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

The mechanisms involved in platelet aggregation by a monoclonal antibody (mAb) P256 specific for the GPIIb-IIIa complex was investigated following metabolic 32P labelling of platelets. When compared with thrombin, inositol phosphates (InsP) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [32P]-Ins(1,4,5)P3 and [32P]-Ins(1,3,4,5)P4, was observed between 20 and 90 s. [32P]-Ins(1,3,4)P3 was also produced with a maximum after 90 s. Addition of the ADP scavenger creatinine phosphate/creatine phosphokinase (CP/CPK) and of the cyclooxygenase inhibitor aspirin together with P256 almost totally abolished InsP formation, whereas platelet aggregation and protein phosphorylation were partially inhibited. F(ab')2 fragments of P256 also aggregated platelets but to a smaller extent than IgG, and without any measurable InsPs. To characterize further P256-induced activation, the phosphorylation of p43, the main substrate of protein kinase C (PKC) and the phosphorylation of tyrosine protein (P-Tyr) was also studied. PKC activation was smaller with P256-IgG than with thrombin but both thrombin and P265-IgG induced a similar profile of P-Tyr involving seven major bands, whereas P256-F(ab')2 only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular weight P-Tyr. The data indicate that the binding of P256 to GPIIb-IIIa, in contrast with thrombin, does not initially lead directly to the activation of the phosphoinositide phospholipase C to produce InsP's but rather involves the activation of protein kinases and also both fragments F(ab')2 and Fc play a specific role in the platelet responses to the mAb. Only the crosstalk between the two pathways evoked by F(ab')2 and Fc respectively allows the activation of all platelet activation systems.
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PMID:Mechanisms involved in platelet activation induced by a monoclonal antibody anti glycoprotein IIb-IIIa: inositol phosphate production is not the primary event. 178 4

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and [3H]inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.
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PMID:The separation of [32P]inositol phosphates by ion-pair chromatography: optimization of the method and biological applications. 275 4

The coumarin derivative, cloricromene, an antithrombotic drug previously indicated as AD6, is known to inhibit the release of radioactive arachidonic acid from human platelets prelabelled with arachidonic acid and stimulated with thrombin. This effect might be due to the drug itself or to its catabolite, cloricromene acid. When added to platelet lysates neither compound inhibited phospholipase A2 activity assayed either with endogenous or with exogenous substrates. However, some inhibition was instead shown when intact platelets were first exposed to cloricromene and then enzyme activity was assayed in the lysate. Preincubation of platelets with the drug caused a dose-dependent inhibition of arachidonic acid mobilization in fluoroaluminate-stimulated platelets. beta-Thromboglobulin (beta-TG) release, a phenomenon previously shown to share common steps with phospholipase A2 activation, was also dose-dependently inhibited by cloricromene. Cloricromene also reduced the radioactivity associated with phosphatidic acid in fluoroaluminate-stimulated platelets but not in platelets stimulated with thrombin. These results are consistent with the hypothesis that cloricromene, or its catabolite, inhibits the production of arachidonic acid in stimulated platelets by interfering with a G-protein mediated activation of phospholipase A2 that is independent from the receptor-activated phosphoinositide phospholipase C.
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PMID:Cloricromene inhibits G-protein-mediated activation of phospholipase A2 in human platelets. 821 5