The role of phospholipids and the factor VII Gla-domain in the interaction of factor VII with tissue factor.
Whether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VIIa and assessed its effect on factor VII:tissue factor interactions. FVII-GP inhibited the activation of factor X by factor VIIa in the presence of either soluble or cell surface tissue factor half-maximally at 0.5 microM and 2.7 microM, respectively. However, FVII-GP failed to inhibit the specific binding of factor VIIa to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor VIIa. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor VIIa to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VIIa was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206.(ABSTRACT TRUNCATED AT 250 WORDS)
Wildgoose
P
P
Biopharmaceuticals Division, NOVO-NORDISK A/S, Gentofte, Denmark.
Jørgensen
T
T
Komiyama
Y
Y
Nakagaki
T
T
Pedersen
A
A
Kisiel
W
W
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis; D005167:Factor VII / Q000737:chemistry*; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D010455:Peptides / Q000302:isolation & purification / Q000502:physiology*; D010743:Phospholipids / Q000502:physiology*; D013925:Thromboplastin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008081:Liposomes; D010455:Peptides; D010743:Phospholipids; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa