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)

Bovine fibrinogen was cleaved with CNBr and the peptide F-CB1 which originates from the amino end of the molecule was purified by chromatographic methods. After reduction and alkylation of F-CB1 three main polypeptide chains could be identified. They were derived from the A alpha chain (F-CB1 alpha), Bbeta chain (F-CB1 beta) and gamma chain (F-CB1 gamma) of fibrinogen and consisted of 54, 143 and 78 amino acid residues, respectively. Thrombin digestion released fibrinopeptide A from F-CB1 alpha and smaller amounts of the peptide Gly-Pro-Arg while fibrinopeptide B was released from F-CB1 beta. Fragment F-CB1 gamma was resistant to thrombin. The sequences of 35 amino acid residues in the portion of F-CB1 alpha adjacent to fibrinopeptide A and of 15 residues in F-CB1 beta beyond fibrinopeptide B were determined. They differ by six and two substitutions, respectively, when compared with the homologous part in human fibrinogen. However, the first 29 amino acid residues in bovine and human F-CB1 gamma were identical. The data show that the cysteine-containing regions and the portions to the right of the site of thrombin cleavage in fibrinogen show much less interspecies variability than that known for the fibrinopeptides.
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PMID:Disulfide-linked cyanogen bromide peptides of bovine fibrinogen. II. Isolation and sequence analysis of the chain constituents from the amino terminal region. 83 81

The erythrocyte membrane inhibitor of the human terminal complement proteins, surface antigen CD59, has previously been shown to enter into a detergent-resistant complex with either the membrane-bound complex of C5b-8 or C5b-9 (Meri, S., Morgan, B. P., Davies, A., Daniels, R. H., Olavesen, M. G., Waldmann, H. and Lachmann, P. J. (1990) Immunology 71, 1-9; Rollins, S. A., Zhao, J., Ninomiya, H., and Sims, P. J. (1991) J. Immunol, 146, 2345-2351). In order to further define the interactions that underlie the complement-inhibitory function of CD59, we have examined the binding interactions between 125I-CD59 and the isolated components of human complement membrane attack complex, C5b6, C7, C8, and C9. By density gradient analysis, we were unable to detect interaction of 125I-CD59 with any of these isolated complement components in solution. Specific binding of 125I-CD59 to C8 and C9 was detected when these human complement proteins were adsorbed to either plastic or to nitrocellulose, suggesting that a conformational change that accompanies surface adsorption exposes a CD59-binding site that is normally buried in these serum proteins. The binding of 125I-CD59 to plastic-adsorbed C8 and C9 was saturable and competed by excess unlabeled CD59, with half-maximal binding observed at 125I-CD59 concentrations of 80 and 36 nM, respectively. No specific binding of 125I-CD59 was detected for surface-adsorbed human C5b6 or C7 nor was such binding observed for C8 or C9 isolated from rabbit serum. Binding of CD59 to human C8 and C9 was not mediated by the phospholipid moiety of CD59, implying association by protein-protein interaction. In order to further define the binding sites for CD59, ligand blotting with 125I-CD59 was performed after separation of C8 into its noncovalently associated subunits (C8 alpha-gamma and C8 beta) and after alpha-thrombin digestion of C9. These experiments revealed specific and saturable binding of 125I-CD59 to C8 alpha-gamma subunit (half-maximal binding at 75 nM), but not to C8 beta, and specific and saturable binding to the 37-kDa fragment (C9b) of thrombin-cleaved C9 (half-maximal binding at 35 nM), but not to the 25-kDa C9a fragment. Partial reduction of C8 alpha-gamma revealed that only C8 alpha polypeptide exhibited affinity for CD59, and no specific binding to the C8 gamma chain was detected.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The human complement regulatory protein CD59 binds to the alpha-chain of C8 and to the "b"domain of C9. 137 90

Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS-polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.
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PMID:Plasma crosslinked fibrin polymers: quantitation based on tissue plasminogen activator conversion to D-dimer and measurement in normal and patients with acute thrombotic disorders. 138 60

The precipitous increase in the number of structurally defined fibrinogen defects in recent years has resulted from application of high performance liquid chromatography in combination with peptide mapping and sequencing procedures. More recently, application of DNA sequence of polymerase chain reaction products has accelerated the pace of identification of mutations. Highly frequent defects are Arg substitutions, accounting for eight mutation sites substituted by Cys or His and less frequently by Ser. Amino acid substitutions at different positions on all three chains have pointed to possible structures with polymerization-related functions. Also, substitutions yielding consensus sequences resulted in extra glycosylations of the appropriate Asn in four different mutation sites; the impaired polymerization was reported associated with undue bleeding in two of these. Among informative defects have been those of homozygous probands with A alpha 16Arg----His and A alpha 16Arg----Cys in that failure of release of peptide A (but not of B), as shown with A alpha 16Arg----Cys, resulted in markedly delayed polymerization of such fibrin monomers, in general agreement with conclusions reached in studies of normal fibrin. This dysfunction, as well as the slow rate of release of A shown with A alpha 16Arg----His, was associated with clinically significant hemorrhagic diathesis (in the homozygous probands), consistent with the known physiologic importance of peptide A cleavage in normal hemostasis. Also, defects on the A alpha 17-19 sequence resulting in impaired polymerization are consistent with the known role of this segment in polymerization. Of similar interest have been defects within a B beta chain span encoded its exon 2. Two defects resulting in impaired polymerization and thrombin binding were associated with clinical thrombosis commencing in early life, and this lends strong support to other evidence suggesting a role in polymerization and in noncatalytic thrombin binding by this B beta chain segment. Thrombosis associated with A alpha 554Arg----Cys in a heterozygous proband with impaired tPA interaction is unique and may shed light on this poorly understood but important interaction among fibrin, plasminogen, and tPA. A group of different defects within the gamma 275-375 sequence have pointed to a polymerization role, evidenced by delayed gelation and impaired binding of mutant D to normal fibrin E. An unusual example is a 15 residue insertion between gamma 350 and 351 resulting in impaired polymerization, gamma chain crosslinking, and platelet aggregation support and is associated with hemorrhagic diathesis and poor healing.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fibrinogen anomalies and disease. A clinical update. 140 80

Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two- to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N-(3-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and gamma chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro two- to fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cell lines.
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PMID:Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. 184 69

Fibrinogen Haifa is a congenital heterozygous fibrinogen variant (gamma 275 Arg----His) characterized by prolonged thrombin and reptilase times and normal fibrinopeptide (FPA, FPB) release. We compared the polymerization rate (by turbidity measurements at 350 nm) and the ultrastructure of Haifa alpha-, beta-, and alpha, beta-fibrin with that of normal. Haifa alpha, beta-fibrin polymerized less rapidly than did normal and formed a highly branched matrix with a smaller mean fiber diameter; this network closely resembled that of normal alpha, beta-fibrin with EDTA added. In the presence of CaCl2 (1 to 10 mM), Haifa alpha, beta-fibrin polymerized more rapidly than in buffer alone and possessed a matrix structure closely resembling that of normal fibrin. From these observations it appears that the functional defect in Haifa fibrin can be related to the inability of the abnormal molecule to effectively utilize available calcium. The polymerization profile of Haifa alpha-fibrin differed only modestly from that of normal alpha-fibrin, whereas that of Haifa beta-fibrin was markedly impaired. This finding plus similarities in the ultrastructure of Haifa and normal alpha-fibrin specimens suggests that the defective gamma chain structure of Haifa fibrinogen results in greater impairment of the carboxy terminal "b" polymerization domain reacting with the site exposed by cleavage of FPB ("B" site) than it does that of the carboxy terminal "a" domain reacting with the site exposed by cleavage of FPA ("A" site). Whether this effect is due to absolute differences in the degree of impairment of these two types of polymerization sites, or whether proper utilization of the "B" to "b" site is dependent upon participation of the "A" to "a" site remains to be determined.
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PMID:The polymerization of fibrin prepared from fibrinogen Haifa (gamma 275Arg----His). 251 77

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.
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PMID:Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper). 253 61

The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG-dependent manner. A first cistron sequence, inserted into the expression vector 5' to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1-42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.
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PMID:Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin. 264 71

Blood platelets are implicated in a series of cellular recognition and adhesion phenomena (adhesion to the subendothelial matrix and platelet aggregation) which are key events in the processes of haemostasis and thrombosis. Platelet aggregation is a model of homotypic cellular adhesion. It is mediated by the binding of bifunctional molecules of fibrinogen to the plasma membrane of adjacent platelets, following stimulation of the platelets by agonists such as ADP, thrombin or collagen, with the fibrinogen serving as an intercellular glue. The platelet receptor for fibrinogen is a macromolecular complex, GPIIb-IIIa, made of two transmembrane glycoproteins, GPIIb (Mr = 142,000) and GPIIIa (Mr = 99,000), assembled into a heterodimer whose conformation depends upon the binding of calcium (Ca2+) to GPIIb. Furthermore, the GPIIb-IIIa complex represents the prototype, and one of the most studied, among a large family of membrane receptors, the integrins, all implicated in various adhesion processes. Binding of fibrinogen to GPIIb-IIIa occurs through interactions between peptide sequences within the receptor and particular adhesive sites within the ligand: thus, GPIIIa can bind to a tetrapeptide sequence of the A alpha chain of fibrinogen, whereas a dodecapeptide of the gamma chain can be preferentially bound to GPIIb. On a physiopathological point of view, qualitative or quantitative defects of GPIIb-IIIa are associated with a rare haemorrhagic syndrome, Glanzmann' thrombasthenia. Its central role in platelet aggregation and thrombogenesis, together with a potential role in tumor cell-platelet interactions and in metastasis, make GPIIb-IIIa, nowadays, an important pharmacological target.
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PMID:[The platelet fibrinogen receptor: a model for the analysis of cellular adhesion mechanisms and their modification in pathology]. 269 68

A congenital dysfibrinogen characterized by impaired fibrin monomer polymerization was found in an asymptomatic 50-year-old woman and her two sons. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) run according to the method of Laemmli, we noticed two gamma chain species in fibrinogen and its plasmic fragments D1 and D2, consisting of a normal species and an apparently lower molecular weight (mol wt) variant in respective fractions. However, in fragment D3 only a single gamma chain remnant was observed. By chromatofocusing of the plasmic-CaCl2 digests of the abnormal fibrinogen, we separately isolated the normal and abnormal D1 species, the latter having been eluted in a slightly higher pH range. As expected, the abnormal D1 species failed to interfere with thrombin clotting of normal fibrinogen and normal fibrin monomer polymerization, whereas the normal D1 species inhibited them markedly. By analyzing the lysyl endopeptidase digests of the isolated gamma chain, we identified a replacement of aspartic acid by tyrosine at position 330 of the mutant gamma chain. The point mutation from an aspartic acid (pK for the beta-carboxyl = 3.86) to a tyrosine (pK for the aromatic hydroxyl = 10.07) may have perturbed the folding gamma chain structure in the D domain of fibrinogen specifically required for polymerization.
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PMID:Fibrinogen Kyoto III: a congenital dysfibrinogen with a gamma aspartic acid-330 to tyrosine substitution manifesting impaired fibrin monomer polymerization. 281 42


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