Molecular recognition sites on factor Xa which participate in the prothrombinase complex

• Published in: The Journal of biological chemistry Vol. 267; no. 17; pp. 12323 - 12329
• Main Authors: CHATTOPADHYAY, A, JAMES, H. L, FAIR, D. S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-06-1992
• Subjects: activation; Amino Acid Sequence; Autoradiography; Binding Sites; Biological and medical sciences; Blood coagulation. Blood cells; coagulation factor Xa; Coagulation factors; Cross-Linking Reagents; derivatives; Electrophoresis, Polyacrylamide Gel; Factor Xa - metabolism; Fundamental and applied biological sciences. Psychology; inhibition; Kinetics; Molecular and cellular biology; Molecular Sequence Data; Peptides - metabolism; prothrombin; Substrate Specificity; Thrombin - metabolism; Thromboplastin - metabolism; Human; Site specificity; Autoprothrombin C; Multienzyme complex; Domain structure; Enzyme; Molecular interaction; Kinetics; Localization; Coagulation factor
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)49842-6

Coagulation factor X, when activated to factor Xa by proteolytic cleavage, itself becomes an active serine protease which
participates as a component of the macromolecular prothrombinase complex along with factor Va, phospholipid, and calcium ions.
To identify specific structural regions on factor Xa responsible for mediating its function in activating prothrombin, we
used 21 synthetic peptides corresponding to 65% of the primary structure of factor X as potential inhibitors of prothrombin
activation. Using purified components, thrombin formation was inhibited by seven peptides in a dose-dependent noncompetitive
manner. Antibodies to selected inhibitory peptides affinity purified on a factor Xa-agarose column inhibited thrombin formation
in a dose-dependent manner, indicating that the corresponding regions on factor Xa are surface-exposed. Kinetic analyses varying
the order of reagent addition suggested that peptides 211-222, 254-269, and 263-274 were highly effective in preventing the
factor Xa-factor Va interaction. Peptides 275-287 and 415-425 were considered to derive from a distal region involved in substrate
binding, based upon mixed inhibition kinetic analyses and assuming that inhibitory peptides not inhibitory in factor Va binding
are related to a specific region of substrate interaction. Cross-linking studies confirmed that peptides 263-274 and 263-276
could bind specifically to the light chain of factor V/Va. These findings provide the basis for further pursuing the precise
definition of interactive sites on factor Xa using site-directed mutagenesis and molecular modeling.