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)

We investigated the role of cytoskeletal components in platelet activation under conditions in which changes in the shape of platelets, their secreting reaction, or both, would occur. When rabbit platelets were stimulated with 0.1 U/ml thrombin, they changed their shape, secreted serotonin, and incorporated actin-binding protein (ABP), myosin, and actin into their cytoskeletons. Platelets treated with 2 microM cytochalasin E and stimulated with 0.1 U/ml thrombin secreted serotonin and assembled myosin and actin into their cytoskeletons, but did not change in shape. Stimulation with a low dose of thrombin (0.001 U/ml) or ADP (10 microM) caused a change in shape and incorporation of ABP, myosin, and actin, but not serotonin secretion. These results suggest that the assembly of myosin and actin into platelet cytoskeletons is related to the secreting reaction, and that ABP, myosin, and actin are all involved in the changes in platelet shape.
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PMID:Platelet cytoskeletal components involved in shape change and secretion. 370 19

Platelets are rapidly activated by several agonists. When phorbol 12-myristate 13-acetate (PMA) was added to washed platelet suspensions 10 s prior to either thrombin or ADP, it caused a dose-dependent inhibition of shape change correlated with decreased myosin association with the cytoskeleton and with inhibition of the calcium transient measured in fura-2-loaded platelets. PMA added 5-10 s after agonists did not reverse shape change or the association of myosin with the cytoskeleton, but markedly increased the rate at which the calcium signal returned to the baseline. The analogue, 4 alpha-phorbol didecanoate did not cause these effects. Our results suggest that one effect of C-kinase activation is to provide negative feedback in sequential responses.
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PMID:Rapid effects of phorbol ester on platelet shape change, cytoskeleton and calcium transient. 375 53

Using the thrombin-cut [68-30 kilodalton (kDa)] myosin subfragment 1 (S-1) whose heavy chain has been selectively split within the central 50-kDa region, at Lys-560, with concomitant specific alterations of the ATPase and actin binding properties [Chaussepied, P., Mornet, D., Audemard, E., Derancourt, J., & Kassab, R. (1986) Biochemistry 25, 1134-1140; Chaussepied, P., Mornet, D., Barman, T., Travers, F., & Kassab, R. (1986) Biochemistry 25, 1141-1149], we have isolated and renatured the COOH-terminal 30-kDa fragment associated with the alkali light chains by the procedure recently described [Chaussepied, P., Mornet, D., Audemard, E., Kassab, R., Goodearl, J., Levine, B., & Trayer, I. P. (1986) Biochemistry 25, 4540-4547]. The 30-kDa peptide preparation was found to exhibit a crucial feature of the native S-1; namely, it interacts with F-actin in an adenosine 5'-triphosphate (ATP)-dependent manner. Studies by ultracentrifugation, turbidity measurements, and chemical cross-linking experiments showed that the acto-30-kDa peptide complex was dissociated almost completely by the gamma-phosphoryl group containing ligands ATP, 5'-adenylyl imidodiphosphate, and pyrophosphate, to a lesser extent by ADP, and not at all by AMP and inorganic phosphate. The maximal dissociating effect is operating with the thrombic 30-kDa entity, whereas the 22-kDa fragment produced by staphylococcal protease is only slightly dissociated. In contrast, the tryptic 20-kDa fragment binds irreversibly to actin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of polyphosphate recognition sites communicating with actin sites on the skeletal myosin subfragment 1 heavy chain. 379 May 30

Heparin and synthetic inhibitors of thrombin are able to decrease the rate of division of porcine vascular smooth muscle cells in primary culture and to increase their myosin content. These effects are abolished by thrombin. Inhibitors of thrombin may therefore be useful in preventing arteriosclerotic lesions.
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PMID:Influence of inhibitors of thrombin on porcine aortic smooth muscle cells in primary culture. 381 9

Platelets circulating in the human blood stream are smooth disk-shaped structures. The disks change within seconds of exposure to ADP or thrombin to irregular spheres bearing filopodia and pseudopodia. It is well-established that platelets also change shape (although more slowly) when chilled to 5 degrees C and revert to disks on rewarming. This cold-induced shape change may be due to the depolymerization of the submembranous microtubule ring. However, we found that chilling in the presence of Taxol, which stabilizes the microtubules, still results in shape change. Chilled platelets show an increase in the amount of myosin in the Triton-X insoluble residue or 'cytoskeleton' which is correlated in time both with phosphorylation of the myosin regulatory light chain and with the induced shape change. We suggest here that the slow cold-induced change from disks to spheres is due primarily to a gradual activation of myosin.
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PMID:Reversible association of myosin with the platelet cytoskeleton. 383 8

1H-NMR experiments on myosin subfragment-1 (S1) isoenzymes, containing either the A1 or the A2 alkali light chains (S1(A1) or S1(A2)), have previously suggested the 41-residue proline, alanine and lysine-rich N-terminal extension of A1 to constitute a mobile 'domain' in solution. This segment of the molecule is immobilised in the presence of actin (Prince et al. (1981) Eur. J. Biochem. 121, 213-219). We now establish that the A1 light chain interacts with actin directly, and furthermore, that the binding site appears to be restricted to the terminal 41 residues. This observation carries important consequences for both the structure of the actomyosin complex and the role of myosin isoenzymes. Using the proteinase, thrombin, a technique has been developed in which the A1 light chain is cleaved, releasing the N-terminal 'tail' from an A2-like fragment. The method is shown to be widely applicable to light chains from a variety of sources. The isolated N-terminal fragments from rabbit skeletal and bovine cardiac muscle have been shown to interact directly with actin by a combination of affinity chromatography and 1H-NMR experiments. The 1H-NMR results are similar to those obtained earlier with S1 (ibid) and suggest the terminal alpha-N-trimethylalanine residue (Henry et al. (1982) FEBS Lett. 144, 11-15) to participate in the interaction.
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PMID:Characterization of the actin-binding site on the alkali light chain of myosin. 402 51

Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.
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PMID:Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets. 409 95

The effects of lysoPC, four other amphiphiles containing a linear 16 carbon alkane tail and chlorpromazine on platelet cytoskeletal assembly were compared. LysoPC and nonmetabolized amphiphiles all caused time-dependent inhibition followed by potentiation of thrombin-induced aggregation, serotonin secretion and cytoskeletal assembly in gel-filtered platelets, a result which ruled out hydrolysis of the amphiphiles as the mechanism of the time dependence. Hexadecanesulfonate was superior as a potentiator and cetyltrimethyl ammonium bromide (CTAB) was a better inhibitor. On the contrary, inhibition of platelet activation by arachidonate was not effected in a time-dependent manner and the actin-crosslinking proteins, actin-binding protein and myosin, were selectively prevented from incorporation into cytoskeletal cores, although protein phosphorylation and actin polymerization still occurred. Chlorpromazine also showed this selective inhibition of cytoskeletal assembly. LysoPC at concentrations which have been reported to cause development of filopodia did increase slightly the amount of actin present in Triton X-100-insoluble cores but not protein phosphorylation or incorporation of actin-crosslinking proteins. The effective concentrations of lysoPC and chlorpromazine can be predicted from the Meyer-Overton-Mullins rule of anesthesia which indicates their general effectiveness, but their specific effects only partially overlap.
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PMID:The effects of lysophosphatidylcholine and related amphiphiles on platelet cytoskeletal assembly. 609 54

Exposure of human platelets to 10 discharges from a 4.5 microF capacitor charged at 3 kV permitted isolation of a stable preparation of permeabilized platelets that, after equilibration with Ca2+ buffers (pCa less than 6) for 15 min at O degrees C, secreted 5-hydroxytryptamine (5-HT) at 25 degrees C. Thrombin enhanced the sensitivity to Ca2+ of the secretion of 5-HT by about 10-fold, whereas Arg -vasopressin and the prostaglandin endoperoxide analogue, U-46619, increased sensitivity to Ca2+ by 3 to 4-fold. This action of thrombin was associated with stimulation of diacylglycerol formation, a marked increase in phosphorylation of protein P47 and a smaller increase in phosphorylation of the P-light chain of myosin. Thrombin exerted these effects at a [Ca2+ free] of 0.1 microM, suggesting that the receptor-activated breakdown of platelet phosphoinositides to diacylglycerol may not require prior Ca2+ mobilization in intact platelets. In both the presence and absence of thrombin, a higher [Ca2+ free] was required for optimal secretion than for maximal phosphorylation of P47 and myosin light-chain, indicating that Ca2+ and possibly diacylglycerol have roles in the secretory mechanism additional to activation of the enzymes that phosphorylate these proteins. Stable GTP analogues such as guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), and to a lesser extent GTP itself, enhanced the Ca2+ sensitivity of the secretion of 5-HT from permeabilized platelets. Moreover, GTP potentiated the stimulatory action of thrombin. These effects of GTP gamma S and GTP were associated with increased diacylglycerol formation and were inhibited by guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) suggesting that a GTP-binding protein may play a role in the receptor-activated breakdown of phosphoinositides. However, as GDP beta S did not inhibit the potentiation of secretion caused by thrombin alone, a GTP-independent pathway of platelet activation may also exist.
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PMID:Receptor-induced diacylglycerol formation in permeabilized platelets; possible role for a GTP-binding protein. 609 73

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


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