Lu/BCAM Expression Defines Functionally Distinct Red Blood Cells

In recent years, blood cells such as monocytes, neutrophils and platelets have been found to be much more heterogenous than previously anticipated, with different subsets of these cells performing slightly different functions. Red blood cells (RBCs) are thought to be phenotypically and functionally highly homogeneous, with strong emphasis on their primary purpose, the transport of oxygen. However, RBCs play crucial roles in other biological processes including coagulation. This prompted us to investigate if there are phenotypical and functional differences between RBCs. One of the proteins that is differentially expressed on RBCs is Lutheran/basal cell adhesion molecule (Lu/BCAM). Lu/BCAM, known for its adhesive properties, is differentially expressed among RBCs, forming two distinct populations; Lu/BCAM-positive and Lu/BCAM-negative cells. Lu/BCAM is typically expressed at the orthochromatic erythroblast stage, the stage at which Lu/BCAM-positive and Lu/BCAM-negative cells are formed. We compared the transcriptome of Lu/BCAM-positive and Lu/BCAM-negative erythroblasts by next generation sequencing and identified a set of differentially expressed genes between the two subsets. Interestingly, gene expression analysis demonstrated significant expression of the protease-activated receptor 1 (PAR1), the thrombin receptor, and genes associated with its signal transduction pathway, in Lu/BCAM-negative cells, indicating a distinct role for Lu/BCAM-negative RBCs in coagulation. Notably, functional assays revealed that Lu/BCAM-negative RBCs bind to fibrin in response to thrombin stimulation, in contrast to Lu/BCAM-positive RBCs. Moreover, clots composed of only Lu/BCAM-negative RBCs exhibited a different distribution of fibrin throughout the clot, compared to normal blood clots. Interestingly, upon analysis of the structure of clots by confocal microscopy, it was evident that Lu/BCAM-negative RBCs interacted with fibrin, which is typically located at the outside of the clot. This observation aligns well with the observed increase in fibrin binding. This finding was confirmed in patient-derived deep-vein thrombi, where Lu/BCAM-negative, but not Lu/BCAM-positive, RBCs co-localized with fibrin. Thus, our results reveal that Lu/BCAM-positive RBCs and Lu/BCAM-negative RBCs are distributed different in blood clots, dependent on their propensity to be activated by thrombin and their subsequent binding to fibrin. This study underscores that Lu/BCAM-negative and -positive RBCs, while phenotypically similar, are functionally distinct in coagulation. To our knowledge, this is the first evidence for the presence of RBC subsets that are functionally different. Further research might reveal the existence of additional functional subsets of RBCs.