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

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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)

Previous work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3--30 minutes of incubation (Sas el al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor =X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into "activated" and "non activated" groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This aggreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.
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PMID:Spectrophotometric determination of factor Xa generation in factor IX concentrates. 57 33

Heterotrimeric factor VIIIa was reconstituted from isolated A2 subunit and A1/A3-C1-C2 dimer of thrombin-activated human factor VIII in a reaction that was sensitive to pH. Maximal levels of reconstituted factor VIIIa at pH 6.0 were as much as 20-fold greater than were values observed at pH 7.5. The presence of factor IXa and phospholipid resulted in a marked increase in factor VIIIa reconstituted at physiologic pH. However, the resultant factor VIIIa was unstable due to slow proteolysis of the A1 subunit. Factor IXa modified by the active site-specific reagent dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone (DEGR-IXa) increased the level of factor VIIIa reconstituted from subunits to a similar extent as was observed for unmodified factor IXa and yielded stable factor VIIIa. This enhancement was saturated above a 1:1 molar ratio of DEGR-IXa to factor VIIIa subunits and could be blocked by an anti-factor IX antibody, suggesting that the DEGR-IXa-dependent increase in factor VIIIa reconstitution correlated with assembly of the factor X-ase complex. At a saturating amount of DEGR-IXa, the level of factor VIIIa reconstitution at pH 7.5 approached values obtained at pH 6.0. Fluorescence polarization measurements indicated that factor VIIIa altered binding of DEGR-IXa to phospholipid. However, neither the A2 subunit nor the A1/A3-C1-C2 dimer alone produced this effect. This result suggested that both A2 and A1/A3-C1-C2 were necessary for association of the cofactor with factor IXa. These results suggest a model in which assembly of the intrinsic factor X-ase complex stabilizes factor VIIIa through inhibition of subunit dissociation.
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PMID:Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer. 174 Apr 24

Factor X Friuli was isolated from plasma by immunoaffinity and ion exchange chromatography and compared with normal factor X purified by the same method. Similar molecular weights were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the intact or activated factor X molecules including their respective heavy and light chains. These data indicated that there were no gross structural differences between the normal and variant proteins. Immunochemical assays employing either polyclonal or 46 monoclonal antibodies (MoAbs) did not reveal any structural deviations. Two-dimensional peptide maps indicated that while the light chains of normal and Friuli factor X were very similar, the heavy chains of the native and activated molecules contained a limited number of differences. These data suggested that the defect in factor X Friuli may be a point mutation which lies within the activated heavy chain defined by the 195-424 amino acid sequence. Activation of factor X Friuli in purified systems showed that Russell's viper venom cleaved the molecule at 70% of the normal rate, while the rate of proteolysis of the variant protein was reduced 98% and 75% when incubated with the extrinsic and intrinsic activation complexes, respectively. These data support the clinical laboratory findings and the hypothesis that the defect associated with the Friuli variant may reflect an abnormal interaction between factor X Friuli and the nonproteolytic cofactors of the extrinsic and intrinsic factor X activation complexes. Fluorescence polarization studies suggested that a bound dansylated inhibitor of factor Xa was not oriented to the same extent within the active site of the variant enzyme relative to normal factor Xa until the addition of phospholipid and factor Va. Activated factor X Friuli generated thrombin from prothrombin in a purified system, but at one third the normal rate that was attributed to the Kcat suggesting a secondary effect of this defect.
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PMID:Isolation and characterization of the factor X Friuli variant. 247 58

Fibrinopeptide A (FPA) levels as an indicator of thrombin activity in the cerebrospinal fluid (CSF) and plasma of 25 patients with subarachnoid haemorrhage (SAH) were measured serially by radioimmunoassay (RIA). FPA levels in CSF were extremely high on days 0-1 (1253 +/- 269 ng/ml, mean +/- standard error) but decreased rapidly (11.3 +/- 3.9 ng/ml on days 2-4, 10.7 +/- 5.9 ng/ml on days 5-7, and 6.3 +/- 1.5 ng/ml on days 8-14). In the controls the FPA concentration in CSF was 1.2 +/- 0.9 ng/ml (mean +/- standard deviation). Plasma FPA levels in patients with SAH showed no statistically significant changes with time. The bradykinin (BK) concentration in CSF and plasma in 27 patients with SAH was measured serially by RIA. The concentrations in CSF were 122.7 +/- 22.7 pg/ml (mean +/- standard error) on day 0, 38.6 +/- 6.1 pg/ml on day 1, 22.7 +/- 6.3 pg/ml on day 2, and 17.1 +/- 3.0 pg/ml or less thereafter. Plasma BK levels in patients with SAH were higher than those in the control group, but there was no statistically significant change over time. From the measurement of FPA it was apparent that the coagulation system in the subarachnoid space is strongly activated in the early stage of SAH. The formation of BK in CSF after SAH is thought to be due to the contact activation of Hageman factor (intrinsic factor) in the subarachnoid space. Trabeculae as collagen bundles in the subarachnoid space were considered to have a possible role in activating the Hageman factor of the coagulation system in SAH.
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PMID:Activation of the coagulation system in the subarachnoid space after subarachnoid haemorrhage: serial measurement of fibrinopeptide A and bradykinin of cerebrospinal fluid and plasma in patients with subarachnoid haemorrhage. 340 55

Activation of coagulation factor X by a complex of factors IXa-VIIIa and prothrombin by a complex of factor Xa.Va is markedly enhanced in the presence of a negatively-charged phospholipid surface. A suitable phospholipid surface is provided by a platelet lysate but not by a suspension of intact platelets, due to the internal localization of phosphatidylserine in the platelet membrane. Upon stimulation of platelets with a combination of collagen and thrombin, or calcium ionophore A23187 or treatment with diamide, alterations in the distribution of membrane phospholipids take place resulting in the exposure of significant amounts of phosphatidylserine at the platelet surface. As a consequence, an increased number of intrinsic factor X and prothrombinase complexes can be assembled at the platelet surface thus leading to an acceleration of factor Xa and thrombin formation. Studies with pathological platelets have shown that neither release nor aggregation are essential to provoke prothrombinase activity. The relatively high prothrombinase activity of non-stimulated Bernard-Soulier platelets is in agreement with the slightly altered phospholipid distribution in these platelets, in which more phosphatidylserine is exposed at the outer surface. Disturbances in the membrane bilayer structure as well as changes in the plasma membrane-cytoskeleton interaction are considered as possible explanations for the increased transbilayer movement of phosphatidylserine.
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PMID:Development of procoagulant binding sites on the platelet surface. 391 34

Factor VIII Leiden is a genetic variant of coagulation factor VIII which has been detected in the plasma of a patient with mild haemophilia A. In this patient's plasma factor VIII procoagulant antigen was in 5-fold excess over factor VIII procoagulant activity, indicating the presence of an abnormal factor VIII molecule. The variant factor VIII was isolated from the patient's plasma, and its functional properties were studied in a factor X-activating system consisting of purified components. The isolated factor VIII Leiden was normally activated by factor Xa and by thrombin, but the activity of the factor VIIIa was about 3% of normal. The defect of factor VIIIa Leiden was studied by comparison with normal factor VIIIa in kinetic experiments of factor Xa formation. The results support the hypothesis that factor VIIIa Leiden has a reduced affinity for phospholipid-bound factor IXa in the intrinsic factor X-activating complex.
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PMID:The functional defect of factor VIII Leiden, a genetic variant of coagulation factor VIII. 393 62

The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.naja phospholipase A2 almost completely abolished their activity in prothrombin and factor X activation. Since no significant cell lysis occurs during phospholipase treatment, this indicates that platelet phospholipids, exposed at the membrane exterior, play an essential role in the interaction of platelets with the proteins of the prothrombin and factor X-activating complexes. The time course of generation of the procoagulant platelet surface was different when the amount of coagulation factors present in the assay systems was varied. At suboptimal concentrations of coagulation factors, maximum platelet activity was reached after a shorter time period than at saturating concentrations. When measured at suboptimal amounts of coagulation factors, the platelet activity in prothrombin and factor X activation is also more sensitive to phospholipase treatment. Experiments with synthetic phospholipid mixtures show that prothrombin and factor X activation are optimal at low mol% phosphatidylserine when high concentrations of factor Va and factor VIIIa are employed. The optimal mol% phosphatidylserine increases when the concentrations of nonenzymatic protein cofactors are lowered. These findings are discussed in relation to a model in which phosphatidylserine, exposed at the outer surface of activated platelets, plays an essential role in prothrombin and factor X activation. It is proposed that this phosphatidylserine is not homogeneously distributed in the platelet outer membrane, but that areas with different phosphatidylserine density participate in coagulation factor activation.
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PMID:The role of activated human platelets in prothrombin and factor X activation. 396 85

The effect of activated protein C on human coagulation factor VIII was evaluated by studying its effect on the intrinsic factor X activation using a system of purified coagulation factors (factor IXa, factor X, factor VIII, activated protein C). Activated protein C had no effect on the activation of factor X by factor IXa in the absence of factor VIII. In the presence of thrombin activated factor VIII the rate of factor X activation was decreased by activated protein C in a dose dependent way. The presence of factor IXa during the preincubation of factor VIII with activated protein C was found to protect the factor VIII against inactivation. The results suggest that activated protein C and factor IXa compete for the same part of the factor VIII molecule.
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PMID:Factor IXa protects activated factor VIII against inactivation by activated protein C. 643 1

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII. 762 Jan 60

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein. 767 Jan 8


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