Abstract
Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities 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, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. 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% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of 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 activation, and Trp(215) appears to very important for clotting and protein C activation.
Highlights
Thrombin is a trypsin-like serine protease that is generated upon vascular injury from its plasma precursor prothrombin following a cascade of zymogen-to-enzyme conversions (1, 2)
Fluorescence Properties of Selected Tryptophan Residues of Thrombin—The emission spectra and individual contributions of Trp60d, Trp96, Trp148, Trp207, and Trp215 to the total fluorescence intensity of thrombin and energy transfer to DAPA were determined by differences between wild-type thrombin and the mutants lacking each of the residues
The analysis was very similar to that described by Meagher et al (41), who used mutagenesis to study the properties of the individual tryptophan residues of antithrombin
Summary
Thrombin is a trypsin-like serine protease that is generated upon vascular injury from its plasma precursor prothrombin following a cascade of zymogen-to-enzyme conversions (1, 2). Thrombin stabilizes the newly formed fibrin clot through the activation of factor XIII (6), a transglutaminase that introduces isopeptide bonds between fibrin protomers, reinforcing the clot structure. The tryptophan residues of thrombin are designated Trp, Trp, Trp60d, Trp, Trp141, Trp148, Trp207, Trp215, and Trp237. Four of these are intimately associated in space with the active site of thrombin. Previous studies indicate that one or more of the tryptophan residues of thrombin are required for activity and are in some way associated with the formation of the active site upon conversion of prothrombin to thrombin. Differences in the environments of some of the tryptophan residues exist, as inferred by
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call