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 subunit and the Me(2+)-linked A1/A3-C1-C2 dimer. Previously we speculated that the acidic region at the COOH terminus of the A1 subunit was involved 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 from A1/A3-C1-C2 dimer plus A2 subunit. A primary component of the inhibitory activity of the peptide was attributed to its acidic nature based upon similar inhibition of factor VIIIa reconstitution using a synthetic 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 13 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 inhibitory activity of FVIII337-372 was attenuated by the presence of Ca2+. Ca2+ also inhibited the reconstitution of factor VIIIa in the absence of peptide and increased the rate and extent of factor VIIIa decay, suggesting that Ca2+ effectively shielded charges important for the intersubunit interactions. The above results support a role for this acidic region in the association of A2 subunit with A1/A3-C1-C2 dimer.
Fay
P J
PJ
Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Haidaris
P J
PJ
Huggins
C F
CF
D000595:Amino Acid Sequence; D001665:Binding Sites; D002122:Calcium Chloride / Q000494:pharmacology; D003619:Dansyl Compounds; D015944:Factor VIIIa / Q000378:metabolism; D015942:Factor VIIa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G; D007700:Kinetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014357:Trypsin
D003619:Dansyl Compounds; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D015944:Factor VIIIa; D015942:Factor VIIa; D014357:Trypsin; D002122:Calcium Chloride