INHIBITION AND ACTIVATION OF THE BLOOD COAGULATION CASCADE BY VENOM AND SALIVA PROTEINS: MECHANISMS AND POTENTIAL CANDIDATES FOR DRUG DESIGN

RAGHUVIR K. ARNI(1)

(1)Structural Biology Group - Department of Physics - IBILCE/UNESP

Haemostasis, an intricate and balanced cascade of reactions that involves a number of serine proteinases and macromolecular cofactors which are responsible for maintaining the fluidity of blood under physiological conditions. Worm, snake, leech, tick and lizard gland secretions contain a wide variety of haemostatically-active proteins, which interfere at diferent levels in the coagulation cascade and fibrinolytic system and serve as promising drugs for the treatment of haemostatic disorders. Nematode anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum has been targeted for the control and regulation of thrombosis and is significantly more potent than low molecular weight heparin. NAPc2 only partially inhibits the amidolytic activity of FXa and prevents the formation of thrombin by FXa at a site distinct and remote from the active site with the resultant binary complex inactivating the TF-FVIIa complex with a Ki = 8.4 pM. The crystal structure of the NAPc2 complexed with human FXa reveals the location of this novel site. The homologous protein NAP5 inhibits FXa by binding at both the active site and at the exosite. Molecular modeling of  Ixolaris, an double Kunitz domain anticoagulant from the hard tick Ixodes scapularis, which is a vector for Lyme´s disease indicates that it binds to the FXa  exosite and inhibits the interaction of FXa with the tissue factor/ F VIIa complex. Structural results indicate that both NAP5, NAPc2 and Ixolaris could serve as the basis for the design of  powerful anticoagulants. Another promising anticoagulant protein for the treatment of haemostatic disorders is the protein C activator from Agkistrodon contortrix contortrix (Protac), a glycosylated single-chain serine proteinase, which activates the protein C pathway without relying on thrombomodulin and due to the direct and fast-action in protein C activation, Protac has found a broad application in diagnostic practice for the determination of disorders in the PC pathway. The crystal structures of native and inhibited Protac indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation.

Acknowledgements: We are grateful to FAPESP, CAPES, CNPq, DAAD, CEPID, Pentapharm Ltd., Wyeth Research and CORVAS International Inc for financial support and samples.