The plasma protein histidine-rich glycoprotein (HRGP), which has been identified as an angiogenesis inhibitor, binds to heparan sulfate (HS) in a Zn 2؉ -dependent manner. We wished to test whether this interaction is mechanistically important in mediation of the anti-angiogenic effect of HRGP. Inhibition of angiogenesis by HRGP is exerted through its central His/Pro-rich domain, which is proteolytically released. A 35-amino-acid residue synthetic peptide, HRGP330, derived from the His/Pro-rich domain retains the inhibitory effect on blood vessel formation in vitro and in vivo, an effect dependent on the presence of Zn 2؉ . We now show that HRGP330 binds heparin/HS with the same capacity as full-length HRGP, and the binding is Angiogenesis, the formation of new capillary blood vessels, is essential during development and physiological conditions, such as wound healing and the menstrual cycle (1). However, prolonged and excessive angiogenesis has been implicated in a number of pathological processes, for instance rheumatoid arthritis, diabetic retinopathy, and tumor growth (2, 3). The adult vasculature is tightly regulated by naturally occurring pro-and anti-angiogenic factors. Fibroblast growth factor (FGF) 3 and vascular endothelial growth factor (VEGF) are well known stimulators of endothelial cells in vitro and in vivo. To date, a number of endogenous factors negatively regulating angiogenesis have also been identified (4). The inhibitors described so far mainly fall into three groups: plasma proteins, basement membrane proteins, and serine protease inhibitors (serpins). A common feature of many of these antiangiogenic molecules is inhibition of endothelial cell chemotaxis in vitro.We have identified histidine-rich glycoprotein (HRGP) as a potent inhibitor of angiogenesis in vivo (5). HRGP is a heparin-binding plasma protein highly conserved through vertebrate species (for review, see Ref. 6). The N-terminal part of the protein contains two cysteine protease inhibitor (cystatin)-like stretches, hence the classification of HRGP as a member of the cystatin superfamily (Fig. 1A). The central domain is rich in histidine and proline residues, and the human form contains 12 more or less conserved tandem repeats of the pentapeptide HHPHG. Multiple binding partners for HRGP have been reported, such as heparin/heparan sulfate (HS), plasminogen, fibrinogen, tropomyosin, and divalent cations (6). The heparin-binding affinity of HRGP can be modulated and is increased in the presence of Zn 2ϩ and at low pH (7), a common environment in hypoxic tumors. The anti-angiogenic effect of HRGP is mediated via its His/Pro-rich domain, which needs to be released from the full-length protein to exert its effects (5). HRGP attenuates endothelial cell migration and adhesion to vitronectin in vitro and tumor vascularization and growth in vivo, by interfering with endothelial cell focal adhesion function. We have narrowed down the minimal active domain of HRGP to a 35-amino-acid (aa) peptide, HRGP330, corresponding to a sequence in the H...