FUNCTIONAL CONSEQUENCES OF MUTATIONS IN SER-52 AND SER-60 IN HUMAN BLOOD-COAGULATION FACTOR-VII

Human blood coagulation factor VII has unique carbohydrate moieties O- glycosidically linked to serine 52 and serine 60 residues in its first epidermal growth factor-like domain. To study the functional role of these glycosyl moieties in factor VII, we constructed, expressed, and purified site-specific recombinant mutants of human factor VII in whic h serine 52 and serine 60 were conservatively replaced with alanine re sidues. S52A factor VIIa (Ser-52 --> Ala), S60A factor VIIa (Ser-60 -- > Ala), and S52,60A factor VIIa (Ser-52, Ser-60 --> Ala) exhibited 56, 73, and 44%, respectively, of the clotting activity of wild-type fact or VIIa using human brain thromboplastin as a source of tissue factor/ phospholipids and 32, 43, and 14% of wild-type factor VIIa using a mix ture of recombinant soluble tissue factor and mixed brain phospholipid s. The tissue factor-dependent and -independent amidolytic activities of these mutants were essentially indistinguishable from that of wild- type factor VIIa. In addition, equilibrium dialysis experiments indica ted that the profiles of Ca-45(2+) binding to these mutants were ident ical with that of wild-type factor VII. In the presence of either Ca2 or EGTA, the K-d values for the interaction of the three factor VIIa mutants to full-length tissue factor were 2- to 5-fold higher than tha t of wild-type factor VIIa, while the K-d values for the interaction o f these mutants to soluble tissue factor were 4- to 15-fold higher tha n that of wild-type factor VIIa. Measurement of the association and di ssociation rate constants for factor VIIa binding to relipidated tissu e factor apoprotein revealed that the association rate constants of th e three factor VII mutants were decreased in comparison with that of w ild-type factor VIIa, while the dissociation rate constants of these t hree mutants were virtually identical to that of wild-type factor VIIa . These findings strongly suggest that glycosyl moieties attached to S er-52 and Ser-80 in factor VII/VIIa provide unique structural elements that are important: for the rapid association of factor VII/VIIa with its cellular receptor and cofactor. (C) 1998 Academic Press.