Matsumoto A, Tahara T, Morita H, Usuki K, Ohashi H, Kokubo-Watarai A, Takahashi K, Shimizu E, Tsunakawa H, Ogami K, Miyazaki H, Urabe A, Kato T
Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan.
Thromb Haemost. 1999 Jul;82(1):24-9.
Thrombopoietin (TPO) isolated from thrombocytopenic plasma of various animal species has previously been shown to comprise only truncated forms of the molecule, presumably generated by proteolysis. Native TPO has now been partially purified from normal human plasma by immunoaffinity chromatography and was confirmed to be biologically active. Gel filtration in the presence of SDS revealed that TPO eluted in two peaks: a major peak corresponding to the elution position of fully glycosylated recombinant human TPO (rhTPO) consisting of 332 amino acid residues, and a minor peak corresponding to a smaller molecular size. Immunoblot analysis also revealed that most plasma-derived TPO migrated at the same position as fully glycosylated rhTPO, corresponding to a molecular size of approximately 80 to 100 kDa. Furthermore, the size distribution of circulating TPO in patients with various haematologic disorders did not differ markedly from that of plasma-derived TPO from healthy individuals. These results indicate that the truncation of circulating TPO is not related to disease pathophysiology, and that the predominant form of TPO in blood is a biologically active approximately 80- to 100-kDa species. The size distribution of TPO extracted from normal platelets was similar to that of TPO in plasma; the proportion of truncated TPO was decreased by prior incubation of platelets with hirudin. indicating that the endogenous truncated TPO, at least in platelet extract, was generated by thrombin-mediated cleavage.
先前已表明，从各种动物物种的血小板减少血浆中分离出的血小板生成素（TPO）仅包含该分子的截短形式，推测是由蛋白水解产生的。现在已通过免疫亲和色谱法从正常人血浆中部分纯化出天然TPO，并证实其具有生物活性。在SDS存在下进行凝胶过滤显示，TPO以两个峰洗脱：一个主峰对应于由332个氨基酸残基组成的完全糖基化重组人TPO（rhTPO）的洗脱位置，一个小峰对应于较小的分子大小。免疫印迹分析还显示，大多数血浆来源的TPO与完全糖基化的rhTPO在同一位置迁移，对应于约80至100 kDa的分子大小。此外，各种血液系统疾病患者循环TPO的大小分布与健康个体血浆来源的TPO的大小分布没有明显差异。这些结果表明，循环TPO的截短与疾病病理生理学无关，并且血液中TPO的主要形式是具有生物活性的约80至100 kDa的物种。从正常血小板中提取的TPO的大小分布与血浆中TPO的大小分布相似；水蛭素预先孵育血小板可降低截短TPO的比例。这表明内源性截短TPO，至少在血小板提取物中，是由凝血酶介导的裂解产生的。
