Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants

A series of six CMTI I variants mutated in the P-2-P-4' region of the canon ical binding loop were used to probe the role of single amino acid substitu tions on binding to the following human proteinases involved in blood clott ing: plasmin, plasma kallikrein, factors X-a and XIIa. The mutants were exp ressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escher ichia coli, purified from inclusion bodies, followed by cyanobromide cleava ge and refolding. The mutants inhibited the proteinases with the associatio n constants in the range 10(3)-10(9) M-1. Inhibition of plasma kallikrein a nd factors X-a and XIIa could be improved up to 30-fold by single mutations . In contrast, neither of the introduced mutations increased inhibitory pro perties of CMTI I against plasmin. Additionally, using two inhibitors of na tural origin, CMTI I (P-1 Arg) and CPTI II (P-1 Lys), we determined the eff ect of Lys --> Arg on binding to four proteinases. With the exception of pl asmin (no effect), P-1 Arg resulted in up to 30-fold stronger binding than P-1 Lys. (C) 2000 Elsevier Science B.V. All rights reserved.