Hematology-Oncology Division, Department of Medicine, University of California, San Diego, La Jolla, CA, USA.
Methods Mol Biol. 2021;2217:237-249. doi: 10.1007/978-1-0716-0962-0_13.
Platelets are small, anucleate cells that play oversized roles in hemostasis, immunity, and inflammation. An important mediator of platelet function is integrin αIIbβ3, which is required for fibrinogen-dependent platelet aggregation during hemostasis. This platelet response is dependent on conformational changes in the integrin induced by "inside-out" biochemical signals that are triggered by platelet agonists. In turn, fibrinogen binding to αIIbβ3 initiates "outside-in" biochemical and mechanical signals that regulate the platelet cytoskeleton and help to promote full platelet aggregation and secretory responses. Without a nucleus, there is a limited range of experimental manipulations that are possible with human platelets to study the molecular basis of integrin signaling in these primary cells. Consequently, many studies of αIIbβ3 function use genetic approaches that rely on heterologous expression systems or platelets from gene-targeted mice, sometimes with uncertain applicability to human platelets. This chapter will detail a method for genetic manipulation of megakaryocytes and platelets derived from human induced pluripotent stem cells for molecular studies of αIIbβ3 signaling and for modeling of human platelet functions potentially relevant to hemostasis, immunity, and inflammation.
血小板是一种无核的小细胞，在止血、免疫和炎症中发挥着重要作用。整合素αIIbβ3是血小板功能的一个重要介质，它在止血过程中纤维蛋白原依赖性血小板聚集中是必需的。这种血小板反应依赖于整合素的构象变化，这种构象变化是由血小板激动剂触发的“内-外”生化信号引起的。反过来，纤维蛋白原与αIIbβ3的结合启动“外-内”生化和机械信号，调节血小板细胞骨架，并有助于促进血小板的完全聚集和分泌反应。由于没有细胞核，因此在研究这些原代细胞中整合素信号的分子基础时，人类血小板能够进行的实验操作范围有限。因此，许多关于αIIbβ3功能的研究使用依赖于异源表达系统或基因靶向小鼠血小板的遗传方法，有时对人类血小板的适用性不确定。本章将详细介绍一种从人诱导多能干细胞中分离的巨核细胞和血小板的遗传操作方法，用于研究αIIbβ3信号转导以及模拟与止血、免疫和炎症相关的人类血小板功能。
