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 developed a new monoclonal antibody directed against platelet myosin (NNKY6-19). Using this antibody, we analyzed platelet cytoskeletal changes related to stimulation with thrombin and to long-term storage. Immunoelectron microscopy showed increased binding of NNKY6-19 to pseudopods and the open canalicular system during treatment with thrombin (0.1 U/ml) and during storage for 7 days. Flow cytometry also showed increased binding to platelets by NNKY6-19 and an antiactin monoclonal antibody during storage. The binding of NNKY6-19 showed an increase greater than that with the antiactin antibody after storage of platelets for 7 days and after thrombin treatment. These findings indicated that the increased binding of NNKY6-19 had some relationship to changes in intracellular myosin and platelet morphology. Thus use of NNKY6-19 allowed analysis of subtle changes related to platelet activation, which differed from those detected by antibodies against platelet glycoproteins or by the antiactin antibody. This antibody appears to provide a simple method for studying changes in platelet cytoskeletal and surface proteins.
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PMID:Flow cytometric analysis of changes in cytoskeletal proteins during platelet destruction and activation using a monoclonal antibody against platelet myosin. 826 14

Exposure of 3T3 fibroblasts to the phosphatase inhibitor, calyculin-A, induces marked morphological changes and the formation of an aggregate of actin and myosin connected to the nucleus by intermediate filaments (Hirano, K., L. Chartier, R. G. Taylor, R. E. Allen, N. Fusetani, H. Karaki, D. J. Hartshorne: J. Muscle Res. Cell Motil. 13, 341-353 (1992)). Vimentin was isolated from this complex and shown to be phosphorylated. At least 4 phosphorylation sites were indicated. These sites were distinct from those phosphorylated by the cAMP-dependent protein kinase. Limited proteolysis was used to define the domains in which phosphorylation occurred. Vimentin was isolated from 32P-labeled calyculin-A-treated cells and digested with thrombin and alpha-chymotrypsin. Proteolysis with thrombin limited the phosphorylation to either the central core or C-terminal domain. Proteolysis with alpha-chymotrypsin indicated that the multiple phosphorylation sites were restricted to the C-terminal domain of vimentin.
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PMID:Phosphorylation of vimentin in the C-terminal domain after exposure to calyculin-A. 826 79

The mechanisms involved in the hemostatic abnormality of uremic patients remain obscure. We have explored the response of normal and uremic platelets to surface activation at the ultrastructural level and analyzed changes in the composition of proteins associated with normal and uremic platelet cytoskeletons after stimulation with thrombin (0.01 and 0.1 U/ml). Cytoskeletons were obtained by extraction with Triton X-100, processed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of cytoskeletal proteins analyzed by densitometry. Under static conditions, uremic platelets spread with difficulty on formvar-coated grids. The percentage of platelets that spread fully on this polymer surface was statistically reduced compared with that of control platelets (11 +/- 1.4 vs. 21 +/- 1.6; P < 0.05). An impairment of cytoskeletal organization was observed in resting uremic platelets but abnormalities were more evident after thrombin activation. The incorporation of actin into the cytoskeletons of thrombin-stimulated uremic platelets was significantly reduced with respect to controls (6 +/- 3% vs. 29 +/- 5%; P < 0.01 after 0.01 U/ml and 28 +/- 9% vs. 59 +/- 10%; P < 0.05 after 0.1 U/ml). Decreased associations of actin-binding protein (P < 0.01), alpha-actinin (P < 0.05), and tropomyosin (P < 0.05) with the cytoskeletons of uremic platelets were also noted. No difference was observed for the incorporation of myosin into the cytoskeletons of activated uremic platelets. These results suggest functional and biochemical alterations of the platelet cytoskeleton in uremia, which may contribute to the impairment of platelet function observed in uremic patients.
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PMID:Abnormal cytoskeletal assembly in platelets from uremic patients. 836 80

Platelets seem to be involved in the pathogenesis of some kidney diseases, but the exact relationships between platelets and the changes in renal function are incompletely known. Mesangial cells (MC) were incubated with platelet-supernatants (PS) and cellular surface area (CSA) and myosin light-chain phosphorylation (MLCP) were measured. CSA of PS-incubated MC (PS-MC) significantly diminished, as compared to control MC (70 +/- 6% vs. 100 +/- 5%). PS induced a significant increase in MLCP with respect to control cells (150 +/- 23% vs. 100 +/- 18%). When platelets were pretreated with indomethacin, the PS-dependent contraction was abolished. Pretreatment with sulotroban (SU) or BN-52021 (BN), a thromboxane A2 (TXA2) and a platelet-activating factor (PAF) receptor blocker respectively, also completely blocked the PS effects. In other experiments, platelets were activated with thrombin (T), adding the so obtained PS to MC. Moreover, cells were also preincubated with T and then added PS. No changes in CSA were observed in either case. It may be concluded that PS contracted cultured MC, and these changes could be related to the decreased glomerular filtration rate (GFR) observed in some diseases in which platelets seem to be involved. TXA2 and PAF may be responsible for this effect. In contrast, T incubation inhibited the effect of PS, perhaps through a direct relaxing effect of T in MC.
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PMID:Direct interactions between platelets and cultured rat mesangial cells. 841 9

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP-280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb-IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb-IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb-IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.
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PMID:Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation. 855 84

The serine protease, thrombin, evokes numerous endothelial cell responses which regulate hemostasis, thrombosis and vessel wall pathophysiology. One such response, the development of intercellular gap formation and vascular permeability is relevant to each of these processes and is a cardinal features of inflammation. Regulation of endothelial cell gap formation and therefore permeability is a function of a dynamic balance between competing adhesive, barrier-promoting tethering forces and contractile, tension-producing forces which result in barrier dysfunction. The key tethering events governing focal endothelial cell adhesion to the extracellular matrix and cell-cell interactions are poorly understood. In contrast, information is rapidly increasing regarding endothelial-specific contractile processes driven by the actomyosin molecular motor. The level of myosin light chain phosphorylation catalyzed by a unique myosin light chain kinase promotes productive actin-myosin interaction and governs the degree of centripetal tension produced. In this review the signal transducing and contractile mechanisms by which thrombin elicits endothelial cellular activation through its specific receptor are addressed. The pathways by which thrombin may alter the balance between contractile and tethering forces to promote endothelial cell gap formation are discussed.
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PMID:Vascular endothelial cell activation and permeability responses to thrombin. 856 32

Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 microM TM or 0.1 microM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the microfilament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the microfilament ring and centralization of granules.
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PMID:Effects of the protein phosphatase inhibitors, tautomycin and calyculin-A, on protein phosphorylation and cytoskeleton of human platelets. 858 89

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces.
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PMID:Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. 861 24

The N-terminal 28-residue peptide of actin whose Cys10 was labeled with 5-iodoacetamido-fluorescein (F3K peptide) was isolated from the fluorescently labeled thrombin digest of actin. The effect of myosin subfragment 1 (S1) on the fluorescence of F3K peptide was examined in the absence of ATP. With increasing concentration of S1 added, the fluorescence intensity of F3K peptide increased by maximally 7.3% with an apparent dissociation constant of 5.7 microM, suggesting a role of this peptide region of actin in acto-S1 binding in rigor. F3K peptide was crosslinked with S1 at 10 mM NaCl using a zero-length crosslinker by the method of Grabarek and Gergely [Anal. Biochem. 185, 131-135 (1990)]. The crosslinking was greatly inhibited by the presence of either 0.2 M NaCl or 5 mM MgATP. The analyses of amino acid compositions and sequences of the fluorescent peptides isolated from a lysylendopeptidase digest of the crosslinked S1 indicated that F3K peptide was mainly crosslinked to residues 637-642 of the S1 heavy chain. The crosslinked S1 was isolated by selectively pelleting the uncrosslinked S1 with F-actin. ATPase activity of the isolated crosslinked S1 alone was twice as high as that of control S1. The actin-activated ATPase activity of the crosslinked S1 was much lower than that of uncrosslinked S1. The estimated Vm and Km values were 1.72 s-1 and 125 microM, respectively. The Vm decreased to less than 1/8, while Km increased only twofold. The results suggest that the N-terminal 28-residue segment of actin may be implicated in the rigor binding of actomyosin and in the actin-activation of myosin ATPase, but may not be the main determinant of actomyosin binding in the presence of ATP.
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PMID:Crosslinking of a 28-residue N-terminal peptide of actin to myosin subfragment 1. 872 Jan 41

The increase in endothelial permeability in response to inflammatory mediators such as thrombin and histamine is accompanied by reversible cell rounding and interendothelial gap formation, suggesting that the predominant transport pathway is a diffusive one (i.e., via cellular junctions (paracellular transport)). However, vesicle-mediated transport (i.e., via albumin-binding protein gp60) may also contribute significantly to the overall increase in permeability. Regulation of paracellular transport in endothelial cells is associated with modulation of actin-based systems, which anchor the cell to its neighbor or extracellular matrix, thus maintaining endothelial integrity. At the cell-cell junctions, actin is linked indirectly to the plasma membrane by linking proteins (e.g., vinculin, catenins, alpha-actinin) to cadherins, which function in homophilic intercellular adhesion. At endothelial focal contacts, the transmembrane receptors (integrins) for matrix proteins are linked to actin via linking proteins (i.e., vinculin, talin, alpha-actinin). In response to inflammatory mediators, second messengers signal two regulatory pathways, which modulate the actin-based systems, and can thus lead to impairment of the endothelial barrier integrity. One critical signal may be based on protein kinase C isoenzyme specific phosphorylation of linking proteins at the cell-cell and cell-matrix junctions. The increased phosphorylation is associated with actin reorganization, cell rounding, and increased paracellular transport. Another important event is the activation of myosin light chain kinase (MLCK), which causes an actin-myosin-based contraction that may lead to centripetal retraction of endothelial cells. Current research is being conducted at identification of protein substrates of protein kinase C isoenzymes, the specific role of their phosphorylation in barrier function, and determination of the precise role of MLCK in modulation of endothelial barrier function. Since mechanisms by which the increased permeability is returned to normal may be regulated at multiple levels (e.g., receptor desensitization, protein kinase C mediated negative feedback pathways, activation of protein phosphatases), it is also important to determine these cellular "off-switch" mechanisms.
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PMID:Mechanisms of increased endothelial permeability. 894 65


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