PLATELET-ENDOTHELIAL CELL INTERACTIONS

据估计，10 - 20%的血友病A或B患者 不明原因的出血时间延长， 循环免疫复合物（CIC）。 我们假设作战信息中心的原因 功能性内皮细胞损伤，导致 前列环素（PGI 2）和前列腺素E2（PGE 2），其净效应是 以削弱血小板栓的形成。 为了评估这个假设， 这项工作的目标是1）观察和量化，对于这些患者， 与正常人一样，局部损伤后血小板粘附-聚集 培养的内皮细胞单层上生长至汇合的部位 胶原包被的盖玻片，并暴露于受控条件下， 血流，2）评估平行内皮细胞功能和可能的 通过下游测量PGI 2、PGE 2和 细胞内酶（乳酸脱氢酶，LDH），和3）相关 出血时间的结果，以及患者组的临床严重程度 出血事件。 实现第一个目标的关键是使用一个 体外流动室，在其中形成单层培养的内皮细胞 一个边界面。 流路厚度（254 μ m）和流速 从充分搅拌的恒温血液储存器中， 辊式泵允许控制剪切应力和剪切速率， 血液-单层界面 该室还可容纳视频显微镜 由此粘附或聚集在损伤部位上的血小板可以成像 通过透射光或EPI-荧光显微镜观察全血。 将通过以下方式监测内皮细胞功能和可能的损伤： 一个特殊的腔室功能：包括一个磁搅拌流动区域 在单层的下游。 在这个流动区域，血液 用于PGI 2和PGE 2（通过特定RIA测量）和LDH的样品允许 PGI 2、PGE 2生产速率的计算（浓度流速） 或LDH。 相差直接观测法 显微镜检查（用缓冲液置换血液后，但在腔室前 拆卸）产生关于内皮细胞的补充信息 脱落和形态学改变。 将在以下方面寻求关系： 一方面，血小板粘附-聚集和单层功能， 另一方面，出血时间和出血事件严重程度评分。 独立的实验变量将包括CIC的浓度， 细胞单层损伤大小（30 - 150 μ m）、剪切速率、剪切应力和 血液流动的时间。

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.