Kinetics of the inhibition of thrombin by hirudin
Research article published in Biochemistry (1986)
Abstract
The dissociation constant for hirudin was determined by varying the concentration of hirudin in the presence of a fixed concentration of thrombin and tripeptidyl p-nitroanilide substrate. The estimate of the dissociation constant determined in this manner displayed a dependence on the concentration of substrate which suggested the existence of two binding sites at which the substrate was able to compete with hirudin. A high-affinity site could be correlated with the binding of the substrate at the active site, and the other site had an affinity for the substrate that was 2 orders of magnitude lower. Extrapolation to zero substrate concentration yielded a value of 20 fM for the dissociation constant of hirudin at an ionic strength of 0.125. The dissociation constant for hirudin was markedly dependent on the ionic strength of the assay; it increased 20-fold when the ionic strength was increased from 0.1 to 0.4. This increase in dissociation constant was accompanied by a decrease in the rate with which hirudin associated with thrombin. This rate could be measured with a conventional recording spectrophotometer at higher ionic strength and was found to be independent of the binding of substrate at the active site.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
The dissociation constant for hirudin was determined by varying the concentration of hirudin in the presence of a fixed concentration of thrombin and tripeptidyl p-nitroanilide substrate.
Why This Matters for Hirudotherapy
This study quantified the binding kinetics between hirudin and thrombin, reporting a dissociation constant of 20 fM at ionic strength 0.125 and showing that this constant increased 20-fold when ionic strength rose from 0.1 to 0.4, accompanied by a decreased association rate. The results also suggested two substrate-competitive binding sites on thrombin. Because hirudin is a potent thrombin inhibitor of direct relevance to the anticoagulant mechanisms underlying hirudotherapy, these precise kinetic parameters provide foundational biochemical detail about that molecular interaction. However, this work is limited to purified in-vitro binding-kinetics measurements performed with a spectrophotometer; it contains no physiological or clinical context, does not involve leeches or salivary secretions, and its connection to therapeutic practice is indirect.
Citation
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