Enhanced
thrombin activity has been associated with
coronary thrombosis and with acute and long-term complications following coronary balloon angioplasty. Blocking
thrombin activity with specific inhibitors is proposed as a promising antithrombotic
therapy. We describe the
anticoagulant and antithrombotic properties of
hirunorm, a novel synthetic 26-aminoacid
peptide thrombin inhibitor, in comparison with r-
hirudin and
hirulog-1.
Hirunorm was equipotent to
hirulog-1 and 1/30 as potent as r-
hirudin in blocking
alpha-thrombin amidolytic activity (IC50 = 10 +/- 2, 15 +/- 1 and 0.3 +/- 0.1 nM, respectively), but it did not affect
trypsin,
plasmin and t-PA activities
at 10 microM. All the compounds inhibited clot-bound
thrombin to clots prepared by
thrombin hydrolysis of purified
fibrinogen in
buffer.
Hirunorm and
hirulog-1 showed similar species-dependent potency in doubling basal in vitro clotting times of human, rat and rabbit plasma (EC200 varied 70 to 200 nM for TT, 0.7 to 16 microM for aPTT and 0.8 to 17 microM for PT), while r-
hirudin was always at least three times more active. When assayed by HPLC or by bioassay of the intact
peptide,
hirunorm was stable against
alpha-thrombin and plasma
hydrolases, but it was catabolized by rat liver and kidney
enzymes.
Venous thrombosis was produced in anaesthetized rats by vena cava
ligation following a procoagulant serum injection. Intravenous and subcutaneous
hirunorm inhibited
venous thrombosis at doses (< or = 0.3 mg/kg) two-three times higher than those of r-
hirudin.
Hirulog-1 was as active as
hirunorm only after i.v. infusion. Arterial
thrombosis was obtained in the anaesthetized rat by chemical (FeCl2) stimulation of a common carotid and i.v. infused
hirunorm (1-3 mg/kg/30 min) inhibited it dose-dependently; r-
hirudin was partly active only at 3 mg/kg, but
hirulog-1 was inactive at either dose. Full antithrombotic doses of
hirunorm did not affect the bleeding time as measured from punctured mesenteric vessels, in anaesthetized rats. In conclusion,
hirunorm is a potent
peptide thrombin inhibitor endowed with antithrombotic activity in models of venous and arterial
thrombosis.