The Role of Membranes in Thrombus Formation In Viva

磷脂酰丝氨酸暴露在细胞膜的外部小叶上对于凝血酶复合物tenase（因子X的激活剂）和凝血酶原酶（凝血酶原的激活剂）在膜表面上的组装是重要的。 人们对微粒子膜表面在引发血液凝固和凝固酶复合物组装中的作用越来越感兴趣。 关于凝血复合物在体外的组装，我们已经知道了很多;体内重要膜表面的身份仍然不确定。 这些研究的目的是建立促凝血膜表面的来源，带负电荷的磷脂的时间和空间暴露，以及凝血酶原酶在体内组装的位点。在具体目标1中，我们将通过大小以及蛋白质和磷脂组成来表征从血小板、单核细胞、中性粒细胞和内皮细胞体外生成的微粒，以基于膜蛋白组成来识别特征，从而通过细胞来源和亚群来区分微粒。 在特定目标2中，我们将表征从人血液中分离的微粒，并将其与体外产生的微粒进行比较。 我们预计，可以识别微粒群体起源细胞的签名可能有助于研究人类血栓形成和炎症状态。 我们还将使用微粒和细胞特征在小鼠血栓形成模型中体内鉴定这些物种。 因此，我们将确定小鼠细胞和微粒的类似特征。 在具体目标3中，我们将确定该模型中氨基磷脂的空间和时间暴露以及凝血酶原酶的组装。 为了进行这些研究，我们开发了一种独特的成像设备，用于采集活体显微镜数据。 该仪器专用于活体动物的活体显微镜检查;以高速（接近视频速率）获取图像;具有多个荧光通道，允许同时观察多达三种荧光染料;具有宽视场和共聚焦能力;分辨率高;允许3D/体积重建;提供数字图像分析。

Phosphatidylserine exposure on the external leaflet of cell membranes is important for the assembly of the coagulation enzyme complexes tenase, the activator of factor X and prothrombinase, the activator of prothrombin on the membrane surface. There is increasing interest in the role of the membrane surfaces of microparticles in initiation of blood coagulation and assembly of coagulation enzyme complexes. A great deal is known about the assembly of the coagulation complexes in vitro; the identity of the important membrane surface(s) in vivo remains uncertain. The goals of these studies are to establish the source of procoagulant membrane surfaces, the temporal and spatial exposure of negatively charged phospholipids, and the site of prothrombinase assembly in vivo. In Specific Aim 1 we will characterize microparticles generated in vitro from platelets, monocytes, neutrophils, and endothelial cells by size and by protein and phospholipid composition to identify a signature based on membrane protein composition to distinguish microparticles by cell source and subpopulation. In Specific Aim 2 we will characterize microparticles isolated from human blood and compare them to microparticles generated in vitro. We anticipate that signatures that can identify the cell of origin of microparticle populations may be useful in studying human thrombotic and inflammatory states. We will also use the microparticle and cell signatures to identify these species in vivo in a thrombosis model in the mouse. Thus we will determine analogous signatures for mouse cells and microparticles. In Specific Aim 3 we will determine the spatial and temporal exposure of aminophospholipids and assembly of prothrombinase in this model. To do these sorts of studies we have developed a unique imaging facility for acquisition of intravital microscopy data. The instrumentation is dedicated to intravital microscopy in living animals; acquires images at high speed (near video rate); has multiple fluorescence channels to allow observation of up to three fluorochromes simultaneously; has both widefield and confocal capabilities; is high resolution; permits 3D/volume reconstruction; provides digital image analysis.