Platelet Nucleation on Arrested Neutrophils Drives Vaso-Occlusion in Sickle Cell Disease

Abstract Introduction: Sickle Cell Disease (SCD) is an autosomal recessive genetic disorder that leads to sickling and hemolysis of RBCs under hypoxic conditions. As a result of chronic hemolysis, SCD is associated with a hyper-inflammatory and hyper-coagulation state, which accounts for enhanced adhesion of leukocytes, platelets, RBCs and vascular endothelial cells leading to vaso-occlusion. Acute vaso-occlusive pain crisis (VOC) is the primary reason for emergency medical care by SCD patients. Although neutrophils have been shown to play a role in the on-set of vaso-occlusion by interacting with sickle RBCs and platelets in cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SCD patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SCD (SS) patients and race matched control subjects was perfused through polydimethylsiloxane (PDMS) based microfluidic flow channels (30 µm x 500 µm) with a glass bottom coated with either human microvascular endothelial cells or a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological 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 using quantitative microfluidic fluorescence microscopy (qMFM)1, which is a combination of quantitative dynamic footprinting1 and epifluorescence microscopy. Results and Discussion: Neutrophils in SS blood were observed to roll, arrest and then capture freely flowing platelets leading to the formation of vaso-occlusive aggregates. RBCs were observed getting trapped within the platelet-neutrophil aggregates. The number of platelet-neutrophil interactions, lifetime of these interactions and the extent of platelet-neutrophil aggregation were several folds higher in SS than control subject blood. Bacterial lipopolysaccharide (LPS; 500 ng/ml) pretreatment led to enhanced platelet-neutrophil aggregations in SS but not control blood. The enhanced platelet-neutrophil aggregations in SS blood (+/-LPS) was attenuated to the level observed in control blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils with functional blocking Abs. Conclusion: Our data demonstrates that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of platelet-neutrophil aggregates and trapping of RBCs in these aggregates. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced platelet-neutrophil aggregation than control blood. Vaso-occlusion can be ameliorated in SS blood by simultaneous inhibition of platelet P-selectin and neutrophil Mac-1. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapies that can 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. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods 7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures No relevant conflicts of interest to declare.