Gaur M, Kamata T, Wang S, Moran B, Shattil S J, Leavitt A D
Department of Laboratory Medicine, University of California, San Francisco, CA 94142, USA.
J Thromb Haemost. 2006 Feb;4(2):436-42. doi: 10.1111/j.1538-7836.2006.01744.x.
The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits alpha(IIb) and beta(3), is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of alpha(IIb)beta(3), thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to alpha(IIb)beta(3) triggers cytoskeletal changes and granule release (outside-in signaling).
Genetic approaches to characterize the molecular pathways involved in alpha(IIb)beta(3) signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs).
alpha(IIb)beta(3) activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, /GPIbalpha(+) cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of alpha(IIb)beta(3) outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of alpha(IIb)beta(3) activation.
Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and alpha(IIb)beta(3) signaling in the native cellular environment.
血小板纤维蛋白原受体是一种由整合素亚基α（IIb）和β（3）组成的异二聚体，是血小板聚集、铺展和止血所必需的。凝血酶和二磷酸腺苷（ADP）等血小板激动剂可导致α（IIb）β（3）活化，从而增强其与纤维蛋白原结合的亲和力和avidity（内向外信号传导）。此外，纤维蛋白原与α（IIb）β（3）的结合触发细胞骨架变化和颗粒释放（外向内信号传导）。
对于无核的血小板，无法采用基因方法来表征参与α（IIb）β（3）信号传导的分子途径。因此，我们建立了一种OP9基质细胞共培养系统，用于从人胚胎干细胞（hESC）生成巨核细胞。
在用已知的血小板激动剂刺激后，通过可溶性纤维蛋白原与hESC衍生的巨核细胞/GPIbalpha（+）细胞的结合来测量的α（IIb）β（3）活化很容易检测到。针对两种血小板凝血酶受体，蛋白酶激活受体1（PAR1）和PAR4的肽激动剂的剂量反应曲线显示出与人类血小板相似的相对反应性，并且肾上腺素可增强次最大ADP反应。此外，当接种在纤维蛋白原包被的表面上时，hESC衍生的巨核细胞会经历片状伪足形成、肌动蛋白丝组装和纽蛋白在粘着斑处的定位，这是α（IIb）β（3）外向内信号传导的特征。通过慢病毒感染进行基因修饰的未分化hESC可以被克隆并维持在未分化状态，然后分化为能够进行α（IIb）β（3）活化的巨核细胞。
利用hESC，我们开发了一种可再生的人类巨核细胞来源，以及一种在天然细胞环境中研究巨核细胞生成和α（IIb）β（3）信号传导的遗传易处理系统。
