The A1 and A2 subunits of factor VIIIa synergistically stimulate factor IXa catalytic activity.
Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Recently, we showed that isolated A2 subunit enhanced the kcat for factor IXa-catalyzed activation of factor X by approximately 100-fold ( approximately 1 min-1), whereas isolated A1 or A3-C1-C2 subunits showed no effect on this rate (Fay, P. J., and Koshibu, K. J. (1998) J. Biol. Chem. 273, 19049-19054). However, A1 subunit increased the A2-dependent stimulation by approximately 10-fold. The Km for factor X in the presence of A2 subunit was unaffected by A1 subunit, whereas the kcat observed in the presence of saturating A1 and A2 subunits ( approximately 15 min-1) represented 5-10% of the value observed for native factor VIIIa (approximately 200 min-1). An anti-A1 subunit antibody that blocks the association of A2 eliminated the A1-dependent contribution to factor IXa activity. Inclusion of both A1 and A2 subunits resulted in greater increases in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa than that observed for A2 subunit alone and approached values obtained with factor VIIIa. These results indicate that A1 subunit alters the A2 subunit-dependent modulation of the active site of factor IXa to synergistically increase cofactor activity, yielding an overall increase in kcat of over 1000-fold compared with factor IXa alone.
Fay
P J
PJ
Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA. Philip_Fay@urmc.rochester.edu
Koshibu
K
K
Mastri
M
M
D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D004789:Enzyme Activation; D015949:Factor IXa / Q000378:metabolism*; D005169:Factor VIII / Q000378:metabolism; D015944:Factor VIIIa / Q000737:chemistry / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism
D000906:Antibodies; D011994:Recombinant Proteins; D015944:Factor VIIIa; C078147:F8 protein, human; D005169:Factor VIII; D005170:Factor X; D015949:Factor IXa; D015951:Factor Xa