Free Hemoglobin Increases Endothelial Adhesiveness to Red Blood Cells via a Toll-like Receptor 4-Dependent Mechanism

Endothelial dysfunction is a major contributor to microvascular failure and organ injury after trauma. Hemolysis releases cell-free hemoglobin (CFH), which drives oxidative stress, endothelial activation, and immune dysfunction. We previously found that trauma produces early CFH concentrations of 10-20 μM in plasma. As endothelial activation and red blood cell (RBC) adhesion can obstruct the microcirculation, we examined whether CFH directly increases endothelial adhesiveness to RBCs through Toll-like receptor 4 (TLR4).

Human aortic endothelial cells were exposed to human hemoglobin (10 or 20 μM), media, or lipopolysaccharide for 1 h. Cells were incubated with fresh whole blood or isolated RBCs from healthy volunteers. Fluorescence-stained endothelial cells and adherent RBCs were counted. Some cells were pretreated with a TLR4 antagonist.

CFH exposure produced a concentration-dependent increase in RBC adhesion. Following whole blood incubation, endothelial cells exposed to 10 μM CFH demonstrated a median of 9 RBCs per endothelial cell versus 2 in controls (P < 0.001), and 20 μM CFH increased adhesion to 18 RBCs per endothelial cell (P < 0.001 versus control). Similar findings occurred with isolated RBCs, demonstrating that the pro adhesive phenotype was independent of plasma proteins or platelets. Pretreatment with TLR4 C34 significantly reduced CFH-induced RBC adhesion across all exposures.

Brief exposure of cultured human aortic endothelial cells to clinically relevant concentrations of CFH markedly increase adhesion of RBCs, even in the absence of plasma proteins or platelets. This pro adhesive endothelial response is partially dependent on TLR4. These findings support a mechanistic link between trauma-induced hemolysis, endothelial activation, and microvascular dysfunction.