Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin

Conservative Trp-to-Phe mutations were individually created in human thromb in at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for th ese residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transf erred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfony larginine-N- (3-ethyl-1,5-pentanediyl)amide efficiently, but residue 207 di d not. Intensities correlated inversely with exposure to solvent, and measu red and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activat ion, inhibition by antithrombin, and the thrombomodulin-dependent activatio n of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All ac tivities of W96F and W207F ranged from 74 to 154% of the wild-type activity . This was also true for W148F, except for inhibition by antithrombin, wher e it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibri nogen clotting, platelet activation, and factor V cleavage (Arg(1006)), res pectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% o f normal levels with protein C and TAFI, respectively. In contrast, W215F w as 25 and 124% of normal levels in these reactions. Thus, many activities o f thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI acti vation, and Trp(215) appears to very important for clotting and protein C a ctivation.