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)

Factor D (C3 proactivator convertase) of human serum has been shown to be absolutely necessary for alternative pathway function, for activation of the C3/C5 convertase of that pathway and not to be a subunit of this enzyme. Factor D was found to be present in human plasma in active form only, at a concentration of 2 microgram/ml, and not to be controlled by plasma protease inhibitors or by spontaneous decay. Unlike trypsin, factor D cleaves and activates factor B only when it is in Mg++-dependent complex with C3b, has no esterolytic activity, and is unable to cleave the B chain of insulin. The alleged functional and antigenic relationship of factor D to alpha-thrombin could not be verified. The results of this study led to the description of the mechanism of action of factor D in terms of the cryptic site hypothesis.
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PMID:Mechanism of action of factor D of the alternative complement pathway. 8 4

The human platelet thrombin receptor is activated when thrombin cleaves its receptor's amino-terminal extension to reveal a new amino terminus that functions as a tethered peptide ligand. Exactly how this "agonist peptide domain" remains cryptic within the uncleaved receptor and becomes functional after receptor cleavage is unknown. In this report we define the structural features of the thrombin receptor's agonist peptide domain important for receptor activation. Studies with mutant thrombin receptors have suggested that agonist peptide domain residues 2-6 contained determinants critical for receptor activation, and the synthetic peptide SFLLR-NH2 representing the 1st 5 amino-terminal residues of the agonist peptide domain was sufficient to specify agonist activity. Acetylating or removing the agonist peptide's amino-terminal ammonium group greatly attenuated agonist activity. Agonist peptide residue Phe2 was vital for agonist function; residues Leu4 and Arg5 individually played less important roles. These structure-function relationships held for both platelet activation and activation of the cloned receptor expressed in transfected mammalian cells. Our studies suggest that structures at the extreme amino terminus of the thrombin receptor's agonist peptide domain, in particular the free ammonium group of Ser1 and the phenyl ring of Phe2, are critical for receptor activation and that the agonist function of this domain is expressed when receptor proteolysis unmasks such determinants. In addition to revealing details of the thrombin receptor's proteolytic triggering mechanism, these studies open avenues to the development of drugs targeting the thrombin receptor and to further definition for the role of the thrombin receptor in cellular regulation.
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PMID:Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. 132 11

In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin independent.
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PMID:Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils. 133 90

Vitronectin (VN, complement S-protein) is a multifunctional protein which participates in cell adhesion, coagulation, fibrinolysis, and protection against complement lysis. VN is incorporated into several complexes, such as the terminal complement complex and thrombin-antithrombin III, and is bound to plasminogen activator inhibitor 1. The present study showed that purified VN spontaneously forms polymers of approximately 1000 kDa with a Stokes radius of 10 nm. The polymers are to a varying extent stabilized by disulfide bonds, but are quite stable even after reduction and alkylation, indicating the importance of noncovalent bonds. Plasma VN circulates mainly as a 65/75-kDa monomer containing a cryptic heparin-binding site which is exposed upon a conformational change induced by different stimuli, such as coagulation, heating, adsorption to surfaces, or exposure to acids, urea, or other denaturating agents. In the present study, VN was demonstrated to expose its heparin-binding site and its conformationally dependent 8E6 epitope when incorporated into the terminal complement complex. We suggest that exposure of the heparin-binding site and a putative hydrophobic binding site of VN are linked events dependent upon the same conformational change. In vivo, complex formation probably induces the heparin-binding site. Such a link might also explain why purified heparin-binding VN spontaneously forms polymers. The heparin-binding site may be involved in the elimination of multimolecular complexes containing VN.
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PMID:Heparin-binding properties of vitronectin are linked to complex formation as illustrated by in vitro polymerization and binding to the terminal complement complex. 138 12

In the presence of a monoclonal antibody raised against the human thrombin-antithrombin III complex, the reaction between thrombin and antithrombin III proceeded to form preferentially a two-chain form of the inhibitor rather than to follow the major pathway of stable acyl complex formation. We thus propose the term "switching antibody" for an antibody that switches the enzyme-inhibitor reaction (Asakura, S., Matsuda, M., Yoshida, N., Terukina, S., and Kihara, H. (1989) J. Biol. Chem. 264, 13736-13739). By analyzing a CNBr fragment of the thrombin-antithrombin III complex that reacts with the antibody we localized the epitope for the antibody to a strongly hydrophobic residue 382-386 peptide segment, Ala-Ala-Ala-Ser-Thr, of the inhibitor, which is also contiguous with a hydrophobic amino acid Ala at its carboxyl terminus. This particular region should be cryptic in nascent antithrombin III, but could have been exposed to provide the reactive site for the antibody at an early stage of the reaction. Thereby a conformational change may have been induced at or near the reactive site of the complex, facilitating hydrolysis of the inhibitor by the enzyme. Interestingly, this hydrophobic region is highly conserved among members of the serpin family.
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PMID:Hydrophobic residues 382-386 of antithrombin III, Ala-Ala-Ala-Ser-Thr, serve as the epitope for an antibody which facilitates hydrolysis of the inhibitor by thrombin. 169 Jul 36

Purification of vitronectin by identical procedures from serum instead of plasma results in the coisolation of an additional protein component with mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 82 kDa. We show that this component is the thrombin-antithrombin III complex based on the following evidence. Similar to a complex constructed using purified thrombin and antithrombin III, the 82-kDa component has a reduced molecular size of 69 kDa if it is not boiled prior to SDS-PAGE. Upon prolonged boiling in SDS it dissociates into 56- and 32-kDa components which co-migrate in SDS-PAGE with purified antithrombin III and thrombin, respectively. The 82- and 56-kDa components react with an antiserum against antithrombin III, and an antiserum prepared against the 82-kDa complex reacts with purified antithrombin III. Thrombin-antithrombin III complex, from either serum or recalcified clotted plasma, bound to vitronectin immobilized on Sepharose or plastic. However, purified antithrombin III which had not reacted with thrombin lacked affinity for vitronectin as did antithrombin III from citrated plasma. Purified antithrombin III acquired affinity for immobilized vitronectin if it was complexed with thrombin or was modified by radioiodination. Binding of vitronectin to antithrombin III coated on plastic was demonstrated using enzyme-linked immunosorbent assay. These results demonstrate that vitronectin binds thrombin-antithrombin III complexes through a cryptic site in antithrombin III which can be exposed when antithrombin III is radioiodinated, bound to plastic, or complexed with thrombin. Since vitronectin can interact with cells, the binding of vitronectin to the thrombin-antithrombin III complex may serve to facilitate the interaction of this complex with cell surfaces.
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PMID:Association of thrombin-antithrombin III complex with vitronectin in serum. 241 21

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains. 282 10

Previous work from this laboratory has indicated that thrombin's influence on cell growth can be negative as well as positive. Addition of enzyme to actively growing or confluent cultures of human skin fibroblasts produced growth stimulation, whereas cultures receiving thrombin at the time of subculture displayed inhibited DNA synthesis and mitosis. The specific binding of [125I]thrombin to cells under stimulatory and inhibitory conditions has been studied. Fibroblasts receiving enzyme at subculture bound about two times more [125I]thrombin than those processed in the same way several hours later. The apparent dissociation constant for both groups was approximately 1.5 x 10(-8) M. In each case binding was saturable, although cells receiving enzyme at subculture showed a much higher rate of binding. Experiments were conducted in which enzyme was added to cells at various times after subculture. It was found that the ability of these fibroblasts to specifically bind [125I]thrombin decreased progressively over a 2-h period after subculture and then remained constant for at least 24 h. Evidence is also presented indicating that the binding of [125I]thrombin in both experimental groups was inversely dependent upon the culture density. The biological effects of elevated thrombin binding in cells receiving enzyme at subculture were examined. It was found that inhibited DNA synthesis and altered cellular morphology were directly to this parameter. This study suggests that fibroblasts may possess cryptic thrombin receptors that become exposed during subculture or after injury in vivo. These possibilities and the relationship of cell shape to the availability of thrombin receptors are discussed.
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PMID:Binding of thrombin to cultured human fibroblasts: evidence for receptor modulation. 625 26

SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.
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PMID:Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates. 764 67

Vitronectin is a structurally labile molecule with a native, non-heparin binding form and a conformationally altered, heparin binding form. To understand the physiological significance of the two conformers of vitronectin, we examined the metabolism of both conformers by cultured human skin fibroblasts. Both native and altered vitronectin bound to confluent fibroblast monolayers. Both conformers of vitronectin competed equally well for the binding of altered vitronectin to the cell layer, suggesting that both conformers bound to the same site in the cell layer. In contrast, 125I-altered vitronectin, but not 125I-native vitronectin, was degraded to trichloroacetic acid-soluble radioactivity by the fibroblast monolayer. Degradation of vitronectin was saturable, sensitive to chloroquine, and occurred intracellularly, suggesting that vitronectin was degraded through a lysosomal pathway. Heparin and thrombospondin inhibited the degradation of altered vitronectin. The degradation of native vitronectin was induced by addition of gamma-thrombin which exposes vitronectin's cryptic heparin-binding domain. These studies suggest that the heparin-binding domain in vitronectin is required for the clearance of vitronectin from the matrix. In addition, these data demonstrate that the conformation of vitronectin regulates its half-life in the matrix. These studies provide the first evidence for a distinct function for the conformers of vitronectin.
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PMID:Receptor-mediated endocytosis of vitronectin is regulated by its conformational state. 768 28


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