Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR-1 or protein C

Citation
D. Arosio et al., Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR-1 or protein C, BIOCHEM, 39(27), 2000, pp. 8095-8101
Citations number
23
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
39
Issue
27
Year of publication
2000
Pages
8095 - 8101
Database
ISI
SICI code
0006-2960(20000711)39:27<8095:MOWCTC>2.0.ZU;2-V
Abstract
W215 is a highly conserved residue that shapes the S3 and S4 specificity si tes of thrombin and participates in an edge-to-face interaction with residu e Fs of the fibrinogen Aa chain. Protein C and the platelet receptor PAR-1 carry an acidic residue at P3 and bind to the active site of thrombin witho ut making contact with W215. This suggested that mutation of W215 could dis sociate the cleavage of fibrinogen from that of protein C and PAR-1. Replac ement of W215 with Phe produces modest effects on thrombin function, wherea s the W215Y replacement compromises significantly the catalytic activity to ward all chromogenic and natural substrates that are tested. Replacement of W215 with Ala almost obliterates Naf binding, reduces the level of fibrino gen 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 th at of fibrinogen and protein C and is the first thrombin derivative to be d escribed that functions as an almost exclusive activator of PAR-1. The envi ronment of W215 influences differentially three physiologically important i nteractions of thrombin, which should assist in the study of each of these functions separately in vivo.