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)

Thrombin and poly-l-lysine alter the incorporation of acetate, glycerol, and fatty acids into the lipids of washed human platelets. Both aggregating agents decrease the incorporation of acetate into all lipid classes other than free fatty acids. Similarly, glycerol incorporation into complex lipids is impaired by both thrombin and polylysine. Thrombin caused marked depression of the incorporation of palmitic acid into both lecithin and triglycerides. By contrast it enhanced the incorporation of oleic acid into lecithin, but not into triglycerides. The data suggest that the process of primary platelet aggregation is associated with a defect in the assembly of complex lipids.
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PMID:Altered lipid metabolism in human platelets after primary aggregation. 468 85

The influence of forskolin, an adenylate cyclase activator, and of dibutyryl cyclic AMP (Bt2cAMP) on [3H]glycerol incorporation into glycerolipids was investigated in human platelets. It was found that preincubation with 2.5 mM Bt2cAMP produced a 2-4-fold increase in thrombin-induced incorporation into phospholipids compared to platelets activated by thrombin alone. Pretreatment with forskolin, which increased cellular cAMP content, also resulted in an increase in thrombin-stimulated [3H]glycerol incorporation into phospholipids. These findings demonstrate that a rise in platelet cAMP can accentuate thrombin-induced de novo synthesis of phospholipids from [3H]glycerol. Since the content of cellular cAMP was correlated with its ability to inhibit platelet activation monitored by serotonin release, it seems likely that glycerolipid, in particular phospholipid biosynthesis, is involved in controlling platelet activation by thrombin.
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PMID:Effects of dibutyryl cyclic AMP and forskolin on phospholipid biosynthesis in thrombin-stimulated human platelets. 609 Dec 92

We found that a small, reproducible amount of calmodulin is present in the cytoskeleton of human platelets. Triton-insoluble materials (cytoskeletons), which were prepared by centrifugation at 1000 X g for 10 min of platelets after lysis by Triton X-100, stimulated cyclic AMP phosphodiesterase activity in the presence of Ca2+ but not in the presence of the calcium chelator, EGTA, or the calmodulin antagonist, trifluoperazine. The activation of the enzyme was also obtained after heating Triton-insoluble materials. An alkaline glycerol polyacrylamide gel electrophoresis of fractions obtained after gel filtration of solubilized Triton residues showed a protein band which had a faster electrophoretic mobility in the absence than in the presence of Ca2+. Upon thrombin activation of platelets, calmodulin in the Triton-insoluble cytoskeletons increased rapidly parallel to actin, actin-binding protein and myosin. With other stimulants such as collagen, epinephrine and ADP, similar results were obtained but with slower association of these proteins with cytoskeletons. However, after treatment with the Ca2+-ionophore A23187, calmodulin, actin and actin-binding protein in Triton residues decreased rapidly, whereas the association of myosin increased. Thus, calmodulin seems to be associated with actin filaments rather than myosin filaments, and may be involved in the generation of contractile force in the cell.
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PMID:Presence of calmodulin in human platelet cytoskeletons and its concentration change upon activation of platelets. 614 9

Platelet phospholipids undergo significant alterations during aggregation induced by thrombin or other agents. There is an early increase in phosphatidic acid, with a decrease in phosphatidyl inositol. De novo synthesis of most phospholipids from 14C-glycerol is decreased. Thrombin stimulates 32P-phosphate incorporation into di- and triphosphoinositides, suggesting increased phosphorylation of phosphatidyl inositol during aggregation. Arachidonic acid for prostaglandin synthesis is released from platelet phospholipids. Thrombin induced aggregation results in release of arachidonic acid primarily from phosphatidyl choline and phosphatidyl inositol. The availability of free arachidonic acid may be regulated by platelet phospholipase A2 activity. The latter activity is stimulated by thrombin, requires calcium ions, and is inhibited by agents which elevate cyclic adenosine monophosphate. Phospholipids are probably an essential component of the platelet surface lipoprotein procoagulant activity known as platelet factor 3. There is evidence that calcium ions may mediate binding between gamma carboxyglutamic acid residues on the amino terminal portion of prothrombin and negatively charged phosphate groups on phospholipid micelles. Binding of prothrombin to phospholipid on the platelet surface may orient the former such as to facilitate the prothrombinase activity of Factor Xa. Platelet phospholipids and platelet factor 3 activity are decreased in some congenital and myeloproliferative disorders. Increases in these factors may be associated with thrombotic and arterial occlusive disorders.
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PMID:The role of phospholipids in platelet function. 625 56

Thrombin has been shown to resynthesize the Arg134-Thr135 peptide bond between the 134- and 57-residue thrombin fragments of reduced and carbamidomethylated human somatotropin at pH 6.0 in 90% (vol/vol) glycerol. The maximal amount of synthesis was about 20% as estimated by sodium dodecyl sulfate gel electrophoresis and radioreceptor assay. The resynthesized polypeptide was isolated and shown to be indistinguishable from the reduced and carbamidomethylated hormone when tested by two receptor-binding assays, radioimmunoassay, and rat tibia test.
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PMID:Human somatotropin: covalent reconstitution of two polypeptide contiguous fragments with thrombin. 627 51

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.
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PMID:The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets. 630 36

Experiments with washed platelets from rabbits demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml) that causes maximal aggregation and partial release of granule contents does not significantly decrease the amount of phosphatidylinositol 4,5-bisphosphate [ PtdIns (4,5)P2] at 10s; this contrasts with ADP stimulation. The amount of PtdIns (4,5)P2 was significantly decreased by a higher concentration of thrombin (0.3 unit/ml). Increased turnover of the PtdIns (4,5)P2 at 60s was indicated by changes in labelling with [3H]glycerol in platelets stimulated with both concentrations of thrombin. An unexpected observation with the lower thrombin concentration was a significant increase in the amount of phosphatidylinositol ( PtdIns ) at 10s. This contrasts with data from other laboratories, which indicate that thrombin causes a significant decrease in PtdIns . At 60s, with the lower concentration of thrombin, PtdIns was significantly decreased. With the higher concentration of thrombin there was a significant decrease in the amount of PtdIns at 10s, in keeping with the data from other laboratories. The initial increase in PtdIns may not have been observed by other investigators because higher concentrations of thrombin were used. The reaction involved in this initial increase in the amount of PtdIns does not appear to be increased degradation of PtdIns4P or PtdIns (4,5)P2, since their total amount was unchanged at 10s. The magnitude of the increase in PtdIns is such that more than the existing pool of phosphatidic acid would have to be converted into PtdIns to account for the increase. It is suggested that synthesis of phosphatidic acid de novo from dihydroxyacetone phosphate and glycerol 3-phosphate might be the source of phosphatidic acid, which leads to increased PtdIns at 10s with the lower concentration of thrombin. Thus it appears that the initial response of platelets to thrombin does not require an early change in PtdIns (4,5)P2 and may involve stimulation of synthesis de novo of PtdIns via phosphatidic acid.
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PMID:Changes in the platelet phosphoinositides during the first minute after stimulation of washed rabbit platelets with thrombin. 632 56

We have examined the temporal relationship between arachidonate release and phosphatidic acid (PA) metabolism in human platelets stimulated with thrombin. Within 1 min of stimulation at 37 degrees C, platelets released up to 16 nmol of arachidonate/10(9) cells from membrane phospholipids and exhibited an increase of severalfold in PA mass. At 23 degrees C, arachidonate release was half-maximal by 10-30 s, but PA remained near control levels for 10-30 s, indicating that increased PA is not necessary for release. [3H]Glycerol-labeled platelets synthesized phospholipids from [3H]PA at a rate of 0.08-0.3 nmol/min/10(9) cells at 37 degrees C. This rate of [3H]glycerol-PA turnover was not enhanced by thrombin stimulation; thus, PA did not turn over rapidly enough to be an intermediate in the release of several nanomoles of arachidonate from phosphatidylinositol in the first few seconds of stimulation. When aspirin-treated platelets were prelabeled with [14C]- or [3H]arachidonate, the specific activity of phospholipid-bound arachidonate pools remained constant after thrombin stimulation. Released arachidonate had specific activity intermediate to that in phosphatidylcholine and phosphatidylinositol. [14C]arachidonate in phosphatidylethanolamine had specific activity 6-fold less than released [14C]arachidonate; this phospholipid could therefore contribute only a small fraction of released fatty acid.
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PMID:Arachidonate release and phosphatidic acid turnover in stimulated human platelets. 640 33

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.
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PMID:Synergistic functions of phorbol ester and calcium in serotonin release from human platelets. 640 48

When human platelets were stimulated by synthetic diacylglycerol such as 1-oleoyl-2-acetyl-glycerol, which was a potent activator in vitro of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) (Mori, T., Takai, Y., Yu, B., Takahashi, J., Nishizuka, Y., and Fujikura, T. (1982) J. Biochem. (Tokyo) 91, 427-431), a protein having Mr approximately 40,000 (40-kilodalton protein) was rapidly phosphorylated, just as it was by natural extracellular messengers such as thrombin. Fingerprint analysis appeared to indicate that protein kinase C was indeed responsible for this 40-kilodalton protein phosphorylation in intact platelets. Under these conditions, neither inositol phospholipid breakdown nor endogenous diacylglycerol formation was observed, indicating that the synthetic diacylglycerol intercalated into the membrane and directly activated protein kinase C without interaction with cell surface receptors. During this process, the diacylglycerol was converted in situ to the corresponding phosphatidate, 1-oleoyl-2-acetyl-glyceryl-3-phosphoric acid. Experiments with the synthetic diacylglycerol and Ca2+ ionophore A23187 suggested that the protein phosphorylation catalyzed by protein kinase C was a prerequisite requirement for the release of serotonin, and that the receptor-linked protein phosphorylation and Ca2+ mobilization acted synergistically to elicit the full physiological cellular response.
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PMID:Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. 640 88


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