Biased agonism of protease-activated receptor-1 regulates thrombo-inflammation in murine sickle cell disease

• APC inhibition exacerbates thrombin generation, inflammation and end-organ damage in a mouse model of sickle cell disease (SCD).

• APC-PAR1 biased agonism reduces inflammation, suggesting that targeting this pathway may mitigate vascular complications of SCD.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOE). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOE, and end-organ damage. Coagulation proteases like thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either pro-inflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and TNFα challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we employed transgenic mice with point mutations at two PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared to PAR1-WT or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.-