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A series of 12 bovine pancreatic trypsin inhibitor variants mutated in the P 4 and P 3 positions of the canonical binding loop containing additional K15R and M52L mutations were used to probe the role of single amino acid substitutions on binding to bovine trypsin and to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X a and XII a , thrombin, and protein C. The mutants were expressed in Escherichia coli as fusion proteins with the LE1413 hydrophobic polypeptide and purified from inclusion bodies; these steps were followed by CNBr cleavage and oxidative refolding. The mutants inhibited the blood-clotting proteinases with association constants in the range of 10 3 -10 10 M ؊1 . Inhibition of plasma kallikrein, factors X a and XII a , thrombin, and protein C could be improved by up to 2 orders of magnitude by the K15R substitution. The highest increase in the association constant for P 3 mutant was measured for factor XII a ; P13S substitution increased the K a value 58-fold. Several other substitutions at P 3 resulted in about 10-fold increase for factor X a , thrombin, and protein C. The cumulative P 3 and P 1 effects on K a values for the strongest mutant compared with the wild type bovine pancreatic trypsin inhibitor were in the range of 2.2-(plasmin) to 4,000-fold (factors XII a and X a ). The substitutions at the P 4 site always caused negative effects (a decrease in the range from over 1,000-to 1.3-fold) on binding to all studied enzymes, including trypsin. Thermal stability studies showed a very large decrease of the denaturation temperature (about 22°C) for all P 4 mutants, suggesting that substitution of the wild type Gly-12 residue leads to a change in the binding loop conformation manifesting itself in non-optimal binding to the proteinase active site.Blood coagulation is a series of proteolytic events resulting in clot formation. Equally important are the processes of anticoagulation and fibrinolysis that are also mediated by proteolytic enzymes. Research during the past decade has resulted in the determination of spatial structures for many of these enzymes, including thrombin, factors VII a , IX a , and X a , protein C, tissue plasminogen activator, and plasmin (1-6). The specificity of a particular enzyme toward its cognate sequence results from well defined subsites on the enzyme surface recognizing not only the scissile peptide bond but often also more extended regions. These structural studies nicely explain earlier data on sequence-specific cleavage of natural substrates and provide a framework for drug design efforts.Because the coagulation/fibrinolysis processes are of vital importance, they are precisely controlled. Protein inhibitors are one of the most essential regulating factors. Interestingly, the scaffold of Kunitz-type inhibitors is used both to control the natural coagulation process in human blood and to prevent blood clotting in a blood-sucking organism, the soft tick. In the former case, tissue factor pathway inhibitor (TFPI) 1 compo...

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