Fischer Thomas H, Merricks Elizabeth P, Bode Author P, Bellinger Dwight A, Russell Karen, Reddick Robert, Sanders William E, Nichols Timothy C, Read Marjorie S
Department of Pathology, 350 Old Fayetteville Road, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.
Hematology. 2002 Dec;7(6):359-69. doi: 10.1080/1024533021000047954.
Stored human platelets are frequently used in hemorrhagic emergencies, but have limited immediate utility for controlling bleeding due to storage lesion and are frequently contaminated with microorganisms. The development of paraformaldehyde-treated, lyophilized and rehydrated (RL) platelets, which are sterile and have a prolonged shelf life (years), ameliorate the efficacy and sterility problems with stored platelets. RL platelets have been shown to have many native functions of fresh platelets in vitro and to mediate hemostasis in vivo in large animal models of hemorrhagic shock and cardiopulmonary bypass induced platelet dysfunction. To further evaluate the functional properties of this transfusion product, we studied the role of RL platelets in three aspects of thrombus formation and lysis. First, the interaction between RL platelets and fibrinogen was investigated. The surface density of unligated GPIIb-IIIa on RL and fresh platelets were, respectively 30000 and 70000 molecules per cell as detected with the monoclonal antibody 10E-5. Freezing, lyophilization and rehydration steps in the preparation of RL platelets resulted in the surface presentation of 120000 molecules of fibrinogen per cell from alpha granule sources. After ADP activation, RL platelets bound exogenous 125I-labeled fibrinogen in a dose-dependent manner with an affinity that is similar to that of fresh platelets and was inhibited by RGD peptides. 125I-Labeled fibrinogen binding to RL and fresh platelets, respectively, saturated at 14000 and 32000 molecules per cell. Scanning electron microscopic ultrastructural analysis showed that fibrin strands interacted with the surface of RL platelets in a normal manner. The second set of studies investigated the ability of RL platelets to catalyze and amplify the clot formation process in an activation-dependent manner. We showed that RL platelets undergo degranulation in fibrin in clots and functioned as thrombogenic surfaces for the generation of activated coagulation factors and fibrin generation. A final set of studies was performed to investigate fibrin of clots that contained RL platelets. RL platelet clots were lysed in the presence of tissue plasminogen activator with a similar time course as clots without platelets, and lysis occurred faster than when fresh platelets were included in the fibrin mass. The results of these three studies demonstrate that RL platelets are capable of mediating thrombus formation and do not inhibit lysis. Our results help explain how RL platelets restore hemostasis in vivo, and indicate that these cells might be a viable alternative to fresh stored platelets in transfusion medicine.
储存的人血小板常用于出血性紧急情况，但由于储存损伤，其控制出血的即时效用有限，且经常被微生物污染。经多聚甲醛处理、冻干和复水（RL）的血小板已研发出来，这些血小板无菌且保质期延长（数年），改善了储存血小板的功效和无菌问题。RL血小板在体外已显示出具有新鲜血小板的许多天然功能，并在出血性休克和体外循环诱导的血小板功能障碍的大型动物模型中，能在体内介导止血。为了进一步评估这种输血产品的功能特性，我们从血栓形成和溶解的三个方面研究了RL血小板的作用。首先，研究了RL血小板与纤维蛋白原之间的相互作用。用单克隆抗体10E-5检测发现，RL血小板和新鲜血小板上未结合的GPIIb-IIIa的表面密度分别为每细胞30000和70000个分子。RL血小板制备过程中的冷冻、冻干和复水步骤导致每个细胞从α颗粒来源呈现120000个纤维蛋白原分子。ADP激活后，RL血小板以剂量依赖的方式结合外源性125I标记的纤维蛋白原，其亲和力与新鲜血小板相似，并被RGD肽抑制。125I标记的纤维蛋白原与RL血小板和新鲜血小板的结合分别在每细胞14000和32000个分子时达到饱和。扫描电子显微镜超微结构分析表明，纤维蛋白丝以正常方式与RL血小板表面相互作用。第二组研究调查了RL血小板以激活依赖的方式催化和放大凝血过程的能力。我们发现RL血小板在凝块中的纤维蛋白中发生脱颗粒，并作为生成活化凝血因子和纤维蛋白生成的促血栓形成表面发挥作用。最后一组研究是为了研究含有RL血小板的凝块的纤维蛋白。在组织纤溶酶原激活剂存在的情况下，RL血小板凝块的溶解时间进程与不含血小板的凝块相似，并且溶解速度比纤维蛋白块中包含新鲜血小板时更快。这三项研究的结果表明，RL血小板能够介导血栓形成且不抑制溶解。我们的结果有助于解释RL血小板如何在体内恢复止血，并表明这些细胞在输血医学中可能是新鲜储存血小板的可行替代品。
