Acute pain is the most common and debilitating symptom of sickle cell disease (SCD), with recurrent vaso-occlusive episodes being the primary cause of hospitalization. While acute pain is attributed to episodic vaso-occlusion, the mechanisms underlying chronic pain remain poorly understood, although inflammation plays a key role. We previously demonstrated that thrombin-mediated cleavage of protease-activated receptor-1 (PAR1) at Arg41 promotes inflammation and vaso-occlusion, whereas cleavage at Arg46 by activated protein C (APC) is protective in sickle cell mice. We have also shown that Parmodulin 2 (PM2), a small molecule PAR1 modulator that inhibits thrombin-induced signaling while promoting APC-mediated cytoprotection, attenuates thrombin generation, inflammation, and endothelial activation in sickle cell mice. Therefore, we hypothesized that PAR1-R41 signaling contributes to pain and neuroinflammation in sickle cell mice.

To test this hypothesis, 8-10 week old male and female Townes HbAA (wild-type) and HbSS (sickle) mice were treated with a standard solubilizing vehicle (20% DMSO, 20% PEG-800, 60% saline) or PM2 (10 mg/kg, intraperitoneal injection) three times per week for 8 weeks. We confirmed the role of PAR1-R41 signaling with the PAR1-R41Q mouse model, which harbors a mutation that renders PAR1 insensitive to thrombin, thus preventing pro-inflammatory signaling. Bone marrow from HbAA or HbSS mice were transplanted into either wild-type PAR1 or PAR1-R41Q recipient mice. Mechanical hyperalgesia was measured either 1 hour after the last PM2 injection, or 8 months post- transplant in chimeric mice. Mechanical hyperalgesia was assessed with Von Frey Filaments, via paw withdrawal frequency (PWF) and mechanical threshold measurements. Plasma samples were collected at the end of the studies to evaluate neuroinflammatory markers including Glial Fibrillary Acidic Protein (GFAP), Calcitonin Gene-Related Peptide (CGRP) and substance P.

We found that PM2 treatment significantly reduced PWF (SS/PM2: 3.98 ± 0.28 vs. SS/Vehicle: 5.73 ± 0.3 in SS/Vehicle, p < 0.001) and improved mechanical threshold (SS/PM2: 0.71 ± 0.1 vs. SS/Vehicle: 0.22 ± 0.02, p < 0.005). Notably, male HbSS mice had a higher analgesic response to PM2 than HbSS females. HbSS mice had elevated levels of neuroinflammation markers compared to HbAA controls. In both male and female mice, PM2 modestly reduced levels of GFAP (SS/PM2: 36 ± 6.6 vs. SS/Vehicle: 46.05 ± 6.8, p<0.57) and CGRP (SS/PM2: 2.59 ± 1.1 vs. SS/Vehicle 9.35 ± 3.2, p<0.3) with no change in substance P. These data suggest that pharmacologic modulation of beneficial PAR1 biased signaling reduces neuroinflammation and mechanical hyperalgesia in SS mice.

We also analyzed mechanical hyperalgesia in HbSS bone marrow chimeric mice with normal PAR1 (SS/WT) and HbSS mice with the thrombin-insensitive PAR1-R41Q mutation (SS/R41Q). SS/R41Q mice had a significant reduction in PWF (2.58 ± 0.15 vs. SS/WT: 4.43 ± 0.28, p < 0.001) and improved mechanical threshold (0.89 ± 0.06 vs. SS/WT: 0.27 ± 0.04, p < 0.01) compared to SS/WT controls. This was accompanied by a marked reduction in GFAP (SS/R41Q: 25.7 ± 2.1 vs. SS/WT: 128.9 ± 23.5, p < 0.001), a modest decrease in CGRP (SS/R41Q: 5.85 ± 1.4 vs. SS/WT: 7.13 ± 3.85), and no change in substance P.

Conclusions: These findings suggest that hyperalgesia in sickle mice is associated with neuroinflammation driven by thrombin-PAR1 signaling. Both pharmacologic modulation via PM2 and genetic disruption of thrombin-PAR1 signaling attenuate pain and neuroinflammation in sickle cell mice. Targeting biased PAR1 signaling may represent a novel strategy to alleviate chronic pain in SCD. Further studies are warranted to fully elucidate the underlying mechanisms.Keywords: sickle cell disease, thrombin, protease activated receptor 1, activated protein c, pain, neuroinflammation

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2025
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