Thrombin acts on many protein substrates during the hemostatic process. Its specificity for these substrates is modulated through interactions at regions remote from the active site of the thrombin molecule, designated exosites. Exosite interactions can be with the substrate, cofactors such as thrombomodulin, or fragments from prothrombin. The relative activity of alpha-thrombin for fibrinogen is 10 times greater than that for protein C. However, the relative activity of meizothrombin for protein C is 14 times greater than that for fibrinogen. Modulation of thrombin specificity is linked to its Na(+)-binding site and residues in autolytic loop-2 that interact with the Na(+)-binding site. Recombinant prothrombins that yield recombinant meizothrombin (rMT) and rMT des-fragment 1 (rMT(desF1)) enable comparisons of the effects of mutations at the Na(+)-binding residue (Asp(554)) and deletion of loop-2 (Glu(466)-Thr(469)) on the relative activity of meizothrombin for several substrates. Hydrolysis of t-butoxycarbonyl-VPR-p-nitroanilide by alpha-thrombin, recombinant alpha-thrombin, or rMT(desF1) was almost identical, but that by rMT was only 40% of that by alpha-thrombin. Clotting of fibrinogen by rMT and rMT(desF1) was 12-16% of that by alpha-thrombin, as already known. Strikingly, however, although meizothrombins modified by substitution of Asp(554) with either Ala or Leu or by deletion of loop-2 had 6-8 and <1%, respectively, of the clotting activity of alpha-thrombin, the activity of these meizothrombins for protein C was increased to >10 times that of alpha-thrombin. It is proposed that interactions within thrombin that involve autolytic loop-2 and the Na(+)-binding site primarily enhance thrombin action on fibrinogen, but impair thrombin action on protein C.