The design of low molecular weight thrombin inhibitors IIa-d (hirutonins) that bind concurrently with the enzyme's catalytic site and auxiliary "anion-binding exosite" for fibrinogen recognition is reported. A practical synthesis of the required homologous ketomethylene arginyl dipeptide inserts [Arg psi CO(CH2)nCO] (n = 1-4) corresponding to the P1-P1' scissile position of hirutonins is described. The substitution of the scissile amide function by a ketomethylene group is compatible with the enzyme active site and conferred complete plasma proteolytic stability. This modification also enhanced enzyme affinity up to 20-fold with hirutonin-4 (IIb, n = 4) displaying highest affinity (Ki = 140 +/- 20 pM). Hirutonins 1-4 exhibited potent inhibition of plasma prothrombin time (PT) and activated partial thromboplastin time (aPTT). The inhibition was biphasic and showed good correlation with the corresponding Ki. Hirutonin-2 inhibited thrombin-mediated platelet aggregation and exhibited a strong antithrombotic effect comparable to r-hirudin in an in vivo rat arteriovenous shunt model (ED15 = 1.20 mg/kg for hirutonin-2 and 1.14 mg/kg for r-hirudin). Lower molecular weight inhibitors were obtained by substituting the six native amino acid residues (Q-S-H-N-D-G), connecting the active site and the auxiliary exosite binding elements with a variable number of interening omega-aminopentenoyl units. In addition, the exosite component was reduced to seven amino acid residues (D-F-E-P-I-P-L). Incorporation of these modifications into the bifunctional format resulted in nanomolar thrombin inhibitory peptides (IIIa-c). The resulting inhibitors were studied by molecular modeling with alpha-thrombin, and the bimolecular interactions served to explain the retention of high enzyme affinity.