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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We evaluated the clinical usefulness of a recently developed semi-automated one-step chromogenic equivalent of activated partial thromboplastin time (APTT; Behring). This simple test is easily adaptable for automation. Generally, the results with this chromogenic one-step APTT were at least as precise as those obtained with comparative coagulometric methods. The chromogenic one-step APTT showed, both in vitro and in vivo, adequate sensitivity to congenital intrinsic factor deficiency but no sensitivity to Factor VII deficiency. Unlike a two-step coagulometric APTT (Dade), the one-step chromogenic APTT seemed sensitive to activation products of the contact system, which are present in immunoadsorbed factor-deficient plasma. The in vitro sensitivity of the chromogenic APTT to heparin was comparable with that of a coagulometric APTT, but the sensitivity to heparin in patients' samples differed slightly. The chromogenic APTT is relatively insensitive to anomalies in the fibrinogen-fibrin conversion. Finally, we observed discrepancies between the chromogenic and coagulometric APTT results for plasma of patients with disseminated intravascular coagulation. We conclude that this one-step chromogenic APTT warrants further evaluation for possible use as a routine test for the clinical laboratory.
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PMID:One-step chromogenic equivalent of activated partial thromboplastin time evaluated for clinical application. 185 96

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

Kinetic studies of prothrombin activation and intrinsic factor X activation carried out in the absence and presence of phospholipids and the protein cofactors Va or VIIIa have provided insight in the mechanism by which the accessory components enhance coagulation factor activation. In intrinsic factor X and prothrombin activation, phospholipids cause a drastic drop of Km for the substrates factor X and prothrombin, whereas the protein cofactors factor Va and factor VIIIa increase Vmax of the prothrombin- and factor X-activating reactions. The mode of action of factor Va in prothrombin activation is however somewhat more complex. Besides its stimulatory effect on the catalytic activity of factor Xa, Factor Va also plays an important role in the assembly of the prothrombin-activating complex at phospholipid surfaces especially when the latter have a low affinity for vitamin-K-dependent coagulation factors. This effect is likely accomplished by promoting the binding of both prothrombin and factor Xa to the procoagulant surface.
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PMID:Role of accessory components in the activation of vitamin K-dependent coagulation factors. 309 36

Mouse Lewis lung (LL) carcinoma cells possess a factor X activator (procoagulant) that is inhibited in vivo by warfarin treatment or diet-induced vitamin K deficiency. This inhibition suggests that vitamin-K-dependent proteins are involved in LL cell activation of factor X. A LL primary tumor clone (LL13) was isolated which contained a warfarin-sensitive vitamin-K cycle of metabolism and expressed factor X procoagulant activity. LL13 cells exposed to media containing warfarin or deficient in vitamin K grew as well as cells in normal media, and activated factor X to similar extents. In contrast, administration of warfarin to mice bearing LL13 cells inhibited factor X procoagulant activity as well as the vitamin K cycle of metabolism in the primary tumors. In relation to LL13 cells grown in media containing fetal bovine serum, those incubated for 20 hr in media containing mouse serum or the sera from LL13-bearing mice exhibited 9- to 10-times higher levels of factor X procoagulant activity. However, LL13 cells exposed to media containing the sera of warfarin-treated LL-13-bearing mice or to barium-sulfate-adsorbed normal mouse serum activated factor X much less efficiently. Collectively, these data suggest that inhibition of vitamin K function in LL cells does not affect the extent of factor X activation and thus the intrinsic factor X procoagulant is not a vitamin-K-dependent protein. They further suggest that both a warfarin-sensitive (vitamin-K-dependent) protein present in normal mouse serum and a LL13 cell component participate in factor X procoagulant activity.
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PMID:Evidence for a warfarin-sensitive serum factor that participates in factor X activation by Lewis lung tumor cells. 357 May 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 purpose of this study was to determine the effects of the dilution of plasma (1/3 in saline) on the kinetics of fibrin generation in the activated partial thromboplastin time (APTT) assay. The diluted APTT is considered to increase the sensitivity of the APTT test however, studies in our laboratory using an electro-optical fibrin detection system failed to show significant differences in APTT values obtained with diluted and undiluted canine plasma. Seventeen plasmas, including plasmas moderately and markedly deficient in intrinsic factor activity were assayed in the undiluted and diluted APTT assay using two methods for fibrin endpoint detection; a visual "tilt-tube" technique and an electro-optical detection system. In the former technique the endpoint was the formation of a visible fibrin web or clot; in the latter procedure the end point was the first detection of a change in optical density of the plasma. Optical density changes during fibrin formation were also recorded ( thrombokinetograms ). The results indicated that the electro-optical fibrin detection system failed to identify a prolongation of the APTT as a result of 1/3 plasma dilution; a prolongation that was consistently observed with the visual fibrin detection technique. Plasma dilution however, did significantly reduce the rate of fibrin production as indicated by the thrombokinetogram profile. It was concluded that the dilution of plasma with saline, as has been used to increase the sensitivity of the APTT assay procedure, has little effect on the time of onset of fibrin formation in a given plasma. The major effect appears to be on the way in which fibrin forms in that the polymerization/crosslinkage events associated with macroscopic fibrin production are delayed.
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PMID:The activated partial thromboplastin time of diluted plasma: variability due to the method of fibrin detection. 672 46

The intrinsic activation of human factor X has been studied in a system consisting of purified factors and in plasma. In both these systems factor Xa stimulated the activation of factor X by factor IXa plus factor VIII. This is due to the activation of factor VIII by factor Xa. When this factor Xa is formed via the extrinsic pathway, the extrinsic factor X activator functions as a stimulator of the intrinsic factor X activator.
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PMID:Activation of human coagulation factor VIII by activated factor X, the common product of the intrinsic and the extrinsic pathway of blood coagulation. 680 97

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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