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 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

The effect of melatonin on thrombin-induced [3H]-arachidonic acid (AA) metabolism to cyclooxygenase derivatives was determined in platelets obtained from normal volunteers at 0830 and 2030 h. Percent conversion of radioactive AA was generally greater at 2030 h than at 0830 h for every cyclooxygenase derivative analyzed. Micromolar or greater concentrations of melatonin decreased significantly the conversion of [3H]-AA to prostaglandin (PG) F2 and thromboxane (Tx) B2, and inhibited slightly the conversion to PGE2 and PGD2. After preincubation of platelets with 1 mM imidazole, the melatonin inhibitory effect was significant for PGF2 only. Melatonin (10(-6) M) showed a significant inhibitory influence on platelet ATP release induced by phorbol-12 myristate-13 acetate (PMA) at 2030 h, an effect inhibited by 1 mM aspirin. These results indicate that at pharmacological concentrations melatonin inhibits human platelet cyclooxygenase.
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PMID:Melatonin effect on arachidonic acid metabolism to cyclooxygenase derivatives in human platelets. 179 20

Stimulation of platelets induces a rapid release of arachidonate from specific phospholipids and subsequent remodeling of arachidonate-containing phospholipids. This process is accompanied by transformation of released arachidonate by cyclooxygenase and lipoxygenase enzymes. We addressed the question of whether the cyclooxygenase and the lipoxygenase products originated from the same arachidonate-containing phospholipids. [14C]Arachidonate prelabeled platelets were stimulated by thrombin or by ionophore A 23187. We monitored the cyclooxygenase pathway by following 12-hydroxy-5,8,10-heptadecatrienoic acid [12(S)-HHT] formation and the lipoxygenase pathway by following 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] formation and compared specific activities. The data showed that the same pool of released arachidonate can be utilized by either cyclooxygenase or by lipoxygenase. Indeed, the specific activity of both products was identical when both enzymes were acting. Since cyclooxygenase was rapidly deactivated while lipoxygenase continued to be active, the specific activity of 12(S)-HETE became lower than the specific activity of 12(S)-HHT when large amounts of 12(S)-HETE were synthesized. Based on comparison of specific activity between phospholipids and oxygenated products, the pools of arachidonate-containing phospholipids involved in the synthesis of oxygenated products are dependent on the amount of arachidonate released.
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PMID:A unique pool of free arachidonate serves as substrate for both cyclooxygenase and lipoxygenase in platelets. 181 90

Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.
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PMID:Different calcium pools in human platelets and their role in thromboxane A2 formation. 183 1

The influence of protein kinase C (PKC) activation on canine coronary vasoreactivity was assessed in vitro. Activation of PKC by phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) caused slow sustained constriction of isolated coronary artery rings. PDBu was a more potent and efficacious constrictor than PMA (169 +/- 21 vs. 81 +/- 7% of maximum KCl constriction). Constriction to PDBu was reduced slightly by deendothelialization and by meclofenamate. Pretreatment with threshold concentrations of PDBu increased constriction to serotonin from 3 +/- 1 to 48 +/- 4% of maximum KCl constriction whether or not the endothelium was present but had no effect on response to the thromboxane analogue U-46619. In addition, in arteries constricted with PDBu, dilations to ADP, thrombin, acetylcholine, and sodium nitroprusside were impaired when compared with arteries constricted with U-46619. These results suggest that activation of PKC in coronary arteries 1) produces potent constriction mediated only in small part by the endothelium and by cyclooxygenase products, 2) potentiates markedly the constrictor response to serotonin by an endothelium-independent mechanism, and 3) attenuates both endothelium-dependent and endothelium-independent vasodilation.
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PMID:Effects of phorbol esters on canine coronary artery constriction and dilation in vitro. 185 25

Because vascular smooth muscle cells (SMC) can be exposed to platelets at sites of significant arterial injury, we studied whether cultured rat aorta SMC can utilize platelet-derived arachidonate and prostaglandin (PG) endoperoxides (PGG2/PGH2) in the synthesis of prostacyclin (PGI2). SMC converted exogenous PGH2 to PGI2, measured by radioimmunoassay (RIA) of 6-keto-PGF1 alpha, despite cyclooxygenase inhibition or PGH2-receptor blockade. SMC produced increasing amounts of PGI2 in the presence of an increasing number of platelets when the two cell types were coincubated with arachidonate. Furthermore, aspirin-pretreated SMC produced PGI2 in response to arachidonate, ionophore A23187, or thrombin in the presence of platelets but not in their absence. SMC, by themselves unresponsive to thrombin, produced PGI2 during coincubation with thrombin-stimulated aspirin-pretreated platelets. Separation of the SMC monolayer and platelets with a filter did not prevent platelet-dependent PGI2 formation by the SMC. Finally, aspirin-pretreated SMC, in cosuspension with platelets, inhibited platelet aggregation in association with PGI2 production. These data indicate that 1) SMC can synthesize PGI2 from exogenously added PGH2 and from platelet-derived arachidonate or endoperoxides, 2) direct cell-cell contact is not required for intercellular endoperoxide transfer, and 3) SMC can inhibit platelet aggregation possibly through PGI2 production from platelet-derived endoperoxides.
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PMID:Platelet interaction with vascular smooth muscle in synthesis of prostacyclin. 190 2

Superoxide dismutase (SOD) triggers activation of human platelets exposed to subthreshold concentrations of arachidonic acid and collagen. The subthreshold concentrations used are not able to activate platelets but "prime" platelets to be activated by SOD. The addition of SOD to arachidonic acid-or collagen-primed platelets induced aggregation, thromboxane A2 production, and release of [3H]serotonin. Superoxide dismutase does not have any effect on resting platelets and ADP-, thrombin-, calcium ionophore A23187-, PAF-, or U46619-stimulated platelets. Furthermore, superoxide dismutase-dependent platelet activation is fully prevented by catalase and/or aspirin, suggesting a role for H2O2 and the involvement of the cyclooxygenase pathway of arachidonic acid in such activation.
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PMID:Superoxide dismutase triggers activation of "primed" platelets. 191 Mar 14

As a source of several vasoactive factors, the endothelium takes part in the regulation of vascular tone. The most important endothelium-derived vasoactive substances are nitric oxide, prostacyclin, endothelin-1 and contracting factors requiring the activity of cyclooxygenase. The endothelium is an obvious target organ of cardiovascular risk factors. Accordingly, functional alterations do occur with aging, hypertension, and lipids. All three conditions are associated with a decreased basal and stimulated release of endothelium-derived nitric oxide. On the other hand, the release of endothelin-1 appears to increase with age, while the sensitivity to the peptide markedly decreases under the same conditions. In the spontaneously hypertensive rat, acetylcholine and stretch evoke the release of cyclooxygenase-dependent endothelium-derived contracting factor, most likely prostaglandin H2. The sensitivity and circulating levels of endothelin-1, on the other hand, are reduced in this experimental model of hypertension. In the porcine coronary circulation, oxidized low-density lipoproteins selectively reduce endothelium-dependent relaxations to aggregating platelets, serotonin, and thrombin which are mediated by nitric oxide. The alterations of endothelial function occurring with aging, hypertension, and hyperlipidemia may have important clinical implications for the pathogenesis of cardiovascular disease.
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PMID:Endothelium-dependent control of vascular tone: effects of age, hypertension and lipids. 195 6

Two platelet mechanisms contribute to haemostasis and thrombosis. (1) Compounds such as thrombin activate glycoprotein IIb/IIIa; fibrinogen is the ligand. The cyclooxygenase pathway is involved and so this process is aspirin sensitive. (2) Shearing forces alone activate a different domain on glycoprotein IIb/IIIa; von Willebrand's factor is the ligand. This process is probably non-enzymatic and is aspirin insensitive. The prevention of shear-induced platelet activation may prove to be more rewarding therapeutically than inhibition of aspirin sensitive pathways.
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PMID:Shear-induced platelet aggregation. 196 70

Erythrocytes are known to influence hemostasis. Bleeding times are prolonged in anemia and corrected by normalizing the hematocrit. We now demonstrate that intact erythrocytes modulate biochemical and functional responsiveness of activated platelets. A two-stage procedure, permitting studies of cell-cell interactions and independently evaluating platelet activation and recruitment within 1 min of stimulation, was developed. Erythrocytes increased platelet serotonin release despite aspirin treatment, enzymatic adenosine diphosphate removal, protease inhibition, or combinations thereof. The data suggested that erythrocyte enhancement of platelet reactivity can reduce the therapeutic effectiveness of aspirin. Erythrocytes metabolically modified platelet arachidonate or eicosapentaenoate release and eicosanoid formation. They promoted significant increases in cyclooxygenase and lipoxygenase metabolites upon platelet stimulation with collagen or thrombin. However, with ionophore, erythrocytes strongly reduced platelet lipoxygenation. These erythrocyte modulatory effects were stimulus-specific. Activated platelet-erythrocyte mixtures, with or without aspirin, promoted 3-10-fold increases in extracellular free fatty acid, which would be available for transcellular metabolism. Erythrocyte-induced increases in free eicosapentaenoate may contribute to antithrombotic and anti-inflammatory effects of this fish oil derivative. These results provide biochemical insight into erythrocyte contributions to thrombosis and hemostasis, and support the concept of thrombus formation as a multicellular event.
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PMID:Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. A new approach to platelet activation and recruitment. 199 40


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