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)

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.
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PMID:Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes. 0 49

Contact of rat platelets with thrombin or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in the secretion of adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterases. Significant association of calcium with platelets occurred during platelet surface contact with thrombin. Thrombin concentration to induce association of calcium virtually agreed with that to release the enzyme. The finding that A-23187 (5 to 20 muM) also provoked a rapid and marked association of extracellular calcium with platelets suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between platelet and calcium. Two different phosphodiesterases, a relatively specific cyclic AMP and a relatively specific cyclic GMP phosphodiesterase were secreted from platelets into the plasma in soluble form. The amounts of the phosphodiesterases secreted were dose- or time-dependent on thrombin (0.1 to 2 units) or A-23187 (5 to 20 muM) within 30 min. The enzyme release by thrombin was completely inhibited by heparin but the release by A-23187 was not. The two phosphodiesterases secreted seemed to correspond to the two enzymes isolated from platelet homogenates in many respects. Rat platelets contained, at least, three cyclic 3':5'-nucleotide phosphodiesterases, namely, two relatively specific cyclic AMP phoshodiesterases and a relatively specific cyclic GMP phosphodiesterase which were clearly separated from each other by Sepharose 6B or DEAE-cellulose column chromatography or sucrose gradient centrifugation. The two platelet cyclic AMP phosphodiesterase (Mr = 180,000 and 280,000) had similar apparent Km values of 0.69 and 0.75 muM with different sedimentation coefficient values of 4.9 S and 7.1 S, respectively. They did not hydrolyze cyclic GMP significantly. A cyclic GMP phosphodiesterase (Mr - 260,000) exhibited abnormal kinetics for cyclic GMP with an apparent Km value of 1.5 muM and normal kinetics for cyclic AMP with a Km of 300 muM. The properties of a platelet cyclic AMP phosphodiesterase (Mr = 180,000) and a platelet cyclic GMP phosphodiesterase were found to agree with those of the two phosphodiesterases released from platelets by thrombin or A-23187. Depletion of extracellular calcium by an addition of citrate, EDTA, or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the blood or platelet suspension resulted in a loss of the activity of the smaller form of platelet cyclic AMP phosphodiesterase (Mr = 180,000) and addition of calcium restored the activity of this cyclic AMP phosphodiesterase. Thus, calcium seemed to be involved in the mechanism of an occurrence of this smaller form of cyclic AMP phosphodiesterase as well as the secretion of this enzyme. Contact of human platelets with thrombin also resulted in the secretion of cyclic nucleotide phosphodiesterase which was dependent on the concentration of calcium. No species difference was observed in this respect.
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PMID:Platelet cyclic 3':5'-nucleotide phosphodiesterase released by thrombin and calcium ionophore. 18 90

Natural compounds have been the first historical source of antithrombotic compounds (heparin, vitamin K antagonists, streptokinase, urokinase); molecules extracted from plants or animals still provide some of the most original and promising approaches for the discovery of new drugs in this class. In this review, we will briefly describe three examples of current research trends that could lead to the development of new antithrombotic drugs of natural origin. Flavonoids have been shown to be inhibitors of cyclic nucleotide phosphodiesterase; this enzymatic activity is one of the main mechanisms of inhibition of aggregation of blood platelets by flavonoids. Some of these compounds could represent templates for the development of new inhibitors of platelet activation. Garlic (Allium sativum) has been shown to inhibit platelet aggregation in vivo and in vitro; a number of active principles has now been identified and their mechanisms of action are currently being explored. An ancient remedy, the medicinal leech (Hirudo medicinalis), has been found to contain several potent anticoagulant proteins. Among them, hirudin, a polypeptide of 65 amino acids, has been identified as one of the most potent inhibitors of thrombin. The production of sufficient amounts of hirudin through molecular biology techniques has now allowed the performance of clinical trials. These three examples show that careful consideration of biochemical, ethnopharmacological, or toxicological properties of natural products can still constitute a valuable basis for the development of new drugs.
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PMID:Old and new natural products as the source of modern antithrombotic drugs. 195 60

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGE1-stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGE1-induced cAMP elevation but this effects seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.
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PMID:Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits. 205 5

A potent platelet antiaggregant action of an antiarrhythmic peptide (AAP) was demonstrated to be a cause of the antithrombotic effect. AAP (10, 20 or 40 mg/kg, i.v.) inhibited ex vivo platelet aggregation induced by collagen in a dose-dependent manner. AAP also inhibited the platelet aggregation of platelet-rich plasma (PRP) induced by collagen, Ca-ionophore A-23187, adenosine diphosphate (ADP), thrombin or arachidonic acid in vitro. The IC50 was 2.5 mM for collagen, 1.7 mM for A-23187, 5 mM for ADP, 0.4 mM for thrombin and 0.15 mM for arachidonic acid. The aggregation inhibitory activity of the peptide on washed platelet (WP), in a Ca2+-free medium, was stronger than on PRP. The IC50 was 1 mM for collagen and 20 microM for A-23187. No significant difference was found between antiaggregant activities of platelet-free plasma (PFP) from AAP-treated rats and PFP from normal rats supplemented with AAP. The direct action of AAP on platelets was also supported by the incorporation of AAP into platelet cytoplasma which caused a decrease of Ca2+-dependent 3':5'-cyclic nucleotide phosphodiesterase (Ca-PDE) activity. It was considered that AAP showed its platelet aggregation inhibitory activity by decreasing intracellular Ca2+ concentration through the inhibition of Ca-PDE activity.
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PMID:Potent platelet antiaggregant action of an antiarrhythmic peptide (AAP). 301 60

The relation of cyclic 3',5'-adenosine monophosphate to platelet function has been studied by investigating the influence of this compound and of its N(6)-2'-0-dibutyryl derivative on platelet aggregation and other aspects of platelet behavior after demonstration of adenyl cyclase activity in disrupted platelets. Dibutyryl cyclic AMP inhibited platelet aggregation induced by ADP, epinephrine, collagen, and thrombin. Cyclic AMP was also inhibitory but was less effective. The platelet "release reaction" was also inhibited; specifically, there was inhibition of the induction of platelet factor 3 activity and of the release of labeled 5-hydroxytryptamine. Platelet swelling produced by ADP was not inhibited. The action of dibutyryl cyclic AMP did not result from contamination with 5'-AMP, nor was it attributable to production of 5'-AMP by plasma enzymes. Dibutyryl cyclic AMP was degraded to 2'-O-monobutyryl cyclic AMP and to cyclic AMP in plasma, but plasma exhibited no cyclic nucleotide phosphodiesterase activity, and the production of 5'-AMP did not occur. The in vitro effects of dibutyryl cyclic AMP were associated with uptake of the compound by platelets. Adenyl cyclase activity of platelet homogenates was demonstrated with production of 9.27 x 10(-11) (+/-2.62 x 10(-11)) mole cyclic AMP per min per 10(10) platelets. The activity was increased by NaF and by prostaglandin PGE(1) and was decreased by epinephrine. The effect of epinephrine was blocked by phentolamine but not by propanolol. Adenyl cyclase activity was also inhibited by collagen, 5-hydroxytryptamine, and thrombin. ADP, dibutyryl cyclic AMP, and cyclic AMP did not alter adenyl cyclase activity. These observations are consistent with the hypothesis that platelet aggregation is favored by a decrease in platelet cyclic AMP and inhibited by an increase in cyclic AMP.
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PMID:Cyclic 3',5'-adenosine monophosphate in human blood platelets. II. Effect of N6-2'-o-dibutyryl cyclic 3',5'-adenosine monophosphate on platelet function. 432 65

Quercetin and 12 other natural flavonoid aglycones inhibit washed human platelet aggregation and secretion of serotonin induced by ADP, collagen or thrombin. The inhibitory effect of flavonoids is of the same order of magnitude as IBMX and dipyridamole. The structural features required for a flavonoid to inhibit human platelet function are similar to those previously reported by us to inhibit cyclic nucleotide phosphodiesterase. The inhibitory effect of flavonoids on human platelet function was diminished by saturation of the C-2, C-3 double bond, lack of the C-4 carbonyl, glycosylation at C-3 and a high number of hydroxyl substituents.
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PMID:Inhibition of aggregation and secretion of human platelets by quercetin and other flavonoids: structure-activity relationships. 618 78

We have previously reported (Proc. Natl. Acad. Sci. 79, 495-499, 1982) that the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), stimulates cyclic AMP accumulation and inhibits prostacyclin (PGI2) production in primary monolayer cultures of human umbilical vein endothelium. The present study was carried out to determine whether these effects are causally related. Incubation of endothelial monolayers with the diterpene, forskolin, increased the intracellular concentration of cyclic AMP by 10-fold. Despite this marked increase in cyclic AMP, neither baseline production of PGI2 nor release in response to stimulation by thrombin or the divalent cation ionophore, A23187, was affected. Both forskolin and isoproterenol were found to potentiate the effect of IBMX on cyclic AMP accumulation without causing further inhibition of PGI2 biosynthesis. Inhibition of cyclic nucleotide phosphodiesterase activity with 2,6-bis-(diethanolamino)-4-piperidinopyrimido-[5,4-d]pyrimidine increased cyclic AMP levels to the same extent as IBMX; however, this agent had no effect on PGI2 biosynthesis. These findings demonstrate that increases in the intracellular concentration of cyclic AMP have no short-term effects on PGI2 biosynthesis in vascular endothelium and suggest that inhibition of PGI2 production by IBMX is the result of some other, cyclic AMP-independent action of the drug.
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PMID:Prostacyclin biosynthesis in vascular endothelium is not inhibited by cyclic AMP. Studies with 3-isobutyl-1-methylxanthine and forskolin. 618 29

Incubation of primary monolayer cultures of human umbilical vein endothelial cells with buffer, thrombin (0.5 unit/ml), ionophore A23187 (10 microM), arachidonic acid (20 microM), prostaglandin H2 (PGH2) (4 microM) resulted in prostacyclin (PGI2) production in nanomolar quantities to the extent of 36 +/- 2, 276 +/- 13, 485 +/- 32, 533 +/- 22, and 532 +/- 22, respectively, as measured by radioimmunoassay of 6-keto-PGF alpha. Preincubation of the endothelium with 1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, an antagonist of cytoplasmic Ca2+, or with 4 mM 1-methyl-3-isobutylxanthine (MIX), an inhibitor of cyclic nucleotide phosphodiesterase activity, blocked PGI2 release induced by thrombin or A23187, decreased arachidonic acid-induced release by approximately 50%, but had no effect on PGH2-induced release. Radioimmunoassay of cAMP in the endothelium showed that the basal level (1.85 +/- 0.14 pmol of cAMP per 4.5 x 10(5) cells) was increased by an average of 3.9-fold with 4 mM MIX. PGI2 (0.4 microM) had no significant effect on cAMP levels in the absence of MIX, but caused a 2-fold increase with 4 mM MIX. The findings suggest that: (i) the stimulation of PGI2 biosynthesis is mediated by Ca2+, (ii) increased cAMP inhibits PGI2 production, and (iii) cAMP phosphodiesterase activity modulates PGI2-induced increases in the intracellular concentration of cAMP.
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PMID:Role of Ca2+ and cyclic AMP in the regulation of the production of prostacyclin by the vascular endothelium. 628 72

Satigrel (E5510, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid) is a potent inhibitor of platelet aggregation. Like cyclooxygenase/prostaglandin H synthase (PGHS) inhibitors such as aspirin, which suppress platelet aggregation by inhibiting thromboxane A2 production, satigrel inhibits collagen- and arachidonic acid-induced aggregation of human platelets. In contrast to other PGHS inhibitors, satigrel, like cyclic nucleotide phosphodiesterase (PDE) inhibitors such as cilostazol, shows inhibitory activity against thrombin-induced platelet aggregation. To investigate the mechanism of the anti-platelet activity of satigrel, we examined the selectivity and potency of satigrel against PGHS isozyme activities and PDE isoform activities. Two isozymes of PGHS are known; constitutive enzyme (PGHS1) and inducible enzyme (PGHS2). Satigrel showed inhibitory activity against PGHS1 (IC50: 0.081 microM) and PGHS2 (IC50: 5.9 microM), suggesting the selective inhibition of PGHS1. Indomethacin, which is a selective inhibitor of PGHS1, showed similar selectivity against PGHS isozymes (IC50: 0.12 microM and 1.4 microM, respectively). These results support that satigrel suppresses thromboxane A2 production by inhibiting PGHS1. It is known that three isozymes of PDE exist in human platelets: Type V, which specifically hydrolyzes guanosine 3',5'-cyclic monophosphate (cGMP), Type III, which mainly hydrolyzes cAMP, and Type II, which hydrolyzes both cGMP and cAMP. We separated these three isozymes from human platelets and examined the inhibitory activity of satigrel against each enzyme. Of the three isozymes, the inhibitory activity of satigrel was the most potent against Type III PDE (IC50: 15.7 microM). The IC50 value for Type III corresponded with that for thrombin-induced platelet aggregation. Type V and Type II were also inhibited by satigrel (IC50: 39.8 and 62.4 microM, respectively). In human platelets, satigrel increased both cAMP and cGMP levels in a dose-dependent manner (100, 300 microM). In conclusion, satigrel inhibits collagen- and arachidonic acid-induced platelet aggregation through preventing thromboxane A2 synthesis by selective inhibition of the target enzyme, PGHS1, which exists in platelets. The anti-aggregating activity of satigrel against thrombin-induced aggregation may be due to elevation of the cyclic nucleotide levels through the inhibition of PDE isozymes.
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PMID:Mechanisms of satigrel (E5510), a new anti-platelet drug, in inhibiting human platelet aggregation. Selectivity and potency against prostaglandin H synthases isozyme activities and phosphodiesterase isoform activities. 879 81


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