Evidence of Human Thrombomodulin Domain as a Novel Angiogenic Factor

Abstract

Background-Thrombomodulin is an anticoagulant, endothelial-cell-membrane glycoprotein. A recombinant thrombomodulin domain containing 6 epidermal growth factor-like structures exhibits mitogenic activity. This study explored the novel angiogenic effects of the recombinant domain using in vitro and in vivo models. Methods and Results-Human recombinant thrombomodulin containing 6 epidermal growth factor-like structures (TMD2) and TMD2 plus a serine and threonine-rich domain (TMD23) were prepared using the Pichia pastoris expression system. Combined with purified TMD2 or TMD23, thrombin effectively activated protein C. TMD23 had higher activity than TMD2 in stimulating DNA synthesis in cultured human umbilical vein endothelial cells. Additionally, TMD23 stimulated chemotactic motility and capillarylike tube formation in human umbilical vein endothelial cells, an effect mediated through phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase and the phosphatidylinositol-3 kinase/Akt/endothelial nitric oxide synthase pathway. TMD23 also stimulated endothelial cell expression of matrix metalloproteinases and plasminogen activators, which mediated extracellular proteolysis, leading to endothelial cell invasion and migration during angiogenesis. Furthermore, TMD23-containing implants in rat cornea induced ingrowth of new blood vessels from the limbus. With the murine angiogenesis assay, TMD23 not only induced neovascularization coinjected with Matrigel and heparin but also enhanced angiogenesis in Matrigel containing melanoma A2058 cells in nude mice. Conclusions-The

An Akt- and Fra-1-Dependent Pathway Mediates Platelet-Derived Growth Factor-Induced Expression of Thrombomodulin, a Novel Regulator of Smooth Muscle Cell Migration

“…Other reports have suggested that exogenous recombinant TM could stimulate expression of mediators of matrix proteolysis in endothelial cells, including matrix metalloproteinases and plasminogen activators. 31 Taken together, these observations suggest that TM may regulate migration through its ability to alter cell association with the microenvironment, including cell-cell and cell-matrix interactions.…”

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

“…A human recombinant TM comprising the six EGF-like repeats (domain 2) and Ser/Thr-rich section (domain 3), subsequently referred to as TMD23, has also been reported to have vasculoprotective properties in animal models. Shi et al (140) illustrate the proangiogenic potential of TMD23 in rat corneal implants, while Wei et al (177) demonstrate the ability of TMD23 to reduce both neointimal formation (C57BL/6 mouse carotid ligation model) and atherosclerotic lesion formation (ApoE…”

Recombinant Thrombomodulin Inhibits Lipopolysaccharide-Induced Inflammatory Response by Blocking the Functions of CD14

“…Methods for the preparation of the TM domains were described previously (18,22,24 (I424A; rTMD23 I424A ) using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) (25). All recombinant TM proteins contained His-tag for purification and c-Myc tag for detection.…”

“…Because rTMD23 has six EGFlike domains, it acts as a novel angiogenic factor by facilitating endothelial cell proliferation and migration (22). rTMD23 also suppresses atherosclerosis in apolipoprotein E-deficient mice by binding to thrombin and inhibiting thrombin-induced endothelial cell activation (23).…”