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)

Unactivated platelets contain about 69% G actin and less than 10% of the contractile proteins in a cytoskeletal core resistant to extraction with 1% Triton X-100. Activation by thrombin leads, within 1 min, to the formation of pseudopodia and contractile gels, accompanied by the reduction of the G-actin content to about 22% and the development of cytoskeletal cores containing 70%-80% of the total actin and 60%-80% of the total myosin and actin-binding protein. Inhibition of pseudopodal formation by pretreatment with cytochalasin B before thrombin activation results in the loss of most of the actin-binding protein and about one third of the actin from the cytoskeletal core. Myosin incorporation and contractile gel formation are unaltered by this treatment. Conversely, activation with phorbol 12-myristate 13-acetate leads to pseudopodal but not contractile gel formation, with cytoskeletal cores containing mostly actin and actin-binding protein. These results demonstrate that there are separable cytoskeletal assembly processes in platelets for pseudopodal and contractile gel formation.
 ...
PMID:Separable assembly of platelet pseudopodal and contractile cytoskeletons. 689 Apr 13

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.
 ...
PMID:Identification of membrane proteins mediating the interaction of human platelets. 689 55

The temporal changes in human platelet actin polymerization, cytoskeletal morphology, and protein content that occur during thrombin-induced platelet activation were investigated by analysis of Triton-extracted platelets. Measurement of the DNase inhibitory activity of control platelets immediately after adding an equal volume of 2% Triton X-100, 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.1 M Tris, pH 7.4, showed that approximately 50% of the actin in unstimulated platelets was filamentous and unable to inhibit DNase-catalyzed hydrolysis of DNA. Activation of platelets with thrombin for 15 s caused the amount of actin in the filamentous form to increase to approximately 65%. Examination of the morphology and protein composition of these filamentous structures showed that the cytoskeletal structures from control platelets consisted of a random array of filaments which contained 14% of the total platelet myosin and 6% of the total actin-binding protein. In contrast, the cytoskeletal structures of thrombin-activated platelets appeared as cytoskeletal structures of individual platelets. The composition of these cytoskeletons varied depending on the time of thrombin activation. Those from platelets activated with thrombin for 15 to 30 s contained 90% of the platelet myosin and 20% of the platelets with thrombin before Triton addition resulted in a decreased association of myosin to 60% with no change in either the actin or actin-binding protein content of the cytoskeletal structures. Since these changes are rapid and precede serotonin secretion, it is suggested that they are involved in the physiological response of the platelet.
 ...
PMID:Changes in the cytoskeletal structure of human platelets following thrombin activation. 689 99

The effect of myosin light chain phosphorylation on the association of myosin with the cytoskeletal structures of platelets was quantitated. In unstimulated platelets, little myosin light chain was phosphorylated and myosin remained in solution when cytoskeletons from Triton X-100 lysates of platelets were sedimented by centrifugation. In platelets activated by thrombin, the calcium ionophore A23187, or collagen, the rate and extent of myosin light chain phosphorylation paralleled the association of myosin with platelet cytoskeletal structures. Dephosphorylation of myosin light chain and myosin dissociation from the cytoskeleton occurred at comparable rates at longer times after addition of the stimulating agents to platelets. Quantitation of radioactive phosphate in the cytoskeleton-associated myosin and in the soluble myosin showed that the phosphorylated myosin light chain was selectively isolated with the Triton-insoluble cytoskeletons, whereas nonphosphorylated myosin was not associated. Inhibition of the light chain kinase with the calmodulin antagonist trifluoperazine inhibited myosin light chain phosphorylation and incorporation of myosin into the platelet cytoskeletons. Inhibition of light chain phosphorylation by prostaglandin E1 and prostacyclin produced similar effects. Thus, phosphorylation of the myosin light chain stabilizes the association of myosin with the contractile structures within platelets.
 ...
PMID:Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets. 704 Mar 79

We have examined platelet protein organization by treatment of intact resting or thrombin-activated platelets with two cross-linking reagents, diamide or dithiobis(succinimidyl propionate) (DTSP). Cross-linked complexes were separated by polyacrylamide gel electrophoresis in the absence of reducing agent and their composition determined after reductive cleavage and analysis in a second-dimensional gel. The most prominent cross-linked species produced by diamide treatment of of resting platelets are (A) cytoskeletal protein homopolymers, such as myosin heavy chain dimer and actin oligomers, and (B) high molecular weight material consisting of homo- or heteropolymers of cytoskeletal proteins and 230,000, 170,000, 100,000, 55,000, and 52,000 dalton proteins. DTSP treatment forms similar complexes and also cross-links membrane glycoproteins IIb and III into high molecular weight material. Thrombin activation of platelets before treatment with diamide or DTSP results in increased cross-linking of myosin and increased incorporation of several proteins, particularly myosin and glycoproteins IIb and III, into high molecular weight material. The results provide evidence for reorganization of cytoskeletal and membrane proteins during platelet function.
 ...
PMID:Platelet protein organization: analysis by treatment with membrane-permeable cross-linking reagents. 705 68

The dynamics of actin polymerization, cytoskeleton formation, and interaction with membrane and cytoplasmic proteins as a result of platelet activation by temperature. ADP, or thrombin were studied. The polymerization of about 30% of platelet DNase I available actin to a nonavailable state is rapid and complete within 10 s after platelet activation with ADP and thrombin. This polymerization might be related to shape change rather than to aggregation or secretion. A similar value of actin polymerization is obtained when platelets are induced to change shape by cooling. This polymerization is partially reversible upon deactivation of the platelets by apyrase, hirudin, or rewarming. Cycles of temperatures-mediated activation and deactivation show a cyclic variation in the state of actin, with a tendency to refractivity to further changes after a couple of cycles. No correlation is observed between microtubule integrity and actin polmerization when studies are performed with platelets pretreated with colchicine. Analysis of the Triton residue composition shows that the cytoskeleton of resting platelets is composed mainly of actin and myosin in a 4.5:1 ratio. Activation with ADP and thrombin leads to the association and incorporation of several other protein (actin binding protein, 95 000 daltons, three to four proteins in the 35 000-dalton region, and two proteins in the 17 000-dalton region with the cytoskeleton). The incorporation of these proteins has a dynamic nature that depends on both the state of aggregation and the reversibility of the activation. Activation leads to a significant increase in the total cytoskeletal proteins, and although low temperature also induces such an increase, the cytoskeletal pattern of cooled platelets is not different from that of resting platelets. A complete reversibility in morphology and amount of protein was observed with temperature cycling. In light of these results, the dynamic nature of the state of actin in platelets is discussed.
 ...
PMID:Dynamics of membrane-cytoskeleton interactions in activated blood platelets. 720 51

The phosphorylation state of myosin in intact platelets has been investigated with alkaline urea-polyacrylamide gel electrophoresis. In gels of control cells, a band was found that co-migrated with the dephosphorylated form of isolated platelet myosin 20,000-dalton light chain. Stimulation of the cells by thrombin produced a dose-dependent shift of this band to the same position as that of the phosphorylated light chain. Conversion to the phosphorylated position was both complete and saturable with respect to thrombin concentration. Two-dimensional polyacrylamide gel electrophoresis was used to confirm the identity of this band as the 20,000-dalton myosin light chain. When (32P)PO4-labeled platelets were used, a direct correlation was found between the position of the light chain on the alkaline urea gel and the radioactivity. Our results demonstrate that in resting platelets myosin exists mainly in the dephosphorylated state and that stimulation by thrombin can produce a shift to the totally phosphorylated state.
 ...
PMID:Myosin phosphorylation in intact platelets. 725 6

Protein phosphorylation may play a critical role in stimulus-coupled secretion of platelets. Some platelet proteins become phosphorylated on exposure to agents such as thrombin and collagen, and the smallest of these phosphoproteins (molecular weight 20,000), has been identified as a light chain of myosin. Phosphorylation of myosin light chain increases the activity of actin-activated myosin ATPase and the resultant contraction of the actomyosin presumably mediates the release reaction. Platelet myosin light chain kinase has been identified as a calcium-dependent enzyme requiring calmodulin for its activity. Calmodulin is a Ca2+-binding protein with a molecular weight of approximately 18,000 which seems to be involved in a wide variety of cellular processes. Although a growing body of evidence suggests that non-muscle actomyosin, such as that isolated from platelets, is regulated by Ca2+-calmodulin-dependent light chain phosphorylation, the precise relationship between the phosphorylation and the function of platelets is not clearly established. We now present pharmacological evidence that a calmodulin-mediated system, such as Ca2+-dependent myosin light chain phosphorylation, also plays an important role in the phenomenon of the release reaction. N-(6-aminohexyl)-5-chloro-1-napthalene-sulphonamide (W-7) (refs 13-15) is shown to bind selectively to calmodulin in vitro and inhibit its biological activity.
 ...
PMID:Ca2+-calmodulin-dependent phosphorylation and platelet secretion. 743 2

The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.
 ...
PMID:Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation. 762 62

Affinity-purified polyclonal antibodies prepared against a synthetic peptide corresponding to sequence 18-29 from the N-terminus of rabbit alpha-skeletal actin reacted with G- and F-actin. Epitope mapping experiments with thrombin and hydroxylamine cleaved actin, and immunochemical assays verified the specificity of antibodies for the 18-29 sequence on actin. The binding of up to 0.5 mol of IgG per mole of actin did not affect the rigor binding of myosin subfragment 1 (S-1) to actin. Similarly, the binding of IgG to actin was not changed by a complete saturation of actin by S-1. In contrast to this, the weak acto-S-1 interactions in the presence of ATP were strongly inhibited by the 18-29 antibodies. At 25 degrees C, the acto-S-1 ATPase activity was inhibited by IgG stronger than the binding of S-1.ATP gamma S to actin. Thus, at this temperature, a catalytic inhibition of the acto-S-1 system appears to account at least in part for the antibody effect. Acto-S-1 ATPase activities at 25 degrees C were inhibited also by F(ab)(18-29). At 5 degrees C, the acto-S-1 ATPase activity and the binding of S-1.ATP to actin were inhibited approximately to the same extent by IgG(18-29). These results are discussed in terms of S-1 binding sites on actin and the possible role of sequence 18-29 in actomyosin interactions.
 ...
PMID:Role of sequence 18-29 on actin in actomyosin interactions. 768 58


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>