Abstract Introduction Microparticles from activated endothelial cells (EMP) are well known to expose tissue factor (TF) and initiate coagulation in vitro. TF coagulant activity is critically dependent on the presence of aminophospholipids, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), but it is unknown whether or not TF-exposing EMP are enriched in such aminophospholipids. Furthermore, despite the fact that EMP have been reported in several pathological conditions, direct evidence for their (putative) coagulant properties in vivo is still lacking. We investigated the phospholipid composition of endothelial MP (EMP) and their thrombogenic properties in vivo. Materials and methods Human umbilical vein endothelial cells (HUVEC; n = 3) were incubated with or without interleukin (IL)-1α (5 ng/mL; 0–72 h). Phospholipid composition of EMP was determined by high-performance thin layer chromatography. The association between EMP, TF antigen and activity was confirmed in vitro (ELISA, Western blot and thrombin generation). Thrombogenic activity of EMP in vivo was determined in a rat venous stasis model. Results Levels of TF antigen increased 3-fold in culture medium of IL-1α-treated cells ( P < 0.0001). This TF antigen was associated with EMP and appeared as a 45–47 kDa protein on Western blot. In addition, EMP from activated cells were enriched in both PS ( P < 0.0001) and PE ( P < 0.0001), and triggered TF-dependent thrombin formation in vitro and thrombus formation in vivo. In contrast, EMP from control cells neither initiated coagulation in vitro nor thrombus formation in vivo. Conclusions EMP from activated endothelial cells expose coagulant tissue factor and are enriched in its cofactors PS and PE.