Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase.
The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.
Buddai
Sai K
SK
Joseph Stokes Research Institute, Children's Hospital of Philadelphia, Pennsylvania 19104, USA.
Toulokhonova
Larisa
L
Bergum
Peter W
PW
Vlasuk
George P
GP
Krishnaswamy
Sriram
S
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D015801:Helminth Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013925:Thromboplastin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015801:Helminth Proteins; C400226:anti-coagulant protein C2, Ancylostoma caninum; D005170:Factor X; D013925:Thromboplastin; D015951:Factor Xa