Reid T J, LaRussa V F, Esteban G, Clear M, Davies L, Shea S, Gorogias M
Division of Medicine, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
Cryobiology. 1999 May;38(3):209-24. doi: 10.1006/cryo.1999.2164.
Cytoskeletal rearrangements and a membrane lipid phase transition (liquid crystalline to gel) occur in platelets on cooling from 23 to 4 degrees C. A consequence of these structural alterations is irreversible cellular damage. We investigated whether platelet membrane integrity could be preserved by (a) previously studied combinations of a calcium chelator (EGTA) and microfilament stabilizer (cytochalasin B) with apparent benefit in protecting platelets from cooling injury or (b) agents of known benefit in protecting membranes and proteins from freezing injury. Platelet function and activation before and after freezing or cooling were measured by agglutination with ristocetin, aggregation with thrombin or ADP, platelet-induced clot retraction (PICR), and expression of P-selectin. Platelets were loaded with 10 nM fluorescein diacetate. After freezing or cooling, the preparations were centrifuged and the supernatant was measured for fluorescein. For cooling experiments, fresh platelets were chilled at 4 degrees C for 1 to 21 days with or without the combination of 80 microM EGTA/AM and 2 microM cytochalasin B (EGTA/AM-CytoB) and then warmed rapidly at 37 degrees C. For freezing experiments, 5% dimethyl sulfoxide (Me2SO) or 5 mM glycerol were added to fresh platelets. The preparations were then frozen at -1 degrees C/min to -70 degrees C and then thawed rapidly at 37 degrees C. Platelet membrane integrity, as measured by supernatant levels of fluorescein, correlated inversely with platelet function. Chilling platelets at 4 degrees C with EGTA/AM-CytoB showed a gradual loss of membrane integrity, with maximum loss reached on day 7. The loss of membrane integrity preceded complete loss of function as demonstrated by PICR. In contrast, platelets chilled without these agents had complete loss of membrane integrity and function after 1 day of storage. Freezing platelets in Me2SO resulted in far less release of fluorescein than did freezing with or without other cryoprotectants (P < 0.001). This result correlated with enhanced function as demonstrated by PICR and supports earlier observations that Me2SO protects platelet membranes from freezing injury. Release of fluorescein into the surrounding medium reflected loss of membrane integrity and function in both cooled and frozen platelets. Membrane cytoskeletal rearrangements are linked to membrane changes during storage. These results may be generally applicable to the study of platelet storage.
血小板从23℃冷却至4℃时会发生细胞骨架重排和膜脂相转变（液晶态到凝胶态）。这些结构改变的一个后果是不可逆的细胞损伤。我们研究了血小板膜完整性是否可以通过以下方式得以保留：(a) 之前研究过的钙螯合剂（乙二醇双四乙酸，EGTA）和微丝稳定剂（细胞松弛素B）的组合，其在保护血小板免受冷却损伤方面具有明显益处；或者 (b) 已知对保护膜和蛋白质免受冷冻损伤有益的试剂。通过用瑞斯托霉素凝集、用凝血酶或二磷酸腺苷（ADP）聚集、血小板诱导的凝块回缩（PICR）以及P-选择素的表达来测量冷冻或冷却前后血小板的功能和活化情况。用10 nM的荧光素二乙酸酯加载血小板。冷冻或冷却后，将制剂离心，测量上清液中的荧光素。对于冷却实验，将新鲜血小板在4℃下冷藏1至21天，添加或不添加80 μM EGTA/AM和2 μM细胞松弛素B（EGTA/AM-细胞松弛素B）的组合，然后在37℃下快速升温。对于冷冻实验，向新鲜血小板中添加5%二甲基亚砜（Me2SO）或5 mM甘油。然后将制剂以-1℃/分钟的速度冷冻至-70℃，然后在37℃下快速解冻。通过荧光素上清液水平测量的血小板膜完整性与血小板功能呈负相关。用EGTA/AM-细胞松弛素B在4℃下冷却血小板显示膜完整性逐渐丧失，在第7天达到最大丧失。如PICR所示，膜完整性丧失先于功能完全丧失。相比之下，未添加这些试剂而冷却的血小板在储存1天后膜完整性和功能完全丧失。在Me2SO中冷冻血小板导致的荧光素释放远少于在添加或不添加其他冷冻保护剂的情况下冷冻（P < 0.001）。这一结果与PICR所示的功能增强相关，并支持了早期的观察结果，即Me2SO可保护血小板膜免受冷冻损伤。荧光素释放到周围介质中反映了冷却和冷冻血小板中膜完整性和功能的丧失。膜细胞骨架重排在储存期间与膜变化相关。这些结果可能普遍适用于血小板储存的研究。
