Kato T, Oda A, Inagaki Y, Ohashi H, Matsumoto A, Ozaki K, Miyakawa Y, Watarai H, Fuju K, Kokubo A, Kadoya T, Ikeda Y, Miyazaki H
Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki, Gunma 370-12, Japan.
Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4669-74. doi: 10.1073/pnas.94.9.4669.
A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low Mr peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin-cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR191-T192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR117-T118 in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.
据报道，血小板生成素（TPO）的分子量（Mr）存在异质性。我们在人、大鼠、小鼠和犬的TPO中发现了几个凝血酶切割位点，还发现人TPO会被凝血酶选择性地进行蛋白水解。重组人TPO（rhTPO）在钙离子存在的情况下与人血小板一起孵育以产生凝血酶，随后被切割成低Mr肽片段。水蛭素可完全抑制这种切割，表明该蛋白水解作用是由凝血酶介导的。在无血小板系统中，使用抗人TPO肽抗体通过免疫印迹分析凝血酶切割情况，结果显示，根据消化时间会选择性地产生四种主要的凝血酶切割肽片段。进一步分析了凝血酶切割多肽的氨基酸序列，确定了两个主要的凝血酶切割位点。其中一个位于TPO C末端结构域的AR191 - T192处，凝血酶首先在此位点进行切割。另一个位于N末端结构域的GR117 - T118处，在凝血酶长时间消化后随后被切割。结果，TPO的生物学活性受到调节。鉴于TPO与血小板相关的代谢过程，凝血酶产生TPO截短形式的现象可能是一个值得关注的事件。
