Regions of hirudin important for its inhibitory activity with thrombin have been examined by site-directed mutagenesis. Since thrombin has a primary specificity for basic amino acids, each of the three basic residues and the histidine in hirudin were mutated to glutamine. Mutation of Lys-47 caused a small increase (9-fold) in the dissociation constant whereas the other mutations were without effect. These results indicate that hirudin is different from most other inhibitors of serine proteases in that interactions with the primary specificity pocket of its target enzyme are not crucial to its inhibitory activity. The acidic nature of the carboxyl region of hirudin was found to be important for its interaction with thrombin. Single and multiple mutations of carboxyl-terminal glutamate residues (57, 58, 61, and 62) to glutamine caused increases in the dissociation constant. This value increased with the number of mutations and reached a maximum of 61-fold when all four glutamate residues were mutated. Kinetic studies indicated that in all cases where an increase in dissociation constant was observed, it was predominantly due to a decrease in the association rate constant.

The performance characteristics suggest that the LDL-P assay is suitable for routine testing in the clinical laboratory on the Vantera Clinical Analyzer, the first automated NMR platform that supports NMR-based clinical assays.

The contributions of various regions of human alpha-thrombin to the formation of the tight complex with hirudin have been assessed by using derivatives of thrombin. alpha-Thrombin in which the active-site serine was modified with diisopropyl fluorophosphate was able to bind hirudin, but its affinity for hirudin was decreased by 10(3)-fold compared to unmodified alpha-thrombin. Modification of the active-site histidine with D-Phe-Pro-Arg-CH2Cl resulted in a form of thrombin with a 10(6)-fold reduced affinity for hirudin. gamma-Thrombin is produced by proteolytic cleavage of alpha-thrombin in two surface loops corresponding to residues 65-83 and 146-150 in alpha-chymotrypsin [Berliner, L. J. (1984) Mol. Cell. Biochem. 61, 159-172; Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. The gamma-thrombin-hirudin complex had a dissociation constant that was 10(6)-fold higher than that of alpha-thrombin. Treatment of alpha-thrombin with pancreatic elastase resulted in a form of thrombin only cleaved in the loop corresponding to residues 146-150 in alpha-chymotrypsin, and this form of thrombin had only a slightly reduced affinity for hirudin. By using limited proteolysis with trypsin, it was possible to isolate beta-thrombin which contained a single cleavage in the loop corresponding to residues 65-83 in alpha-chymotrypsin. This form of thrombin had a 100-fold decrease in affinity for hirudin. Kinetic analysis of the binding of hirudin to beta-thrombin indicated that the 100-fold decrease in affinity was predominantly due to a decrease in the rate of association of the two molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryChanges in characteristics of optical transmittance data from coagulation assays were examined as a function of concentration of coagulation proteins or anticoagulants. Transmittance data were collected for activated partial thromboplastin time (APTT) and prothrombin time (PT) assays from: 1) plasmas prepared by mixing normal plasmas with deficient plasmas to give varying levels of coagulation proteins; 2) plasmas containing added heparin; and 3) 200 specimen plasmas that were also assayed for fibrinogen, coagulation factors, and other components. Optical profiles were characterized using a set of parameters describing onset and completion of coagulation, magnitude of signal change, rate of coagulation and other properties. Results indicated that parameters other than those typically reported for APTT and PT are associated with individual deficiencies, but that diagnosis of specimen status on the basis of optical data is complex. These results suggest possibilities for expanded interpretation of PT/APTT optical data for clinical or research applications.