BOTHROJARACIN AS ANTITHROMBOTIC AGENT

ZINGALI, R.B.(1), MONTEIRO R.Q.(1), CASTRO H.C.(1)

(1)Departamento de Bioquímica Médica, ICB/CCS, UFRJ, Rio de Janeiro, Brazil.

Bothrojaracin (BJC) first isolated from Bothrops jararaca venom is a 27 kDa heterodimer. It binds  specifically to -thrombin with a K_D=0.7 nM and influences but does not block the proteinase catalytic site, although it inhibits thrombin biological activities such as fibrinogen clotting, platelet aggregation, protein C activation. BJC also binds the inactive thrombin precursor, prothrombin. Gel filtration chromatography (Superose 12) showed that fluorescein-labeled BJC ([5F]BJC) forms a 1:1 Ca²⁺-independent, non-covalent complex (100,000 Da) with prothrombin. On the other hand, [5F]BJCdid not interact with prothrombin. fragments1 or 2. Isothermal titration calorimetry (ITC) showed that the binding of prothrombin to BJC is endothermic, indicating that the binding is entropicallydriven with anK_D = 76 nM. BJC efficiently displaced fluorescein-labeled Hir^54-65(SO^3-) from complexes formed with -thrombin or prothrombin, suggesting that both BJC and Hir^54-65(SO^3-) compete for the same binding sites in those molecules. These data indicate that BJC binds the anion-binding exosite I precursor (proexositeI) on human prothrombin..BJC also greatly inhibited thrombin formation induced by O. scutellatus venom, suggesting a direct interference in the prothrombin activation. BJC strongly inhibited the zymogen activation by factor Xain the presence but not absence of factor Va. The same effect was observed in the presence of phospholipids or platelets, suggesting a specific interference on the cofactor activity. It is proposed that BJC has two independent mechanisms for anticoagulation: 1) inhibition of exositeI-dependent activities on -thrombin, and 2) inhibition of prothrombin activation through interaction with proexosite I.

Supported by FAPERJ, CNPq and Finep IFS contract F3156.

CORRESPONDENCE TO:

ZINGALI, R.B., Departamento de Bioquímica Médica, ICB/CCS, UFRJ, 21000-000, Rio de Janeiro Brazil,  Email: lzingali@bioqmed.ufrj.br