability was not restored by prolonged incubation with CaCl2, adding normal plasma, or washing the platelets. Its loss was temperature and pH dependent, occurring in 2 min at 43 degrees C but not in 7 min at 30 degrees C, and at pH 7.8 but much less at pH 7.2. The defect was not associated with an increase in platelet cyclic AMP, a decrease in metabolic ATP, or the presence of free ADP.
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PMID:Nonreversible loss of platelet aggregability induced by calcium deprivation. 67 68

1. The 2',3'-dialdehyde derivative of ADP (oADP) at concentrations approaching the millimolar range induces human blood platelets to undergo the transition from discoid to globular morphology (the 'shape change') but is incapable of inducing aggregation. 2. When incubated with platelets for 1 min before addition of the agonist, oADP acts as a competitive inhibitor of shape change and aggregation induced by ADP. Under these conditions secretion and hence aggregation induced by low concentrations of collagen; and secretion and hence secondary aggregation induced by adrenaline, thrombin and vasopressin are also inhibited by this analogue. In addition, oADP stimulates the rate of primary aggregation induced by adrenaline and causes partial inhibition of primary aggregation induced by thrombin or vasopressin. When longer preincubation times are employed the extent of inhibition with respect to all agonists, except for high concentrations of collagen, is increased and the competitive character of the inhibition with respect to ADP is no longer apparent. 3. Incubation of human platelets with the 2',3'-dialdehyde derivative of ATP (oATP) causes effects similar to those described for oADP except that the analogue neither induces platelet shape change, nor stimulates the rate of primary aggregation induced by adrenaline. In addition oATP fails to cause significant inhibition of platelet shape change induced by serotonin. The extent and character of inhibition caused by addition of oATP is not a function of the time of incubation. 4. The 2',3'-dialcohol derivatives of ADP and ATP and orATP) effect the aggregation properties of human blood platelets in a manner generally resembling those observed for the 2',3'-dialdehyde analogues. However, orADP is only weakly effective in causing platelet shape change and stimulating the rate of primary aggregation induced by adrenaline and does not inhibit secretion induced by adrenaline, collagen, thrombin and vasopressin. The extent of inhibition by orADP increases only slightly with increased time of incubation. 5. The data suggest that oADP acts as a partial agonist, and oATP as an antagonist, at the platelet ADP receptor, but that platelet membrane stabilisation also results from interaction with these dialdehyde analogues. Such membrane stabilisation does not complicate the interaction of platelets with orADP, which appears to act as a classical antagonist for the ADP receptor.
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PMID:Interaction of human blood platelets with the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of adenosine 5'-diphosphate and adenosine 5'-triphosphate. 68 37

1. The A23187-induced secretion of preabsorbed serotonin from human blood platelets at 37 degrees C is studied. Preincubation at the same temperature before the addition of ionophore is necessary for maximal release induction. When total incubation time is kept constant, longer time with ionophore results in a smaller decrease in the level of metabolic ATP and increase in metabolic ATP and increase in metabolic IMP. This coincides with the reduction in secretion, but statistical treatment of the results suggests that the reduced secretion only partially explains the reduced drop in metabolic ATP, and that therefore a resynthesis of metabolic ATP from IMP may have taken place. 2. In some experiments induction of secretion takes place over a very narrow range of ionophore concentration. 3. When K+ substitutes for Na+ in the extracellular medium, the need for preincubation for maximal secretion becomes less evident, and at times is abolished, while there is still a significant increase in the metabolic ATP level by prolonged incubation with ionophore. 4. A reduction in secretion is observed with metabolic blockers when the ionophore is added after preincubation, but to a much less degree than when secretion is induced by thrombin, in spite of a great reduction in the level of metabolic ATP. This may partly be explained by the increase in secretion induction by A23187 in the presence of inhibitors when the ionophore is added in the cold, suggesting that the inhibitor may cause "weakening" of the platelets' "resistance" to induction of secretion by ionophore. 5. When the effect of Ca2+ and of Mg2+ on the level of intermediates of the TP leads to hypoxanthine conversion is studied, it is evident that the addition of Ca2+ causes enhanced IMP accumulation and a reduction in the level of inosine plus hypoxanthine, while Mg2+ has the opposite effect. This suggests that the two metals affect the enzymes of the IMP leads to hypoxanthine conversion differently. 6. Indomethacin inhibits secretion induced by A23187, suggesting that prostaglandin intermediates may amplify the ionophore-induced release. The adenine nucleotide metabolism is not affected. 7. The results indicate that there is an indirect, rather than direct, link between the major metabolic changes and the secretion induced by A23187, but that the ionophore may cause intracellular changes which are not connected to its effect as release inducer.
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PMID:Metabolic aspects of the secretion of stored compounds from blood platelets. V. Effect of ionophore A23187 on washed platelets. 79 62

Per cent aggregation, release and content of adenine nucleotides, and specific radioactivity were evaluated in citrated platelet-rich plasma (PRP) prepared from paired samples of maternal and cord blood. Platelets of newborn infants aggregated normally in response to highdose ADP (20 muM), strong collagen suspensions, and thrombin; however, when compared with PRP from the mothers or from normal adults, per cent aggregation in response to lower concentrations of ADP (2 muM), weak collagen, and part particularly epinephrine was markedly reduced. Nucleotide release after stimulation of the newborns' PRP with the latter two inducers was also impaired. ATP and ADP content of the newborns' platelets was also significantly less than that of their mothers or of normal adults, but specific activity was normal. The data suggest that the impairment of ADP release in the platelets of newborn infants is due to decreased sensitivity to external stimuli. Since metabolic ATP is necessary for the platelet release reaction, it is postulated that the platelet dysfunction results from a lack of metabolic ATP.
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PMID:Newborn platelet dysfunction: a storage pool and release defect. 103 10

Citrated canine platelet rich plasma stimulated with ADP, thrombin, collagen and E. coli endotoxin demonstrated platelet aggregation when measured with Payton aggregometer. Following incubation with hydrocortisone sodium succinate (Solu-Cortef) the same platelet rich plasma did not undergo platelet aggregation when stimulated by these agents. Solu-Cortef added to platelet rich plasma during ATP, thrombin, collagen and endotoxin stimulated platelet aggregation resulted in a reversal of the optical density suggesting deaggregation. These in vitro observations support the beneficial effects observed when steroids are administered in vivo to experimental or clinical shock.
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PMID:In vitro inhibition of endotoxin induced platelet aggregation with hydrocortisone sodium succinate (Solu-Cortef). 110 67

[3H]-adenine-labeled human platelets in plasma were incubated with or without nonradioactive serotonin. Release reaction was then induced by ADP, epinephrine, collagen, or thrombin. Platelets that had been incubated with serotonin released four times as much serotonin as platelets incubated without serotonin. The specific radioactivities of the ATP and ADP released to plasma during release reaction induced with all four inducers were the same in both systems. This shows that when serotonin is taken up by human platelets, it enters the compartment containing nonmetabolic, granula-stored ATP, and not the compartment with metabolic extragranular ATP. These results suggest that the mechanism of serotonin storage in human platelets is similar to that in other species investigated, i.e., rabbit, guinea pig, and pig.
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PMID:Short communication: possible association of newly absorbed serotonin with nonmetabolic, granule-located adenine nucleotides in human blood platelets. 111 36

When platelets take part in the formation of hemostatic plugs and thrombi in vivo, electron microscopic evidence indicates that some of the platelets not only release their granule contents but also undergo lysis. In the present study we have examined, in vitro, the relation between the release reaction of platelets and platelet lysis in response to the release-inducing agents thrombin and collagen. Release was measured by determining the amounts of 14C-serotonin and adenine nucleotides that appeared in the ambient fluid of prelabeled platelets. Lysis was measured by determining the amount of either lactate dehydrogenase or 14C-labeled cytoplasmic ATP from platelets incubated with -14C-adenosine. Washed platelets prepared from rabbit, pig, or human blood lost some lactate dehydrogenase and 14C-ATP upon exposure to thrombin, but the amounts of lactate dehydrogenase and 14C-ATP lost from rabbit platelets were much greater than from pig or human platelets. The reason for this species difference is not aparent. The platelet release reaction appeared to be necessary for lysis to occur. Reduction of the extent of the release reaction by preincubation of rabbit platelets with metabolic inhibitors to deplete metabolic ATP reduced the extent of lysis. In addition, it was apparent that the fall in platelet metabolic pool ATP caused by thrombin was not responsible for platelet lysis. Lack of calcium, addition of prostaglandin E(1), OR Increasing the albumin concentration of the suspending medium of rabbit platelets inhibited platelet lysis. These conditions may prevent the loss of material that causes lysis, inhibit the action of this lost material, or inhibit the lytic reaction. Release and lysis may occur together and release can occur without detectable lysis, but lysis in response to a release-inducing agent does not take place unless the release reaction occurs.
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PMID:Conditions influencing platelet lysis. 112 14

Separation of platelets from plasma is achieved by adding ADP (final concentration 10-5 M) to platelet-rich plasma and allowing aggregates to form. Aggregates are removed quickly by brief, gentle centrifugation, washed two to three times with 0.9% NaCl (saline), and then incubated for 10 minutes in the presence of apyrase, albumin and calcium. Platelet aggregates deaggregate completely during this incubation period. The platelet suspension is then subjected to 1100g for 12 minutes, gently resuspended in a small volume of saline, and finally diluted with an appropriate medium to the desired concentration. The entire separation procedure requires approximately 30 minutes. Platelets obtained by this procedure are a) comparable in aggregability to the platelet preparations obtained by gel filtration, b) have normal intracellular amounts of ATP and ADP, and c) except for slight dilatation of the surface-connected canalicular system, have normal ultrastructural appearance. When suspended in an appropriate medium, these separated platelets take up serotonin 14-C and subsequently release it in nearly normal quantities when exposed to thrombin, collagen or ADP.
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PMID:Characterization of human platelets separated from blood by ADP-induced aggregation. 112

The levels of four acid hydrolases, beta-N-acetyl glucosaminidase, beta-glucuronidase, beta-galactosidase, and acid phosphatase, and the extent of their release (release II) by thrombin was determined in platelets from nine normal subjects, nine patients with storage pool disease, and in normal platelets which had been exposed to aspirin. The levels of all four hydrolases were normal in patients with SPD. However, release of three of these hydrolases (acid phosphatase was an exception) by low concentrations of thrombin (0.015 and 0.04 U/ml) was decreased in the patients as a group, although considerable variation in the extent of release of each enzyme was noted. In contrast, aspirin failed to inhibit release II in normal platelets (except for a slight impairment in the release of beta-N-acetyl glucosaminidase), although release I (serotonin, ATP and ADP) was inhibited. All release defects could be overcome by using higher concentrations of thrombin (0.2 U/ml). The normal levels of acid hydrolases in the platelets of patients with SPD (who are deficient in the platelet dense granules) suggest that these enzymes are not normally stored in the dense granules, but rather in alpha-granules. The findings also support the conclusions of previous studies that the release reaction is impaired in SPD. This release defect appears to be different from that seen in normal platelets after exposure to aspirin.
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PMID:Content and thrombin-induced release of acid hydrolases in gel-filtered platelets from patients with storage pool disease. 113 24

The mechanism of stimulation of platelets by thrombin and other proteases was studied by following kinetics of secretion of Ca2+ or ATP. The progress-time curves of secretion were analyzed for rate and total amount released. The reaction of thrombin was perturbed by addition of hydroxylamine or a competitive inhibitor and by variation of pH and it was compared with the reactions of other proteases. Trypsin and papain, with specificities for arginyl residues, induced secretion with a time course that was nearly identical with that induced by thrombin when saturating levels of enzyme were used. At low levels of enzyme, trypsin and papain gave extended lags in the progress-time curves. Higher concentrations of trypsin and papain were required for saturation of the measured parameters. Human plasmin (lysly specificity) and bovine chymotrypsin (aromatic amino acid specificity) failed to induce platelet secretion. Active site inhibited thrombin was also ineffective. Both yield and kinetics depended on pH, with the pH profile for each enzyme similar to its profile for hydrolysis of synthetic substrates. Studies at low pH also showed that the early part of the reaction undergoes a change in rate-determining step from enzyme dependent at low enzyme to enzyme indepdenent at high enzyme. Hydroxylamine, a nucleophile that would be expected to accelerate hydrolytic reactions, actually decreased both the rate of initial reactions and yield. A competitive inhibitor of thrombin also decreased both rate and yield; a calculated inhibition constant was in agreement with the value for a synthetic substrate, suggesting that the interaction of thrombin with platelets is analogous to reaction with substrates. A modification of our previous model is proposed in order to accommodate the results described here and to reaoncile the apparent contradictions that enzyme was found not to turn over in the reaction (Detwiler, T. C., and Feinman, R. D. (1973), Biochemistry 12, 282), that catalytic activity is required (Davey, M. G., and Luscher, E. F. (1967), Nature (London) 216, 875; this paper), and that the reaction is characterized by an apparent equilibrium binding (Tollefsen, D. M., Feagler J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). The essential feature is a reversible catalytic step with no dissociation of enzyme from product. This is followed by irreversible, thrombin-independent platelet processes leading to secretion, with yield dependent on the equilibrium concentration of the thrombin product. The model thus has aspects of catalysis, stoichiometry, and an agonist-receptor equilibrium.
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PMID:Platelet stimulation by thrombin and other proteases. 116 69


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