ECOTIN IS A POTENT ANTICOAGULANT AND REVERSIBLE TIGHT-BINDING INHIBITOR OF FACTOR-XA

Ecotin, a serine protease inhibitor found in the periplasm of Escheric hia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The e cotin gene was cloned by PCR, highly expressed in E. coli, and purifie d from the E. coli periplasm. The binding of ecotin to FXa was stoichi ometric with an equilibrium dissociation constant K(i) of 54 pM. The a ssociation rate constant was 1.35 X 10(6) M-1 s-1, and the dissociatio n rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 X 10(-5) s- 1. Ecotin prolonged clotting time ca. 10-fold at 0.3 muM and at 2 muM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma prot eases thrombin, tissue factor-factor VIIa, factor XIa, activated prote in C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine tryp sin and chymotrypsin. Coincubation of ecotin and FXa at 10 muM each re sulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration wh ich yielded a K(d) of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with K(i) values of 11 and 21 pM, respective ly. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor-fac tor VIIa, t-PA, or HLE.