title: Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.
abstract: Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. 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 protein. 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 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.
authors.affiliation: Department of Biomedical Engineering, University of Connecticut, Storrs, CT, USA; Department of Hematology, Division of Experimental Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Maciaszek
authors.affiliation: Department of Mechanical Engineering, University of Connecticut, Storrs, CT, USA.
authors.forename: Kostyantyn
authors.initials: K
authors.lastname: Partola
authors.affiliation: Department of Mechanical Engineering, University of Connecticut, Storrs, CT, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Adult Sickle Cell Disease Center, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Biree
authors.initials: B
authors.lastname: Andemariam
authors.affiliation: Department of Biomedical Engineering, University of Connecticut, Storrs, CT, USA; Department of Mechanical Engineering, University of Connecticut, Storrs, CT, USA. Electronic address: gelyko@engr.uconn.edu.
authors.forename: George
authors.initials: G
authors.lastname: Lykotrafitis
mesh_terms: D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D004912:Erythrocytes / Q000502:physiology*; D064368:Healthy Volunteers; D006801:Humans; D007797:Laminin / Q000502:physiology*; D008188:Lutheran Blood-Group System / Q000502:physiology; D008562:Membrane Glycoproteins; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: C090228:BCAM protein, human; D015815:Cell Adhesion Molecules; D007797:Laminin; D008188:Lutheran Blood-Group System; D008562:Membrane Glycoproteins; D000242:Cyclic AMP
keywords: Atomic force microscopy; BCAM/Lu; Cytoadhesion; Erythrocyte; Single molecule force spectroscopy