Ahmad Syed S, Walsh Peter N
The Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Biochemistry. 2005 Oct 25;44(42):13858-65. doi: 10.1021/bi0511033.
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.
凝血因子X（FX）的最佳激活速率需要凝血因子IXa（FIXa）、凝血因子VIII(a) [FVIII(a)]和FX与活化血小板受体结合。为了确定介导血小板相互作用的FVIIIa结构域，将经凝血酶受体肽SFLLRN（25 microM）激活的白蛋白密度梯度洗涤、凝胶过滤血小板（3.5 x 10(8)/mL）与125I标记的FVIII C2结构域、或125I-FVIIIa、或125I-FVIII((LC))、或来自C2结构域区域的肽一起孵育，有或没有抗C2结构域单克隆抗体（MoAb）ESH4或ESH8。FVIIIa（解离常数约为1.7 nM）、FVIII((LC))（解离常数约为3 nM）和C2结构域（解离常数约为16 nM）均与每个血小板约700 - 800个结合位点相互作用。与FVIIIa不同，C2结构域在存在过量EGR-FIXa（45 nM）和FX（1.5 microM）时，结合化学计量和亲和力并未增强。MoAb ESH4和对应于FVIII残基2303 - 2332的合成肽（FVIII MoAb ESH4的表位）均抑制FVIIIa与血小板的结合，而MoAb ESH8和对应于残基2248 - 2285的C2结构域肽（FVIII MoAb ESH8的表位）未能抑制FVIIIa与血小板的结合。因此，一个主要的血小板结合位点位于FVIIIa的C2结构域中残基2303 - 2332内，并且残基2248 - 2285内的另一个位点仅在FIXa和FX存在时增加FVIIIa与活化血小板结合的化学计量和亲和力，但不直接介导FVIIIa与血小板表面的结合。
