Darbepoetin-alpha does not promote microvascular thrombus formation in mice: role of eNOS-dependent protection through platelet and endothelial cell deactivation.

OBJECTIVE Erythropoietin (EPO) treatment has become the standard treatment of renal anemia. Though a link between hematopoiesis-stimulating drugs and thrombosis has not been proven, it is generally assumed that systemic application of EPO and its analogues increases the risk for thrombotic events. METHODS AND RESULTS Here we show in C57BL/6J mice that 4-week treatment with the long-lasting EPO analogue darbepoetin-alpha (DPO) at a dose of 10 microg/kg/week induces a reduction of platelet reactivity using flow cytometry and Western blot analysis of tyrosine-specific platelet phosphorylation. Additionally, immunohistochemistry of endothelial adhesion molecule expression and ELISA of circulating endothelial activation markers demonstrated a reduced endothelial activation. Immunohistochemistry and RT-PCR analysis revealed a significant (P<0.05) increase of eNOS expression. Further, DPO did not exert prothrombogenic effects in a murine intravital microscopic thrombosis model of the cremaster muscle. The role of eNOS in prevention of DPO-mediated microvascular thrombosis is further underlined by a significantly accelerated thrombus formation on DPO treatment in eNOS (-/-) mice. CONCLUSION Thus, DPO-related erythropoiesis with a raised hematocrit is not associated with an increased risk for thrombosis as long as endothelial NO production serves as compensatory mechanism.