The contribution of factor Xa to exosite-dependent substrate recognition by prothrombinase.
Kinetic studies support the concept that protein substrate recognition by the prothrombinase complex of coagulation is achieved by interactions at extended macromolecular recognition sites (exosites), distinct from the active site of factor Xa within the complex. We have used this formal kinetic model and a monoclonal antibody directed against Xa (alphaBFX-2b) to investigate the contributions of surfaces on the proteinase to exosite-mediated protein substrate recognition by prothrombinase. alphaBFX-2b bound reversibly to a fluorescent derivative of factor Xa (K(d) = 17.1 +/- 5.6 nm) but had no effect on active site function of factor Xa or factor Xa saturably assembled into prothrombinase. In contrast, alphaBFX-2b was a slow, tight binding inhibitor of the cleavage of either prethrombin 2 or meizothrombin des-fragment 1 by prothrombinase (K(i)(*) = 0.55 +/- 0.05 nm). Thus, alphaBFX-2b binding to factor Xa within prothrombinase selectively leads to the inhibition of protein substrate cleavage without interfering with active site function. Inhibition kinetics could adequately be accounted for by a kinetic model in which prethrombin 2 and alphaBFX-2b bind in a mutually exclusive way to prothrombinase. These are properties expected of an exosite-directed inhibitor. The site(s) on factor Xa responsible for antibody binding were evaluated by identification of immunoreactive fragments following chemical digestion of human and bovine Xa and were further confirmed with a series of recombinantly expressed fragments. These approaches suggest that residues 82-91 and 102-116 in the proteinase domain contribute to alphaBFX-2b binding. The data establish this antibody as a prototypic exosite-directed inhibitor of prothrombinase and suggest that the occlusion of a surface on factor Xa, spatially removed from the active site, is sufficient to block exosite-dependent recognition of the protein substrate by prothrombinase.
Wilkens
Matthias
M
Joseph Stokes Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Krishnaswamy
Sriram
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D015943:Factor Va / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D008969:Molecular Sequence Data; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015943:Factor Va; D005170:Factor X; D013925:Thromboplastin; D015951:Factor Xa