Role of the C2 domain of factor VIIIa in the assembly of factor-X activating complex on the platelet membrane.
Optimal rates of factor X (FX) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated platelet receptors. To define the FVIIIa domains that mediate platelet interactions, albumin density gradient washed, gel-filtered platelets (3.5 x 10(8)/mL) activated by the thrombin receptor peptide, SFLLRN (25 microM), were incubated with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 domain region, with or without anti-C2 domain monoclonal antibodies (MoAb), ESH4 or ESH8. FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approximately 3 nM), and the C2 domain (Kd approximately 16 nM) all interacted with approximately 700-800 binding sites/platelet. Unlike FVIIIa, the C2 domain did not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding stoichiometry and affinity. Both the MoAb ESH4 and a synthetic peptide corresponding to FVIII residues 2303-2332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2 domain peptide corresponding to residues 2248-2285 (epitope for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding. Thus, a major platelet-binding site resides within residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.
Ahmad
Syed S
SS
The Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Walsh
Peter N
PN
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D015944:Factor VIIIa / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry*
D009363:Neoplasm Proteins; D015944:Factor VIIIa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant