Human Red Blood Cell Adhesion to Laminin Measured by Atomic Force Microscopy

Abstract

Introduction: Red blood cells (RBCs) express several surface adhesion receptors which modulate cellular function and mediate cell-cell interactions, or adhesion, on both healthy and pathological RBCs. In this study, we established an in vitro single-cell force spectroscopy (SCFS) technique to study human RBC adhesion to substrates coated with laminin protein. Additionally, in order to establish the sensitivity of the assay, we investigated changes in the detachment force between single RBCs and the laminin substrate in the presence of pharmacological modulators of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway.Methods and Results: Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin. SCFS records force-distance curves of an approach/retraction cycle between the RBC probe to and from the laminin-functionalized substrate. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm2 areas of the RBC membrane via single-molecule force spectroscopy (SMFS). Finally, RBCs were treated with the PKA activator-forskolin (FSK), PKA inhibitor-KT5720 and epinephrine to study the variations in the adhesion of BCAM/Lu to laminin due to pharmacologic modulation of the cAMP signaling pathway.Conclusion: We established that SCFS can detect variations in the detachment force of RBCs to laminin based primarily on the number of active BCAM/Lu receptors, which was modulated via biochemicals affecting the cAMP-PKA pathway. This study shows important implications for AFM-based SCFS measurements in understanding and evaluating the pharmacologic response of adhesion receptors on RBCs. Further, this method can easily be employed to measure the adhesive interactions of various cell types with functionalized substrates or other cells.