Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site

RESEARCH ARTICLE Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site Nataliya Bohdan1☯, Salvador Espín1☯, Sonia Águila1, Raúl Teruel-Montoya1,2, Vicente Vicente1,2, Javier Corral1,2, Irene Martínez-Martínez1,2* 1 Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, Murcia, Spain, 2 Grupo de investigación CB15/ 00055 del Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain a11111 ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Bohdan N, Espín S, Águila S, TeruelMontoya R, Vicente V, Corral J, et al. (2016) Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site. PLoS ONE 11(6): e0157834. doi:10.1371/journal.pone.0157834 Editor: Nikos K Karamanos, University of Patras, GREECE Received: February 2, 2016 Accepted: June 6, 2016 Published: June 20, 2016 Copyright: © 2016 Bohdan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by CP13/00126 (Instituto de Salud Carlos III) and PI12/00657 (Instituto de Salud Carlos III & Fondo Europeo de Desarrollo Regional), URL: http://www.isciii.es/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin. Introduction Heparanase is an endoglycosidase able to cleave heparan sulphate side chains at a limited number of sites, yielding heparan sulphate fragments of still appreciable size (~5–7 kDa) [1–3]. Heparanase is expressed as an enzymatically inactive proheparanase that is later converted into an active enzyme following processing by proteases, such as cathepsin-L [4]. Its activity PLOS ONE | DOI:10.1371/journal.pone.0157834 June 20, 2016 1 / 11 Heparanase Activates Antithrombin Abbreviations: AT, antithrombin; LMWH, low molecular weight heparin; UFH, unfrationated heparin; TF, tissue factor; TFPI, tissue factor pathway inhibitor. correlates with the metastatic potential of tumor cells, attributed to enhanced cell dissemination as a consequence of heparan sulphate cleavage and remodeling of the extracellular matrix barrier [5,6]. Heparanase is secreted by platelets after degranulation, leukocytes or endothelial cells and it has multiple roles. It can aid cell invasion by degrading heparan sulphate proteoglycans and can release growth factors bound to heparan-sulphate that initiate angiogenesis, such as VEGF, or activate tissue repair by releasing FGF. Heparan sulphate-disaccharides liberated by heparanase also inhibit TNF production by macrophages with direct consequences as a negative regulator of inflammation [4]. Up-regulated expression of heparanase has been noted in essentially all human tumors examined, as well as in inflammation, wound healing, and diabetic nephropathy [5–7]. Besides, heparanase has been recently revealed as an important modulator of blood coagulation. Heparanase is able to activate tissue factor (TF) in a non-enzymatic manner, and up-regulates TF expression [8]. Heparanase also interacts with tissue factor pathway inhibitor (TFPI) on the cell surface, leading to dissociation of TFPI from the cell membrane of endothelial and tumor cells, resulting in decreased anticoagulant capacity at the cell surface [9]. Antithrombin is the main inhibitor of the coagulation system and its main targets are FXa and FIIa or thrombin [10]. The binding of glycosaminoglycans to the heparin-binding site causes a conformational transition in antithrombin that closes the β-sheet A, which expels the reactive center loop, activating the molecule [11]. This control allows a slight delay in the antithrombin anticoagulant function and restrains it to the site of vascular injury. Therefore, alteration of heparin affinity results in a defective inhibitory function of this serpin. Several studies have reported the procoagulant state derived from mutations affecting the antithrombin heparin binding [12–15]. Under physiological conditions, heparanase is not over-expressed and it does not exert endoglycosidase activity. However, it has been demonstrated that there is an increased heparanase procoagulant activity in healthy shift working nurses in comparison to healthy daytime working nurses [16]. In this study we have addressed the potential effect of heparanase on antithrombin inhibitory function under physiological pH in order to better know its role in coagulation. Materials and Methods Materials Low molecular weight heparin (LMWH; Bemiparin) and unfractionated heparin (UFH) were from Rovi (Madrid, Spain). FXa was from Enzyme Research Laboratories (Swansea, United Kingdom) and FIIa was from (Merck Millipore, Madrid, Spain). Culture media were obtained from Gibco (Fisher Scientific, Madrid, Spain). Rec (...truncated)