Abstract
A thrombin receptor has recently been cloned and the sequence deduced. The sequence reveals a thrombin cleavage site that accounts for receptor activation. The receptor also has an acidic region with some similarities to the carboxyl-terminal region of the leech thrombin inhibitor, hirudin. Synthetic peptides corresponding to the receptor cleavage site (residues 38-45), the hirudin-like domain (residues 52-69), and the covalently associated domains (residues 38-64) were evaluated for their ability to bind to thrombin. Peptides 38-45 and 38-64 were competitive inhibitors of thrombin's chromogenic substrate activity (Ki = 0.96 mM and 0.6 microM, respectively. Residues 52-69 altered the chromogenic substrate specificity, resulting in accelerated cleavage of some substrates and inhibited cleavage of others. The same peptide binds to thrombin and alters the fluorescence emission intensity of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-thrombin in which the dansyl is attached directly to the active site serine (Kd = 32 +/- 7 microM). Residues 52-69 displace the carboxyl-terminal peptide of hirudin, indicating that they share a common binding site in the anion exosite of thrombin. These data suggest that the thrombin receptor has high affinity for thrombin due to the presence of the hirudin-like domain and that this domain alters the specificity of thrombin. This change in specificity may account for the ability of the receptor to serve as an excellent thrombin substrate despite the presence of an Asp residue in the P3 site, which is normally inhibitory to thrombin activity.
Highlights
From the Departmentsof $Biochemistry and I(Pathology, University of Oklahoma Health Sciences Center, the the circulation, thrombin can be inhibited by antithrombin I11 or heparin cofactor 2
Synthetic peptides correspond- tension.Thisextracellularextensioncontains a thrombin ing to the receptor cleavage site, the cleavage site (DPR/SFLL) resembling the known thrombin hirudin-like domain, and the cova- cleavage site found in the thrombin-activatedzymogen, prolently associated domains were eval- tein C (DPR/V) ( 5 ) .Structure-activity studieswith the cloned uated for their ability to bind to thrombin
The concentration of fluorescein-Hirugen was calculated based oanmolar was examined for its ability to bind to thrombin and inhibit chromogenic substrate hydrolytic activity
Summary
The same peptide binds to thrombin and alters the fluorescence emission intensity of 5dimethylaminonaphthalene-1-sulfonyl (dansy1)thrombin in which the dansyl is attached directly to thrombin cleavage site was shown to be afull agonist for thrombin receptor activation [4]. Indicating that they share a common binding site in the WEDEE) that grossly resembles the carboxyl tail of hirudin, anion exosite of thrombin. A peptide mimicking the carin specificity may accountfor the ability of the receptor boxyl-terminal tail of hirudin, termed Hirugen,’ is known to to serve as an excellent thrombin substrate despite the block thrombin-inducedplateletactivation[9], suggesting presence of an Asp residue in the P3 site, which is that thrombin’s anionexosite might play a role in thrombin/. Thrombin plays a major regulatory role in blood coagulabin/receptor interaction In this studyw, e demonstrate thata peptide, corresponding. Thrombin Interaction with IRtseceptor the receptor peptide acts by binding to thrombin’sanion exosite
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