Zhang Jing, Abiraman Krithika, Jones Sasia-Marie, Lykotrafitis George, Andemariam Biree
Department of Mechanical Engineering, University of Connecticut, Storrs, Connecticut.
Department of Biomedical Engineering, University of Connecticut, Storrs, Connecticut.
Biophys J. 2017 Jan 10;112(1):143-152. doi: 10.1016/j.bpj.2016.11.3204.
Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvβ3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD. A better understanding of the mechanisms that control RBC adhesion to endothelium may lead to novel approaches to both prevention and treatment of vaso-occlusive episodes in SCD. One important mechanism of ICAM-4 activation occurs via the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA)-dependent signaling pathway. Here, we employed an in vitro technique called single-molecule force spectroscopy to study the effect of modulation of the cAMP-PKA-dependent pathway on ICAM-4 receptor activation. We quantified the frequency of active ICAM-4 receptors on WT-RBC and SS-RBC membranes, as well as the median unbinding force between ICAM-4 and αvβ3. We showed that the collective frequency of unbinding events in WT-RBCs is not significantly different from that of SS-RBCs. This result was confirmed by confocal microscopy experiments. In addition, we showed that incubation of normal RBCs and SS-RBCs with epinephrine, a catecholamine that binds to the β-adrenergic receptor and activates the cAMP-PKA-dependent pathway, caused a significant increase in the frequency of active ICAM-4 receptors in both normal RBCs and SS-RBCs. However, the unbinding force between ICAM-4 and the corresponding ligand αvβ3 remained the same. Furthermore, we demonstrated that forskolin, an adenylyl cyclase activator, significantly increased the frequency of ICAM-4 receptors in WT-RBCs and SS-RBCs, confirming that the activation of ICAM-4 is regulated by the cAMP-PKA pathway. Finally, we showed that A-kinase anchoring proteins play an essential role in ICAM-4 activation.
人类健康（野生型（WT））和纯合镰刀型（SS）红细胞（RBC）表达大量介导红细胞之间以及红细胞与白细胞、血小板和内皮细胞之间细胞黏附的表面受体。在镰状细胞病（SCD）中，红细胞与内皮细胞的异常黏附由细胞间黏附分子-4（ICAM-4）介导，ICAM-4出现在红细胞膜上并与内皮αvβ3整合素结合。这是血管闭塞性发作起始的关键因素，血管闭塞性发作是SCD的标志。更好地理解控制红细胞与内皮细胞黏附的机制可能会带来预防和治疗SCD血管闭塞性发作的新方法。ICAM-4激活的一个重要机制是通过环磷酸腺苷 - 蛋白激酶A（cAMP-PKA）依赖性信号通路发生的。在此，我们采用一种称为单分子力谱的体外技术来研究cAMP-PKA依赖性通路的调节对ICAM-4受体激活的影响。我们量化了野生型红细胞和镰状细胞红细胞膜上活性ICAM-受体的频率，以及ICAM-4与αvβ3之间的中位解离力。我们表明，野生型红细胞中解离事件的总体频率与镰状细胞红细胞的总体频率没有显著差异。共聚焦显微镜实验证实了这一结果。此外，我们表明，用肾上腺素孵育正常红细胞和镰状细胞红细胞，肾上腺素是一种与β-肾上腺素能受体结合并激活cAMP-PKA依赖性通路的儿茶酚胺，会导致正常红细胞和镰状细胞红细胞中活性ICAM-4受体的频率显著增加。然而，ICAM-4与相应配体αvβ3之间的解离力保持不变。此外，我们证明，腺苷酸环化酶激活剂福司可林显著增加了野生型红细胞和镰状细胞红细胞中ICAM-4受体的频率，证实ICAM-4的激活受cAMP-PKA通路调节。最后，我们表明A激酶锚定蛋白在ICAM-4激活中起重要作用。
