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Background: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) for the inactivation of activated factors V and VIII(1). We identified an anti-PS single-domain antibody (PS003biv) that surprisingly enhanced the APC-cofactor activity of PS and exerted an in vivo antithrombotic effect(2). A moderate decrease in PS plasma levels is frequently observed in sickle cell disease (SCD) patients, with a further reduction during vaso-occlusive crises (VOC) (3). We hypothesized that PS003biv might limit VOC in SCD. Methods: HbSS-Townes mice were intravenously injected with 10 mg/kg PS003biv or vehicle (control). Mice were placed in a hypoxic atmosphere chamber (8% O2) for 3 h, after which they were returned to room air. Spleen, liver and plasma were collected. The intensity of vaso-occlusion was quantified by Ter-119 immunofluorecence staining of red blood cells (RBC) clogging the vessels in the liver and spleen. Bilirubin, free heme, and thrombin-antithrombin complexes were measured in plasma. Results: Quantifying Ter-119 staining density in liver and spleen showed that PS003biv decreased RBC accumulation and VOC (liver control 13.57 ± 1.46 × 106 versus PS003biv 7.28 ± 0.38 × 106 AU; and spleen control 5.64 ± 0.21 × 107 vs PS003biv 5.03 ± 0.03 × 107 AU, p < 0.05). Biomarkers for hemolysis were lower in PS003biv-treated mice: bilirubin control 6.87 ± 0.21 vs PS003biv 4.88 ± 0.60 mg/dl; and free heme control 133.0 ± 10.4 vs PS003biv 77.5 ± 5.3 μM. PS003biv decreased thrombin-antithrombin complexes (control: 10.14 ± 0.72 vs PS003biv 7.47 ± 0.33 ng/mL, p < 0.05). Conclusions: PS003biv showed beneficial properties in the context of a hypoxia/reoxygenation murine model of SCD, with reduced vasoocclusion, hemolysis, and coagulation activation. The mechanism of action of PS003biv needs to be determined. [ABSTRACT FROM AUTHOR]

In children with sickle cell disease (SCD), splenectomy is immediately beneficial for acute sequestration crises and hypersplenism (ASSC/HyS) but portends a long-term risk of asplenia-related complications. We retrieved peripheral and splenic red blood cells (RBCs) from 17 SCD children/teenagers undergoing partial splenectomy for ASSC/HyS, 12 adult subjects without RBC-related disease undergoing splenectomy (controls), five human spleens perfused ex vivo with Hb SS - and Hb AA -RBC, and quantified abnormal RBC by microscopy, spleen-mimetic RBC filtration, and adhesion assays. Spleens were analyzed by immunohistochemistry and transmission electron microscopy (TEM). In circulating blood of SCD and control subjects, dysmorphic (elongated/spherocytic) RBCs were <2%, while proportions of pocked-RBC were 4.3-fold higher in SCD children than in controls. Compared to controls, splenic RBCs were more frequently dysmorphic (29.3% vs. 0.4%), stiffer (42.2% vs. 12.4%), and adherent (206 vs. 22 adherent RBC/area) in SCD subjects. By TEM, both polymer-containing and homogenous RBC contributed to spleen congestion, resulting in 3.8-fold higher RBC population density in SCD spleens than in control spleens, predominantly in the cords. Perfused spleens with normal function displayed similar congestion and retention of dysmorphic RBC as SCD spleens. The population density of active macrophages was similar in SCD and control spleens, with a relative deficit in phagocytosis of polymer-containing RBC. Despite the existence of hyposplenism, splenectomy in SCD children removes an organ that still efficiently filters out potentially pathogenic altered RBC. Innovative treatments allowing fine-tuned reduction of RBC retention would alleviate spleen congestion, the major pathogenic process in ASSC/HyS, while preserving spleen protective functions for the future., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)