Platelet-Neutrophil Aggregates Promote Pulmonary Arteriole Microembolism in Sickle Cell Disease

Abstract

Introduction: Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ~100,000 Americans and millions of people worldwide. The acute chest syndrome (ACS), a form of acute lung injury, is a major cause of morbidity among SCD patients. The current treatment for ACS is primarily supportive and the molecular mechanism remains largely unknown. SCD patients hospitalized with vaso-occlusive pain crisis (VOC) often develop ACS within the ensuing days, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular and molecular mechanism and the anatomical site of pulmonary vaso-occlusion are still elusive.Materials and Methods: Intravenous (IV) bacterial lipopolysaccharide (LPS) was used to induce VOC in SCD mice. Intravital multiphoton excitation (MPE) fluorescence microscopy was used to study the blood cell trafficking within the pulmonary microcirculation of live SCD or control mice. Fluorochrome-conjugated anti-mouse Ly-6G, Ter-119, and CD49b antibodies were administered IV for in vivo staining of circulating neutrophils, red blood cells and platelets, respectively. Cellular trafficking was recorded at baseline and 2 hours after IV challenge with LPS. Image sequences were analyzed to identify vaso-occlusion, which was defined as cellular aggregation and stasis of blood flow within the pulmonary blood vessels.Results and Discussion: Preliminary data using MPE imaging in transgenic SCD mice revealed that vaso-occlusion was absent at baseline in unchallenged SCD mice and the cellular trafficking within the pulmonary microcirculation was comparable in SCD and control mice. Doses of IV LPS (0.01-5 mg/kg of body weight), which were innocuous to control mice were found to be lethal to SCD mice. Remarkably, MPE imaging of the lung microcirculation revealed that IV LPS led to microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates in SCD but not control mice. The microembolism involved either entrapment of circulating platelet-neutrophil aggregates or in situ aggregation through sequential steps of neutrophil arrest on the arteriolar endothelium, followed by platelet nucleation on arrested neutrophils and microthrombus formation.Conclusions: Initial findings demonstrate that pulmonary vaso-occlusion in SCD mice involves microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates. Future studies will determine the molecular interactions responsible for pulmonary arteriolar microembolism.Acknowledgments: This study is supported by the 11SDG7340005 from the American Heart Association (P.S.) and the VMI startup account (P.S.). M.F.B is supported by the NIH NHLBI VMI T32 training grant T32HL110849. DisclosuresNo relevant conflicts of interest to declare.