The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor.
The intrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor to form a catalytic binary complex. Both the activation of factor VIIa and the expression of serine protease activity of factor VIIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the interaction of zymogen factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure factor VIIa functional activity, a two-stage chromogenic assay was used; an assay which measures the factor Xa generated by the activation of factor VII to factor VIIa. Purified immunoglobulin from murine monoclonal antibody 231-7, which was shown to be reactive with amino acid residues 51-88 of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIIa in a dose-dependent manner. The mechanism of this inhibition was demonstrated using a novel solid-phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtiter wells. Thus, the binding of factor VII zymogen to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dependent manner. Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the beta-galactosidase fusion proteins containing amino acid residues 51-88 (exon 4) of human factor VII. We conclude therefore that the exon 4-encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.
Department of Pathology, McMaster University Medical Center, Hamilton, Ontario, Canada.
B J
BJ
Clarke
F A
FA
Ofosu
S
S
Sridhara
R D
RD
Bona
F R
FR
Rickles
M A
MA
Blajchman
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D005167:Factor VII / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004815:Epidermal Growth Factor; D005167:Factor VII; D013925:Thromboplastin