Pasha R, Benavides M, Kottke-Marchant K, Harasaki H
Department of Biomedical Engineering, Cleveland Clinic Foundation, Ohio, USA.
Lab Invest. 1995 Sep;73(3):403-8.
Hyperthermic temperatures exist from the heat dissipation of the implantable energy source of an artificial heart. This procedure as well as therapies for cancer and thermal injuries pose a new medical problem. Among many reported effects of heat on biologic systems, platelet functions such as maximal aggregation and adhesion are known to be reduced. Using flow cytometry, we have studied platelet dysfunction at elevated temperatures and have gained a mechanistic comprehension of the loss of platelet function.
Platelet rich plasma was incubated at differing temperatures for 1 hour. Immediately after, the platelets were stained using mAb against glycoprotein IIb/IIIa (GPIIb-IIIa) (CD41a) and other platelet surface glycoproteins (GP) involved in aggregation and adhesion. Relative fluorescence intensity was measured using single-labeled, laser flow cytometry to determine changes in GP surface expression. In addition, scanning electron microscopy was used to evaluate morphologic changes.
Hyperthermic temperatures between 40 and 44 degrees C significantly lowered the mAb cell surface binding in vitro of GP that participate in aggregation and adhesion. The most dramatic temperature-dependent loss of mAb binding was demonstrated by anti-GPIIb-IIIa, the mAb against the fibrinogen receptor. mAb binding to this receptor at 44 degrees C was decreased to 6.2% of a base-line fluorescence intensity of 654 (arbitrary units). The ADP-induced aggregation of platelets incubated at the same temperature also decreased to 2.1% of maximum aggregation. Other mAb, such as those against the von Willebrand factor receptor (GPIb) (CD42b), the thrombospondin receptor (GPIV) (CD36), and GPIIIa (CD61), also showed statistically significant reduction of mAb binding but to a lesser degree. Finally, scanning electron microscopy as well as side-scatter density plots from flow cytometry revealed that platelets became more spherical after incubation at 44 degrees C.
The significant reduction in mAb binding correlates with functional impairment exhibited during hyperthermic incubation. Our results support the loss of binding ability of surface GP that are involved in aggregation and adhesion as a mechanism of platelet dysfunction upon heating. GPIIb-IIIa appeared the most susceptible to heat and the principal agent in thermal induced loss of platelet function. Significant morphologic changes at 44 degrees C, the critical temperature at which ADP-induced aggregation ceases, may contribute as well.
人工心脏植入式能量源散热会产生高温。该过程以及癌症和热损伤治疗引发了一个新的医学问题。在众多关于热对生物系统影响的报道中，已知血小板功能如最大聚集和黏附会降低。我们使用流式细胞术研究了高温下的血小板功能障碍，并对血小板功能丧失的机制有了一定理解。
富含血小板血浆在不同温度下孵育1小时。之后，立即使用针对糖蛋白IIb/IIIa（GPIIb-IIIa）（CD41a）以及其他参与聚集和黏附的血小板表面糖蛋白（GP）的单克隆抗体（mAb）对血小板进行染色。使用单标记激光流式细胞术测量相对荧光强度，以确定GP表面表达的变化。此外，使用扫描电子显微镜评估形态学变化。
40至44摄氏度的高温显著降低了参与聚集和黏附的GP在体外的mAb细胞表面结合。抗GPIIb-IIIa（针对纤维蛋白原受体的mAb）显示出最显著的温度依赖性mAb结合丧失。在44摄氏度时，该受体的mAb结合降至基线荧光强度654（任意单位）的6.2%。在相同温度下孵育的血小板经ADP诱导的聚集也降至最大聚集的2.1%。其他mAb，如针对血管性血友病因子受体（GPIb）（CD42b）、血小板反应蛋白受体（GPIV）（CD36）和GPIIIa（CD61）的mAb，也显示出mAb结合的统计学显著降低，但程度较小。最后，扫描电子显微镜以及流式细胞术的侧向散射密度图显示，在44摄氏度孵育后血小板变得更呈球形。
mAb结合的显著降低与高温孵育期间表现出的功能损害相关。我们的结果支持参与聚集和黏附的表面GP结合能力丧失是加热时血小板功能障碍的一种机制。GPIIb-IIIa似乎对热最敏感，是热诱导血小板功能丧失的主要因素。在44摄氏度（ADP诱导聚集停止的临界温度）出现的显著形态学变化可能也起了作用。
