Neutrophil-Platelet Aggregation Enables Vaso-Occlusion in Sickle Cell Disease

Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-genetic disorder that leads to sickling and hemolysis of red blood cells (RBCs). Acute vaso-occlusive pain crisis (VOC) is the predominant pathophysiology faced by SCD patients and the primary reason for emergency medical care. Although neutrophils have been shown to play a role in vaso-occlusion by interacting with sickle RBCs in the cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SS patients is not completely understood.

Materials and Methods: Freshly collected heparinized blood from steady-state SS patients and race matched control (AA) subjects was perfused through silicone based microfluidic flow channels with a glass bottom coated a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological wall shear stress (6 dyn cm^-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded at a single cell-resolution using quantitative microfluidic fluorescence microscopy (qMFM)¹, which allows quantitative assessment of vaso-occlusive events at an unprecedented single cell resolution².

Results: Vaso-occlusion in the microfluidic channel involved neutrophil arrest followed by nucleation of platelets on arrested neutrophils, formation of neutrophil-platelet-aggregates (NPA) and partial occlusion of the microfluidic flow channel. Remarkably, the number of platelet-neutrophil interactions and the lifetime of these interactions were several folds higher in SS patient than control AA blood. Surprisingly, preincubation with 250 ng/ml of bacterial lipopolysaccharide (LPS) led to a significant increase in the number and lifetime of platelet-neutrophil interactions in SS but not AA blood. This enhanced NPA formation in SS patient blood was attenuated to the level observed in AA blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils as well as pretreatment with a small molecule inhibitor of toll-like-receptor-4 (TLR4) signaling pathway.

Conclusion: Our data shows that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of large NPAs and obstruction of blood flow. Platelet-neutrophil aggregation can be ameliorated by the simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced neutrophil-platelet aggregation than control blood. The enhanced platelet-neutrophil aggregation in SS blood is dependent on activation of TLR-4 pathway. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapeutics to prevent VOC in SS patients.

References:

1 Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in sickle cell disease. Haematologica. 2015;100(10):e390-e393. doi:10.3324/haematol.2015.126631.

2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods7, 821-824, doi:10.1038/nmeth.1508 (2010).