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Volume 271, Number 27, Issue of July 5, 1996 pp. 16126-16134
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Role of the Activation Peptide Domain in Human Factor X Activation by the Extrinsic Xase Complex

(Received for publication, February 27, 1996)

From the Division of Hematology/Oncology, Department of Medicine, Emory University, Atlanta, Georgia 30322

The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg⁵²-Ile⁵³ peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1-52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase  (ARH), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated X_des1-34, X_des1-43, and X_des1-49. The products were purified to yield X_{des 1-49} and a mixture of X_{des 1-34} and X_{des 1-43}. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since X_{des 1-49} lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1-49 of the activation peptide or with carbohydrate structures attached to these residues.

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