METH-1/ADAMTS1 is a member of a newly described family of genes that contain metalloprotease, disintegrin, and thrombospondin-like motifs. We have recently shown that METH-1 protein is a potent inhibitor of angiogenesis. Here, we demonstrate that secreted human pro-METH-1 is processed in two consecutive steps to release both p87 and p65 active forms. The p87 form lacks the N-terminal prodomain and p65 results from an additional processing event in the C-terminal end. Generation of p87 was blocked with specific inhibitors of furin, and incubation of pro-METH-1 with purified furin released the p87 fragment but not p65. Generation of p65 required preformation of p87 and was suppressed by inhibitors of matrix metalloproteases. We demonstrate that matrix metalloproteases 2, 8, and 15 were able to release p65 when p87 was used as substrate. This second processing step removes two thrombospondin repeats from the carboxyl-terminal end of p87-METH-1 and alters the affinity of the protein to heparin and endothelial cultures. Furthermore, this deletion was associated with a reduced activity upon suppression of endothelial cell proliferation. We hypothesize that METH-1 processing is relevant for the modulation of the anti-angiogenic properties displayed by the protein.METH-1/ADAMTS1 belongs to the recently described metallospondin/ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) family of proteins (1). The mouse AD-AMTS1 gene was first identified as a transcript expressed highly in a cachexigenic colon tumor cell line (2), and it has been suggested to be an active metalloprotease by means of the proteinase trapping mechanism of ␣ 2 -macroglobulin (3). More recently, it has been shown that targeted disruption of the mouse ADAMTS1 gene resulted in growth retardation and defects in fertility and morphogenesis (4). We cloned the human ortholog of ADAMTS1 (named METH-1) and a second homologous gene (METH-2/ADAMTS8) (1). Recombinant METH-1 and -2 proteins display anti-angiogenic properties and inhibit endothelial but not smooth muscle or fibroblast proliferation (1). Other members of the ADAMTS family have been implicated in processing of extracellular matrix components (5-7), and gonadal morphogenesis of Caenorhabitis elegans (8). Additional cDNAs showing conservation of the METH-1 domain structure have been cloned (ADAMTS5, -6, and -7), but their function remains unknown (9).The structure of all ADAMTS members include a signal sequence for targeting to the secretory pathway, a prodomain, a catalytic motif related with the reprolysin subfamily of metalloproteases, a putative disintegrin domain, and a carboxylterminal region containing a variable number of type I repeats (properdin-or thrombospondin-like). Several members of this family appear to be processed upon secretion (1,3,6,7). By analogy to proteins of the metalloprotease family, it is likely that this processing is required for functional activation. For example, all matrix metalloproteases (MMPs) 1 are synthesized as inactive zymogens in which the...

The differentiation of human peripheral blood monocytes into resident macrophages is driven by colony-stimulating factor-1 (CSF-1), which upon interaction with CSF-1 receptor (CSF-1R) induces within minutes the phosphorylation of its cytoplasmic tyrosine residues and the activation of multiple signaling complexes. Caspase-8 and -3 are activated at day 2 to 3 and contribute to macrophage differentiation, for example, through cleavage of nucleophosmin. Here, we show that the phosphatidylinositol-

alpha1 Proteinase inhibitor (alpha1PI), a natural inhibitor of the serine proteinase leukocyte elastase, is also an intravenous therapeutic agent used to treat hereditary emphysema and may be useful in other respiratory disorders. However, to achieve sustained suppression of leukocyte elastase, alpha1PI must be given frequently and in large amounts, thus limiting its clinical use. We hypothesized that conjugating alpha1PI with polyethylene glycol (PEG) at Cys(232) could extend the in vivo half-life of alpha1PI in blood and lung. We present evidence that site-specific conjugation with either 20 or 40 kD PEG at Cys(232) of nonglycosylated recombinant human alpha1PI (rhalpha1PI) results in an active inhibitor with prolonged in vivo stability. In addition, 72 h after airway instillation PEG-rhalpha1PI was found to be significantly better than glycosylated alpha1PI in protecting the lung against leukocyte elastase-mediated lung hemorrhage. We conclude that thiol-specific PEGylation markedly improves the in vivo pharmacokinetic profile of rhalpha1PI and represents a simple, novel strategy to address the therapeutic goal of human leukocyte elastase inhibition.