A-Kinase Anchoring Proteins Mediate Healthy and Sickle Red Blood Cell Adhesion To Endothelial Laminin-5

Healthy and pathological human red blood cells (RBCs) express surface adhesion receptors which mediate interactions with the endothelium, platelets, and white blood cells. The adhesive properties of RBCs are defined by the density and distribution of adhesion receptors, and the binding force to specific ligands. In sickle cell disease (SCD), cytoadherence of RBCs to the vascular endothelium and subendothelial matrix is thought to be a major contributor to and possibly the primary cause of vasculopathy in SCD¹. Painful vaso-occlusive episodes (VOEs), the hallmark of SCD, result from the blockage of small vessels. Here, we establish a molecular-level technique, based on atomic force microscopy (AFM), to measure the binding force between a specific ligand and its corresponding receptor on a single RBC. Using AFM, we can compare the frequency and localization of adhesion receptors present on the membrane of RBCs collected from healthy human volunteers and SCD patients. We focused on the interaction between basal cell adhesion molecule and its isoform Lutheran (BCAM/Lu) on the RBC and the subendothelial matrix protein laminin-5. Sickle RBCs (SS RBCs) overexpress BCAM/Lu and are more adherent to laminin-5 than healthy RBCs. Importantly, by using a flow adhesion assay, Hines et al² demonstrated that the interaction between BCAM/Lu and laminin-5 is mediated by cyclic adenosine monophosphate (cAMP) which activates protein kinase A (PKA) and BCAM/Lu receptors. Because PKA binding can be mediated by A-kinase anchoring proteins (AKAPs), which sequester PKA to subcellular sites³, here we investigate whether AKAPs play a role in BCAM/Lu receptor activation on RBCs from healthy volunteers and SCD patients.

Peripheral blood RBCs were collected from consenting healthy volunteers (n=5) and individuals with SCD (n=4). RBCs were separated from whole blood using centrifugation. AFM was employed to quantify the frequency of active BCAM/Lu receptors on a single RBC. Molecular interactions were quantified by recording force v. distance curves between a laminin-5 functionalized probe and a 1μm² area of the RBC surface with a lateral resolution of 30nm. Detected binding forces represent adhesive events between BCAM/Lu and laminin-5, which translate to the percentage of active BCAM/Lu receptors on the RBC surface. To detect the presence of AKAPs, RBCs were treated with St-Ht31 inhibitor peptide (83nM bath), which inhibits the interaction between the regulatory subunits of PKA and AKAP in cells. As a negative control, RBCs were treated with St-Ht31P control peptide (83nM bath), which is the inactive form of the St-Ht31 inhibitor peptide.

Our experiments reveal that following treatment with St-Ht31, the frequency of active BCAM/Lu receptors is significantly lower on healthy RBCs (from 5.71±1.23% to 3.15±1.09%; p<0.05) and even more in SS RBCs (from 7.19±1.42% to 2.02±0.37%; p<0.0001). Treatment with St-Ht31P control peptide did not change the frequency of active BCAM/Lu receptors on healthy RBCs (5.92±1.09%) or SS RBCs (6.49±1.27%) as compared to baseline measurements.

Importantly, using St-Ht31, we confirm the presence of AKAPs as scaffold proteins which can modulate the adhesion of BCAM/Lu adhesion receptors on the SS RBC to laminin-5. PKA binding can be mediated by AKAPs, which sequester PKA to specific subcellular sites through binding to regulatory subunits³. Each AKAP contains two classes of binding sites: an anchoring motif, which binds the regulatory subunit of PKA, and a targeting domain, which directs the subcellular localization of the PKA-AKAP complex through association with structural proteins, membranes, or other components. This finding provides evidence for a new paradigm in AKAP adhesive signaling in RBCs and a potential new pharmacologic target for the prevention and treatment of VOEs in SCD.

References:

1. Hillery CA, Panepinto JA. Pathophysiology of stroke in sickle cell disease. Microcirculation. 2004;11(2):195-208. 10.1080/10739680490278600.

2. Hines PC, Zen Q, Burney SN, et al. Novel epinephrine and cyclic amp-mediated activation of bcam/lu-dependent sickle (ss) rbc adhesion. Blood. 2003;101(8):3281-3287. 10.1182/blood-2001-12-0289.

3. Benovic JL. Novel β2-adrenergic receptor signaling pathways. Journal of Allergy and Clinical Immunology. 2002;110(6, Supplement):S229-S235. http://dx.doi.org/10.1067/mai.2002.129370.

No relevant conflicts of interest to declare.