PLATELET-ENDOTHELIAL CELL INTERACTIONS

据估计，所有血友病A或B患者中有10%至20%患有 原因不明的出血时间延长，并伴有血浆中 循环免疫复合物(CIC)。我们假设中情局的理由是 功能性内皮细胞损伤导致血管生成增加 前列环素(PGI2)和前列腺素E2(PGE2)，其净作用是 以损害血小板堵塞的形成。为了验证这一假设， 这项工作的目标是：1)对此类患者进行观察和量化 与正常人一样，局部损伤时的血小板黏附-聚集 培养的内皮细胞单层上的部位生长为融合 覆盖胶原蛋白的玻璃和暴露在受控条件下的整体 血流，2)评估平行内皮细胞的功能和可能性 前列腺素I2、前列腺素E_2和前列腺素E_2的下游测定对人体的伤害 细胞内酶(乳酸脱氢酶，LDH)和3)与 结果到出血时间，对于患者组，临床严重程度 出血发作。这些目的中的第一个关键是使用in 形成单层培养内皮细胞的体外流室 一个边界曲面。流道厚度(254Mum)和流率 从搅拌良好的恒温储血池通过 滚柱泵允许控制切应力和切变率 血液单层界面。这个房间还容纳了一个视频显微镜。 由此可以对附着或聚集在损伤部位的血小板进行成像 通过透射光或EPI荧光显微镜在全血中检测。 将通过以下方式监测内皮细胞功能和可能的损伤 一个特殊的腔体特征：包括磁力搅拌流动区 就在单层的下游。在这个流动区域，血液 PGI2和PGE2的样本(通过特定的RIA测量)和LDH许可证 前列腺素I_2、前列腺素E_2产率的计算(浓度流量) 或LDH作为一个整体的单分子膜。相衬直接观察法 显微镜(用缓冲液置换血液之后，但在腔室之前 拆卸)产生关于内皮细胞的补充信息 剥离和形态改变。双方将寻求建立关系 一方面，血小板黏附-聚集和单层功能，以及 另一方面，出血时间和出血发作严重程度的评分。 独立实验变量将包括CIC的浓度， 细胞单层损伤的大小(30-150 Mum)、剪切率、剪应力和 血液流动的持续时间。

An estimated 10 to 20% of all patients with hemophilia A or B have unexplained prolonged bleeding times together with elevation in levels of circulating immune complexes (CIC's). We hypothesize that the CIC's cause functional endothelial cell injury leading to increased production of prostacyclin (PGI2) and prostaglandin E2 (PGE2), the net effect of which is to impair platelet plug formation. To evalutate this hypothesis, objectives of this work are 1) to observe and quantify, for such patients as well as normal subjects, platelet adhesion-aggregation upon local injury sites on cultured endothelial cell monolayers grown to confluence on collagen-coated cover glasses and exposed to controlled conditions of whole blood flow, 2) to assess parallel endothelial cell function and possible injury by downstream measurement of production of PGI2, PGE2 and intracellular enzymes (lactate dehydrogenase, LDH), and 3) to relate findings to bleeding time and, for the patient group, clinical severity of bleeding episodes. Key to the first of these aims is the use of an in vitro flow chamber in which a monolayer of cultured endothelial cells forms one bounding surface. Thickness of the flow path (254Mum) and rate of flow from a well-stirred, constant temperature blood reservoir by means of a roller pump allow for control of shear stress and shear rate at the blood-monolayer interface. The chamber also accomodates a video microscope whereby platelets adherent to or aggregated upon injury sites can be imaged in whole blood by either transmitted light or EPI-fluorescent microscopy. Endothelial cell function and possible injury will be monitored by means of a special chamber feature: inclusion of a magnetically stirred flow region immediately downstream from the monolayer. In this flow region, blood samples for PGI2 and PGE2 (measured by specific RIA's) and LDH permit calculation (concentration flow rate) of rate of production of PGI2, PGE2 or LDH by the monolayer as a whole. Direct observation by phase contrast microscopy (after displacing blood by buffer, but before chamber disassembly) yields complimentary information concerning endothelial cell detachment and morphologic change. Relationships will be sought between platelet adhesion-aggregation and monolayer function, on the one hand, and bleeding time and a score for severity of bleeding episodes, on the other. Independent experimental variables will include concentration of CIC's, size (30 to 150Mum) of cell monolayer injury, shear rate, shear stress and duration of blood flow.