Location of the platelet binding site in zymogen coagulation factor IX

The assembly of the tenase complex on the surface of the platelet is an ess ential step in maintaining normal hemostasis as evidenced by the serious he morrhagic diathesis associated with either factor IX (FIX) or factor VIII d eficiencies. Understanding the regions and or residues of FIX crucial for p roper binding to platelets has important clinical implications. The ability of FIX to bind activated platelets in the presence of 4 mmol/l CaCl2 was e xamined using electrophoretic light-scattering experiments. Wild-type FIX b inds to activated platelets with dissociation constant K-d = 7.9 nmol/l. Ac tivated FIX binds to activated platelets with K-d = 2 nmol/l. Activated fac tor VII does not bind activated platelets at physiological concentrations. The Gla domain of FIX is important for the binding of FIX to activated plat elets since a chimera with a factor VII (FVII) template and FIX Gla [FVII(F IXGla)] has K-d = 9.6 nmol/l, and a chimera with a FVII template and FIX Gl a, A and the first epidermal growth factor domain (EGF1) [FVII(FIXGla,A,EGF 1)] has K-d = 9.7 nmol/l, but a chimera with a FIX template and a FVII Gla [FIX(FVIIGla)] does not bind activated platelets. Altering the fifth residu e of FIX from a lysine to an alanine (Lys5 --> Ala) abolishes the mutant fr om binding to collagen but does not affect FIX binding to the activated pla telet (K-d = 9.8 nmol/l). Point mutations involved with residues 4 and 5 (G ly4 --> Phe and Lys5 --> no residue), residue 9 (Phe9 --> Ala), residue 10 (Val10 --> Lys) and residues 9-11 (Phe9 --> Met). (C) 2001 Lippincott Willi ams & Wilkins.