Bode A P, Read M S, Reddick R L
Department of Pathology and Laboratory Medicine, East Carolina University School of Medicine, Greenville, North Carolina, USA.
J Lab Clin Med. 1999 Feb;133(2):200-11. doi: 10.1016/s0022-2143(99)90013-6.
The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.
采用鲍姆加特纳灌注技术，测试复水后的冻干人血小板在高剪切条件下黏附于剥脱动脉血管段致血栓形成表面并发生血小板激活的能力。20份洗涤血小板制剂在Virtis 600冻干机中冻干前，通过1小时或2小时暴露于多聚甲醛来进行稳定处理。将这些固定干燥制剂的反应与新鲜枸橼酸盐全血中未固定血小板的反应进行比较。通过用针对糖蛋白Ib（CD42）或糖蛋白IIbIIIa（CD41a）的单克隆抗体进行免疫荧光染色后，在显微照片中通过形态计量学估计血管段被黏附血小板覆盖的面积百分比，对每个灌注实验的结果进行量化。通过流式细胞术检测非黏附血小板中CD62和GP53表达，以检查激活证据。此外，通过放射免疫测定法测量血栓素B2，作为灌注期间血小板对激活条件反应性的指标。在重组全血中观察到的冻干血小板的血管覆盖百分比略低于新鲜全血中的血小板（分别为39%和73%）。在使用未剥脱血管段进行的其他灌注实验中，两种血小板制剂的覆盖百分比均降低了一半或更多。扫描电子显微镜证实，冻干血小板不黏附于完整内皮区域。此外，在这些灌注实验中，冻干血小板的CD62或CD63激活抗原表达有小幅但显著的升高，并且产生血栓素B2的速率约为新鲜血小板的三分之一。通过用吲哚美辛或PGE-1预处理，新鲜或冻干血小板制剂在灌注期间的血栓素生成受到抑制。我们将这些数据解释为证据，证明我们的冻干血小板制剂能够至少部分地对生理刺激做出反应，并黏附于适当的致血栓形成部位以支持止血。
