Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Coagulation factor X, when activated to factor Xa by proteolytic cleavage, itself becomes an active serine protease which participates as a component of the macromolecular prothrombinase complex along with factor Va, phospholipid, and calcium ions. To identify specific structural regions on factor Xa responsible for mediating its function in activating prothrombin, we used 21 synthetic peptides corresponding to 65% of the primary structure of factor X as potential inhibitors of prothrombin activation. Using purified components, thrombin formation was inhibited by seven peptides in a dose-dependent noncompetitive manner. Antibodies to selected inhibitory peptides affinity purified on a factor Xa-agarose column inhibited thrombin formation in a dose-dependent manner, indicating that the corresponding regions on factor Xa are surface-exposed. Kinetic analyses varying the order of reagent addition suggested that peptides 211-222, 254-269, and 263-274 were highly effective in preventing the factor Xa-factor Va interaction. Peptides 275-287 and 415-425 were considered to derive from a distal region involved in substrate binding, based upon mixed inhibition kinetic analyses and assuming that inhibitory peptides not inhibitory in factor Va binding are related to a specific region of substrate interaction. Cross-linking studies confirmed that peptides 263-274 and 263-276 could bind specifically to the light chain of factor V/Va. These findings provide the basis for further pursuing the precise definition of interactive sites on factor Xa using site-directed mutagenesis and molecular modeling.
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PMID:Molecular recognition sites on factor Xa which participate in the prothrombinase complex. 160 96

: Coagulation factor X and factor VII (FVII) are both very important components in blood coagulation. To study the molecular pathogenic mechanism of the inherited factor X and FVII deficiency, the factor X activity (FX:C) and FVII activity were tested with one-stage clotting methods. The factor X antigen and factor FVII antigen were tested with ELISA. All the exons, intron-exon boundaries and 5',3'-flanking regions of F10 and F7 genes were amplified by PCR with direct sequencing. The ClustalX software was used to analyze the conservative property of the mutation sites. The PolyPhen-2 and Sorting Intolerant From Tolerant (SIFT) online bioinformatics softwares were taken to predict whether the point mutation could affect protein function. The software Swiss-pdb Viewer was brought to analyze the impact of mutations on the structure and function of the protein. The thrombin generation tests were applied to evaluate whether there were obstacles in producing thrombin about the mutant protein. The FX:C and FVII activity of the proband were reduced to 35 and 42%, and the factor X antigen and factor FVII antigen were decreased to 43 and 55%, simultaneously. Correspondingly, a FX:Cys81Arg (Cys81 by Arg) mutation and a FVII:Arg353 replaced by Gln polymorphism were detected in the proband. The Cys81 of factor X was conserved among homologous species, but the Arg353 of FVII was not. All softwares analysis results indicated protein features and structures might be affected by the mutation and the polymorphism. And the thrombin generation tests showed that the mutant protein had obstacles in thrombin generation. We identified a FX:Cys81Arg mutation and a FVII:Arg353 replaced by Gln polymorphism in the proband. And they accounted for the decrease of the activity and antigen of factor X and FVII. Of note, the Cys81Arg of factor X was first reported in the world.
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PMID:A novel factor X mutation Cys81 by Arg and a reported factor VII polymorphism Arg353 replaced by Gln co-occured in a patient. 2925 40