Hemextin AB Complex, a Unique Anticoagulant Protein Complex fromHemachatus haemachatus(African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity
Journal of Biological Chemistry
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 TF⅐FVIIa 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
... 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 TF⅐FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF⅐FVIIa 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 TF⅐FVIIa 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 ؋ 10 5 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/TF⅐FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation. 3 The abbreviations used are: We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts. PAGE 36848: In the Abstract, lines 19 -21, this sentence should read: "These mutations did not decrease ␣1,3 activity but reduced the ␣1,4 activity to 66.9, 55.6, and 3.1% of wild type level, respectively."