Summary
The carboxy-terminal region of hirudin (residues 54-65) has previously been shown
to inhibit thrombin clotting activity without binding to the catalytic site of the
enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction
with specified platelet proteins has been investigated. Hirudin 54-65 was found to
inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner.
Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet
activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a
platelet membrane substrate for thrombin, was only partially inhibited by hirudin
54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets
during cross-linking experiments, it was found to inhibit the specific binding of
thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin
has previously been reported to bind near the trypsin-catalyzed β cleavage site, we
have analyzed the consequences of a to β-thrombin conversion on both thrombin-hirudin
54-65 interaction and thrombin activity toward platelets. The β cleavage induced a
decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether,
our results indicate that thrombin recognition sites for hirudin 54-65 and platelet
membrane glycoprotein Ib share common structures located near the β cleavage site
at Arg 73 on the thrombin B chain.
