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

The hemophilia A mutation database lists more than 160 missense mutations: each represents a molecular defect in the FVIII molecule, resulting in the X-linked bleeding disorder hemophilia A with a clinical presentation varying from mild to severe. Without a three-dimensional FVIII structure it is in most cases impossible to explain biological dysfunction in terms of the underlying molecular pathology. However, recently the crystal structure of the homologous human plasma copper-binding protein ceruloplasmin (hCp) has been solved, and the A domains of FVIII share approximately 34% sequence identity with hCp. This advance has enabled the building of a molecular model of the A domains of FVIII based on the sequence identity between the two proteins. The model allows exploration of predictions regarding the general features of the FVIII molecule, such as the binding-sites for factor IXa and activated protein C; it has also allowed the mapping of more than 30 selected mutations with known phenotype from the database, and the prediction of hypothetical links to dysfunction in all but a few cases. A computer-generated molecular model such as that reported here cannot substitute for a crystal structure. However, until such a structure for FVIII becomes available, the model represents a significant advance in modeling FVIII; it should prove a useful tool for exploiting the increasing amount of information in the hemophilia A mutation database, and for selecting appropriate targets for investigation of the structure-function relationships via mutagenesis and expression in vitro.
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PMID:A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin. 911 85

To find if there is a relation between levels of haemostatic variables at low and high hormonal levels (oestradiol and progesterone) in an individual, blood samples were drawn from 12 women repeatedly during one menstrual cycle (Study I) and from 14 women undergoing in vitro fertilization, before hormonal stimulation and daily during the periovulatory period (Study II). Regression coefficients were calculated between minimum (independent) and maximum (dependent) values in both studies. In Study II highly significant regression coefficients were found between oestradiol minimum (pretreatment) and maximum (median 105 and 4730 pmol/l, respectively) for coagulation factors FVIII, von Willebrand Factor (antigen), FVII (activity and antigen), fibrinogen, protein C, protein S (free), antithrombin, plasminogen and plasminogen activator inhibitor-1; furthermore, between progesterone-minimum at day -3 or -2 (related to ovum pick up) and maximum (median 4.7 and 98 nmol/l, respectively) for FVIII, von Willebrand Factor, FVII (activity and antigen), protein C, protein S (free), and plasminogen. In Study I, where much lower hormonal levels were obtained at maximum (oestradiol median 297 pmol/l and progesterone 47 nmol/l), the same pattern was observed especially for FVII, FX, fibrinogen, plasminogen and plasmin inhibitor. Thus, the concentration of a haemostatic variable at a low oestradiol or progesterone level can predict the level at a high hormonal level.
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PMID:Prediction of changes in levels of haemostatic variables during natural menstrual cycle and ovarian hyperstimulation. 918

Activated Protein C (APC) resistance, one of the most common genetic risk factors for venous thrombosis, is caused by a single base mutation (G1691-->A) in the factor V (FV) gene resulting in the replacement of Arg506 by Gln at a predominant cleavage site for APC. Great progress in understanding the mechanism of downregulation of FVa activity via the protein C pathway has been achieved by studying APC-mediated inactivation of FVa purified from homozygous APC-resistant individuals. This review briefly summarizes the role of FVa in prothrombin activation and the structure-function relationship of FV and FVa. Subsequently, APC-dependent inactivation of FVa and FVa Leiden and its modulation by protein S and factor Xa in model systems containing purified proteins is discussed. FV also has a function in increasing the inactivation of FVIII/VIIIa by APC. This cofactor activity appears diminished in FV Leiden. Thus, an intricate mechanism of regulation of thrombin formation via the protein C pathway is starting to emerge. Extensive studies in plasma milieu will be needed to gain more insight into the relation between the presence of FV Leiden and impaired downregulation of thrombin formation in APC-resistant individuals.
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PMID:Regulation of thrombin formation by activated protein C: effect of the factor V Leiden mutation. 924 9

We have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the Al subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1(336)/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.
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PMID:Interaction of the A1 subunit of factor VIIIa and the serine protease domain of factor X identified by zero-length cross-linking. 975 21

High FVIII:C levels have previously been shown to be an independent risk factor for thrombosis with 4.8 times higher potential risk of thrombosis in individuals with FVIII:C levels greater than 1.5 u/ml. Recently, we found that raised FVIII:C levels are largely attributable to elevated FVIII:Ag levels. The determinants of FVIII:Ag levels are unclear and might be partly genetic. The promoter of the F8 gene has recently been characterised we therefore investigated the promoter and the 3' terminus of the F8 gene for possible polymorphisms associated with raised FVIII:Ag levels in 62 selected individuals with a thrombotic tendency. We confirm previous reports that raised FVIII:C levels are largely attributable to an elevation in FVIII:Ag and this is also associated with elevation of vWF; non-O blood group: relatively short APTT and relatively low APC ratio. We screened 1140 bp of the proximal promoter including the protein binding sites identified by DNase I footprint analysis by SSCP, however no polymorphisms were identified. Direct DNA sequence analysis of the region -542 to +165 failed to identify any sequence polymorphisms. The recently described polymorphism in the polyadenylation cleavage site in the prothrombin gene associated with increased prothrombin activity prompted us to screen the region surrounding the 3' terminus of the F8 gene for polymorphisms but we found none.
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PMID:Analysis of the F8 gene in individuals with high plasma factor VIII: C levels and associated venous thrombosis. 979 69

Three model systems have been used to study the dynamics of the blood clotting process initiated by tissue factor (TF): synthetic plasma mixtures prepared with purified coagulation proteins and inhibitors; mathematical models based on the reaction constants, stoichiometries and thermodynamics of individual catalyst and inhibitor reactions; and contact suppressed whole blood induced to clot in vitro by the addition of exogenous TF. In the three models, the generation of thrombin can be described in terms of an initiation phase in which pmol/l concentrations of the coagulation serine proteases are generated and the cofactor proteins factor V (FV) and FVIII are activated. Subsequently, explosive thrombin generation occurs during a propagation phase. The complementary inhibitory pathways extinguish the generation of thrombin. Tissue factor pathway inhibitor (TFPI), present in low concentrations, primarily influences the duration of the initiation phase and has little influence on the propagation phase. Antithrombin III (ATIII), present in higher concentrations, has little influence during the initiation phase, but decreases the rate of thrombin generation during the propagation phase. The protein C pathway cannot act in the absence of thrombin and therefore only influences the duration of the propagation phase by inactivating activated FV. Thus combinations of TFPI plus ATIII and TFPI plus protein C pathway components contribute to the synergistic inhibitory processes. As a consequence of the roles of pro, and anti-coagulants, the generation of thrombin by the TF pathway becomes a threshold limited process.
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PMID:The role of the tissue factor pathway in initiation of coagulation. 981 22

Activated protein C (APC) resistance caused by the factor V Leiden mutation is associated with an increased risk of venous thrombosis. We investigated whether a reduced response to APC, not due to the factor V point mutation, is also a risk factor for venous thrombosis. For this analysis, we used the Leiden Thrombophilia Study (LETS), a case-control study for venous thrombosis including 474 patients with a first deep-vein thrombosis and 474 age- and sex-matched controls. All carriers of the factor V Leiden mutation were excluded. A dose-response relationship was observed between the sensitivity for APC and the risk of thrombosis: the lower the normalized APC sensitivity ratio, the higher the associated risk. The risk for the lowest quartile of normalized APC-SR (<0.92), which included 16.5% of the healthy controls, compared with the highest quartile (normalized APC-SR > 1.05) was greater than fourfold increased (OR = 4.4; 95% confidence interval, 2.9 to 6.6). We adjusted for VIII:C levels, which appeared to affect our APC resistance test. The adjusted (age, sex, FVIII:C) odds ratio for the lowest quartile was 2.5 (95% confidence interval, 1.5 to 4.2). So, after adjustment for factor VIII levels, a reduced response to APC remained a risk factor. Our results show that a reduced sensitivity for APC, not caused by the factor V Leiden mutation, is a risk factor for venous thrombosis.
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PMID:A reduced sensitivity for activated protein C in the absence of factor V Leiden increases the risk of venous thrombosis. 994 70

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.
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PMID:Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII. 1041 Mar 9

Inherited resistance to activated protein C (APC) is the most common genetic risk factor of venous thrombosis. It is caused by a single point mutation in the factor (F)V gene which predicts replacement of Arg506 with a Gln (FVR506Q, FV: Q506 or FV Leiden). This mutation affects the function of the protein C system, a physiologically important natural anticoagulant pathway. APC inhibits coagulation by cleaving a limited number of peptide bonds in both intact and activated forms of factor V (FV/FVa) and factor VIII (FVIII/FVIIIa). Degradation of FVa by APC is stimulated by protein S, whereas inactivation of FVIIIa requires the synergistic cofactor function of protein S and FV proteolytically modified by APC. Thus, FV has the potential to express opposing functions, as a procoagulant after cleavage by thrombin or FXa and as an anticoagulant after cleavage by APC. The FVR506Q mutation not only confers partial resistance of FVa to APC but also impairs the degradation of FVIIIa because APC-mediated cleavage of FV at Arg506 is required for expression of the anticoagulant activity of FV. The impaired degradation of both FVIIIa and FVa yield a hypercoagulable state conferring a lifelong increased risk of thrombosis. The FV mutation is common in Caucasians, whereas it is rarely found among other groups worldwide. In patients with severe thrombophilia having other inherited defects such as deficiencies of protein S, protein C, or antithrombin, APC resistance is often found as a contributing genetic risk factor. Individuals with combined genetic defects have a high risk of thrombosis, and it is now generally accepted that thrombophilia is a multigenetic disease.
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PMID:Activated protein C resistance and thrombosis: molecular mechanisms of hypercoagulable state due to FVR506Q mutation. 1044 59

Thromboembolism is a serious complication after Fontan operation, which may be caused by alterations of the coagulation system. We therefore investigated pro- and anticoagulant factors in 20 patients aged 4 to 21 years, 4 to 63 months following total cavopulmonary connection. Furthermore we compared markers of thrombin activation and fibrinolysis and in vitro clotting and clot-lysis to age-matched healthy subjects. Compared to results of age-matched controls, the Fontan operated individuals had significant decreases in levels of protein C (0.88 U/ml in controls, 0.67 U/ml in patients; p <0.001) and protein S (1.05 in controls, 0.93 U/ml in patients; p <0.05). Moreover, half of the patients had high values of FVIII (>1.5 IU/ml), which are associated with an increased thrombotic risk. These changes may result in enhanced generation of thrombin and plasmin, indicated by our finding of increased thrombin-antithrombin III (TAT) and plasmin-antiplasmin (PAP) levels and a similar trend in prothrombin fragments F1+2. Clot lysis tests, global coagulation tests, red blood cell count, liver enzymes AST, ALT, but not GGT, were generally within the normal ranges.
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PMID:Hemostatic changes following the modified Fontan operation (total cavopulmonary connection). 1181 32


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