UMLS:C0272170 - FACTA Search

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Query: UMLS:C0272170 (SDS)
50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A model system for the analysis of intracellular events governing the modification of individual vitamin K-dependent (VKD) proteins by the carboxylase has been developed using recombinant VKD protein-transfected cell lines. When untransfected 293 cells were analyzed by in vitro carboxylation followed by SDS-PAGE, endogenous VKD proteins were not detected. With 293 cells stably-transfected with recombinant native factor IX, most (> 95%) of the carboxylase was in complex with the factor IX, as assayed by adsorption of carboxylase activity to immobilized anti-factor IX antibody. In contrast, with 293 cells stably-transfected with recombinant factor IX deleted in the propeptide sequence (amino acids -18 to -4, delta pro factor IX), no association of factor IX with the carboxylase was observed. This observation was used to specifically isolate and identify the human carboxylase, and carboxylase-associated protein. When the carboxylase was purified from solubilized microsomes from either native factor IX, or delta pro factor IX, stably-transfected 293 cells, a single 98 kDa band was specifically obtained from native factor IX microsomes, but not from delta pro factor IX microsomes. This band was subsequently shown by Western and microsequencing analysis to comprise both the carboxylase and carboxylase-associated protein. This isolation, which represents the first isolation to near homogeneity of both the human carboxylase and the carboxylase from cell lines, will be valuable in isolating enzymatically active recombinant carboxylase, which has been refractile to other purification attempts. This system was also used to show that the human carboxylase in 293 cells is capable of binding and modifying two different liver-derived proteins. Protein C-producing 293 cells were generated from the same 293 progenitor cell line used to created the factor IX-expressing cells. With both factor IX- and protein C-transfected 293 cells, the secreted proteins were almost completely carboxylated, and in microsomes from each cell line the carboxylase was found in near quantitative complex with the two different VKD proteins. Thus the carboxylase modifies both VKD proteins. The approach described here for the analysis of the carboxylase from recombinant VKD protein-transfected cell lines should provide an important new system for studying protein carboxylation and VKD protein-carboxylase interaction.
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PMID:Isolation of the human gamma-carboxylase and a gamma-carboxylase-associated protein from factor IX-expressing mammalian cells. 867 78

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.
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PMID:Inhibition of coagulation factors by recombinant barley serpin BSZx. 884 56

The bactericidal activity of normal human serum (NHS) and treated NHS to avoid either the classical complement pathway (CPC) or the alternative complement pathway (APC) was studied with four strains identified as Acinetobacter baumanii. Three of them were sensitive to serum whereas only one was serum resistant. The serum sensitive strains showed different susceptibility mechanisms: one strain was sensitive to both the CPC and the APC and the others were sensitive only to APC. Serum sensitive and serum resistant strains showed on PAGE-SDS and silver strain incomplete profiles of lipopolysaccharides (LPS). In all cases the carbohydrate percentage of LPS was lower than the value corresponding to Salmonella enteritidis with complete profiles of LPS. The strain sensitive to CPC and APC had a lower carbohydrate content. The serum sensitivity of the three strains could be attributed to the presence of incomplete LPS, while the serum resistance could be assigned either to small variations in the LPS composition or to some particular membrane component.
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PMID:Behaviour of Acinetobacter strains with normal human serum. 888 57

Ecamulin, a novel prothrombin activating enzyme, has been isolated and purified 63-fold with a 57% yield from the venom of the Middle-Asian sand viper Echis multisquamatus using three-step ion-exchange chromatography. The enzyme was shown to activate prothrombin similarly to Ecarin, a prothrombin-converting enzyme from Echis carinatus venom, however, differing from the latter by structural and physico-chemical properties. The enzyme is a Zn-proteinase: it contains 1 mol Zn per 1 mol of protein. The molecular mass of the enzyme as determined by Sephacryl S-200 chromatography is 93 +/- 2 kDa. Upon SDS-PAAG electrophoresis ecamulin produces two bands with Mr of 67 and 27 kDa under non-reducing conditions, and three bands with Mr of 67, 14 and 13 kDa in the presence of DTT. During native PAGE without SDS, the activator yields one slow mobility band: two bands are observed after addition of DTT or EDTA. Carbohydrates containing N-acetyl-alpha-D-glucosamine residues are localized in the 67 kDa chain. Ecamulin has two isoforms, S2 and S3, that are distinguished by the charge and partial coagulation activities: form S2 has 250 NIH units/mg, while the S3 form has 524 NIH units/mg. The amino acid sequences of the both isoforms are similar but the more active S3 form has 4 times higher content of Gln and 4 times less of Gly than the S2 form. The isoelectric point is 4.3-4.5; E280 of 1% solution is 10.2. Forms S2 and S3 of ecamulin hydrolyze chromogenic substrates of plasma kallikrein S2302 and glandular kallikrein 2266. Ecamulin does not hydrolyze BAEE, TAME, LEE, thrombin substrates Chromozym TH and S2160, factor Xa-S2222, protein Ca-Chromozym PCa and Plasmin S2251. The amidase activity is nonreversibly inhibited by EDTA, o-phenanthroline (the activity is recovered by addition of Zn2+), Cys or DTT, EGTA, DFP, PMSF or pCMB do not inhibit the enzyme activity. Ecamulin converts prothrombin to alpha-thrombin passing by a shunt via the meizothrombin stage. The reaction of prothrombin activation does not require Ca2+, phospholipids of factor Va. Part of this work was presented at the International Conference "Fibrinogen and fibrinolysis", Yalta, September 23-28, 1995.
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PMID:[Isolation and characteristics of ekamulin--a prothrombin activator from multiscaled viper (Echis multisquamatus) venom]. 901 Dec 45

Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor V, there was no effect of heparin on inactivation of thrombin-activated factor Va by APC. Based on SDS-PAGE analysis, heparin enhanced the rate of proteolysis of factor V but not factor Va by APC. Coagulation assays using immunodepleted plasmas showed that the enhancement of heparin action by APC was independent of antithrombin III, heparin cofactor II, and protein S. Experiments using purified proteins showed that heparin did not inhibit factor V activation by thrombin. In summary, heparin and APC showed significant anticoagulant synergy in plasma due to three mechanisms that simultaneously decreased thrombin generation by the prothrombinase complex. These mechanisms include: first, heparin enhancement of antithrombin III-dependent inhibition of factor V activation by thrombin; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of membrane-bound factor V by APC, which is enhanced by heparin.
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PMID:Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C. 916 95

Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.
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PMID:Intrinsic specificity of the reactive site loop of alpha1-antitrypsin, alpha1-antichymotrypsin, antithrombin III, and protease nexin I. 919 29

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458, 468, 489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458-->Gln, Ser460-->Gly; Asn468-->Gln, Thr470-->Gly; Asn489-->Gln, Thr491-->Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of approximately 78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by alpha-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser480) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.
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PMID:The effect of N-linked glycosylation on molecular weight, thrombin cleavage, and functional activity of human protein S. 924 50

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with alpha-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.
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PMID:Human neutrophil elastase activates human factor V but inactivates thrombin-activated human factor V. 924 37

Platelet factor 4 (PF4) is an abundant platelet alpha-granule heparin-binding protein. We have previously shown that PF4 accelerates up to 25-fold the proteolytic conversion of protein C to activated protein C by the thrombin.thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the gamma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 binds to GAG+ but not GAG- forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcoreTM biosensor system confirms the extremely high affinity of PF4 for heparin (KD = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a KD of 31 nM and to protein C with a KD of 0.37 microM. In contrast, PF4 had no measurable interaction with GAG- thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG- domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin.thrombomodulin complex for protein C and thereby promote the generation of activated protein C.
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PMID:Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C. 939 24

The triglyceride (TG) concentration in plasma is an independent risk factor for coronary heart disease. There is evidence that TG-rich lipoprotein (TGRLP), ie, chylomicrons (CMs), chylomicron remnants (CMRs), and VLDLs associate with factor VII and prothrombin and that the association enhances a platelet factor Xa-mediated prothrombin activation when the CM-prothrombin complex is exposed to platelets. In this study, we examined the association of the vitamin K-dependent coagulation factors VII, IX, X, and prothrombin, as well as the anticoagulation protein C and its cofactor protein S, in plasma lipoproteins obtained from human fasting and postprandial plasma. We also analyzed some other proteins that are related to the coagulation system but not to vitamin K-dependent proteins, including factor V, serum amyloid P component (SAP), C4b binding protein (C4BP), and thrombomodulin (TM), and as a control, Ig G. Human TGRLP (d < 1.006 kg/L), LDL (d = 1.006 to 1.063 kg/L), and HDL (d = 1.063 to 1.210 kg/L) were separated from normal subjects both in fasting and 2 to 3 hours after the ingestion of a meal containing 100 g fat. The different coagulation proteins, SAP, C4BP, TM, and Ig G were determined by SDS-polyacrylamide gel electrophoresis combined with Western blotting, using specific polyclonal or monoclonal antibodies, and were visualized by peroxidase staining. All the vitamin K-dependent proteins associate with TGRLP in both fasting and postprandial plasma, but not with LDL or HDL. Factor V, SAP, TM, and Ig G were not found in any lipoprotein classes. C4BP, which is a regulatory protein of the classic pathway of the complement system and which binds protein S in vivo to regulate blood coagulation, was present in TGRLP, especially postprandial, but not in LDL or HDL. The amounts of prothrombin, protein S, and C4BP in postprandial TGRLP were larger than those in fasting TGRLP. Vitamin K-dependent procoagulation and anticoagulation proteins, as well as C4BP, could be associated with TGRLP in vivo. If the association enhances prothrombin activation, this effect may thus be counteracted by simultaneous binding of protein S.
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PMID:Association of vitamin K-dependent coagulation proteins and C4b binding protein with triglyceride-rich lipoproteins of human plasma. 944 53

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