Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin

Takashi Kato, Atsushi Oda, Yoshimasa Inagaki, Hideya Ohashi, Atsushi Matsumoto, Katsutoshi Ozaki, Yoshitaka Miyakawa, Hiroshi Watarai, Kazumi Fuju, Atsuko Kokubo, Toshihiko Kadoya, Yasuo Ikeda, Hiroshi Miyazaki

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Abstract

A heterogeneity in the molecular weight (M(r)) 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 M(r) 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.

Original languageEnglish
Pages (from-to)4669-4674
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number9
DOIs
Publication statusPublished - 1997 Apr 29
Externally publishedYes

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    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. (1997). Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin. Proceedings of the National Academy of Sciences of the United States of America, 94(9), 4669-4674. https://doi.org/10.1073/pnas.94.9.4669