Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Translational Research, Hemoglobinopathies, Diseases
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.
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