Non-apoptotic externalization of phosphatidylserine (PS) can act as a reactive surface for the efficient assembly of the prothrombinase complex leading to thrombin generation and coagulation. Here we show that extracellular ATP, acting at the macrophage P2X(7) receptor, drives the rapid Ca(2+)-dependent formation and release of PS-rich microvesicles that enhance the assembly of the prothrombinase complex and subsequent formation of thrombin. Incubation with P2X(7) receptor antagonists (KN-62 and Brilliant Blue G) attenuates ATP induced prothrombotic responses. Consistent with the hypothesis that exposed PS enhances prothrombinase activity; pre-incubation with annexin V blocks the increase in thrombin formation. The rapid translocation of PS and formation of pro-thrombotic microvesicles occurs in the absence of cell lysis. These data demonstrate that the pro-inflammatory P2X(7) receptor can also support and propagate rapid increases in thrombin formation.