Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR-1 or protein C.
W215 is a highly conserved residue that shapes the S3 and S4 specificity sites of thrombin and participates in an edge-to-face interaction with residue F8 of the fibrinogen Aalpha chain. Protein C and the platelet receptor PAR-1 carry an acidic residue at P3 and bind to the active site of thrombin without making contact with W215. This suggested that mutation of W215 could dissociate the cleavage of fibrinogen from that of protein C and PAR-1. Replacement of W215 with Phe produces modest effects on thrombin function, whereas the W215Y replacement compromises significantly the catalytic activity toward all chromogenic and natural substrates that are tested. Replacement of W215 with Ala almost obliterates Na(+) binding, reduces the level of fibrinogen cleavage 500-fold, but decreases the levels of protein C activation and PAR-1 cleavage only 3- and 25-fold, respectively. The W215A mutant cleaves PAR-1 with a specificity constant that is more than 13-fold higher than that of fibrinogen and protein C and is the first thrombin derivative to be described that functions as an almost exclusive activator of PAR-1. The environment of W215 influences differentially three physiologically important interactions of thrombin, which should assist in the study of each of these functions separately in vivo.
Arosio
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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, Missouri 63110, USA.
Ayala
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YM
Di Cera
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D000595:Amino Acid Sequence; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; D011486:Protein C; D005340:Fibrinogen; D017346:Protein Serine-Threonine Kinases; D013917:Thrombin