FUNCTION OF FACTOR VIII ON THE PLATELET MEMBRANE

因子VIII a-因子IXa酶复合物的重要性是 以血友病为例， 危及生命的出血 在我们过去5年的调查中 我们发现，在低摩尔分数的酸性膜上， 磷脂，类似于细胞膜，因子VIII表现出 对含磷脂酰-L-丝氨酸结合立体选择性亲和力 网站. 含磷脂酰丝氨酸（PS）的位点具有高度特异性 因此，没有血浆蛋白将有效地与因子VIII竞争， 约束力 我们还发现因子VIIIa与因子IXa结合， 在没有磷脂膜的情况下具有中高亲和力，但 因子VIIIa-因子IXa复合物的催化效率为 通过与含PS的膜结合而大大增强。 这些发现 这就引出了以下三个假设。 第一，膜结合因子 VIIIa为因子IXa提供高亲和力的膜结合位点， 高亲和力结合需要因子的正确构象 IXa活性位点。 我们将测量IXa因子的结合，衍生于 通过荧光素标记的三肽底物（Glu-Gly- Arg-氯甲基酮），与结合到磷脂酰- 含L-丝氨酸的合成膜与血小板。 为了比较 因子IXa将用非生理性三肽衍生 改变活性的底物（D-Phe-Pro-Arg氯甲基酮） 凝血酶和显然是因子IXa的位点构象。 第二、 单个磷脂分子或磷脂的小聚集体 激活因子VIII a-因子IXa复合物，引起的变化， 酶复合物的构象。 我们建议用可溶性物质进行实验 磷脂和与磷脂混合胶束，以区分 活化来自那些的因子VIII a-因子IXa复合物的部分 它们是膜基质的惰性成分。 实验 因子IXa的磷脂结合结构的抗体， 因子VIIIa和因子X的修改形式将表明 复合物是否在远离膜的部位发生改变。 第三，含PS的膜对因子VIIIa的高特异性 结果来自膜磷脂自发排列成 作为血小板特异性结合位点的微区 膜。 我们将制备单克隆抗体片段， 含PS的微区，以便更好地表征微区 并研究它们作为因子的功能受体的重要性， 八. 完成拟议研究的人数将大大增加 我们对血小板磷脂作用的了解 激活因子VIII a-因子IXa复合物。

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.