Kelton J G, Warkentin T E, Hayward C P, Murphy W G, Moore J C
Department of Medicine, McMaster University Medical Centre, Hamilton, Ontario, Canada.
Blood. 1992 Nov 1;80(9):2246-51.
Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium-dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.
血栓性血小板减少性紫癜（TTP）的特征是血小板减少以及在整个微血管系统中出现弥散性血小板血栓。我们团队的研究表明，急性TTP患者康复后，其血清中不再能检测到钙依赖性蛋白水解活性（钙蛋白酶）。本研究的目的是调查TTP的蛋白酶活性在血浆中是否可检测到，因此并非血清形成后的体外现象。此外，我们在患者样本中寻找活性蛋白酶与膜结合的证据，这将解释其在血浆抑制剂存在的情况下活性持续存在的原因。从10例TTP患者中采集急性TTP样本，包括血清和血浆。使用酶活性生物测定法以及用抗钙蛋白酶单克隆抗体（MoAb）进行免疫印迹检测蛋白质来测量钙蛋白酶。在所有情况下，急性TTP血清和血浆中均可在功能和抗原方面检测到钙蛋白酶。在任何对照中均未检测到钙蛋白酶活性，尽管在一名接受体外循环手术的患者的一个样本中可检测到抗原性钙蛋白酶。为了研究钙蛋白酶是否与血浆中的微粒相关，对TTP血浆样本进行超滤和超速离心。活性不会因通过0.2微米滤膜而丧失，但仅在超速离心后的沉淀中可检测到。使用Triton X-100溶解也证明了微粒中钙蛋白酶与膜的结合。免疫沉淀研究表明，钙蛋白酶活性可被抗血小板膜糖蛋白（IX和IIb/IIa）的MoAb去除，但不能被抗红细胞膜血型糖蛋白的MoAb去除。这些研究表明，活性钙蛋白酶与TTP患者血浆中的血小板微粒相关。
