Involvement of the Mural Thrombus as a Site of Protease Release and Activation in Human Aortic Aneurysms

Abstract

Acquired abdominal aortic aneurysms are usually associated with a mural thrombus through which blood continues to flow. Some early data suggest that aneurysmal evolution correlates with the biological activity of the thrombus. Our hypothesis was therefore that the thrombus could adsorb blood components and store, release, and participate in the activation of proteases involved in aneurysmal evolution. For this purpose, we have explored both the metalloproteinase and fibrinolytic systems in the thrombus and the wall of human aneurysms. We have first investigated blood clot formation and lysis in vitro. Spontaneous clotting induces a release of promatrix metalloproteinase (pro-MMP)-9 into the serum that was fourfold higher than in paired control plasma (P < 0.001). Fibrinolysis progressively released more MMP-9 in a time-dependent manner (P < 0.01). After selective isolation, we demonstrated that polymorphonuclear leukocytes are the main source of MMP-9 release during clot formation. Protease content was then analyzed in 35 mural thrombi and walls of human abdominal aortic aneurysms sampled during surgical repair. In 15 aneurysms, the liquid phase at the interface between the thrombus and the wall was sampled separately. Both thrombus and wall contained MMP-2 and MMP-9 but the ratio MMP-9/MMP-2 was higher in the thrombus than in the wall. The liquid interface also contained active MMP-9. Immunohistochemistry of the thrombus confirmed these findings, showing the presence of polymorphonuclear leukocytes at the luminal pole of the thrombus, co-localizing with MMP-9 storage. In contrast, MMP-3 and MMP-7 were only present in the aneurysmal wall. Plasminogen was present in the mural thrombus but plasmin activity was present in both thrombus and wall. In the liquid interface, plasmin-alpha(2)-anti-plasmin complexes were detected demonstrating in vivo the activation of plasminogen. In contrast, u-PA and t-PA were detectable only in the wall, suggesting that plasminogen present in the thrombus could be activated by factors secreted by the arterial wall. This was demonstrated in vitro, in which co-incubation of thrombus and wall extracts generated plasmin in the presence of a fibrin matrix and activated MMPs. In conclusion, our study strongly suggests that the mural thrombus, by trapping polymorphonuclear leukocytes and adsorbing plasma components could act as a source of proteases in aneurysms that may play a critical role in enlargement and rupture.

Role of Leukocyte Elastase in Preventing Cellular Re-Colonization of the Mural Thrombus

“…41,42 In a recent study, we have shown that the mural thrombus in human atherosclerotic aneurysms is a site of storage and release of MMP-9 and that polymorphonuclear leukocytes trapped within the thrombus are the main cellular source of this MMP-9. 22 Recent luminal clots are sites of platelet deposition 43 and fibrin-fibronectin copolymerization, 44 and represent an optimal substrate for PMN adhesion. In the present work, we have extended these data using an ex vivo approach to study proteases bound to and released by the mural thrombus in human aneurysms.…”

“…22 Samples containing 5 g and 15 g of proteins were loaded on gelatin and casein gels, respectively. Gelatin and casein gels were incubated at 37°C for 19 hours and 72 hours, respectively.…”

“…21 We have recently shown that, compared to the deeper layers, the luminal layer of the aneurysmal thrombus is enriched with PMNs and releases gelatinase B (MMP-9). 22 In parallel, it has been reported that elastase released by PMN degranulation is able to induce loss of cell anchorage and apoptosis (anoikis) of cultured adherent cells. 19,23 It thus appears probable that elastase released by PMNs trapped within the fibrin matrix may play a role in preventing the colonization of the mural thrombus by mesenchymal cells.…”

Anoikis: an emerging hallmark in health and diseases

“…These pericellular proteolytic events are claimed to play a predominant role in the initiation of atherosclerosis and during the consequent athero-thrombotic complications [149,150]. A similar proteolytic cascade involving pericellular plasminogen activation and fibronectin degradation is instrumental in the disappearance of smooth muscle cells during aneurysm and varicose vein development [151,152]. In the latter case, release of proteases by polymorphonucleated leukocytes, recruited and activated in response to blood stasis and hypoxia, is also involved in endothelial cell detachment and disappearance [152,153].…”

Morphological aspects of mural thrombi deposition residual lumen route in infrarenal abdominal aorta aneurisms

“…10 Recent studies suggest that the ILT is an active pathogenic factor for AAA. [11][12] Adolph et al, 11 have demonstrated the biological activity of the thrombus by the presence of activated cells and by the relative permeability of the aneurismatic wall to formed elements of the blood. Fontaine et al 12 have indicated the enzymatic activity concentrated in mural thrombi as a likely relevant factor in the course of aneurisms.…”