Activation of a recombinant human factor VII structural analogue alters its affinity of binding to tissue factor.
A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC50) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor.
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
S
S
Sridhara
S
S
Chaing
K A
KA
High
M A
MA
Blajchman
B J
BJ
Clarke
D001665:Binding Sites; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000235:genetics; D005167:Factor VII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D005167:Factor VII; D013925:Thromboplastin