Isolation and characterization of the circulating form of human endostatin

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Abstract
Recently, fragments of extracellular proteins, including endostatin, were defined as a novel group of angiogenesis inhibitors. In this study, human plasma equivalent hemofiltrate was used as a source for the purification of high molecular weight peptides (10–20 kDa), and the isolation and identification of circulating human endostatin are described. The purification of this C‐terminal fragment of collagen α1(XVIII) was guided by MALDI‐MS and the exact molecular mass determined by ESI‐MS was found to be 18 494 Da. N‐terminal sequencing revealed the identity of this putative angiogenesis inhibitor and its close relation to mouse endostatin. The cysteine residues 1–3 and 2–4 in the molecule are linked by disulfide bridges. In vitro biological characterization of the native protein demonstrated no anti‐proliferative activity on different endothelial cell types. These data indicate that human endostatin, which is a putative angiogenesis inhibitor, is present in the circulation.