Cooling and freezing damage platelet membrane integrity
Cooling and freezing damage platelet membrane integrity
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DOI：
10.1006/cryo.1999.2164
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发表时间：
1999-05-01
期刊：
影响因子：
2.7
通讯作者：
Gorogias, M
中科院分区：
文献类型：
--
作者：
Reid, TJ;LaRussa, VF;Gorogias, M
在23℃到4℃的温度下，血小板发生细胞骨架重排和膜脂相变(液晶到凝胶)，这些结构变化的结果是不可逆转的细胞损伤。我们研究了血小板膜的完整性是否可以通过(A)先前研究过的钙螯合剂(EGTA)和微丝稳定剂(细胞松弛素B)的组合来保存，这些组合在保护血小板免受冷损伤方面有明显的好处，或者(B)在保护膜和蛋白质免受冷冻损伤方面具有已知的好处。冰冻或降温前后用瑞斯托菌素凝集、凝血酶或ADP聚集、血小板诱导凝块回缩(PICR)和P-选择素表达等方法检测血小板功能和活化情况。血小板负载10 nM的二乙酸荧光素。冷冻或冷却后，将制备的制剂离心，并测定上清液中的荧光素。在降温实验中，新鲜血小板在4℃下冷却1~21d，加入或不加入80亩M EGTA/AM和2亩M细胞松弛素B(EGTA/AM-CytoB)，然后在37℃快速升温。在冷冻实验中，在新鲜血小板中加入5%二甲基亚砜(Me2SO)或5 mM甘油。然后将这些制剂在-1摄氏度/分钟到-70摄氏度的温度下冷冻，然后在37摄氏度下快速解冻，以荧光素上清液水平衡量的血小板膜完整性与血小板功能呈负相关。用EGTA/AM-CytoB在4℃冷藏血小板时，膜完整性逐渐丧失，第7天达到最大值。如PICR所示，膜完整性丧失先于功能完全丧失。相反，在没有这些试剂的情况下，冷冻的血小板在储存1天后完全失去了细胞膜的完整性和功能。与使用或不使用其他冷冻保护剂的冷冻相比，在Me2SO中冷冻血小板导致的荧光素释放要少得多(P<；0.001)。这一结果与PICR证明的增强功能相关，并支持了早期关于Me2SO保护血小板膜免受冷冻损伤的观察结果。荧光素向周围介质的释放反映了冷却和冷冻的血小板膜完整性和功能的丧失。膜细胞骨架重排与贮藏过程中膜的变化有关。这些结果可能普遍适用于血小板储存的研究。
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 mu M EGTA/AM and 2 mu M 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.