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)

Platelet membranes play a key role in all stages of the haemostatic mechanism. Four of these in particular are considered here: adhesion to subendothelium, which involves an interaction between the glycoprotein I complex in the platelet membrane (deficient in the Bernard-Soulier syndrome) and plasma factor VIII; aggregation, involving the membrane glycoprotein IIb/IIIa complex (deficient in thrombasthenia), plasma fibrinogen and divalent cations; platelet factor 3 availability, a function of surface membrane phospholipids; and thromboxane synthesis, a function of the phospholipids of the membrane of the dense tubular system. The glycoprotein I complex also carries binding sites for thrombin and for drug-dependent antibodies, and glycoprotein IIb/IIIa is the site of the P1A1 antigen and of alpha-actinin.
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PMID:[The platelet membrane: some aspects of the pathophysiology of haemostasis]. 39 4

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.
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PMID:The presence of a covalently cross-linked matrix in human platelets. 200 80

Actin polymerization is an essential component of platelet activation. Since actin appears to polymerize at its membrane-associated end, knowledge of the structural relationship of actin filaments to membrane is an important part of understanding that polymerization process. A membrane-associated actin-containing cytoskeleton has been described in human platelets biochemically and is composed, at least in part, by an association between glycoprotein Ib and the actin-binding protein originally isolated from macrophages. Many other actin-associated proteins with known sub-membranous localization in other systems have been found in platelets, including alpha-actinin, vinculin, and low levels of spectrin and the red cell protein Band 4.1. Because of the density of the platelet cytoplasm, the structure of the membrane-skeleton has not yet been visualized. We have used quick freeze-deep etch techniques to observe the sub-membranous cytoplasm and report visualization of a periodic, submembranous filament system not before seen in the platelet. This filamentous system was more easily observed in thrombin-stimulated platelets, but appeared to be present in resting, discoid cells as well. The filaments could also be readily observed when platelets are lysed after fixation, stained with tannic acid, and embedded for thin-sectioning. This membrane cytoskeleton was composed of 9 nm thick filaments lying 15 nm apart, and 15 nm from the membrane. The filaments appeared to lie in parallel and to encircle the cell. Similar filaments could be seen associated with intracytoplasmic membrane systems in activated cells.
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PMID:Platelet membrane skeleton revealed by quick-freeze deep-etch. 236 21

Blood platelets are particularly rich in cytoskeletal proteins and respond to stimulation and activation by changes in shape. We examined the effect of blood platelet activation on the subcellular distribution of the cytoskeletal proteins, actin, myosin, alpha-actinin and actin-binding protein. These studies were performed with immunofluorescent staining on thin cryosections of paraformaldehyde-fixed platelets and by immunogold labeling of ultrathin cryosections of glutaraldehyde-fixed blood platelets. Platelets were studied immediately at blood collection (resting platelets), in platelet-rich plasma and after gel filtration (partially activated platelets), and after gel filtration and thrombin activation (0.5 U/ml, 10 min, 37 degrees C) (activated platelets). Resting platelets were disk-shaped and showed homogeneous distribution of cytoskeletal proteins. Partially activated platelets were more spherical and showed at least one protrusion. Immunofluorescence and immunogold labeling showed a more intense staining of the peripheral 0.2 to 0.3 micron of cytoplasm of these platelets. In the immunofluorescence photographs this resulted in the appearance of small fluorescent rings with staining at the periphery of cross-sectioned cells. Activated platelets showed an irregular outline composed of broad based pseudopods. Cell centers were composed of poorly delineated electron-dense material, interspersed with profiles of surface-connected tubules. The broad based pseudopods stained uniformely for actin, alpha-actinin and actin-binding protein. The cell center stained poorly for these proteins. Myosin staining was found in the peripheral cortex, but also in the cell center. Partially activated platelets that had returned to the disk shape after incubation at 37 degrees C showed increased submembranous concentration of microfilament proteins. These data reveal the profound cytoskeletal rearrangements that already occur upon minimal platelet activation and emphasize that platelets that have returned to the disk shape are not identical to resting platelets.
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PMID:Immunoelectron microscopic localization of actin, alpha-actinin, actin-binding protein and myosin in resting and activated human blood platelets. 274 1

The kinetic parameters and some enzymatic characteristics of human platelet and chicken gizzard transglutaminases were determined. Activity of the transglutaminases was regulated by calmodulin. These enzymes co-isolated with alpha-actinin and were dissociated from alpha-actinin by gel filtration and absorption onto a calmodulin affinity column. Silver-stained polyacrylamide gels showed that the protein peak eluted by EGTA from this column contained polypeptides of Mr approximately 58,000 and 63,000. The transglutaminases required Ca2+ for incorporation of monodansylcadaverine into casein and actin substrates. Activity was enhanced 3-fold by calmodulin with a biphasic effect, showing stimulation at 10-200 nM and inhibition at concentrations higher than 300 nM. In the presence of 200 nM calmodulin, half-maximal transglutaminase stimulation was obtained with 2.5 microM free [Ca2+]. Chlorpromazine inhibited calmodulin enhancement of the transglutaminases. Activity of the transglutaminases was independent of proteolytic activation, since inhibitors for Ca2+-dependent proteases failed to inhibit filamin cross-linking. For comparison, factor XIIa, a plasma and platelet transglutaminase, required both Ca2+ and thrombin for activation and was insensitive to calmodulin. The cross-linking pattern of fibrin, fibrin monomers, and fibrinogen by the calmodulin-regulated transglutaminases showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, disappearance of fibrinogen alpha-chains with no decrease of beta- and gamma-chains or formation of gamma-gamma dimers. By autoradiography, cross-linked products of 125I-fibrinogen revealed heavily labeled high molecular weight polymers and polypeptides of Mr 98,000, 116,000, and 148,000; the latter appeared to be a transient species. However, when fibrin, fibrin monomers, and fibrinogen were used as factor XIIIa substrates, gamma-gamma dimers and alpha-polymers were formed. Formation of gamma-gamma dimers was slower with fibrinogen than with fibrin. Iodoacetamide blocked activity of factor XIIIa but not of the calmodulin-regulated transglutaminases.
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PMID:Catalytic properties of a calmodulin-regulated transglutaminase from human platelet and chicken gizzard. 286 89

Actin filament formation during early stages of platelet activation by stimulants was analyzed by determining the distributions of cytoskeletal proteins to a low speed centrifuge pellet (TP), ultracentrifuge pellet (UP), and ultracentrifuge supernatant (US) after lysing platelets with 1% Triton X-100. TP contained actin binding protein, myosin, alpha-actinin and actin, and UP contained these cytoskeletal proteins and other proteins. During thrombin activation, total protein and actin in TP increased with time, while they decreased in UP. Aggregating agents (ADP, PMA, thrombin) and, to a small extent, colchicine and nocodazole, microtubule inhibitors, increased cytoskeletal proteins and total protein in TP, while cytochalsin B, an inhibitor of actin polymerization, had the opposite effect. When the amounts of the respective proteins in TP and UP were summed, these values were not affected by agonists and inhibitors, except in the case of thrombin stimulation. These data suggest that the actin in UP is a form intermediate between the actin filaments in TP and actin monomer in the soluble fraction, and there may be a dynamic conversion between these forms upon platelet activation. UP was also characterized by immunostaining with antibodies against fibrinogen, tubulin, and glycoprotein Ib after SDS-PAGE/electrophoretic transfer to nitrocellulose sheets.
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PMID:Platelet cytoskeletal protein distributions in two triton-insoluble fractions and how they are affected by stimulants and reagents that modify cytoskeletal protein interactions. 341 29

The interaction of the cytoskeleton with plasma membranes may be mediated by vinculin, alpha-actinin and other proteins; alpha-actinin can interact specifically with model membranes only if they contain diacylglycerol and palmitic acid. On stimulation of platelets by thrombin, which leads to a reorganization of the cytoskeleton, diacylglycerol is produced rapidly, simultaneously with the disappearance of phosphatidylinositol. One important function of the diacylglycerol produced in platelets may be the activation of the Ca2+-and phospholipid-dependent protein kinase C. We show here that, in the presence of diacylglycerol and palmitic acid, a supramolecular complex between alpha-actinin and actin is formed in vitro. In the electron microscope, this complex displays substructures similar to those of microfilament bundles in vivo. Furthermore, such alpha-actinin/lipid complexes can also be formed in situ during the stimulation of blood platelet aggregation. Thus, alpha-actinin may be one of the proteins directly involved in structures connecting the cytoskeleton to cell membranes.
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PMID:Diacylglycerol in large alpha-actinin/actin complexes and in the cytoskeleton of activated platelets. 403 39

Control and stimulated platelets were analyzed by two-dimensional polyacrylamide gel electrophoresis to determine whether proteins are altered during platelet activation. Platelets were stimulated with thrombin, collagen, or the calcium ionophore A23187, and aggregation was brought about by stirring in the presence of Ca2+. These activated platelets contained at least three polypeptides not found in control platelets: 1) Mr = 200,000, pI between 6.2 and 6.4; 2) Mr = 100,000, pI = 6.3; and 3) Mr = 91,000, pI = 6.1. An additional polypeptide, polypeptide 4, with Mr = 97,000 and pI = 5.9, was present only in platelets activated by thrombin. When aggregation was prevented, either by adding 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to the platelet suspension or by incubating the platelet suspension without stirring, polypeptides 1-3 were not formed. Partial hydrolysis of polypeptides 2 and 4 with Staphylococcus aureus V8 protease yielded distinct sets of peptide hydrolytic fragments. These differed from those produced by the hydrolysis of alpha-actinin, a major platelet protein, which has a molecular weight similar to polypeptides 2 and 4. Polypeptides 1-3 were also produced during incubation of platelet lysates in the presence of Ca2+. Generation of these polypeptides in lysates was prevented either by chelation of Ca2+ with EGTA or by the addition of N-ethylmaleimide, leupeptin, or mersalyl, inhibitors of the calcium-dependent protease. These data show that the calcium-dependent protease is activated during aggregation of platelets by physiological agents and suggest that this protease could have a role in platelet response to stimulation.
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PMID:Calcium-dependent proteolysis occurs during platelet aggregation. 630 92

Cytoskeletal proteins were isolated from chicken gizzard smooth muscle and from platelets and antibodies prepared against them. It was shown by indirect immunofluorescence technique that actin, alpha-actinin, and vinculin are not present on the surface of platelets. Physiologic concentrations of thrombin (0.04 to 0.5 U/ml) that induce platelet aggregation and release in the presence of calcium from freshly isolated platelets do not induce platelet changes resulting in the availability of the cytoskeleton to antibodies. Because the F(ab')2 fragments of anti-cytoskeletal proteins IgG do not inhibit thrombin-induced aggregation of platelets, the direct role of these proteins in thrombin-induced platelet aggregation, as with ADP and collagen, may be rejected. However, when freshly isolated platelets are treated with thrombin (1 U/ml), antibodies to actin, alpha-actinin, and vinculin stained the platelets; therefore, this demonstrates that thrombin at this high and probably nonphysiologic concentration induces a reorganization of the membrane components with the subsequent exposure of the proteins of the cytoskeleton. We demonstrate interaction between isolated actin and alpha-actinin but not vinculin with fibronectin. After stimulation of platelets by thrombin, certain cytoskeletal proteins may interact with subendothelial fibronectin and thereby promote and consolidate platelet adhesion.
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PMID:Platelet cytoskeleton: immunofluorescence studies on thrombin-activated platelets. 638 93

Platelets were activated by receptor and non receptor-mediated reagents. The effect of these reagents on aggregation, secretion, cytoskeleton formation, interaction of alpha-actinin and other membrane proteins with the cytoskeleton was studied. Results show that receptor-mediated activation (e.g. ADP or thrombin activation) leads to a high extent of association of alpha-actinin with the cytoskeleton while non receptor-mediated activation (e.g. ionophore A-23187, arachidonic acid) leads to a low association between the two species. The degrees of aggregation, secretion and total amount of protein in the two modes of activation were the same.
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PMID:Receptor and non receptor-mediated activation of blood platelets. Effect on membrane-cytoskeleton interaction. 642 2


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