Proposed Mechanism Of Angiostatin Protein Generation

What is the mechanism for the phenomenon of the inhibition of tumor growth by tumor mass? The working hypothesis put forward by O'Reilly and Folkman proposes that the presence of the primary tumor inhibits the growth of metastases. This occurs because the primary tumor generates the inhibitors of metastases. Attempts at detecting AP transcripts in LLC-LM cells, freshly resected from mice or after 4 passages in vitro by RT-PCR, or by Northern blotting, have been unsuccessful, indicating that primary LLC-LM tumors do not express AP per se (Sim et al., unpublished). Recent work by Gately et al. (19) and Dong et al. (20), provided an explanation for the hypothesis that tumors could generate their own inhibitors of angiogenesis. Metalloelastase produced by tumor-infil trating macrophages can generate AP from plasminogen (20). Also, several human prostate carcinoma cell lines were shown to express a serine protease(s) that could convert plasminogen to AP (19). More recently, it was shown that AP activity could be generated by the proteolysis and reduction of plasmin (21). In the generation of plasmin, plasminogen is first converted to the active serine protease plasmin by hydrolysis/cleavage of the Arg561-Val562 peptide bond, and an autoproteolytic release of the amino-terminal peptide by cleavage of the Lys77-Lys78 peptide bond (Fig. 1). The five K domains (heavy chain) of plasmin are covalently attached to the serine proteinase module (light chain) by two disulfide bonds. Stathakis et al. (21) suggest that a cell-derived plasmin reductase, generated, in their case, by Chinese hamster ovary or HT1080 cells, then reduces the disulfide bonds and releases the heavy from the light chain. They then show that this reduction of plasmin disulfide bond(s) is followed by the autoproteolysis of the heavy chain, resulting in the K1-4, active AP (21). Several sequential steps must occur for the generation of active AP: the generation of plasmin, the reduction of plasmin, the auto proteolysis to generate K1-4 from the heavy chain that is released, and the constant presence of free sulfydryls. The generation of plasmin can occur by a variety of methods: Urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), or urokinase cleave the Arg561-Val562 peptide bond to generate plasmin. Recent studies by Gately et al. (17) show that human prostate carcinoma cells (PC-3) release uPA, which functions this way. They also show that PC-3 cells produce sulfydryl donors, which, essentially in combination with released uPA, can by themselves generate AP (17). This work provides the explanation for the working hypothesis that the primary tumor, even though not expressing AP itself, controls the production of AP by being responsible for the mechanism that generates AP.

What is the in vivo mechanism for the inhibition of endothelial cells by AP? Does the resultant K1-4 then bind to a specific endothelial cell receptor(s) and/or is further processing and/or internalization required? It is known that plasminogen can bind to a variety of cell-surface receptors, including alpha enolase, a glycolytic enzyme on monocytes (22); receptors on lymphocytes; granulocytes, as well as on adherent fibroblasts (23); and platelets (24). Annexin II, a phospholipid-binding protein, has also been identified as an endothelial cell receptor for plasminogen (25,26). The K domains have been implicated in the receptor-binding interactions of plasminogen (25). The role of the K domains of plasminogen, in the regulation of activation and overall conformational changes of plasminogen, has been well documented (reviewed in ref. 27), but the role of the K domains in the proteolytic cascade caused by plasmin, has not been established. Can proteolysis and reduction of plasminogen to generate AP occur on receptor-bound plasminogen? Or is there a multistep process of further processing of smaller K fragments that bind to separate cellular receptors and stimulate other signal processes, to bring about endothelial cell inhibition?

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