Time dependent apoptotic rates in the evolving coronary thrombus mass of myocardial infarction patients.

Aim: To study the rate of apoptotic cell death in the process of thrombus evolution after plaque rupture in myocardial infarction.

Methods: Paraffin embedded thrombosuction aspirates of 63 patients were stained with haematoxylin & eosin (H&E) to assess histologically the age of the thrombi: fresh (intact blood cells; <1day old), lytic (necrosis; 1-5days old) or organized (ingrowth of cells; >5days old). Presence of plaque constituents (atheroma including foam cells, cholesterol crystals calcifications and fibrous cap tissue) was also recorded. Immunohistochemical (double) stains with anti-caspase-3-antibody were used to visualize apoptosis and the cells involved. For the latter caspase-3 antibody was combined with cell-specific markers MPO (granulocytes), CD68 (macrophages), CD34 (endothelial cells), SMA-1 (smooth muscle cells) and a Feulgen stain (DNA). Second, the rate of apoptosis was evaluated in relation to the age of the thrombi. Platelet apoptosis was further evaluated with the use of TEM.

Results: From a total of 63 aspirates, plaque constituents were found in 33 of the aspirates, and in 15 of them lipid rich plaque tissue was the sole component. Age classification of all thrombus containing aspirates (n=48) resulted in 12 fresh (25%), 18 lytic (37.5%) and 18 organized (37.5%) thrombi. Apoptosis was more extensive in lytic thrombi than in fresh or organized thrombi (P<0.0001). Plaque-containing aspirates showed more apoptosis than aspirates without plaque (P<0.05). Immuno staining with caspase-3 antibody in combination with cell-specific markers showed that apoptosis was most extensive in MPO+ granulocytes. Caspase-3-positive platelets (CD61+ anucleate particles) were most abundant in lytic thrombi. Apoptosis in platelets was confirmed by ultrastructure.

Conclusion: This study demonstrated a significant association between thrombus age and occurrence of apoptosis of granulocytes and also platelets, with highest rates in (fragile) lytic thrombi. We propose that apoptotic cell death in athero thrombosis could potentially serve as a biomarker for thrombus instability.