To explore possible mechanisms responsible for the absence of cell re-colonization of mural thrombi in aneurysms, we analyzed the release and storage of leukocyte proteases in the most luminal layer versus intermediate and abluminal layers of 10 mural thrombi of human abdominal aortic aneurysms. The luminal layer contained many polymorphonuclear leukocytes (PMNs), which released pro-matrix metalloproteinase (MMP)-9 and MMP-8. Leukocyte elastase was also stored and released by the luminal layer (immunohistochemistry, activity on synthetic substrates, and casein zymography). Acid buffer allowed extraction of leukocyte elastase from the luminal layer, which was inhibited by elastase inhibitors. Casein zymography of luminal extracts and conditioned medium from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMNs exhibited a similar lysis pattern, corresponding to elastase activity. Smooth muscle cell (SMC) seeding resulted in colonization of the intermediate thrombus layer ex vivo but not of the luminal layer. Extracts of the luminal layer induced loss of anchorage of both cultured human smooth muscle cells and stromal cells of bone marrow origin (anoikis). This anoikis was prevented by preincubation of the extracts with serine protease inhibitors. Moreover, adhesion of human SMCs and stromal bone marrow cells on fibrin gels was strongly inhibited when the gel was preincubated with pure elastase, medium of fMLP-stimulated PMNs, or extracts of luminal layers of mural thrombi. This loss of cell anchorage was prevented by the preincubation of the medium or extracts with alpha(1)-antitrypsin, but not when alpha(1)-antitrypsin was added after binding of elastase to the fibrin gel. In conclusion, elastase released by PMNs trapped within the mural thrombus impairs the spontaneous anchorage of mesenchymal cells to a fibrin matrix. This phenomenon could be one mechanism by which cellular healing of the mural thrombus in aneurysms is prevented.