Murphy W G, Moore J C, Kelton J G
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Blood. 1987 Nov;70(5):1683-7.
Plasma and serum from patients with thrombotic thrombocytopenic purpura (TTP) can cause activation and aggregation of normal human platelets in vitro. It is possible that this platelet-activating factor contributes to the disease. In this report we describe studies designed to identify the platelet-activating factor in TTP. Platelet activation by sera from 15 patients with TTP was inhibited by leupeptin, iodoacetamide, and antipain but not by phenylmethylsulphonylfluoride, epsilon-aminocaproic acid, soybean trypsin inhibitor, aprotinin, and D-phenylanyl-1-prolyl-1-arginine chloromethyl ketone. These studies suggested that the platelet-activating factor in TTP serum was a cysteine protease. We confirmed that a calcium-dependent cysteine protease (CDP) was present in the sera of each of the 15 patients when we used an assay based on the ability of CDP to proteolyse platelet membrane glycoprotein 1b (GP1b) and hence to abolish the ability of CDP-treated normal platelets to agglutinate in the presence of ristocetin and von Willebrand factor. This proteolytic activity was inhibited by EDTA, leupeptin, antipain, iodoacetamide, and by N-ethyl-maleamide (NEM) but not by the serine protease inhibitors. Activity was detected in 15 of 15 patients with TTP tested before therapy was begun. In contrast, no activity was detected in the serum of any of five of the TTP patients tested in remission or in any of the sera from 36 patients with thrombocytopenia and 423 nonthrombocytopenic controls. To look for in vivo CDP activity in patients with TTP, we studied platelets from two patients with acute TTP (drawn into acid-citrate-dextrose, NEM, and leupeptin). These platelets showed a loss of GP1b from the platelet surface. Both patients were also studied in remission: GP1b on the platelet surface had returned to normal. These studies provide evidence that CDP is present in the sera of patients with TTP, that it is specific to this disease, and that is is active in vivo as well as in vitro. We postulate that a disorder of CDP homeostasis plays a major role in the pathophysiology of TTP.
血栓性血小板减少性紫癜（TTP）患者的血浆和血清可在体外引起正常人血小板的活化和聚集。这种血小板活化因子有可能促成了该疾病。在本报告中，我们描述了旨在鉴定TTP中血小板活化因子的研究。15例TTP患者血清引起的血小板活化受到亮抑酶肽、碘乙酰胺和抑肽酶的抑制，但不受苯甲基磺酰氟、ε-氨基己酸、大豆胰蛋白酶抑制剂、抑肽酶和D-苯丙氨酰-1-脯氨酰-1-精氨酸氯甲基酮的抑制。这些研究表明，TTP血清中的血小板活化因子是一种半胱氨酸蛋白酶。当我们使用基于钙依赖性半胱氨酸蛋白酶（CDP）蛋白水解血小板膜糖蛋白1b（GP1b）的能力并因此消除经CDP处理的正常血小板在存在瑞斯托菌素和血管性血友病因子时的凝集能力的检测方法时，我们证实15例患者的血清中均存在CDP。这种蛋白水解活性受到EDTA、亮抑酶肽、抑肽酶、碘乙酰胺和N-乙基马来酰胺（NEM）的抑制，但不受丝氨酸蛋白酶抑制剂的抑制。在开始治疗前检测的15例TTP患者中均检测到活性。相比之下，在5例处于缓解期的TTP患者的血清中未检测到活性，在36例血小板减少症患者和423例非血小板减少症对照者的血清中也均未检测到活性。为了寻找TTP患者体内的CDP活性，我们研究了2例急性TTP患者（采集到含酸-枸橼酸盐-葡萄糖、NEM和亮抑酶肽的采血管中）的血小板。这些血小板显示血小板表面的GP1b丢失。这2例患者在缓解期也进行了研究：血小板表面的GP1b已恢复正常。这些研究提供了证据，证明CDP存在于TTP患者的血清中，它是该疾病所特有的，并且在体内和体外均具有活性。我们推测，CDP稳态失调在TTP的病理生理学中起主要作用。
