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A Novel Anticoagulant Protein – Hemextin A, from the Venom of Hemachatus Haemachatus (African Ringhals Cobra) That inhibits the Extrinsic Tenase Activity and Its Synergistic Interaction with A Three-finger Toxin, Hemextin B
Abstract number: P0154
Banerjee1 Y, Kini1,2 RM
1,2 11Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117 543
During injury or trauma the blood coagulation is initiated by the interaction of the serine protease factor VIIa (FVIIa) present in the blood with the freshly exposed tissue factor (TF) to form the extrinsic tenase complex. Unwanted clot formation, however, can lead death and debilitation due to vascular occlusion and hence anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextin A and hemextin B, from the venom of Hemachatus haemachatus (African Ringhals cobra). Amino terminal sequences indicate that these proteins belong to the three-finger toxin family of snake venom proteins. Individually, only hemextin A exhibits mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B forms a synergistic complex with hemextin A and enhances its anticoagulant potency. Using gel filtration chromatography, circular dichroism and isothermal titration calorimetry, we have shown the formation of a 1 : 1 complex between these two proteins and the complex formation is important for the anticoagulant activity. Hemextin is the first heterodimeric complex consisting of three-finger toxins. Using ‘dissection approach’ we have shown that hemextin and hemextin A prolong clotting by inhibiting extrinsic tenase activity. This was further confirmed by studying their effect on the reconstituted extrinsic tenase complex. Thus mechanistically, these proteins mediate their anticoagulant effect by specifically inhibiting the Factor Xa (FXa) formation by the extrinsic tenase complex. Therefore, the understanding of the molecular details of interaction of hemextin complex with the extrinsic tenase complex will open a new paradigm in the search for anticoagulants.
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