Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.
The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs
Duffy
E J
EJ
Department of Medicine, Emory University, Atlanta, Georgia 30322.
Parker
E T
ET
Mutucumarana
V P
VP
Johnson
A E
AE
Lollar
P
P
D000818:Animals; D015949:Factor IXa / Q000737:chemistry / Q000378:metabolism*; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D015944:Factor VIIIa / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine
D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D015944:Factor VIIIa; D005169:Factor VIII; D015949:Factor IXa