The endothelial protein C receptor (EPCR) binds to protein C (PC) and increases the rate of activated protein C (APC) generation by the thrombin-thrombomodulin complex at the endothelial cell surface. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects, many of which are EPCR-dependent. The physiologic importance of EPCR is also demonstrated in EPCR knockout mice, which show placental thrombosis and early embryonic lethality. EPCR is also highly expressed on hematopoietic stem cells (HSC), however there is no known biological role for EPCR in this cell type.
Currently, there are no animal models to study the biological role of EPCR independent of its interaction with PC/APC. In this study, we generated a knock-in mouse model harboring a variant of EPCR (R84A) which lacks ability to bind to PC/APC. We hypothesize that loss of PC/APC binding to EPCR will result in a procoagulant and pro-inflammatory phenotype.
EPCR
R84A/R84A
mice are viable, have a normal lifespan and show no evidence of spontaneous thrombosis. Histological analysis of EPCR
R84A/R84A
mice identified a splenic disorder, characterized by splenomegaly and extramedullary hematopoiesis. Flow cytometric analysis of the spleen from EPCR
R84A/R84A
mice revealed a significant increase in the percentage of CD34+ cells, representing HSCs (2.4 ± 0.1% for EPCR
R84A/R84A
mice and 1.4 ± 0.2% for WT mice; P<0.05). To initiate thrombin generation in mice, an intravenous injection of FXa was administered. Plasma APC levels of FXa challenged EPCR
R84A/R84A
mice were reduced by 150% and thrombin-antithrombin levels (TAT) (an indicator of thrombin generation) were increased by 100% when compared to WT mice. The elevated TAT levels in FXa challenged EPCR
R84A/R84A
mice were accompanied by an increase in the size and number of fibrin clots in the lungs.
EPCR
R84A/R84A
mice are viable suggesting that defects in EPCR that impair PC binding do not affect embryogenesis or development. Introduction of the R84A mutation in EPCR results in impaired PC activation, and a procoagulant phenotype upon thrombotic challenge. Enlargement of the spleen in EPCR
R84A/R84A
mice suggests that EPCR may play a biological role in the regulation of hematopoiesis.