Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain

Abstract

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Herein we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro. The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from Gpr15 knock-out (KO) mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal EC numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. The use of TME5C may be a promising treatment strategy to prevent or treat lethal complications, such as sinusoidal obstruction syndrome, whose pathogenesis is based on endothelial insults.

The Biological and Clinical Relevance of G Protein-Coupled Receptors to the Outcomes of Hematopoietic Stem Cell Transplantation: A Systematized Review

“…For example, recombinant thrombomodulin (rTM) is approved in Japan to treat disseminated intravascular coagulation (DIC) and has been shown to reduce SOS and the occurrence of thrombotic microangiopathy in HSCT patients [162,163]#. In two murine SOS models, one using monocrotaline (MCT) and the other using busulfan/cyclophosphamide conditioning followed by HSCT, rTM’s cytoprotective effect was demonstrated to depend on its fifth epidermal growth factor-like region (TME5) [164,165]#. A murine model of tacrolimus-induced vascular injury showed that the pro-angiogenic functions of TME5 depended on its binding to G protein-coupled receptor (GPR) 15 [165,166]#.…”

“…In two murine SOS models, one using monocrotaline (MCT) and the other using busulfan/cyclophosphamide conditioning followed by HSCT, rTM’s cytoprotective effect was demonstrated to depend on its fifth epidermal growth factor-like region (TME5) [164,165]#. A murine model of tacrolimus-induced vascular injury showed that the pro-angiogenic functions of TME5 depended on its binding to G protein-coupled receptor (GPR) 15 [165,166]#. rTM was able to mitigate aGvHD in mice in a GPR15-dependent manner [167]#.…”

C‑type lectin family XIV members and angiogenesis: A review

“…By contrast, the recombinant thrombomodulin fragment TMD23 (with a 6-tandem EGF-like domain and O-glycosylation site-rich domain) was reported to stimulate angiogenesis (63,74). The C loop of the C-terminal sub-domain of the fifth EGF-like domain of TMD23 has pro-angiogenic and cytoprotective effects, in a G protein-coupled receptor 15-dependent manner (75). Angiogenesis was also mediated by the phosphorylation of ERK1/2, p38, protein kinase B (Akt) and Endothelial nitric oxide synthase (eNOS) (74), and by Fibroblast growth factor receptor 1-A (76).…”

C‐type lectin domain group 14 proteins in vascular biology, cancer and inflammation

“…A subsequent study revealed that this cytoprotective outcome was triggered by thrombomodulin EGF5 binding to G protein‐coupled receptor‐15 (GPR15) on endothelial cells, leading to the activation of endothelial nitric oxide synthase and extracellular signal‐regulated kinase (ERK) signalling, an effect that was abolished in GPR15‐deficient mice . Recently, the minimal fragment of thrombomodulin EGF5 necessary for binding to GPR15 and promoting proangiogenic function was identified as a 19‐amino acid peptide, that includes an intramolecular disulfide bond which adopts a loop structure similar to that observed for the prototypical EGF . This peptide exhibited proangiogenic function and extended survival in mouse models of sinusoidal obstruction syndrome, a condition that is associated with injury of liver sinusoidal endothelium .…”

“…Recently, the minimal fragment of thrombomodulin EGF5 necessary for binding to GPR15 and promoting proangiogenic function was identified as a 19‐amino acid peptide, that includes an intramolecular disulfide bond which adopts a loop structure similar to that observed for the prototypical EGF . This peptide exhibited proangiogenic function and extended survival in mouse models of sinusoidal obstruction syndrome, a condition that is associated with injury of liver sinusoidal endothelium . However, whether thrombomodulin can bind to GPR15 while attached to the cell membrane, or if proteolytic processing is essential, is yet to be determined.…”