Disturbed Arterial Flow As a Driver of Perturbed RBC Physiology and Thromboembolic Events in Sickle Cell Disease

Introduction:

Sickle cell disease (SCD) is a genetic condition marked by abnormal red blood cell morphologies and high levels of hemolysis leading to chronic and severe thromboembolic complications. The biomechanics of the sickle red blood cell (sRBC) has primarily been studied within the context of venous circulation where RBCs adopt their characteristic “sickled” shape. However, much remains to be understood about how the sRBC reacts to the distinct flow patterns found in arterial circulation and its relation to the significant burden of arterial disease observed in SCD.

While healthy arterial blood flow is typically laminar, many large and bifurcated arterial pathways display disturbed laminar and even turbulent flow regimes. Disturbed arterial flow is even more likely under anemic conditions, as reduced blood viscosities, due to lower hematocrits, have been demonstrated to have higher susceptibilities to turbulent flow states. As established in the literature, chronic exposure to disturbed arterial flow contributes to the damage of healthy RBCs and the surrounding vascular endothelium. However, this relationship has not yet been explored in sRBCs. Given the severe chronic anemia associated with SCD and the fragile nature of sRBCs, it is essential to study how and the extent to which these disturbed flow regimes impact the sRBC. This study aims to investigate how disturbed flow regimes in arterial circulation affects sRBC morphology, hemolysis, and viscoelastic properties of clots formed in sickle whole blood, and to contextualize their unique behavior within the broader framework of serious arterial pathologies associated with SCD.

Methodology:

Utilizing our previously developed bench-top paradigm for simulating turbulence, the morphologic and hemolytic responses of healthy and sickle RBC donor suspensions were evaluated under four arterial flow regimes: laminar, disturbed laminar, transitional, and turbulent. The preparation of these samples included isolating the RBCs from the whole blood, eliminating confounders to the study, and suspending them in a physiological buffer to clinically relevant hematocrit ranges. Hemolysis was quantified via measured absorbance values of free hemoglobin present in the suspension following the exposure to each respective flow regime. In addition to utilizing complete blood count analysis to obtain the mean corpuscular volume of the RBCs following flow regimes, we prepared and imaged blood smears for each condition. The resulting images were analyzed using our published software, iCLOTS, to quantify RBC circularity and area. Finally, Rotational Thromboelastometry (ROTEM) was employed to assess the implications of various flow regimes on the viscoelastic properties and clot formation process in sickle cell whole blood.

Results:

These studies have demonstrated that sickle RBCs exposed to disturbed flow regimes (disturbed laminar, transitional, and turbulent) experience significantly higher levels of hemolysis compared to healthy RBCs under the same conditions. From assessing the impact of various SCD treatment regimens (hydroxyurea, voxelotor, and both combined) on the hemolytic patterns of sRBC's exposed to turbulence, we notably observed less hemolysis among patients treated with both drugs compared to either alone. Additionally, sickle RBC suspensions exhibit a marked increase in mean corpuscular volume and a decrease in circularity following exposure to turbulence. Our ROTEM studies have shown that sickle whole blood exposed to turbulence clotted significantly faster and clot dissolution was slower than observed in healthy whole blood exposed to turbulence.

Conclusion:

These results detail the responsive behavior of sRBCs to disturbed arterial flow regimes in terms of sRBC damage, sRBC subtype distribution, and its implications on subsequent thrombotic processes in SCD. Our ongoing endothelial studies are revealing the downstream effects of turbulence on the vascular endothelium and its contribution to arterial disease. The ultimate aim of this work is to underscore the importance of understanding the biomechanical relationship between sRBCs and the unique regimes of flow present in arterial circulation. In doing so, we can then identify regions within the body where turbulence is likely to occur in SCD patients and optimize the implementation of interventions to reduce the burden of thromboembolic events in SCD.

Lam:Sanguina, Inc.: Current equity holder in private company.