Introduction The multidomain protein, HPRG, is an abundant plasma protein (1.5 μM) with pleiotropic functions. We recently reported that the anti-angiogenic activity of HPRG is mediated through its H/P domain (1), which has recently been independently con- firmed (2). Although the physiological role of HPRG is not completely understood, it is thought to be involved in coagula- tion and fibrinolysis, immune regulation and angiogenesis (1, 3, 4). HPRG binds to a variety of ligands that can be grouped into several categories, including those belonging to the coagulation and fibrinolytic systems (fibrinogen (5), plasminogen (6), the extracellular matrix (thrombospondin (7), heparin (8, 9)), as well as small ligands such as transition metals and heme (10). One of the best characterized roles of HPRG is as a plasmino- gen transporter and cofactor (3, 6). Approximately 50% of plas- minogen in serum is bound to the C-terminal domain Lys 518 of HPRG (11). HPRG has been proposed to bridge plasminogen to © 2004 Schattauer GmbH, Stuttgart Histidine-Proline Rich Glycoprotein (HPRG) binds and transduces anti-angiogenic signals through cell surface tropomyosin on endothelial cells Xiaojun Guan 1 , Jose C. Juarez 1 , Xiaoping Qi 3 , Natalya V. Shipulina 2 , David E. Shaw 1,4 , William T. Morgan 2 , Keith R. McCrae 3 , Andrew P. Mazar 1 , Fernando Doñate 1 1 Attenuon, LLC, San Diego, California, 2 Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, 3 Division of Hematology-Oncology, Case Western Reserve University, School of Medicine, Cleveland, Ohio and 4 D. E. Shaw Research and Development, LLC, New York, New York, USA Thromb Haemost 2004; 92: 403–12 immobilized tropomyosin in a Zn 2+ or pH-dependent manner, and that this interaction is mediated by the H/P domain of HPRG. At least two binding sites for HPRG, tropomyosin and heparan sulfate proteoglycans (HSPs), were identified on the surface of FGF-2–activated endothelial cells. Translocation of tropomyosin to the surface of HUVEC occurred in response to FGF-2, and the anti-angiogenic activity of HPRG in a Matrigel plug model was partially inhibited by soluble tropomyosin. These results suggest that HPRG binds to endothelial cell sur- face tropomyosin which at least partially mediates the anti- angiogenic effects of HPRG. Keywords HPRG, angiogenenesis, endothelial cells, tropomyosin Summary The anti-angiogenic properties of the histidine-proline–rich (H/P) domain of HPRG have recently been described (Juarez JC, et al. Cancer Research 2002; 62: 5344-50). However, the binding site that mediates these properties is unknown. HPRG is evolutionarily,functionally and structurally related to cleaved high molecular weight kininogen (HKa), an anti-angiogenic poly- peptide that stimulates apoptosis of proliferating endothelial cells through binding to cell-surface tropomyosin (Zhang J-C, et al. Proc Natl Acad Sci USA 2002; 99: 12224-9). In this study, we demonstrate that HPRG binds with high affinity to FGF-2–stim- ulated human umbilical vein endothelial cells (HUVEC) and Endothelium and Vascular Development 403 Correspondence to: Fernando Doñate. Ph. D. Attenuon, LLC 10130 Sorrento Valley Rd. Suite B San Diego, CA 92121 USA Tel: (858) 622-0510, Fax: 858-622-0517 E-mail: donate@attenuon.com Received February 5, 2004 Accepted after resubmission May 6, 2004 Prepublished online June 4, 2004 DOI: 10.1160/TH04-02-0073 For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2018-04-14 | ID: 1001066444 | IP: 54.70.40.11