SIGNIFICANCE OF SICKLE CELL PHOSPHOLIPID ASYMMETRY

尽管镰状细胞性贫血是一种由血红蛋白引起的遗传性疾病 突变，镰刀红细胞膜上的一些独特的变化 血细胞已经被鉴定出来。其中之一就是改变 磷脂组织。镰刀后，磷脂酰丝氨酸(PS)， 通常局限于脂质双层的内部小叶，存在于 暴露在等离子体环境中的外层小叶。我们假设 这种磷脂组织的扭曲，类似于 激活后发生在血小板膜上，赋予促- 向红细胞注入某些类血小板的红细胞的凝血特性 止血特点。基于由以下因素引起的膜质量 循环中的红细胞和从红细胞中释放出的小泡，我们的 计算表明，可供选择的膜PS的量 止血反应超过或等同于 激活的血小板。因此，我们假设PS包含 镰状细胞病患者红细胞和小泡表面可破坏 止血平衡，导致高凝状态，并导致 特殊的血管闭塞并发症。我们在这项提案中的目标是 调查导致止血后果的机制 由镰刀亚群磷脂不对称性丢失所致 细胞和红血球衍生的小泡。此外，我们将调查 含PS的红细胞膜表面活化假说 凝血，并有助于镰状细胞病的病理生理学。至 为了追求这些目标，我们制定了三个具体目标：L。 探讨磷脂(PS)损失的机制 镰刀状红细胞的不对称性，2.确定镰状红细胞 镰刀状红细胞产生的小泡会导致“高凝”。 镰状细胞病的状态，3.将PS的量与 外周循环中的红细胞衍生双层表面 镰刀型高凝状态的检测及临床表现 细胞疾病。荧光膜联蛋白V结合流式细胞术的新方法 将用于识别和分离具有异常的单个细胞 磷脂组织。PS暴露将通过体外实验进行确认 凝血酶原酶试验。磷脂结构异常的细胞 将通过(共聚焦)荧光显微镜进行表征并选择 通过磁珠分离从种群中分离出来。分子的跨双层运动 磷脂将通过自旋标记的运动来确定 磷脂。PS暴露在体内的止血足迹将是 通过凝血酶原片段1+2(F1.2)的标准测定， 凝血酶抗凝血酶复合体(TAT)和纤维蛋白肽A(FPA)。结果 将与镰状细胞病的临床表现相关联 尤其是在急性胸腔综合征(ACS)患者和 经颅多普勒(TCD)确定为高风险的发展 我们的研究结果应该会为后续中风提供新的信息 磷脂不对称镰状细胞丢失的机制及其意义 潜在的生理影响。

Although sickle cell anemia is a genetic disease due to a hemoglobin mutation, a number of unique changes in the membrane of the sickle red blood cell have been identified. One of these is an alteration in phospholipid organization. Following sickling, phosphatidyl serine (PS), normally confined to the inner leaflet of the lipid bilayer, is present in the outer leaflet, exposed to the plasma environment. We hypothesize that this distortion of phospholipid organization, an event similar to that which occurs in the platelet membrane following activation, confers pro- coagulant properties on the red cell imbuing it with certain platelet-like characteristics in hemostasis. Based upon the membrane mass resulting from circulating red cells and vesicles released from red cells, our calculations suggest that the quantity of membrane PS available for hemostatic reactions either exceeds, or is equivalent to, that provided by activated platelets. Therefore, we hypothesize that the PS containing surfaces on red cells and vesicles in sickle cell disease can disrupt the hemostatic balance, result in a hypercoagulable state, and lead to specific vaso-occlusive complications. Our goal in this proposal is to investigate the mechanisms responsible for, and hemostatic consequences resulting from, loss of phospholipid asymmetry in subpopulations of sickle cells and red cell derived vesicles. Furthermore, we will investigate the hypothesis that red cell derived membrane surfaces containing PS activate clotting and contribute to the pathophysiology of sickle cell disease. To pursue these goals, we have developed three specific aims: l. To investigate the mechanisms responsible for loss of phospholipid (PS) asymmetry in sickle red blood cells, 2. To determine if sickle red cells and vesicles generated from sickle red cells cause a "hypercoagulable" state in sickle cell disease, 3. To correlate the amount of PS- erythrocyte-derived-bilayer-surface in the peripheral circulation with measurements of hypercoagulability and clinical manifestations of sickle cell disease. A new method using fluorescent annexin V and flowcytometry will be used to identify and isolate individual cells with an abnormal phospholipid organization. PS exposure will be confirmed with an in vitro prothrombinase assay. Cells with an abnormal phospholipid organization will be characterized by (confocal) fluorescent microscopy and selected from the population by magnetic bead separation. Transbilayer movement of phospholipids will be determined by the movement of spin labeled phospholipids. Hemostatic footprints of PS exposure in vivo will be determined by standard assays for prothrombin fragment 1+2 (F1.2), thrombin anti thrombin complex (TAT) and Fibrinopeptide A (FPA). Results will be correlated with the clinical manifestations of sickle cell disease in particular in patients with acute chest syndrome (ACS) and patients identified by Transcranial Doppler (TCD) to be at high risk for developing subsequent stroke Our research findings should provide new information on the mechanism of loss of phospholipid asymmetry sickle cells, and its potential physiologic effects.