Introduction: Sickle cell disease (SCD) occurs due to a mutation in the β-subunit of hemoglobin, causing stiffening of red blood cells (RBCs) and leading to RBC sickling and vaso-occlusive crises (VOC) in SCD patients. While sickled RBCs remain a hallmark of SCD, they are prone to lysis and represent a small fraction of the total RBCs present in patients at a given time. The remaining RBCs maintain a normal, discoid shape and are either healthy or stiff due to polymerization of the hemoglobin β-globin subunit. In healthy blood flow, RBCs form a core in the center of the vessel and the remaining cells, platelets and white blood cells (WBCs), marginate towards the endothelium. However, the increased stiffness of RBCs in SCD disrupts this neat segregation of blood cells to different areas of the blood vessel and can contribute to VOC, the root cause of many acute and chronic complications for SCD patients. Despite the presence of normally shaped, stiffened RBCs in SCD patients, the impact of these RBCs on other cell types in blood flow is currently not well understood. Our laboratory previously demonstrated that the presence of artificially rigidified RBCs leads to an expansion of the RBC core and significantly decreases WBC adhesion to an inflamed endothelium in vitro. Here, we examine the impact of stiffened RBCs on platelet adhesion to a damaged endothelium in vitro by first using a model system with artificially rigidified RBCs and second, utilizing SCD patient blood to further support our model and understand platelet-RBC interactions in SCD patients.

Methods: In our model system, we artificially rigidified RBCs taken from healthy donors and reconstituted them into whole blood before perfusing the mixture over an activated, damaged endothelium using a parallel plate flow chamber. We quantified platelet adhesion to the endothelium in comparison to healthy, non-rigidified controls using fluorescent microscopy. To determine if our model findings translated to SCD, we recruited a cohort of hemoglobin SS and SC patients during routine visits and similarly perfused their whole blood over the same damaged endothelium and quantified platelet adhesion.

Results and conclusions: The inclusion of artificially rigidified RBCs in otherwise healthy subject blood flow significantly increased platelet adhesion to a damaged endothelium with a maximum increase in platelet adhesion of six-fold over a healthy, non-rigid control in our model system. Both RBC rigidity and the percentage of RBCs that were artificially rigidified had a large impact on the increase in platelet adhesion. SCD platelet adhesion to the damaged endothelium model varied from donor to donor based on variables such as treatment method and disease severity. Overall, this work experimentally elucidates the biophysical impact of stiffened RBCs on platelet adhesion using both an artificial model utilizing healthy blood as well as SCD blood, which can help determine the mechanism of action causing VOC in SCD.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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