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)

Snake venoms, particularly those belonging to Crotalidae and Viperidae families, are known to possess fibrinogenolytic proteases which strongly affect the blood coagulation system. Three fibrinogenolytic enzymes, named as TM-1, TM-2 and TM-3, were purified from the venom of Taiwan habu using anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. These enzyme fractions were shown to be homogeneous as judged by SDS-gel electrophoresis. Further characterization of these purified fractions with fibrinogenase activity indicated that they are single-chain proteases possessing basic isoelectric points and molecular masses of approximately 27 kDa. These enzymes cleaved alpha-chain of fibrinogen first and then beta-chain, with relatively low activity on gamma-chain. Fibrinogenolytic activities of these enzymes were not significantly affected at extreme pH values, activity loss occurred only at temperatures higher than 65 degrees C. Enzyme activities were strongly inhibited by some metal chelators such as EDTA or 1,10-phenanthroline, suggesting that these fibrinogenases belong to metalloproteases. The closely similar amino-acid compositions coupled with identical cleaved products of similar molecular masses observed on the autolysis of TM-1 and TM-2 suggest that these two proteases are similar in primary structures and probably mutually related.
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PMID:Characterization of three fibrinogenolytic proteases isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus). 819 88

Peptide analogs of the N-terminal portion of fibrin alpha-chain were prepared and their inhibitory effects on fibrinogen/thrombin clotting were examined. Of the synthetic peptides, H-Gly-Pro-Arg-Pro-Pro-NH2 exhibited the most potent inhibitory effect.
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PMID:Amino acids and peptides. XVIII. Synthetic peptides related to N-terminal portion of fibrin alpha-chain and their inhibitory effect on fibrinogen/thrombin clotting. 833 44

The association of factor XIII a-subunits with fibrin was characterized using recombinant human placental factor XIII (rFXIII) and native fully hydrated fibrin clots formed from purified fibrinogen and thrombin. Binding was assessed using small columns containing fibrin and perfusing them with radioiodinated rFXIII. Results show that thrombin activation of rFXIII led to fibrin binding. The association was partially reversible since much of the bound enzyme could be removed by percolating clots with more buffer. Binding was blocked by antibody directed against the COOH-terminal part of fibrinogen A alpha-chain (A alpha 389-402) and also by the COOH-terminal A alpha-chain peptide fragment A alpha 241-476 (Hi2-DSK).
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PMID:Fibrin--recombinant human factor XIII a-subunit association. 836 76

When amino acids Pro60B, Pro60C, and Trp60D are deleted from thrombin, the resulting mutant (des-PPW) exhibits (compared to the wild-type enzyme): a similar second order rate constant of inhibition (k(on)) for diisopropyl fluorophosphate, and a comparable inhibition constant (K(i)) for benzamidine, suggesting that the charge stabilizing system and the primary binding pocket are little altered, if at all, by the mutation. As predicted from the x-ray structure, des-PPW is remarkably sensitive to the bovine pancreatic trypsin inhibitor, with a K(i) over 3 x 10(3) times tighter relative to thrombin, but des-PPW is also markedly less susceptible to inactivation by antithrombin III, with a k(on) that is over 100-fold lower. The catalytic constant (kcat) for most p-nitroanilide substrates tested is preserved or even increased, but the Michaelis constant (Km) increases. In contrast, the Km for the fibrinogen A alpha-chain is essentially unchanged, whereas kcat decreases approximately 50-fold. Unlike thrombin, the rate of fibrinopeptide B release becomes, following a lag phase, comparable to that of fibrinopeptide A. Inasmuch as des-PPW cleaves an additional peptide bond in the bovine fibrin alpha-chain, it remains a highly specific serine protease, which releases a single peptide from denatured casein (versus two with thrombin). Protein C activation by des-PPW is approximately 30 times slower than by thrombin in the absence, as well as in the presence, of calcium and thrombomodulin. Although this study confirms that the B-insertion restricts access to the active site cleft, it also suggests that other motifs and/or discrete amino acids are mainly responsible for the narrow specificity of thrombin.
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PMID:The role of thrombin's Tyr-Pro-Pro-Trp motif in the interaction with fibrinogen, thrombomodulin, protein C, antithrombin III, and the Kunitz inhibitors. 839 26

Ancistron-H and Ancistron-B--two novel thrombin-like serine proteinases--have been purified 188- and 194-fold with a 95% recovery from the venoms of two Middle Asian subspecies of the pit viper--Agkistrodon halys halys and Agkistrodon halys Blomhoffii, using one-step affinity chromatography on agarose with an immobilized dye--Cibacron Blue F3GA (Blue-Sepharose 6B CL). The purified enzymes are one-chain glycoproteins with molecular masses of 34 and 29 kDa, pI of 6.6 and 6.3 and specific activities of 410 and 110 NIH units/mg protein, respectively. Their major amino acids are Gly, Val, Ser and Asp for Ancistron-H and Glu, Gly, Ser and Asp for Ancistron-B. During incubation with fibrinogen the enzymes cleave only the fibrinopeptide A from the A alpha-chain, leaving the B beta- and gamma-chains intact. Both enzymes hydrolyse arginine esters and thrombin-specific chromogenic peptide substrates, and display a weak caseinolytic activity but no fibrinolytic activity.
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PMID:[Isolation and characteristics of alpha-specific thrombin-like enzymes from venoms of the common pit viper (Agkistrodon halys halys) and the eastern pit viper (the central Asian subspecies Agkistrodon halys blomhoffii)]. 839 70

Thrombin displays remarkable specificity, effecting the removal of fibrinopeptides A and B of fibrinogen through the selective cleavage of two Arg-Gly bonds between the 181 Arg/Lys-Xaa bonds in fibrinogen. Significant advances have been made in recent years towards understanding the origin of the specificity of cleavage of the Arg16-Gly17 bond of the A alpha-chain of human fibrinogen. We have previously proposed a model for the bound structure of fibrinopeptide A7-16 (FPA), based upon NMR data, computer-assisted molecular modeling and the synthesis and study of peptidomimetic substrates and inhibitors of thrombin. We now report the structure of the ternary complex of an FPA mimetic (FPAM), hirugen and thrombin at 2.5 A resolution (R-factor = 0.138) and specificity data for the inhibition of thrombin and related trypsin-like proteinases by FPAM. The crystallographic structures of FPA and its chloromethyl ketone derivative bound to thrombin were determined. Although there are differences between these structures in the above modeled FPA structure and that of the crystal structure of FPAM bound to thrombin, the phi, psi angles in the critical region of P1-P2-P3 in all of the structures are similar to those of bovine pancreatic trypsin inhibitor (BPTI) in the BPTI-trypsin complex and D-Phe-Pro-Arg (PPACK) in the PPACK-thrombin structure. A comparison between these and an NMR-derived structure is carried out and discussed.
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PMID:The structure of a designed peptidomimetic inhibitor complex of alpha-thrombin. 841 74

beta 3 Integrin derived peptides 217-230 (DAPEGGFDAIMQAT) and 217-231 (Y) (DAPEGGFDAIMQATVY) at 100 microM inhibited 125I-fibrinogen binding to ADP-stimulated platelets and platelet aggregation. Peptide 217-231 (Y) (100 microM) significantly inhibited the binding of 125I-albolabrin (a disintegrin with a single RGD sequence) to ADP- and thrombin-activated platelets while it had only a slight effect on albolabrin binding to resting platelets. The 125I-beta 3 217-231 (Y) cross-linked selectively to the fibrinogen A alpha-chain. The interaction of the RGD sequence in the A alpha-chain of fibrinogen with beta 3 217-231 sequence appears to play a significant role in the events leading to platelet aggregation.
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PMID:Beta 3 integrin derived peptide 217-230 inhibits fibrinogen binding and platelet aggregation: significance of RGD sequences and fibrinogen A alpha-chain. 842 38

In healing wounds and many solid tumors, locally increased microvascular permeability results in extravasation of fibrinogen and its extravascular coagulation to form a fibrin gel, with concomitant covalent cross-linking of fibrin by factor XIIIa. Subsequently, inflammatory cells, fibroblasts, and endothelial cells migrate into the gel and organize it into granulation tissue and later into mature collagenous connective tissue. To gain insight into some of the cell migration events associated with these processes, we developed a quantitative in vitro assay that permits the study of fibroblast migration in fibrin gels. Early passage human or rat fibroblasts were allowed to attach to tissue culture dishes and then were overlaid with a thin layer of fibrinogen that was clotted with thrombin. Fibroblasts began to migrate upwards into the fibrin within 24 hours and their numbers and the distance migrated were quantified over several days. The extent of fibroblast migration was affected importantly by the nature of the fibrin clot. Fibroblasts migrated optimally into gels prepared from fibrinogen at concentrations of -3 mg/ml; ie, near normal plasma fibrinogen levels. Migration was greatly enhanced by extensive cross-linking of the fibrin alpha-chains by factor XIIIa, as occurs when clotting takes place in vivo. When fibrinogen was clotted in Dulbecco's modified Eagle's medium, gamma-chains were cross-linked, but alpha-chain cross-linking was strikingly inhibited, and fibroblasts migrated poorly. Gels prepared from factor XIII-depleted fibrinogen exhibited neither alpha-nor gamma-chain cross-linking and did not support fibroblast migration. Further purification of fibrinogen by anion exchange high pressure liquid chromatography depleted fibrinogen of fibronectin, plasminogen, and other impurities; this purified fibrinogen clotted to form fibrin gels that supported reproducible fibroblast migration.
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PMID:Fibroblast migration in fibrin gel matrices. 842 60

High molecular weight, low molecular weight and very low molecular weight fibrinogen were purified from human plasma, and converted partially or completely to fibrin by the action of thrombin or batroxobin. The stimulatory effects of these fibrin(ogen) preparations on plasminogen activation by tissue plasminogen activator were studied. When only 3-30% of the fibrinogen molecules were converted to fibrin, the high molecular weight fibrin had a greater stimulatory effect on plasminogen activation than equal amounts of low and very low molecular weight fibrin. In completely converted fibrin preparations, the plasminogen activating capacity of high molecular weight fibrin was either equal to (thrombin treated preparations) or greater than (batroxobin-treated preparations) that of very low molecular weight fibrin. These findings suggest that degradation of the A alpha-chain of fibrin does not per se increase its plasminogen activating capacity.
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PMID:The stimulatory capacity of soluble fibrin prepared from high and low molecular weight fibrinogen on plasminogen activation. 845 41

A novel murine monoclonal antibody against the fibrin alpha-chain N-terminus is presented, which reacts with desAA- and desAABB-fibrin. In immunoblot procedures, the antibody reacted with fibrin degradation products X and Y of non-crosslinked fibrin, and fragment E. No binding was observed to the fibrin fragment D-dimer, and fibrinogen fragments D and E. Minor binding to fibrinogen fragments X, and Y, and desBB-fibrin were presumably due to minor contamination with (desAA)-fibrin. A prerequisite for binding was release of fibrinopeptides A (FpA), the binding site being a fibrin-specific neo-epitope. No binding was observed to fibrinogen or to thrombin-treated dysfibrinogen MANNHEIM III (A alpha 16 Arg-->Cys) molecules, which do not release FpA. The antibody bound to abnormal fibrin molecules prepared from dysfibrinogen MANNHEIM I (A alpha 19 Arg-->Gly), albeit to a lesser extent than to normal fibrin. Binding of the antibody to the fibrin epitope was greatly enhanced by denaturation, e.g. by heat, or by treatment with chaotropic ions. Soluble fibrin in clinical samples is generally found as a complex with fibrinogen, since polymerization sites 'A' exposed by release of FpA react with complementary binding sites 'a' on the D-domains of other fibrin and fibrinogen molecules. Treatment of samples with NaSCN caused dissociation of fibrin monomer complexes. Reassociation was prevented by denaturation of both polymerization sites 'A' and 'a'. The antibody in combination with NaSCN-treatment of samples was useful for specific detection of fibrin monomer in plasma samples. Measurement was not influenced by fibrinogen degradation products, whereas fibrin degradation products at very high concentration caused some underestimation of fibrin monomer concentration.
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PMID:Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site 'A': effects of fibrinogen and fibrinogen derivatives, and pretreatment of samples with NaSCN. 845 57


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