Adhesive Characteristics of Hemoglobin SC Red Blood Cells.
Authors: Chien Ai C. , Zennadi Rahima , De Castro Laura M. , Telen Marilyn J. 
Published: April 2020

Abstract
Hemoglobin (Hb)-S containing red blood cells (RBCs) adhere abnormally to the vascular wall; this phenomenon is postulated to contribute to vaso-occlusive crisis and serious tissue damage. Patients homozygous for Hb S experience the most severe form of SCD, whereas heterozygous patients carrying Hb S and another hemoglobin variant, Hb C, experience somewhat milder clinical courses. Laminin is the extracellular matrix protein to which SS RBCs bind most avidly and is the ligand for the BCAM/Lu receptor. αvβ3 integrin is one of the major integrins on endothelial cells (ECs) and is the counter-receptor for LW on SS RBCs. Epinephrine activates both LW and BCAM/Lu on SS RBCs and thereby induces increased SS RBC adhesion to both ECs and laminin via a cAMP-dependent pathway. We have now compared the adhesive properties of Hb SC RBCs to SS RBCs. We measured the expression of RBC adhesion receptors, BCAM/Lu and LW, and assayed adhesion of SC RBCs to laminin and ECs (with and without prior stimulation of RBCs by epinephrine). Blood samples from Hb SC and SS patients in steady state and from normal controls (Hb AA) were collected into citrate tubes and were washed to remove plasma and buffy coat before use. The levels of BCAM/Lu and LW expression on RBCs were measured by flow cytometry using monoclonal antibodies to BCAM/Lu and anti-LW. SC RBCs expressed higher levels of BCAM/Lu (MFI 476.2, n=13) than did SS RBCs (MFI 332.3, n=33) and AA RBCs (MFI=225.5, n=16), but none of these differences were statistically significant (all p values &gt;0.05). The levels of LW expression were also similar on AA, SC, and SS RBCs. Adhesion to laminin and ECs was measured as previously described, in a graduated height flow chamber. Non-treated, sham-treated, and epinephrine-treated RBCs were each infused into a flow chamber fitted with a slide coated with laminin or ECs. After washing at a constant rate, adherent RBCs were quantitated at points of different shear stresses. Adhesion of SC RBCs to laminin (mean of 53.1 cells/mm2 at 1 dyne/cm2, n=9) was lower than that of SS RBCs (mean of 69.0 cells/mm2 at 1 dyne/cm2, n=6, p=0.459) but was markedly higher than the adhesion seen with AA RBCs (mean of 0.03 cells/mm2 at 1 dyne/cm2, n=3, SC vs AA p=0.011). A positive correlation was found between BCAM/Lu expression and SC RBC adhesion to laminin (r=0.638, p=0.047). While epinephrine induced an increase in SC RBC adhesion to laminin in only 10% of all SC patient samples tested (compared to 50% of SS patients), epinephrine upregulated SC RBC adhesion to ECs approximately 10-fold in all samples tested (n=3). We conclude that SC RBCs represent an intermediate adhesive phenotype compared to AA and SS RBCs. While BCAM/Lu-mediated adhesion to laminin was lower on SC RBCs than on SS RBCs, unstimulated BCAM/Lu adhesive function was strikingly enhanced in relation to AA RBCs. Most importantly, epinephrine uniformly increased SC RBC adhesion to ECs, suggesting that in Hb SC disease, physiological stress may induce SC RBC adhesion and vaso-occlusive crises by mechanisms similar to those postulated to occur as a result of stress in Hb SS disease.