When stimulated in vitro, human platelets release matrix metalloproteinase-2 (MMP-2) that, in turn, potentiates platelet activation. The present study investigated if MMP-2 is released from activated platelets in vivo in humans and whether aspirin inhibits this release. MMP-2 levels were measured by zymography, immunoblotting, flow-cytometry and an activity assay system, in plasma prepared from blood emerging from a skin wound inflicted for the measurement of the bleeding time (shed blood) and simultaneously from venous blood in 27 healthy human volunteers. In a subgroup, the same measurements were carried out before and 1 h after aspirin intake. MMP-2 was significantly higher in shed blood than in venous blood and increased progressively, consistent with ongoing platelet activation. A significant correlation was evident between platelet number and MMP-2 in shed blood; platelet MMP-2 content in shed blood was lower than that of platelets in venous blood. The level of active MMP-2 released by activated platelets in vivo was within the range of concentrations that potentiate platelet activation. Aspirin did not reduce MMP-2 release in vivo. In conclusion, MMP-2 is released from platelets in vivo in humans at a localised site of vessel wall damage in amounts sufficient to potentiate platelet aggregation; aspirin does not reduce this release.