The Protein C Pathway in Human and Murine Sickle Cell Disease: Alterations in Protein C, Thrombomodulin (TM), and Endothelial Protein C Receptor (EPCR) at Baseline and during Acute Vaso-Occlusion
Authors: Guo Yihe , Uy Teresa , Wandersee Nancy , Scott J. Paul , Weiler Hartmut , Holzhauer Sandra , Retherford Dawn , Foster Thomas , Hillery Cheryl 
Published: April 2020

Abstract
The coagulation system is activated in sickle cell disease (SCD) and acute vaso-occlusion may heighten hypercoagulability. Protein C, a natural anticoagulant, has been reported to be low in individuals with SCD. Therefore, the natural anticoagulation pathway may be disrupted in SCD. The objective of this study is to more fully evaluate the protein C pathway in murine and human SCD by examining levels of:
coagulation activation; protein C activity; thrombomodulin (TM); and endothelial protein C receptor (EPCR). In order to assess the level of activation of the coagulation system, we measured plasma thrombin/antithrombin (TAT) complex levels in humans and mice. TAT levels were elevated in 22 humans with SCD versus 9 healthy controls at baseline, and levels increased further in 15 individuals with SCD during acute vaso-occlusive events (5.6±1.2 vs. 2.4±0.2 vs. 9.2±1.8ug/L respectively, p=0.02). In order to study acute vaso-occlusive events in mice, we developed a model of acute vaso-occlusion by exposing Berkeley SCD mice to 3 hours of hypoxia (FI02 8–10%) followed by 2, 4, or 21 hours of reoxygenation in room air (HR2, HR4, HR21). In support of our human findings, TAT was elevated in SCD mice compared to HbA mice at baseline, and increased further in SCD mice exposed to HR2 (n=5–14 per group, p&lt;0.001). Assessment of protein C activity levels in plasma revealed that humans (n=8) with SCD have lower protein C activity levels than healthy controls (n=10) (78%±8.7% vs. 107%±5.3%, p=0.01). Additionally, we are the first to report that protein C activity levels decrease further during acute vaso-occlusive events (paired samples in 7 individuals, p=0.01). Another key protein of the PC pathway is TM, an endothelial-bound protein which activates protein C.
TM is elevated in several chronic inflammatory diseases and acutely decreases in meningococcemia. We evaluated TM in mouse liver, an organ susceptible to vascular congestion, infarction, and inflammation in SCD mice. We first measured TM in mouse liver homogenates by ELISA. All SCD mice, at baseline and after HR, expressed elevated liver TM levels compared to HbA mice (n=6 per group, 1.7 to 2.9-fold increases in SCD livers, p&lt;0.05). Exposure to HR in SCD mice increased hepatic inflammation and ischemia and decreased hepatic TM levels compared to SCD mice at baseline (HR2 84%, HR4 60%, and HR21 85% of baseline SCD liver TM). In preliminary experiments, Western Blot analysis confirmed high TM expression in mouse SCD livers compared to HbA livers at baseline and after HR. Immunohistochemistry demonstrated widespread, increased TM staining in hepatic parenchymal vessels of SCD mice compared to HbA mice both at baseline and after HR, with decreased staining within mature infarcts. 4) Finally, we studied EPCR, a membrane-bound protein that binds circulating protein C and promotes both anti-thrombotic and anti-inflammatory functions. Similar to TM, immunohistochemical staining for EPCR was more prominent in hepatic parenchymal vessels of SCD mice compared to HbA mice at baseline and after HR (preliminary studies, n=3 per group). In summary, these data confirm that SCD is a prothrombotic state and suggest that the protein C pathway is altered in SCD. TAT levels are elevated in human and murine SCD, and increase further during vaso-occlusion, illustrating that SCD is a hypercoagulable state. Protein C activity levels are low in human SCD and decrease further during vaso-occlusive events, suggesting that protein C may be consumed both chronically and acutely. In SCD mice at baseline, elevated expression of TM and EPCR suggests that there may be a chronic, compensatory up-regulation of these proteins in SCD. Finally, an acute consumptive process could account for the transient, decreasing trend of these natural anticoagulant proteins in SCD mice after exposure to HR. Thus, targeted administration of activated protein C may provide a novel therapy to minimize tissue injury during acute vaso-occlusive events in SCD.