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1046 Protease Activated Receptor-1 (PAR-1) Inhibition By Parmodulin 2, but Not Vorapaxar, Protects Sickle Cell Mice from Heme-Induced Acute Chest Syndrome

Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Sickle Cell Disease, Translational Research, Hemoglobinopathies, Diseases
Saturday, December 10, 2022, 5:30 PM-7:30 PM

Kailyn Lowder1*, Nirupama Ramadas, PhD1*, Rafal Pawlinski, PhD2 and Erica Sparkenbaugh, PhD1

1Blood Research Center, Division of Hematology, University of North Carolina Chapel Hill, Chapel Hill, NC
2Blood Research Center, Division of Hematology, University of North Carolina, Chapel Hill, NC

Background: Acute chest syndrome (ACS) is a leading cause of ICU admission and mortality in sickle cell patients, characterized by hypoxemia, fever, and significant chest pain. The main cause for ACS in SCD is hemolytic anemia, which elevates circulating heme levels. A model of ACS identified that excess heme induces acute lung injury (ALI) dependent on TLR4 and endothelial p-selectin in sickle cell mice. Despite increased understanding of the etiology of ACS, current treatments are either supportive or not targeted. We have shown that thrombin-dependent protease activated receptor-1 (PAR-1) signaling contributes to heme-induced vascular stasis via upregulation of endothelial p-selectin. Thrombin/PAR-1 signaling is cytotoxic and pro-inflammatory, whereas activation of PAR-1 by activated protein C (APC) is cytoprotective. We hypothesize that PAR-1 signaling contributes to the pathogenesis of ACS.

Objective: To investigate the role of PAR-1 in a mouse model of heme-induced ACS using two PAR-1 inhibitors: vorapaxar or parmodulin-2 (PM2).

Methods: Townes wild type (AA) and sickle (SS) mice (3-4 months) were used in three experiments. 1) AA females (n=4), SS males (n=6) and SS females (n=11) received 140 µmol/kg heme via retro-orbital injection. 2) SS females received vehicle (n=6) or Vorapaxar (105 µmolar/kg BW, oral gavage) (n=7) daily for 5 days, then treated with heme (140 µmol/kg). 3) SS females received vehicle (n=5) or PM2 (10 mg/kg, intraperitoneal, n=5) 30 minutes prior to heme (140µmol/kg). In all experiments, mice were monitored for signs of ALI (endpoint criteria: severe discomfort with disability) and lungs were collected for histopathology at endpoint or 120 mins after heme infusion.

Results: Heme infusion caused 11/11 of SS females to reach endpoint (median survival = 18 min, p=0.0006 vs AA and SS males), whereas only 2/6 of SS males and 0/4 AA mice reached endpoint. Correlation analysis for RBCs and endpoint time showed that SS females with RBC counts less than 7 x 1012/L were vulnerable to heme-induced ACS; yet even males with significant anemia did not reach endpoint. The lung histology showed alveolar thickening, edema and hemorrhage in heme-treated SS females. Hence, only female SS mice were used for the remaining studies. Interestingly, vorapaxar did not protect SS mice from heme-induced ACS (5/7 mice, median survival 17 min) compared to vehicle treatment (4/6 mice, median survival 16 min). In contrast, PM2 treatment significantly protected the SS mice from heme induced ACS with 0/5 reaching endpoint, compared to 4/5 of vehicle -treated SS mice reaching endpoint (p=0.013), and prevented lung damage observed by histology.

Conclusions: We found that female mice are more susceptible to heme-induced ACS than males. Vorapaxar, an irreversible antagonist of PAR-1 which blocks all PAR-1 signaling pathways, did not protect SS mice against heme-induced ACS. In contrast, PM2 completely alleviated heme-induced lung injury and death in SS mice. This could be attributed to the fact that PM2 allosterically blocks thrombin/PAR-1 signaling while preserving cytoprotective signaling similar to that mediated by APC. Targeting PAR-1 by preserving (or inducing) beneficial signaling might be effective in preventing the development of ACS.

Disclosures: Pawlinski: CSL Behring: Consultancy, Research Funding.

*signifies non-member of ASH