Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adult respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC) are serious complications of sepsis. Thrombomodulin, an important endothelial anticoagulant, binds thrombin to generate activated protein C (APC). To determine whether thrombomodulin purified from human urine (urinary thrombomodulin, UTM) is useful for the treatment of DIC and ARDS in sepsis, we examined the effect of UTM on endotoxin (ET)-induced coagulation abnormalities and pulmonary vascular injury in rats. Intravenous administration of UTM prevented the ET-induced pulmonary accumulation of leukocytes and the increase in pulmonary vascular permeability, as well as ET-induced histological changes such as leukocyte infiltration and pulmonary interstitial edema. On the other hand, dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, did not prevent these effects of ET. UTM did not prevent ET-induced pulmonary accumulation of leukocytes and pulmonary vascular injury in rats pretreated with DEGR-Xa. Our findings suggest that UTM attenuates ET-induced coagulation abnormalities and pulmonary vascular injury. Furthermore, the latter effect may be dependent on the capacity of UTM to activate protein C.
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PMID:Effect of human urinary thrombomodulin on endotoxin-induced intravascular coagulation and pulmonary vascular injury in rats. 903 85

Human protein S (HPS) is a vitamin K dependent plasma glycoprotein involved in the regulation of activated protein C and possibly fibrinolysis. Its c-DNA sequence shows three N-glycosylation consensus sequences (Asn-X-Ser/Thr). In order to study influence of N-linked glycosylation on HPS function, set of mutants of HPS was constructed. Mutants were generated, starting from an SV40/Adeno derived pD5HPS2 expression vector, using PCR enabled, site specific methodology. They included single amino acid substitutions at each of three N-glycosylation consensus sequences: Asn458-->Gln, Ser460-->Gly, Asn468-->Gln, Thr470-->Gly, Asn489-->Gln, Thr491-->Gly. Variant HPS were expressed in stable 293 human kidney cell lines in the presence of vitamin K1 (we did not succeed in expressing variant Asn489-->Gln) and purified from conditioned media using pseudoaffinity chromatography on QAE-Sepharose. Variant Asn468-->Gln showed decreased gamma-carboxyglutamate content. All of the mutants were active in a clotting type assay based on factor Va inactivation, and they were compared to wt-HPS and plasma HPS. In conclusion, we have constructed, expressed and purified set of HPS mutants useful in studying the role of N-glycosylation in HPS function.
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PMID:Construction, expression and preliminary characterization of glycosylation mutants of human protein S. 911 51

Temperature and salt dependence studies of thrombin interaction with thrombomodulin, with and without chondroitin sulfate, and two fragments containing the EGF-like domains 4-5 and 4-5-6 reveal the energetic signatures and the mechanism of recognition of this physiologically important cofactor. Binding of thrombomodulin is affected drastically by the particular salt present in solution and is positively linked to Na+ binding to thrombin and the conversion of the enzyme from the slow to the fast form, but is opposed by Cl- binding to the fibrinogen recognition site and especially to the heparin binding site. Binding of thrombomodulin has an unusually large salt dependence (gamma(salt) = -4.8) contributed mostly by the polyelectrolyte-like nature of the chondroitin sulfate moiety that binds to the heparin binding site and increases the affinity of the cofactor by almost 10-fold. On the other hand, the chondroitin sulfate has no effect on the deltaCp of binding, which is determined predominantly by contacts made by the EGF-like domains 5 and 6 with the fibrinogen recognition site. The modest heat capacity change (-0.2 kcal mol(-1) K(-1)) observed when thrombomodulin binds to the fast form suggests a rigid-body association of the cofactor with the enzyme. In the slow form, however, the heat capacity change is significantly more pronounced (-0.5 kcal mol(-1) K(-1)) and signals the presence of a conformational transition of the enzyme linked to binding of the cofactor that mimics the slow-->fast conversion. These results demonstrate that recognition of thrombomodulin by thrombin is steered electrostatically by the highly charged regions of the fibrinogen recognition site and the heparin binding site, to which the chondroitin sulfate moiety binds and enhances the affinity of the interaction. The recognition event also involves conformational changes of the enzyme in the slow form mediated by binding of the EGF-like domains 5-6 to the fibrinogen recognition site. Consistent with this model, binding of thrombomodulin to the fast form has only a small effect on the hydrolysis of nine chromogenic substrates carrying substitutions at P1, P2, and P3 aimed at probing the environment of the specificity sites S1, S2, and S3 of the enzyme. Binding to the slow form, on the other hand, enhances the specificity toward all substrates up to 15-fold. For substrates carrying a Gly at P2, binding of thrombomodulin changes the relative specificity of the slow and fast forms and makes the slow form more specific. Interestingly, these effects are not specific of thrombomodulin and depend solely on binding to the fibrinogen recognition site of the enzyme. In fact, they are also observed with the hirudin C-terminal fragment 55-65. The characterization of the mechanism of thrombin-thrombomodulin interaction and the effects of the cofactor on the hydrolysis of chromogenic substrates probing the interior of the catalytic pocket bear on the thrombomodulin-induced enhancement of protein C cleavage by thrombin. We propose that this enhancement is due predominantly to an effect of thrombomodulin on the bound protein C in the ternary complex. Therefore, thrombomodulin would carry out its physiological function by making protein C a better substrate for thrombin, rather than making thrombin a better enzyme for protein C.
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PMID:Energetics of thrombin-thrombomodulin interaction. 918 47

A recent study indicated that Tyr99 (chymotrypsin numbering) of factor Xa and Thr99 of activated protein C are S2 subsite residues that determine the P2 specificity of their substrates and inhibitors. To investigate the contribution of Leu99 to the P2 binding specificity of thrombin, three mutants of thrombin were prepared in which Leu99 was substituted with Tyr (L99Y), Thr (L99T), or Gly (L99G). Kinetic analysis indicated that antithrombin (AT with P2 Gly) inhibited thrombin L99Y, 14.1- and 5.5-fold slower than thrombin in the absence and presence of heparin, respectively. The L99Y mutation increased the stoichiometry of AT inhibition in the presence of heparin from approximately 1.6 to approximately 4.6, indicating that L99Y recognized AT as a substrate. The inhibition rates of L99T and L99G by AT, respectively, were 500.0- and 916.7-fold slower than thrombin in the absence of heparin but only 41.8- and 64.5-fold slower than thrombin in the presence of heparin. Resolution of the two-step reactions of AT with the mutant thrombins revealed that the impaired reactivities occurred in the second reaction step in which a non-covalent AT-thrombin encounter complex is converted to a stable, covalent complex. In reactions with protein C inhibitor (PCI with P2 Phe), L99Y was inhibited 3.5-fold slower than thrombin, L99T was inhibited at a similar or faster rate, and L99G was inhibited 23.9-fold faster than thrombin. The epidermal growth factor-like domains 4-6 of thrombomodulin (TM4-6) accelerated the PCI inhibition of wild-type and L99G thrombins 73.9- and 5.3-fold, respectively. Further studies indicated that the fibrinogen clotting and protein C activation rates by the mutants were impaired, but the cofactor function of TM was not affected as TM4-6 bound to wild-type [Kd(app) = 5.9 nM] and mutant thrombins with similar affinities [Kd(app) = 4.4-6.9 nM] and enhanced protein C activation rates by all mutants effectively. These results indicate that (1) Leu99 of thrombin is critical for determination of the P2 specificity of serpins, AT and PCI, (2) increasing the polarity of the S2 pocket of thrombin by introduction of a hydrophilic residue into this pocket is detrimental for reaction with AT, but it is tolerated in reaction with PCI, so that only the size of the S2 pocket of thrombin determines the P2 specificity of PCI, and (3) the thrombomodulin-induced conformational change that results in acceleration of thrombin inhibition by PCI involves Leu99.
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PMID:Role of Leu99 of thrombin in determining the P2 specificity of serpins. 920 Jun 92

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

We have analyzed 83 unrelated Hong Kong Chinese for the presence of genetic variants of factor V gene. Forty-three of them had a history of deep vein thrombosis. The DNA sequence variations of exons 7, 10, and 13, where the codons for Arg306, Arg506, and Arg679 are located, respectively, were studied by denaturing gradient gel electrophoresis. The G1691-->A (Arg 506-->Gln) mutation in exon 10 was not detectable in any of the 83 subjects. However, a high allelic frequency for the G1628-->A (Arg 485-->Lys) substitution was detectable in the same exon. We have also identified a novel DNA sequence mutation (A1090-->G) in exon 7 that resulted in Arg 306-->Gly substitution in 2 thrombotic patients and 1 nonthrombotic subject. Fresh blood samples were available from one of them for analysis of activated protein C resistance and the result was negative. Variation of DNA sequence was not found in exon 13 in any of our 83 subjects. The results of this study showed that, although the Arg 506-->Gln mutation was rarely found in the Hong Kong Chinese population, a different mutation site such as A 1090-->G in exon 7 of the factor V gene (Arg 306) may be of clinical importance.
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PMID:A novel mutation of Arg306 of factor V gene in Hong Kong Chinese. 945 41

Human factor Xa specifically cleaves the anticoagulant protein S within the thrombin-sensitive domain. Amino-terminal amino acid sequencing of the heavy chain cleavage product indicates cleavage of protein S by factor Xa at Arg60, a site that is distinct from those utilized by alpha-thrombin. Cleavage by factor Xa is unaffected by the presence of hirudin and is completely blocked by tick-anticoagulant-peptide and D-Glu-Gly-Arg-chloromethyl ketone, the latter two being specific inhibitors of factor Xa. The cleavage requires the presence of phospholipid and Ca2+, and is markedly inhibited by the presence of factor Va. Factor Xa-cleaved protein S no longer possesses its activated protein C-dependent or -independent anticoagulant activity, as measured in a factor VIII-based activated partial thromboplastin time clot assay. The apparent binding constant for protein S binding to phospholipid (Kd approximately 4 nM +/- 1.0) is unaffected by factor Xa or thrombin cleavage, suggesting that the loss of anticoagulant activity resulting from cleavage is not primarily due to the loss of membrane binding ability. Cleavage and inactivation of protein S by factor Xa may be an additional way in which factor Xa exerts its procoagulant effect, after the initial stages of clot formation.
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PMID:Human protein S cleavage and inactivation by coagulation factor Xa. 956 66

Factor Va inactivation by activated protein C is associated with cleavages at Arg306, Arg506, and Arg679 with Arg306 cleavage causing the major activity loss. To study functional roles of the Arg306 region, overlapping 15-mer peptides representing the sequence of factor Va residues 271-345 were synthesized and screened for anticoagulant activities. The peptide containing residues 311-325 (VP311) noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution with a Kd of 70 microM. Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa or prothrombin bound to immobilized VP311 with relatively high affinity. These results support the hypothesis that residues 311-325, which are positioned between the A1 and A2 domains of factor Va and likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by activated protein C at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311-325, thereby down-regulating the binding of factor Xa to factor Va.
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PMID:Binding site for blood coagulation factor Xa involving residues 311-325 in factor Va. 961 93

Snake venom toxins are now regularly used in the coagulation laboratory for assaying haemostatic parameters and as coagulation reagents. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen and fibrinogen breakdown product assay as well as detecting dysfibrinogenaemias. Significantly, because SVTLE are not inhibited by heparin, they can be used for defibrinating samples that contain the anticoagulant before assay of haemostatic variables. Prothrombin activators are found in many snake venoms and are used in prothrombin assays, for studying dysprothrombinaemias and preparing meizothrombin and non-enzymic prothrombin. Russell's viper (Daboia russelli) venom (RVV) contains a number of compounds useful in the assay of factors V, VII, X, platelet factor 3 and lupus anticoagulants. Activators from the taipan, Australian brown snake and saw-scaled viper have been used to assay lupus anticoagulants. Protein C and activated protein C resistance can be measured by means of RVV and Protac, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with Botrocetin from Bothrops jararaca venom. Finally, phospholipase A2 enzymes and the disintegrins, a family of Arg-Gly-Asp (RGD)-containing proteins found in snake venoms, show great potential for the study of haemostasis including, notably, platelet glycoprotein receptors GPIIb/IIIa and Ib.
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PMID:Use of snake venom fractions in the coagulation laboratory. 971 87

Snake venoms are complex mixtures containing many different biologically active proteins and peptides. A number of these proteins interact with components of the human hemostatic system. This review is focused on those venom constituents which affect the blood coagulation pathway, endothelial cells, and platelets. Only highly purified and well characterized snake venom proteins will be discussed in this review. Hemostatically active components are distributed widely in the venom of many different snake species, particularly from pit viper, viper and elapid venoms. The venom components can be grouped into a number of different categories depending on their hemostatic action. The following groups are discussed in this review: (i) enzymes that clot fibrinogen; (ii) enzymes that degrade fibrin(ogen); (iii) plasminogen activators; (iv) prothrombin activators; (v) factor V activators; (vi) factor X activators; (vii) anticoagulant activities including inhibitors of prothrombinase complex formation, inhibitors of thrombin, phospholipases, and protein C activators; (viii) enzymes with hemorrhagic activity; (ix) enzymes that degrade plasma serine proteinase inhibitors; (x) platelet aggregation inducers including direct acting enzymes, direct acting non-enzymatic components, and agents that require a cofactor; (xi) platelet aggregation inhibitors including: alpha-fibrinogenases, 5'-nucleotidases, phospholipases, and disintegrins. Although many snake venoms contain a number of hemostatically active components, it is safe to say that no single venom contains all the hemostatically active components described here. Several venom enzymes have been used clinically as anticoagulants and other venom components are being used in pre-clinical research to examine their possible therapeutic potential. The disintegrins are an interesting group of peptides that contain a cell adhesion recognition motif, Arg-Gly-Asp (RGD), in the carboxy-terminal half of their amino acid sequence. These agents act as fibrinogen receptor (integrin GPIIb/IIIa) antagonists. Since this integrin is believed to serve as the final common pathway leading to the formation of platelet-platelet bridges and platelet aggregation, blockage of this integrin leads to inhibition of platelet aggregation regardless of the stimulating agent. Clinical trials suggest that platelet GPIIb/IIIa blockade is an effective therapy for the thrombotic events and restenosis frequently accompanying cardiovascular and cerebrovascular disease. Therefore, because of their clinical poten tial, a large number of disintegrins have been isolated and characterized.
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PMID:Snake venoms and the hemostatic system. 983 63


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