DOI: 10.1073/pnas.94.9.4669. Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin

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

10.1073/pnas.94.9.4669

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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 AR 191 -T 192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR 117 -T 118 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.

Bibliography

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 ofâthrombopoietin. Proceedings of the National Academy of Sciences, 94(9), 4669â4674.

Authors 13

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

References 31 Referenced 29

10.1182/blood.V86.2.419.bloodjournal862419
10.1038/369533a0
10.1073/pnas.91.23.11104
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10.1182/blood.V86.2.540.bloodjournal862540
Y Shimada, T Kato, K Ogami, K Horie, A Kokubo, Y Kudo, E Maeda, Y Sohma, H Akahori, K Kawamura, H Miyazaki Exp Hematol 23, 1388–1396 (1995). / Exp Hematol by Shimada Y (1995)
D J Kuter, H Miyazaki, T Kato Thrombopoiesis and Thrombopoietins: Molecular, Cellular, Preclinical, and Clinical Biology, eds D J Kuter, W P Sheridan, P Hunt, D Zucker-Franklin (Humana, Totowa, NJ), pp. 143–164 (1996). / Thrombopoiesis and Thrombopoietins: Molecular, Cellular, Preclinical, and Clinical Biology by Kuter D J (1996)
10.1182/blood.V86.1.23.bloodjournal86123
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10.1182/blood.V85.10.2720.bloodjournal85102720
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10.1111/j.1432-1033.1985.tb09091.x
10.1016/0014-5793(94)01008-0
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10.1016/0378-1119(95)00125-P
10.1038/369565a0
10.1016/S0968-0004(00)88945-8
P Hunt, M M Hokom, B Wiemann, R M Leven, T Arakawa Exp Hematol 21, 372–381 (1993). / Exp Hematol by Hunt P (1993)
10.1097/00001721-199310000-00013
10.1046/j.1365-2141.1996.d01-1741.x
10.1073/pnas.85.15.5409
10.1016/0014-5793(96)01047-2
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10.1016/S0021-9258(18)83213-6
10.1016/S0021-9258(18)82348-1
Kato T. (1996) Stem Cells 14 Suppl. 139–147. (10.1002/stem.5530140718)
Matsumoto A. Tahara T. Morita H. Usuki K. Ohashi H. Kokubo A. Matsumoto E. Tsunakawa H. Ogami K. Miyazaki H. Urabe A. & Kato T. (1996) Blood 88 Suppl. 544a (abstr.).

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:35 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 3:56 p.m.)
Indexed	4 days, 14 hours ago (Aug. 23, 2025, 12:58 a.m.)
Issued	28 years, 3 months ago (April 29, 1997)
Published	28 years, 3 months ago (April 29, 1997)
Published Online	28 years, 3 months ago (April 29, 1997)
Published Print	28 years, 3 months ago (April 29, 1997)

Funders 0

None

BibTeX

@article{Kato_1997, title={Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin}, volume={94}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.94.9.4669}, DOI={10.1073/pnas.94.9.4669}, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Kato, Takashi and Oda, Atsushi and Inagaki, Yoshimasa and Ohashi, Hideya and Matsumoto, Atsushi and Ozaki, Katsutoshi and Miyakawa, Yoshitaka and Watarai, Hiroshi and Fuju, Kazumi and Kokubo, Atsuko and Kadoya, Toshihiko and Ikeda, Yasuo and Miyazaki, Hiroshi}, year={1997}, month=apr, pages={4669–4674} }

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