ROLE OF THE COOH-TERMINAL ACIDIC REGION OF A1-SUBUNIT IN A2-SUBUNIT RETENTION IN HUMAN FACTOR-VIIIA

Factor VIIIa is a heterotrimer of A1,A2 and A3-C1-C2 subunits which is labile due to a relatively weak affinity interaction between the A2 s ubunit and the Me2+-linked A1/A3-C1-C2 dimer. Previously we speculated that the acidic region at the COOH terminus of the A1 subunit was inv olved with the A2 subunit retention. This region, delineated by factor VIII residues 337-372, was chemically synthesized. Both the peptide, designated FVIII337-372, and an IgG fraction prepared from rabbit anti -FVIII337-372 antiserum inhibited the reconstitution of factor VIIIa f rom A1/A3-C1-C2 dimer plus A2 subunit. A primary component of the inhi bitory activity of the peptide was attributed to its acidic nature bas ed upon similar inhibition of factor VIIIa reconstitution using a synt hetic polymer of aspartic acid. Trypsin cleaved the peptide at Arg359 and the resultant two fragments were isolated. Inhibitory activity was associated with the NH2-terminal fragment which contained 10 of the 1 3 acidic residues present in the original peptide. The fluorescence of a dansylated FVIII337-372 was enhanced 2-fold by A2 subunit and this effect was reversed by addition of excess unmodified peptide. The inhi bitory activity of FVIII337-372 was attenuated by the presence of Ca2. Ca2+ also inhibited the reconstitution of factor VIIIa in the absenc e of peptide and increased the rate and extent of factor VIII(a) decay , suggesting that Ca2+ effectively shielded charges important for the intersubunit interactions. The above results support a role for this a cidic region in the association of A2 subunit with A1/A3-C1-C2 dimer.