Aggregates of Reticulocytes, Monocytes and Platelets Exist in Whole Blood of Sickle Cell Patients: Roles for Plasma Fibronectin, α4 Integrins and PSGL-1.

Abstract Leukocyte and platelets are understudied contributors to the overall pathology of sickle cell disease (SCD). Elevated leukocyte counts are common in these patients and correlate inversely with patient lifespan and overall disease severity. For example, a drop in neutrophil count typically predicts a patient's response to hydroxyurea, while increased monocyte counts correlate directly with increased reporting of pain crises. Moreover, both RBCs and WBCs have been detected as components in vaso-occlusive blockages in mouse models, where adhesive RBCs appear to interact directly with WBCs at the vaso-occlusive site. Platelets are activated in SCD and are thought to promote the hypercoagulability in these patients. Despite the potential contribution of all blood cells to the pathology of sickle cell disease, neither a mechanism of adhesion between the WBC and RBC nor a role for soluble matrix proteins in this interaction has been elucidated in humans. To detect potential adhesive interactions between the blood cells in SCD, we collected whole blood into anticoagulants that spare divalent cations (PPACK or factor Xa inhibitor) and assayed for heterotypic cell associations by two and three color flow cytometry. Our results indicate that RBCs, WBCs and platelets exist in heterotypic, multi-cellular aggregates in blood from SCD patients but not unaffected (AA) individuals. By detecting monocyte specific markers, we determined that the primary WBC component of these aggregates was the monocyte, and the primary RBC was the young SS “stress” reticulocyte. Using both in vitro RBC/monocyte adhesion studies and whole blood samples, we demonstrate that α4-containing integrins on both SS RBCs and WBCs mediate this interaction by interacting directly with endogenous plasma fibronectin. Furthermore, we show that the α4 integrin on SS RBCs binds to the RGDS site in fibronectin, whereas the α4 integrin on monocytes binds to the CS-1 site in the molecule, suggesting a novel mechanism of interaction between SS RBCs and monocytes via a fibronectin bridge. Antibodies against the CS-1 binding site in fibronectin substantially disrupt the monocyte/RBC interaction in whole blood, further underscoring the role of fibronectin as a linker between the two cell types. However, platelet incorporation in the aggregate was insensitive to inhibition of the α4 integrin, but was sensitive to inhibition of PSGL-1, suggesting that platelet inclusion likely occurs via a P-selectin/PSGL-1-mediated interaction between the platelet and the monocyte. Interestingly, similar aggregates were also detected in two patients with chronic hemolysis and brisk reticulocytosis, potentially extending the relevance of such aggregates beyond SCD. Taken together our results suggest a new adhesive paradigm for SS RBCs and monocytes as central components of heterotypic blood cell aggregates that include platelets and that are present in whole blood of patients with SCD. Our data therefore illustrate a potentially pathological interaction of all major blood cell types in SCD patients that may impact vaso-occlusion and contribute to other erythrocyte disorders.