FUNCTION OF FACTOR VIII ON THE PLATELET MEMBRANE

因子VIIIa-因子IXa酶复合体的重要性是 由血友病说明，在这种血友病中，两种蛋白质的缺失都会导致 有生命危险的出血。在我们过去5年的调查中 我们已经发现，在酸性摩尔分数较低的膜上 磷脂，类似细胞膜，第八因子展示 含结合的磷脂酰丝氨酸-L-丝氨酸的立体选择性亲和力 网站。含有磷脂酰丝氨酸(PS)的部位具有高度的特异性 因此没有血浆蛋白能有效地与第八因子竞争 有约束力的。我们还发现，因子VIIIa与因子IXa结合 在没有磷脂膜的情况下亲和力中等高，但 因子VIIIa-因子IXa复合体的催化效率为 通过与含有PS的膜结合而大大增强。这些发现 将我们引向以下三个假设。第一，膜结合因子 VIIIA为FIXa和FIXa提供高亲和力的膜结合部位 高亲和力结合需要正确的因子构象 IXA活动站点。我们将测量因子IXa的结合，衍生于 荧光素标记的三肽底物(Glu-Gly-)的活性部位 精氨酸氯甲基酮)，与磷脂酰基结合的因子VIIIa- L-含丝氨酸的合成膜与血小板。以供比较 因子IXa将被一种非生理性三肽衍生 改变活性的底物(D-Phe-Pro-Arg氯甲基酮) 凝血酶和凝血因子IXa的位置构象。第二, 单个磷脂分子或磷脂小聚集体 激活因子VIIIa-因子IXa复合体 酶复合体的构象。我们建议用可溶物质进行实验 磷脂与磷脂在混合胶束中的区分 激活因子VIIIa-因子IXa复合体的部分 它们是膜基质的惰性成分。实验： 针对因子IXa和磷脂结合结构的抗体 因子八a以及修正形式的因子X将表明 复合体是否在远离膜的位置发生改变。 第三，含PS膜对因子VIIIa的高度专一性 膜磷脂自发排列的结果 作为血小板上特异性结合部位的微区 膜。我们将制备识别的单抗片段 含有PS的微区，以便更好地表征微区 并研究它们作为因子功能受体的重要性 。拟议研究的完成将大幅增加 我们对血小板磷脂在血管紧张素转换酶中作用的认识 激活因子VIIIa-因子IXa复合体。

The importance of the factor VIIIa-factor IXa enzyme complex is illustrated by hemophilia in which the absence of either protein causes life-threatening bleeding. In our investigations over the past 5 yrs we have found that on membranes with low mole fractions of acidic phospholipid, resembling cell membranes, factor VIII exhibits stereoselective affinity for phosphatidyl-L-serine containing binding sites. The phosphatidylserine (PS)-containing sites are highly specific so that no plasma proteins will compete efficiently with factor VIII for binding. We have also found that factor VIIIa binds to factor IXa with moderately high affinity in the absence of phospholipid membranes but the catalytic efficiency of the factor VIIIa-factor IXa complex is greatly enhanced by binding to PS containing membranes. These findings lead us to the following three hypotheses. First, membrane-bound factor VIIIa provides high affinity membrane binding sites for factor IXa and high affinity binding requires the correct conformation of the factor IXa active site. We will measure binding of factor IXa, derivatized in the active site by a fluorescein-labeled tripeptide substrate (Glu-Gly- Arg-chloromethyl ketone), to factor VIIIa that is bound to phosphatidyl- L-serine-containing synthetic membranes vs. platelets. For comparison factor IXa will be derivatized with a non-physiologic tripeptide substrate (D-Phe-Pro-Arg chloromethyl ketone) that alters the active site conformation of thrombin and apparently of factor IXa. Second, individual phospholipid molecules or small aggregates of phospholipids activate the factor VIIIa-factor IXa complex by causing a change in the conformation of the enzyme complex. We propose experiments with soluble phospholipids and with phospholipids in mixed micelles to distinguish moieties that activate the factor VIIIa-factor IXa complex from those that are inert constituents of the membrane matrix. Experiments with antibodies to the phospholipid-binding structures of factor IXa and factor VIIIa, and with modified forms of factors X, will indicate whether the complex is altered at a site distant from the membrane. Third, the high specificity of PS-containing membranes for factors VIIIa results from spontaneous arrangement of membrane phospholipids into microdomains which function as specific binding sites on platelet membranes. We will prepare monoclonal antibody fragments that recognize PS-containing microdomains in order to better characterize microdomains and to investigate their importance as functional receptors for factors VIII. Completion of the proposed studies will substantially increase our understanding of the role of the platelet phospholipids in activating the factor VIIIa-factor IXa complex.