Molecular recognition sites on factor Xa which participate in the prothrombinase complex.
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.
Chattopadhyay
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Department of Biochemistry, The University of Texas Health Center, Tyler 75710.
James
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HL
Fair
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D000595:Amino Acid Sequence; D001345:Autoradiography; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D010455:Peptides; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa