BINDING OF FACTOR-VIIIA AND FACTOR-VIII TO FACTOR-IXA ON PHOSPHOLIPID-VESICLES

被引:0
作者
DUFFY, EJ
PARKER, ET
MUTUCUMARANA, VP
JOHNSON, AE
LOLLAR, P
机构
[1] EMORY UNIV,DEPT MED,DIV HEMATOL ONCOL,DRAWER AJ,1003 WOODRUFF MEM BLDG,ATLANTA,GA 30322
[2] UNIV OKLAHOMA,DEPT CHEM & BIOCHEM,NORMAN,OK 73019
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中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:17006 / 17011
页数:6
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