Red blood cell entrapment in thrombi formed under pathological flow: Stiffness and binding antigens impact thrombus...

Thrombosis, or pathological blood clot formation, is a dangerous and potentially fatal event that is often associated with implanted medical devices. Thrombi are comprised of platelets, plasma proteins, red blood cells (RBCs) and other molecular and cellular components. Extensive research has been conducted on the dynamics of platelet aggregation and deposition; however, the process of RBC entrapment remains under-explored. In this study, we used a microfluidic device and fluorescence microscopy to concurrently image platelets and RBCs during thrombus formation under flow and biomaterial conditions representative of implanted devices. We designed and applied MATLAB algorithms to track thrombi as they grew over time and interrogated thrombus growth and RBC accumulation trends. To probe the contributions of the unique mechanical properties of RBCs, we characterized the influence of induced RBC stiffness on thrombus forming behavior via imaging. To study the binding effects of known RBC surface antigens, we visualized thrombus formation with inhibition of glycoprotein (GP) IIb/IIIa and integrin-associated protein (IAP) respectively. We found that the accumulation and clustering of RBCs within the thrombi exhibited an inverse relationship with stiffness. Fibrillar structures were also present in these thrombi. Stiff RBCs enhanced platelet aggregate growth in all directions. Inhibition of surface binding proteins GPIIb/IIIa and IAP decreased the stability of thrombi. The results show that RBC deformability impacts entrapment location, fibril growth and spatial distribution. In turn, RBC binding stabilizes and anchors aggregates to surfaces. These findings will inform improved device design and multi-phase models of thrombosis that incorporate RBCs. STATEMENT OF SIGNIFICANCE: Thrombosis, the pathological formation of blood clots, is linked to cardiovascular disease and implanted devices. This study explores the mechanisms by which red blood cells (RBCs) become trapped in clots and their impact on clot properties. Using a microfluidic model, we show that RBC stiffness affects their accumulation and distribution within thrombi, influencing thrombus structure and composition. We also find that RBC surface proteins, such as GPIIb/IIIa and integrin-associated protein, contribute to thrombus stability. These findings will enhance thrombosis models and inform research on the interaction between biomaterials, including blood-contacting devices, and biological systems at the cellular and molecular levels.