Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.