Maagdenberg Carlijn G, de Boer Onno J, Li Xiaofei, Mackaay Claire, Niessen Hans W, de Winter Robbert J, Van der Wal Allard C
Academic Medical Center, Department of Pathology, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands.
VU Medical Center, Department of Pathology, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
Thromb Res. 2016 Sep;145:12-7. doi: 10.1016/j.thromres.2016.07.003. Epub 2016 Jul 11.
To study the rate of apoptotic cell death in the process of thrombus evolution after plaque rupture in myocardial infarction.
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.
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.
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.
研究心肌梗死斑块破裂后血栓演变过程中凋亡性细胞死亡的发生率。
对63例患者的石蜡包埋血栓抽吸物进行苏木精-伊红（H&E）染色，以组织学评估血栓的年龄：新鲜血栓（完整血细胞；<1天）、溶解血栓（坏死；1-5天）或机化血栓（细胞长入；>5天）。还记录了斑块成分（动脉粥样硬化，包括泡沫细胞、胆固醇结晶、钙化和纤维帽组织）的存在情况。使用抗半胱天冬酶-3抗体进行免疫组织化学（双重）染色，以观察凋亡及相关细胞。对于后者，半胱天冬酶-3抗体与细胞特异性标志物髓过氧化物酶（MPO，粒细胞）、CD68（巨噬细胞）、CD34（内皮细胞）、平滑肌肌动蛋白-1（SMA-1，平滑肌细胞）和福尔根染色（DNA）相结合。其次，根据血栓年龄评估凋亡率。使用透射电子显微镜进一步评估血小板凋亡情况。
在总共63份抽吸物中，33份抽吸物中发现了斑块成分，其中15份中富含脂质的斑块组织是唯一成分。对所有含血栓的抽吸物（n=48）进行年龄分类，结果为12份新鲜血栓（25%）、18份溶解血栓（37.5%）和18份机化血栓（37.5%）。溶解血栓中的凋亡比新鲜或机化血栓更广泛（P<0.0001）。含斑块的抽吸物比无斑块的抽吸物显示出更多的凋亡（P<0.05）。半胱天冬酶-3抗体与细胞特异性标志物联合免疫染色显示，MPO+粒细胞中的凋亡最为广泛。半胱天冬酶-3阳性血小板（CD61+无核颗粒）在溶解血栓中最为丰富。通过超微结构证实了血小板中的凋亡。
本研究表明血栓年龄与粒细胞及血小板凋亡的发生之间存在显著关联，在（脆弱的）溶解血栓中发生率最高。我们提出动脉粥样硬化血栓形成中的凋亡性细胞死亡可能作为血栓不稳定性的生物标志物。
