Crizanlizumab Therapy Is Associated with Lower Levels of Circulating Extracellular Vesicles in Sickle Cell Disease Patients
Authors: de Souza Cristiane Maria , Lanaro Carolina , dos Santos Irene Pereira , Olatunya Oladele , Olalla Saad Sara T , Benites Bruno Deltreggia , Campos Paula De Melo , Fertrin Kleber Yotsumoto , Costa Fernando Ferreira 
Published: November 2021

Abstract
Extracellular vesicles (EVs) are submicron structures released in blood circulation by different cell types which have been found to be increased in sickle cell disease (SCD) and are associated with clinical complications. The most abundant EVs in SCD patients derive from platelets, endothelial cells, and red blood cells (RBCs) and EVs have been explored as biomarkers of clinical severity.
Crizanlizumab is a monoclonal antibody against P-selectin, an adhesion molecule expressed in activated platelets and endothelial cells. P-selectin facilitates the formation of heterocellular aggregates and is implicated in the pathophysiology of vaso-occlusive episodes (VOEs) in SCD.
This study aimed to investigate the circulating levels of EVs in patients with SCD on standard of care or treated with crizanlizumab.
We collected peripheral blood samples from 20 adults with SCD (Non treated group: 7 patients on hydroxyurea treatment and 7 without it. Treated group: 6 patients undergoing treatment with crizanlizumab in combination with hydroxyurea). Patients received the last dose of crizanlizumab at least a month prior to the study. EVs were identified by lactadherin+calcein stain and quantified by flow cytometry to determine the immunophenotype of their parent cell (platelet, endothelial cell, and RBC, with CD41+; CD146+/CD45-; CD235+, respectively). EV quantification was calculated in number per ml of blood as previously described by our group (Olatunya et al., 2019).
We found that patients on crizanlizumab had lower total circulating EV counts than patients not receiving the drug (62.670.000,00 ± 15.600.000,00 vs 13.100.000,00 ± 3.513.000,00/mL, respectively, p=0,0076). The difference was statistically significant in platelet-derived EVs levels (5.397.000,00 ± 953.875,00 vs 2.413.000,00 ± 745.165,00/mL, p=0,0169), but not in endothelium-derived or RBC-derived EVs (345714 ± 101817 vs 220000 ± 64291, and 2.189.000,00 ± 1.648.000,00 vs 1.013.000,00 ± 572775, respectively).
Crizanlizumab therapy has been shown to reduce the incidence of VOEs in SCD. EVs have been recognized as bio-effectors involved in VOEs, contributing to a hypercoagulable state, chronic inflammation, and endothelial damage. Our findings show an association between the use of crizanlizumab and lower EV levels, particularly of the platelet-derived type. While the anti-P-selectin activity of crizanlizumab could be expected to help remove platelets from circulation, clinical studies have not reported a reduction in platelet counts in patients treated with crizanlizumab. Therefore, we speculate that crizanlizumab may decrease the release of EV by activated platelets, reduce platelet activation, or contribute to EV removal from circulation.
Our findings suggest that crizanlizumab therapy may modulate EV levels in the plasma of SCD patients and provide, for the first time, data to support exploring the use of extracellular vesicles as biomarkers to monitor the clinical response to this drug in patients. Further studies on EV expression of P-selectin and how crizanlizumab interacts with EVs and platelets may help clarify this particular effect of this drug.

Disclosures
Benites: Novartis: Honoraria. Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.