INTERACTIONS BETWEEN LEFAXIN AND FACTOR X MOLECULES

Faria, F.(1), Bezeaud, A.(2), Guillin, M.C.(2), Anglés-Cano, E.(3), Sampaio, M.U.(4), Chudzinski-Tavassi, A.M.(1)

(1)Laboratório de Bioquímica e Biofísica, Instituto Butantan (Center for Applied Toxinology CAT, CEPID, FAPESP), São Paulo, Brazil, (2)E9907, INSERM, Paris, France, (3)U143, INSERM, Paris, France, (4)Departamento de Bioquímica, UNIFESP, EPM, São Paulo, Brazil.

The salivary glands of Haementeria depressa leech produce some proteins that induce unclotable blood. The purification and partial characterization of a Factor Xa inhibitor, named lefaxin (leech factor Xa inhibitor), was previously described by FARIA et al (1999). Lefaxin is a single chain glycoprotein, Mr30 kD, pI 5.7, the inhibition of FXa was determined first by inhibition of FXa amidolytic activity determined with chromogenic substrate (S2765) with a Ki about 4nM and second by reduction of FXa ability to activate prothrombin, in the prothrombinase complex (EC₅₀ 10nM).

The aim of this study is to characterize the inhibition by the Dixon plot, and to determine the interaction site of FX molecule.

Lefaxin is a competitive FXa inhibitor with a Ki about 2nM, this inhibitor was able to bind to FX molecule immobilized on the CM5 chip in a BIACORE system, with a Kd about 4nM. The interaction with FXa using the FX (zymogen) as competitor showed that lefaxin is able to bind to the zymogen (Kd about 6nM). Binding to aFXa active site blocked (FXa-GGACK) was observed (Kd about 30nM).

These results indicate that lefaxin, as well as rTAP (JORDAN et al, 1992) and antistasin (LAPATTO et al, 1997), can bind to FX via an exosite, although lefaxin isn’t aFXa inhibitor antistasin-like.

Financial supported by: FAPESP, INSERM, and CAPES

CORRESPONDENCE TO:

Fernanda Faria, Rua Ofélia 324, Apto. 102-A, São Paulo, SP, CEP: 05423-110, Brasil, Email: fe_faria@terra.com.br