Department of Medicine, Veterans Administration Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
Blood. 2011 Mar 17;117(11):3181-9. doi: 10.1182/blood-2010-08-301663. Epub 2010 Dec 14.
Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the V(max) for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the V(max) of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase complex.
VIII 因子通过其串联的凝集素同源结构域 C1 和 C2 与含有磷脂酰丝氨酸 (PS) 的膜结合。然而，C1 结构域与膜结合的细节尚未阐明。我们制备了 4 种定位在假定的膜相互作用表面的 VIII 因子 C1 突变体（Arg2090Ala/Gln2091Ala、Lys2092Ala/Phe2093Ala、Gln2042Ala/Tyr2043Ala 和 Arg2159Ala）。使用含有 15% PS 的膜（模拟由凝血酶加胶原刺激的血小板）和 4% PS 的膜（模拟由凝血酶刺激的血小板）测量了膜结合和辅助因子活性。所有突变体与 4% PS 的膜的亲和力至少降低了 10 倍，并且 3 个突变体也降低了与因子 X 的表观亲和力。针对 C2 结构域的单克隆抗体与野生型 VIII 因子相比，对突变体产生了不同的相对损伤。单克隆抗体 ESH4 降低了所有突变体的 V(max)，但仅降低了野生型 VIII 因子的膜亲和力。单克隆抗体 BO2C11 使野生型 VIII 因子的 V(max)降低了 90%，但使 3 个突变体的活性降低了 98%以上。这些结果确定了 VIII 因子 C1 结构域的一个膜结合面，表明 C1 结构域对 VIII 因子与因子 X 的结合有影响，并表明 C1 和 C2 结构域之间的协同作用是因子 Xa 复合物充分活性所必需的。
