title: Structural and functional characterization of platelet receptor-mediated factor VIII binding.
abstract: Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.
authors.affiliation: The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Ahmad
authors.forename: J M
authors.initials: JM
authors.lastname: Scandura
authors.forename: P N
authors.initials: PN
authors.lastname: Walsh
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D015944:Factor VIIIa / Q000737:chemistry / Q000378:metabolism*; D015943:Factor Va / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: C408471:5-dimethylaminonaphthalene-1-sulfonyl glutamyl-glycyl-arginyl chloromethyl ketone inactivated factor IXa; D017304:Annexin A5; D014841:von Willebrand Factor; D015943:Factor Va; D015944:Factor VIIIa; D005169:Factor VIII; D005170:Factor X; D015949:Factor IXa