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 alternative pathway of complement is regulated on the surface of homologous blood cells at the C3 amplification step by the membrane protein decay-accelerating factor, as well as by the plasma protein factor H. We have reported elsewhere that platelets from patients with paroxysmal nocturnal hemoglobinuria regulate the activity of the C3 convertase C3bBb, even though they lack decay-accelerating factor. We now report that normal human platelets contain factor H, which was released from the platelet in response to complement deposition or thrombin stimulation. Factor H was localized to the platelet alpha granules by immunocytochemical techniques. As determined by a solid-phase radioimmunoassay, thrombin-stimulated platelets released approximately equal to 54 ng of factor H per 10(8) platelets. The release of factor H in response to complement or thrombin was inhibited by treating the platelets with metabolic inhibitors. Such inhibition resulted in a 3-fold increase in the activity of C3bBb. Platelets that released factor H bound only half as many molecules of radiolabeled factor B to platelet-bound C3b than platelets that could not release factor H. Treatment of platelets with anti-decay-accelerating factor antibody had no effect on the activity of C3bBb unless the release of factor H was blocked. Therefore, so far as we know, human platelets have a unique mechanism for the regulation of the alternative pathway of complement.
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PMID:Regulation of the activity of platelet-bound C3 convertase of the alternative pathway of complement by platelet factor H. 295 7

Hexapeptides mimicking the partial amino acid sequence of factor B surrounding the bond that is cleaved by factor D have been synthesized. These peptides have been assessed for their ability to inhibit factor D enzymatic activity and for their susceptibility to serine proteases. The synthetic peptides were cleaved by bovine trypsin and C1s but not by alpha-thrombin and factor D. The peptides inhibited factor B cleavage and fluid-phase or cell-bound alternative pathway C3 convertase activation by factor D. Altogether, these results suggest that peptides analogous to factor B specifically inhibit factor D enzymatic activity. Thus, they constitute an interesting tool for study of alternative pathway activation and can be of use when attempting to manipulate this pathway, since factor D is an essential component for alternative pathway initiation and amplification.
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PMID:Inhibition of alternative pathway factor D by factor B-related synthetic hexapeptides. 692 Feb 99

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
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PMID:A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. 827 48

El mouse is a mutant which has epileptic convulsions after tossing-up stimulations and has a hippocampal dysfunction. Platelet-derived growth factor B-chain homodimer has been reported to be a trophic factor of hippocampal neurons. We found that a recombinant PDGF-BB suppressed the convulsions of El mice in a dose-dependent manner. Furthermore, thrombin-treated mice manifested no convulsions, but thrombin receptor activating peptide-treated ones had convulsions. These findings suggest that an abnormality in PDGF-BB release may make El mice susceptible to tonic-clonic convulsions.
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PMID:Platelet-derived growth factor B-chain homodimer suppressing a convulsion of epilepsy model mouse El. 866 Mar 79

Cerebral ischemia is caused by reduced blood supply at the microcirculatory level. In the microvessels, the main elements of the reperfusion injury following brain ischemia are the transformation of endothelial cell-surface from anticoagulant to procoagulant property, leukocyte adhesion, sludge or clot formation. There is a paucity of information on how hemostatic factors, cytokines, lipoprotein(a) (Lp(a)) and endothelin-1 (ET-1), being responsible for ischemic/reperfusion injury, interact with human brain microvessel endothelium (HBEC). There are no data furthermore about the expression of complement proteins of HBEC influenced by cytokines or fibrinolytic factors. Previously we established optimal conditions for culturing HBEC. Cell contraction induced by thrombin, plasmin, miniplasmin was recorded. The reassembly of F-actin was observed after thrombin treatment. ICAM-1 upregulation was measured following TNF-alpha, IL-1-alpha and thrombin incubation. Plasmin and miniplasmin downregulated the ICAM-1 in our cell culture system. Lp(a) modulated the thromboresistant cell-surface by reduction of t-PA and u-PA, but PAI-1 remained unchanged. Lp(a) modulated the ET-1 production by early increasing and late decreasing, in a bimodal manner. The increased secretion of ET-1 by cytokines (TNF-alpha, IL-1-alpha) was reduced in the presence of Lp(a). Gradual increase of complement proteins (factor H, factor B, C4) was induced by cytokines. Plasmin and miniplasmin augmented a rapid increase of C4. Some factors of complex relationship between regulators and modulators of endothelial adhesion molecules have been demonstrated in a human cell culture system prepared from brain microvessel endothelium. A unified concept of sequential events of ischemia/reperfusion in the brain has not yet developed.
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PMID:Human brain microvessel endothelial cell culture as a model system to study vascular factors of ischemic brain. 889 62

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.
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PMID:Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity. 975 54

Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of (125)I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.
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PMID:Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B. 1047 73

Cell mediated responses to Equid herpesvirus 1 (EHV-1) are of short duration in vivo and require considerable expansion to be detected in vitro. Raised serum levels of active transforming growth factor B (TGF-B1) have been shown to depress proliferative T cell responses in experimental infections with EHV-1 in ponies. The present work indicates that latent transforming growth factor B (TGF-B1) is present in circulating platelets, lymph node, bronchial epithelium and alveolar macrophages. Activation of platelets in vitro by thrombin resulted in the release of latent TGF-B1 from platelets, with a pg level of conversion to active TGF-B1, but virus alone did not activate TGF-B1. Exposure of circulating leucocytes to EHV-1 in vivo or in vitro does not result in detection of active TGF-B1 above residual levels that could be attributed to activation of platelets by manipulation. However, alveolar macrophages obtained by lavage at autopsy yield both latent and active TGF-B1 in ng quantities. Bronchial epithelium, and mesenteric lymph node leucocytes had equivalent levels of latent TGF-B1, but horses varied as to whether these tissues were a source of activated TGF-B1 and as to whether EHV-1 activated TGF-B1.
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PMID:Equid herpesvirus 1: platelets and alveolar macrophages are potential sources of activated TGF-B1 in the horse. 1088

Chitosan is a polycationic and biocompatible polysaccharide composed of glucosamine and N-acetyl glucosamine that is chemotactic for neutrophils and stimulates wound repair through mechanisms that remain unclear. It was previously shown that chitosan depletes complement proteins from plasma, suggesting that chitosan activates complement. Complement activation leads to cleavage of C5 to produce C5a, a neutrophil chemotactic factor. Here, we tested the hypothesis that chitosan generates C5a in human whole blood, citrated plasma, and serum. C5a fragment appeared in coagulating whole blood, and mixtures of chitosan-glycerol phosphate/whole blood, in parallel with platelet and thrombin activation. However, in plasma and serum, thrombin and chitosan-GP failed to generate C5a, although native C3, C5, and factor B adsorbed noncovalently to insoluble chitosan particles incubated in citrated plasma, serum, EDTA-serum and methylamine-treated plasma. By surface plasmon resonance, pure C3 adsorbed to chitosan. The profile of serum factors associating with chitosan was consistent with a model in which anionic blood proteins with a pI lower than the pK(0) 6.78 of chitosan (the upper limit of chitosan pK(a)) associate electrostatically with cationic chitosan particles. Zymosan, a yeast ghost particle, activated complement in serum and citrated plasma, but not in EDTA-serum or methylamine plasma, to generate fluid-phase C5a, while C3b formed covalent cross-links with zymosan-associated proteins and became rapidly cleaved to iC3b, with factor Bb stably associated. These data demonstrate that chitosan is a nonreactive biomaterial that does not directly activate complement, and provide a novel basis for predicting anionic serum protein-chitosan interactions.
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PMID:C3, C5, and factor B bind to chitosan without complement activation. 1992 29


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