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. 2018 Mar 6;8:4080. doi: 10.1038/s41598-018-21728-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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(A) Time course of the proteolysis of prothrombin (ProT), prethrombin-2 (Pre2) and thrombin (T) by subtilisin, under non-reducing conditions, in the absence and presence of the active site inhibitor argatroban. Subtilisin cleaves A470 in the autolysis loop (arrow) only when prethrombin-2 is generated. Argatroban affects proteolysis of prothrombin and prevents accumulation of prethrombin-1 (P1) but not fragment 1 (F1, Gla domain plus kringle-1). (B) Exponential time course of the proteolytic digestion of prothrombin, prethrombin-2 and thrombin by subtilisin obtained by densitometric analysis of the gels in the absence (open circles) and presence (filled circles) of argatroban. Binding of argatroban drastically (>10-fold) enhances the rate of prothrombin digestion but has no effect on prethrombin-2 and thrombin, in line with the shift in the E*-E distribution observed by rapid kinetics (Fig. 4). Gels were not cropped and originals are shown in the Supplementary Information.