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J. Biol. Chem., Vol. 280, Issue 52, 42601-42611, December 30, 2005

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Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity^*

Yajnavalka Banerjee1, Jun Mizuguchi, Sadaaki Iwanaga, and R. Manjunatha Kini¶2

From the Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore Singapore 117543, the Blood Products Research Department, The Chemo-Sero-Therapeutic Research Institute, Kumamoto 869-1298, Japan, and the ^¶Department of Biochemistry, Virginia Commonwealth University Medical Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF·FVIIa complex. However, unwanted clot formation can lead to 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, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a "dissection approach," we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF·FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF·FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF·FVIIa amidolytic activity, with a K_i of 50 nM. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 x 10⁵ M^–1. The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF·FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

Received for publication, August 15, 2005 , and in revised form, September 28, 2005.

^* This work was supported in part by the Biomedical Research Council, Agency for Science and Technology, Singapore. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a National University of Singapore research scholarship.

² To whom correspondence should be addressed. Tel.: 65-6874-5235; Fax: 65-6779-2486; E-mail: dbskinim{at}nus.edu.sg.

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