Abstract
Protease activated receptor 1 (PAR1) couples the coagulation cascade to endothelial inflammation in many diseases, including sickle cell disease (SCD). PAR1 is a GPCR activated by proteolytic cleavage of an extracellular N-terminus, and demonstrates biased signaling. Activation of PAR1 by thrombin stimulates prothrombotic and proinflammatory signaling, whereas activation by activated protein C (APC) stimulates cytoprotective and anti-inflammatory pathways. The ideal PAR-1 targeted therapy would inhibit detrimental signaling while preserving beneficial signaling. 3K3A-APC is a signaling selective variant of APC that activates cytoprotective PAR1 signaling with minimal anticoagulant effects, demonstrated to have a beneficial role in stroke models. Parmodulin 2 (PM2) is a small molecule allosteric inhibitor of PAR1 that simultaneously blocks detrimental signaling and activate beneficial cytoprotective signaling similar to that induced by APC. We previously demonstrated that endogenous APC-PAR1 signaling contributes to thrombin generation and inflammation in a mouse model of SCD, whereas thrombin-PAR signaling enhances these pathways. We hypothesize that inducing cytoprotective PAR1 signaling with either 3K3A-APC and PM2 will reduce thrombo-inflammation in sickle cell mice at steady state.
Townes wild type (HbAA) and sickle (HbSS) mice (male and female, 4 months old) were used in the studies. In the first experiment, HbAA and HbSS (n=4-5 per group) mice were treated daily with either saline (SAL) or 3K3A-APC (0.2mg/kg, BW) for 4 days. Samples were collected 1 hour after final dose. As previously demonstrated, plasma levels of thrombin-anti thrombin (TAT) complexes, the proinflammatory cytokine IL-6, high mobility group box 1 (HMGB1), and, soluble vascular cell adhesion molecule (sVCAM), were increased in HbSS mice compared to HbAA controls. Surprisingly 3K3A-APC treatment did reduce any of these parameters in HbSS mice. APC and its variant 3K3A-APC require binding to the endothelial protein C receptor (EPCR) to activate PAR1; it is known that SCD causes increased EPCR shedding. Indeed, we found that both SAL- and 3K3A-APC treated HbSS mice had elevated sEPCR levels compared to HbAA controls. Together, these data indicate that 3K3A-APC does not affect thrombo-inflammation in HbSS mice at steady state most likely due to by reduced ECPR expression on the endothelium.
In the second experiment, HbAA and HbSS (male and female, 4 months old) mice (n=8-11 per group) were treated with vehicle (20% DMSO, 20% PEG800, 60% PBS) or PM2 (10 mg/kg, BW) every other day for 1 week. The last dose was administered on the 8th day 1 hour prior to collecting plasma samples. As expected, HbSS mice had elevated plasma TAT levels compared to HbAA mice (11.04 ± 1.4 vs 45.19 ± 6.3, p<0.05) which was significantly attenuated by PM2 treatment in SS mice (16.17 ± 2, p<0.05). Similar results were observed for IL-6 (AA: 2.06 ± 0.7 vs SS: 29.12 ± 9.6 vs SS+PM2: 5.83 ± 2.9; p<0.05); sVCAM (AA: 330.6 ± 31.5 vs SS: 571.4 ± 50.4 vs SS+PM2: 403.3 ± 41.22; p<0.05) and HMGB1 (AA: 11.46 ± 1.1 vs SS: 25.37 ± 1.5 vs SS+PM2: 19.12 ± 1.7; p<0.05). PM2 had no effect on markers of anemia in HbSS mice including RBC, hematocrit, and reticulocyte counts.
In the present study ,3K3A-APC did not attenuate thrombin generation and cytokine production in HbSS mice; this might be due to the shedding of EPCR which is important co-receptor for the protective APC-PAR1 signaling. In contrast, PM2-mediated allosteric inhibition of thrombin/PAR1 and simultaneous induction of APC-like cytoprotective signaling attenuated levels of TAT, IL-6, HMGB1, and sVCAM in HbSS mice. Future studies towards evaluating the effect of PM2 on downstream targets of PAR1 signaling in sickle mice will provide more insight into the mechanism of this protective effect.
Disclosures
Mosnier:Coagulant Therapeutics: Research Funding. Pawlinski:CSL Behring: Consultancy, Research Funding.
Author notes
Asterisk with author names denotes non-ASH members.
