The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor

Abstract

The intrinsic pathway of coagulation is initiated when zymogcn factor VII binds to its cell surfxe receptor tissue factor to form a catalytic binary complex. Both the activation of factor VII to factor Vlla and the expression ofserine protuase activity of factor VlIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the intcractian of zymogcn 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 fnctor Vllti functional activity, a two-stage chromogenic assay was used; an nsstly which measures the factor Xa generated by the activation of factor VII to factor Vlla. Purilicd immunoglobulin from murine moncclonal antibody 231-7. which was shown to bc reactive with amino acid residues 5l-SE of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIla 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 microtitcr wells. Thus, the binding of factor VII rymogcn to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dcpcndent manner, Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the fl-galactosidase fusion proteins containing amino acid residues 51-88 (exon 4) of human factor VII. WC conclude therefore that the CXOII 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.

Interspecies exchange mutagenesis of the first epidermal growth factor‐like domain of human factor VII

“…Purified rFVII was quantitatively converted to rFVIIa as previously described in detail [25]. Amidolytic activity of unpurified rFVIIa in transient expression experiments was determined indirectly using the FXa chromogenic peptide substrate S‐2222 as previously described [27]. Amidolytic activity of purified rFVIIa was determined directly using the FVIIa chromogenic peptide substrate Pefachrome FVIIa in a reaction mixture containing 50 m m Tris‐HCl, pH 8.0, 100 m m NaCl, 5 m m CaCl 2 , 50 ng FVIIa, human thromboplastin and 400 μ m Pefachrome FVIIa essentially as described by the manufacturer.…”

Ca2+ Binding to the First Epidermal Growth Factor-like Domain of Factor VIIa Increases Amidolytic Activity and Tissue Factor Affinity

“…The first EGF-like domain of fVIIa is known from biochemical studies (35)(36)(37) as well as from the x-ray crystallographic structure of the fVIIa-sTF complex (13) to interact with TF, but the importance of the Ca 2ϩ site in this domain is unclear. Based on Ca 2ϩ structures of EGF-like domains (8,9) and their homology and high degree of sequence identity with the corresponding domain in fVIIa, the Asp residues in positions 46 and 63 in fVIIa were replaced by Asn to abolish and investigate the role of Ca 2ϩ binding to the first EGF-like domain.…”

Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions

“…The fact that the amidolytic activity of factor VIIa is stimulated by TF and Ca z+ [6] seems to indicate binding of these ligands to the catalytic domain, and substantiation of this interpretation is obtained by several independent observations [5,[7][8][9][10][11]. TF interaction with the catalytic domain does, however, not exclude additional involvement of other regions, and the EGF domains of factor VIIa have been implicated as possible TF binding sites [12][13][14]. While it has long been known that ?-carboxylation of the factor VII Gla-domain is crucial for manifestation of full biologic activity, it is not until recently that the Gla-domain has been directly implicated in TF binding.…”