Role of LW and AKAP79 in β-Adrenergic Receptor Signaling-Induced Sickle Red Blood Cell Adhesion.
Authors: Zennadi Rahima , Moeller Benjamin J. , Dewhirst Mark W. , Telen Marilyn J. 
Published: April 2020

We have previously demonstrated that stimulation of β-adrenergic receptors (βARs) by epinephrine ex vivo induced human sickle (SS) but not normal (AA) erythrocytes (RBCs) adhesion to vascular endothelium and vaso-occlusion in nude mice. However, the molecular level at which RBC βAR signaling differentially regulates SS vs AA RBC adhesion has remained unexplored, and the adhesion molecule(s) activated by such pathways to mediate vaso-occlusion in vivo has not been defined. Since we have shown LW to mediate SS RBC adhesion to αvβ3 integrin in vitro, we first examined whether LW on SS RBCs mediated adhesion to endothelium in vivo and whether this interaction had pathophysiological significance. Window chambers were implanted into the dorsal skin folds of nude mice. To avoid endothelial activation, boluses of fluorescently labeled human SS RBCs were exposed to 20 nM epinephrine ex vivo, extensively washed, and then infused into mice after IV injection 30 min earlier of 500 μg of soluble RBC adhesion molecules LW (sLW) or CD44 (sCD44). In contrast to sCD44, IV sLW administration abrogated adhesion of subsequently injected epinephrine-treated SS RBCs to postcapillary vessels and significantly reduced vaso-occlusion in small diameter venules. Circulation of epinephrine-treated SS RBCs was noticeably improved by sLW, with fluxes of 4833±1954 and 18618±6092 circulating RBCs/min in animals receiving either sCD44 or sLW, respectively (p&lt;0.001). These data strongly argue that activated SS RBC adhesion to endothelium is mediated in large part through LW, supporting the pathophysiological significance of βAR-induced LW activation in SCD. We then investigated at what molecular level this pathway responded differently in SS vs AA RBCs. Epinephrine at 20 nM and forskolin at 80 μM induced variable degrees of cAMP production in SS RBCs among patients, while cAMP production was minimal in epinephrine- or forskolin-stimulated AA RBCs. βAR density on SS and AA RBCs varied similarly between 15 and 70 fmol/mg protein and did not significantly correlate with increased cAMP production. The expression of adenylate cyclase also varied among donors in both SS and AA RBCs but was unrelated to cAMP response. Immunoblots of total RBC membrane proteins showed that the amount of membrane-associated PKA catalytic (PKAc) and its subtype PKAβc varied similarly among healthy donors and patients. However, the amount of membrane-bound PKA regulatory IIβ (PKARIIβ) was higher in AA RBCs when compared to the amount bound to the membranes of SS RBCs, suggesting that PKAβc is already activated in SS RBCs. Forskolin induced a significant further reduction in membrane-bound PKARIIβ in SS RBCs with either high or low cAMP production, but not in AA RBCs, demonstrating that PKARIIβ dissociated from PKAβc in SS RBCs even when adenylate cyclase was only modestly activated. These data suggest that there is a threshold above which cAMP production is capable of activating downstream kinases such as PKA in SS but not in AA RBCs. We then examined the A-kinase anchoring protein 79 (AKAP79), which can tether to the PKA type II regulatory subunit and is known to be involved in β2AR signaling. After forskolin stimulation, AKAP79 dissociated completely from membranes of SS but not AA RBCs. These data suggest that AKAP79 associates with PKARIIβ in RBCs and that differential regulation of AKAP79 binding to PKARIIβ may lead to increased activation of PKAβc and thus increased LW-mediated adhesion of SS but not AA RBCs in response to stimulation.