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

---

Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution curves obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution - plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for the measurement of vitamin K-dependent protein S: the effect of antibody immunoreactivity on plasma protein S antigen determinations. 138 63

Vitamin K-dependent protein S is an anticoagulant plasma protein serving as cofactor to activated protein C in degradation of coagulation factors Va and VIIIa on membrane surfaces. In addition, it forms a noncovalent complex with complement regulatory protein C4b-binding protein (C4BP), a reaction which inhibits its anticoagulant function. Both forms of protein S have affinity for negatively charged phospholipids, and the purpose of the present study was to elucidate whether they bind to the surface of activated platelets or to platelet-derived microparticles. Binding of protein S to human platelets stimulated with various agonists was examined with FITC-labeled monoclonal antibodies and fluorescence-gated flow cytometry. Protein S was found to bind to membrane microparticles which formed during platelet activation but not to the remnant activated platelets. Binding to microparticles was saturable and maximum binding was seen at approximately 0.4 microM protein S. It was calcium-dependent and reversed after the addition of EDTA. Inhibition experiments with monoclonal antibodies suggested the gamma-carboxyglutamic acid containing module of protein S to be involved in the binding reaction. An intact thrombin-sensitive region of protein S was not required for binding. The protein S-C4BP complex did not bind to microparticles or activated platelets even though it bound to negatively charged phospholipid vesicles. Intact protein S supported binding of both protein C and activated protein C to microparticles. Protein S-dependent binding of protein C/activated protein C was blocked by those monoclonal antibodies against protein S that inhibited its cofactor function. In conclusion, we have found that free protein S binds to platelet-derived microparticles and stimulates binding of protein C/activated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles. 146 47

Protein S and C4b-binding protein (C4BP) form a tight complex (Kd approximately 0.6 nM) the physiologic purpose of which is unknown. The participation of protein S in this complex was investigated using site-specific mutagenesis. Normal recombinant human protein S (rHPS) and five specifically mutated protein S analogs were expressed in transformed human kidney 293 cells and the following properties were characterized: solution-phase C4BP binding, ability to be cleaved by thrombin, ability to act as a cofactor in the activated protein C-catalyzed inactivation of factor Va, and gamma-carboxyglutamic acid content. In some cases, beta-hydroxyaspartic acid plus beta-hydroxyasparagine content was also determined. Binding studies indicated that while clearly important for a high affinity interaction, the amino acid sequence Gly605-Ile614 identified by Walker (Walker, F J. (1989) J. Biol. Chem. 264, 17645-17648) does not account for all the binding energy of the HPS-C4BP interaction. All mutants perturbed in this region or lacking it altogether displayed reduced C4BP binding, and some retained anticoagulant cofactor function. Neither human factor X nor human steroid-binding protein had any measurable ability to compete with plasma HPS for C4BP binding. Furthermore, bovine protein S and a rHPS analog with bovine sequence from Gly597-Trp629 bound to human C4BP with the same affinity as did HPS, and both proteins substituted effectively for HPS as a cofactor for activated protein C in an otherwise human anticoagulation system. Together these results suggest that optimal binding of protein S to C4BP requires the putative alpha-helix Gly605-Ile614, as well as other undetermined regions of protein S, and that the regions of HPS responsible for C4BP binding and activated protein C cofactor function are structurally isolated.
...
PMID:Binding of protein S to C4b-binding protein. Mutagenesis of protein S. 153 19

We measured concentrations of the natural anticoagulant protein C; its cofactor, protein S; and the carrier protein C4b-binding protein (C4BP), in 24 patients with severe infection and 13 with septic shock. Decreased antithrombin III levels were found in 16 of 24 infection patients and all shock patients; high thrombin-antithrombin (TAT) complexes were present in 16 of 24 infection and 12 of 13 shock patients. Protein C concentrations were significantly reduced compared to healthy blood donors, to 60 +/- 14% (infection) and 47 +/- 20% (septic shock) (mean +/- 1 SD). Total protein S levels were not reduced (119 +/- 36.7 and 88 +/- 20.0%, normal value 96 +/- 15%). Free protein S was also normal (27 +/- 9.4 and 30 +/- 8.7%, normal value 29 +/- 9%). The percentage free of total protein S was normal in shock patients (35 +/- 8.5%), but significantly reduced in patients without shock (23 +/- 5.3%). C4BP was significantly higher than normal in the latter group (135 +/- 43%), but not in the shock group (118 +/- 40%), possibly due to increased consumption. Thus, no deficiency of total or free protein S was found in these patients, who had evidence of activated coagulation but no clinical DIC.
...
PMID:Protein C, protein S and C4b-binding protein in severe infection and septic shock. 182 15

The protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF) epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 gamma-carboxy glutamic acid residues in the vitamin K-dependent domain, a beta-hydroxylated aspartic acid in the first EGF-like domain and a beta-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical alpha-chains and one beta-chain. The alpha- and beta-chains are linked by disulphide bridges. The cDNA cloning of the beta-chain showed the alpha- and beta-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the beta-chain to contain the single protein S binding site on C4BP, whereas each of the alpha-chains contains a binding site for the complement protein, C4b. As C4BP lacking the beta-chain is unable to bind protein S, the beta-chain is required for protein S binding, but not for the assembly of the alpha-chains during biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Protein S and C4b-binding protein: components involved in the regulation of the protein C anticoagulant system. 183 51

Among the vitamin K-dependent plasma proteins, only protein S contains the post-translationally modified amino acid erythro-beta-hydroxyasparagine (Hyn). Protein S also contains erythro-beta-hydroxyaspartic acid (Hya). The function of these unusual amino acids, located in the epidermal growth factor-like domains, is unknown. To determine if these post-translational modifications contribute to the functional integrity of human protein S (HPS), recombinant human protein S lacking Hya and Hyn (rHPSdesHya/Hyn) was purified from the medium of human kidney 293 cells that were transfected with HPS cDNA and grown in the presence of the hydroxylase inhibitor 2,2'-dipyridyl. Solution-phase equilibrium binding studies revealed that rHPSdesHya/Hyn binds C4b-binding protein (C4BP) in a manner indistinguishable from recombinant HPS and plasma-derived HPS, exhibiting a Kd in the presence of 2 mM CaCl2 of approximately 0.7 nM and a Kd in the presence of 4 mM EDTA approximately 10-fold higher. In a purified component system, rHPSdesHya/Hyn displayed normal anticoagulant cofactor activity in the activated protein C-catalyzed inactivation of coagulation factor Va bound in the prothrombinase complex. In addition, digestion of rHPSdesHya/Hyn with thrombin in the presence of EDTA appeared normal, and 2 mM CaCl2 prevented the cleavage. Together these results suggest that the post-translational modifications of Asn and Asp residues are not necessary for the macromolecular or Ca2+ interactions associated with the anticoagulant and C4BP binding characteristics of HPS.
...
PMID:beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein. 183 48

Protein S is an anticoagulant vitamin-K-dependent plasma protein functioning as a cofactor to activated protein C in the degradation of factors Va and VIIIa. A murine monoclonal antibody, HPS 7, specific for a calcium-stabilized epitope in human protein S, is described. The epitope was available in intact protein S, both in its free form and when protein S was bound to C4b-binding protein. It disappeared upon reduction of disulfide bridges and also after thrombin of chymotrypsin cleavage of protein S. Thrombin cleaves protein S close to the calcium-binding region containing gamma-carboxyglutamic acid (Gla). The cleaved protein still contains the Gla region, linked by a disulfide bridge, but it has a lower affinity for calcium and no protein C cofactor activity. The thrombin-mediated cleavage of protein S could be inhibited by HPS 7. The Ka for the interaction between protein S and the monoclonal was estimated to be approximately 0.7 X 10(8) M-1. Half-maximal binding between HPS 7 and protein S was observed at a calcium concentration of 0.50 mM, indicating that saturation of the Gla region with calcium was required for the interaction. The recently reported Gla-independent high-affinity calcium binding did not induce the epitope. The calcium-dependent binding of protein S to phospholipid vesicles as well as the protein C cofactor activity was inhibited by HPS 7. The data suggests that the epitope for HPS 7 is located in the Gla region of protein S or in the closely positioned thrombin-sensitive region.
...
PMID:Inhibition of human vitamin-K-dependent protein-S-cofactor activity by a monoclonal antibody specific for a Ca2+-dependent epitope. 243 12

The profile of blood coagulation and fibrinolysis was studied in detail in eight patients with acute thrombotic thrombocytopenic purpura (TTP). In the majority of the patients, fibrinogen, factor XIII, antithrombin III, alpha 2-plasmin inhibitor, plasminogen, and alpha 2-macroglobulin were normal, whereas FDP, plasmin-alpha 2-plasmin inhibitor complex, and tissue-type plasminogen activator antigen were marginally or moderately elevated. Low fibronectin values were observed in four patients. Protein C and C4b-binding protein were nearly normal, whereas total protein S and free protein S were reduced in five and six patients, respectively. A positive correlation was found between total protein S and C4 and between free protein S and C3. von Willebrand factor antigen (vWf:Ag) and ristocetin cofactor (RCof) were either normal or elevated, but RCof/vWf:Ag ratio was decreased in seven patients. Crossed immunoelectrophoresis and sodium dodecyl sulfate (SDS)-agarose gel electrophoresis revealed that the large vWf multimers were either absent from or relatively decreased in all patients except one. In addition, one patient had unusually large vWf multimers, and a low-molecular-weight vWf fragment was apparently observed in three patients. These findings indicate that the intravascular generation of thrombin and plasmin was minimal in TTP and suggest that the alterations of the vWf molecule were caused not only by consumption through its participation in platelet thrombus formation but also by accelerated proteolysis. Low protein S values would be related to the immunological abnormalities underlying TTP.
...
PMID:Coagulation studies in thrombotic thrombocytopenic purpura, with special reference to von Willebrand factor and protein S. 252 Dec 76

Four mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/l CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate adsorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/l CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Monoclonal antibodies directed to the calcium-free conformation of human protein S. 253 Jun 47

C4b-binding protein, C4bp, is a regulatory factor of the complement system and is also known to be a binding protein of vitamin K-dependent coagulation factor, protein S. Whereas the C4b-binding site is known to be located in the middle part of the subunit chains of C4bp, the location and properties of protein S-binding site are uncertain. Therefore, we have examined the characteristics of the interaction between human protein S and C4bp. Proteolysis of C4bp-protein S complex with chymotrypsin yielded N-terminal-derived 48-kDa fragments of C4bp subunit chains and a C-terminal-derived 160-kDa core fragment of C4bp, to which protein S was still bound. This result suggested that the protein S-binding site is located in the core domain of C4bp. Gel filtration of guanidine-treated C4bp-protein S complex in the absence of guanidine resulted in the separation of C4bp and protein S. Binding assay with 125I-labeled protein S showed that the guanidine-treated C4bp lacked the protein S-binding activity. This result suggests that the protein S-binding site in C4bp is denatured irreversibly by guanidine treatment and therefore seems to be dependent on a specific conformation of C4bp. The C4bp-binding site of protein S was lost upon thrombin treatment, suggesting that the N-terminal thrombin-sensitive region of protein S may be related to the C4bp-binding site. Although free protein S was susceptible to chymotrypsin, leukocyte elastase, and cathepsin G, C4bp-bound protein S was found to be resistant to these proteases.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Characterization of the interaction between human protein S and C4b-binding protein (C4bp). 296 95


1 2 3 4 5 Next >>

---