Sevuparin Blocks Sickle Blood Cell Adhesion and Sickle-Leukocyte Rolling on Immobilized L-Selectin in a Dose Dependent Manner

Background: The cause and continuation of vaso-occlusion in sickle cell disease (SCD) are fueled by the sickle-Red Blood Cells interactions with multiple other cell populations, promoting inflammation, obstructing the vasculature, and injuring the endothelium, leading to broad manifestations that affect most vital organs. Recent studies have identified multiple cellular components and molecular factors that contribute to the pathophysiology of SCD as reviewed by Zhang et al 2016 in Blood. It is likely that a multi-targeted approach for addressing SCD vaso-occlusion will be required to achieve the best clinical outcome. Sevuparin (DF02), a novel drug in Phase 2 for acute treatment of vaso-occlusive crisis in SCD (NCT02515838), is a polysaccharide blocking abnormal adhesion and thereby normalizing obstructed blood flow. In vitro and in vivo studies have shown potent anti-adhesive effects with a multimodal mechanism of action. In this study, we evaluate the effects of sevuparin on the adhesion of sickle whole blood from individual patients to endothelial cells (HUVECs) and vascular cell adhesion molecule-1 (VCAM-1) (Flow Firm Adhesion) and sickle-leukocyte rolling adhesion on L-selectin (Flow Dynamic Adhesion) using a standardized microfluidic flow-based adhesion assay.

Methods: Blood was obtained from homozygous SCD patients (n = 12, age range 15-25yrs) in sodium citrate after obtaining informed consent. A comprehensive assessment of the effect of sevuparin on whole blood adhesive properties during simulated blood flow was assessed using standardized Flow Firm Adhesion and Flow Dynamic Adhesion assays (Functional Fluidics, Detroit MI). Flow Firm Adhesion: Whole blood firm adhesion was measured during physiologic flow in microfluidic channels (Fluxion-Bioflux 1000, San Francisco, CA) coated with either VCAM-1 or cultured HUVECs. HUVECs were activated by TNF-alpha (25ng/mL x 24 hrs.) and Histamine (100mM x 10min) prior to the assay. Whole blood was treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of whole blood adhesion index (cells/mm²) to sevuparin was measured. Flow Dynamic Adhesion: Rolling adhesion of isolated sickle-leukocytes on an L-selectin coated microfluidic channel was measured during physiologic flow. Isolated sickle-leukocytes were treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of rolling cell density (cells/mm²), rolling cell percentage (%), and average rolling velocity (µm/s) to sevuparin was assessed. Cell identification and tracking of rolling were digitally analyzed.

Results:Statistically significant inhibition of sickle whole blood adhesion to HUVECs was observed at 3.0 µg/mL of sevuparin (p<0.001). In the same manner, statistically significant inhibition of sickle whole blood adhesion to VCAM-1 was observed at 200 µg/mL of sevuparin (p=0.033, absolute adhesion, p=0.001, % baseline adhesion). Each patient sample demonstrated a reduction in adhesion. Sevuparin also demonstrated a statistically significant dose-dependent reduction of sickle leukocyte rolling cell density (cells/mm²), rolling cell percentage (%), and an increase in average rolling velocity (µm/s) on L-selectin. Patient-to-patient variability in sevuparin response was observed.

Conclusions: Sevuparin blocks both sickle whole blood and isolated sickle-leukocyte adhesive interactions under physiologic flow at clinically relevant concentrations. The blocking of adhesion to VCAM-1 indicates that sevuparin acts in the same manner as other heparinoids in vitro, and block the interaction with VLA-4. L-selectin is another possible target for sevuparin therapy now confirmed at the cellular level. Clinically, Okpala et al 2002 has shown that L-selectin expression by monocytes is increased in vaso-occlusive crises, compared to steady state and that both mononuclear cell and neutrophil L-selectin expression is also higher in patients with certain complications of SCD. Here we show that sevuparin acts in a multicellular manner, blocking both SS-RBC firm adhesion and L-selectin-mediated rolling adhesion of sickle-leukocytes, as well as functionally interacting with yet another key adhesion receptor VCAM-1. This further adds to sevuparin's multimodal action and its potential clinical benefits in treating the complex mechanisms manifested in vaso-occlusion and complications in SCD.