P-Selectin Deficiency Reduces Acute Vascular Complications but Not Chronic Organ Damage in Sickle Cell Mice
Authors: Sparkenbaugh Erica , Kasztan Malgorzata , Miller Megan D , Binning Elizabeth A , Abrams Christina M , Reeves Brandi , Sundd Prithu , Pawlinski Rafal 
Published: November 2021

Abstract
P-selectin (Psel) is an adhesion molecule expressed on platelets and endothelial cells, which interacts with its ligand PSGL-1 on leukocytes and a PSGL-1-like molecule on sickle red blood cells (RBCs). Psel mediates the formation of the multicellular aggregates that promote vaso-occlusive crisis (VOC) and acute painful episodes in sickle cell disease (SCD). Crizanlizumab, a monoclonal antibody blocking Psel, reduces the frequency of VOC in SCD patients. In SCD mice, Psel contributes to heme-induced microvascular stasis, and thrombus and platelet-neutrophil aggregate formation in the lung and liver. Unexpectedly, Psel deficiency promotes liver senescence in a mouse model of SCD, suggesting a possible detrimental effect of Psel inhibition. In this study, we further investigated the long-term effects of Psel deficiency on thromboinflammation and end-organ damage in murine SCD.
We used Townes wild type (AA) and sickle (SS) mice (n=12-27) that either express Psel (Psel +/+) or lack Psel (Psel -/-) at 11-12 months of age. Kidney damage was evaluated by histology and urine analysis. Heart function was determined using echocardiography. Complete blood counts and plasma biomarkers of thrombin generation (thrombin anti-thrombin [TAT] complexes) and inflammation (interleukin [IL]-6) were also analyzed. In addition, in a pilot experiment, we analyzed the effect of Psel deficiency on experimental venous thrombosis in SS mice.
SS Psel +/+ mice exhibited renal damage, urinary concentrating defect, and reduced creatinine clearance, which was not improved in SS Psel -/- mice. Psel deficiency attenuated glomerular (GLM) congestion (0.17±0.05 vs 0.39±0.06, p&lt;0.05) but failed to prevent GLM injury, as evidenced by similar extent of GLM sclerosis, hypertrophy and podocyte loss in SS mice. Interestingly, only SS P-sel -/- mice presented withdistinct differences in GLM structure, demonstrated by extensive hypercellularity, mesangial expansion and mesangiolysis. We also observed tubular injury concomitant with brush border loss and interstitial fibrosis in SS mice, regardless of Psel expression. Additionally, there was a significant reduction in CD3 + T cells (5.0±1.2 vs 9.5±1.6 cells/field; p&lt;0.05) and macrophages (1.0±0.1 vs 1.6±0.2 % of area; p&lt;0.05) infiltration in the kidney cortex of SS Psel -/- compared to SS Psel +/+ mice. This corresponded to a greater renal iron accumulation in SS Psel -/- mice (59.9±4.2 vs 48.2±3.2 Mpix/μm; p&lt;0.05).
We observed cardiac hypertrophy (elevated heart weight to tibia length ratio) in SS mice, regardless of Psel expression. Echocardiography of left ventricle (LV) revealed that SS mice had increased LV mass and LV internal diameter with no change in ejection fraction or fractional shortening compared to AA mice. None of these parameters were affected by Psel expression. Furthermore, hypertrophy observed in other organs (kidney, liver, lung and spleen) of SS Psel +/+ mice was also not affected by Psel deficiency.
Consistent with previous studies, SS Psel +/+ mice had elevated plasma levels of TAT and IL-6 compared to AA Psel +/+ mice. Psel deficiency significantly reduced IL-6 (47±15 vs 20±10 ng/mL, P&lt;0.05), but had no effect on elevated plasma TAT levels in SS mice. Despite the lack of effect of Psel deficiency on systemic thrombin generation in SS mice, femoral vein thrombi formed after electrolytic injury exhibited a strong trend in reduced fibrin and platelet content compared to SS Psel+/+ mice (n=5 per group).
As expected, SS Psel +/+ mice exhibited anemia as shown by reduced RBC number, hemoglobin (Hb) and hematocrit (Hct) compared to AA Psel +/+ animals. Interestingly, these parameters were further decreased in SS Psel -/- mice (p&lt;0.01 for all); RBC (5.6±0.2 vs 4.4±0.4 x 10 6/mL), Hg (6.7±0.2 vs 5.7±0.2 g/dL) and Hct (25.1±1.1 vs 19.7±1.1%). This was accompanied by a significant increase in mean corpuscular Hb concentration (26.9±0.5 vs 29.2±0.5 g/dL p&lt; 0.01) but no changes in RBC mean corpuscular volume in SS Psel -/-mice compared to SS Psel +/+ mice.
Our data suggest that despite improving acute vascular pathologies, including VOC and thromboinflammation, long term deficiency of Psel does not prevent end-organ damage. If fact, it may contribute to organ dysfunction and enhanced anemia. More mechanistic studies are needed to better understand the long-term effects of anti-Psel treatment in SCD patients.

Disclosures
Reeves: Incyte Corporation: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Pharma Essentia: Consultancy, Honoraria. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.