Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissuetype plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA͞FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cellmediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.Because tumor growth and metastases are dependent upon angiogenesis (1-3), the identification of agents that inhibit angiogenesis now represents a potential therapeutic approach for the control of cancer (4-7). Angiostatin, consisting of the first four of five kringle domains of plasminogen (8), is one of a number of angiogenesis inhibitors that are internal fragments of larger nonangiogenic precursor proteins (8-14); however, the mechanisms by which these fragments are generated in vivo remains unknown. Although the activity sufficient to cleave plasminogen to angiostatin is present in tumor-bearing animals and serum-free conditioned medium (SFCM) of human prostate carcinoma cells (9), the cancer-dependent mechanism of angiostatin generation has remained unknown. Recently, macrophage-derived metalloelastase was shown to produce angiostatin from plasminogen and may contribute to angiostatin generation in the murine Lewis lung carcinoma model (15). We now describe the enzymatic mechanism for the direct generation of human angiostatin from plasminogen by human prostate cancer cells and demonstrate the generation of bioactive angiostatin from human plasminogen in a defined cell-free system. MATERIALS AND METHODSAngiostatin Generation. Angiostatin was generated from PC-3 cell SFCM as described (9). To generate angiostatin in a cell-free system, human plasminogen (0.2 M) was incubated with 0.2 nM recombinant human urokinase (uPA; Abbott), 1.0 nM recombinant human two-chain tissue-type plasminogen activator (tPA; a gift from Henry Berger, Glaxo-Wellcome), or 8.0 nM streptokinase (Sigma) and with 100 M N-acetyl-L-cys...

Exposure of blood to tissue factor (TF) activates the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation. In this study, we found that mice expressing low levels of human TF (Ϸ1% of wild-type levels) in an mTF ؊/؊ background had significantly shorter lifespans than wild-type mice, in part, because of spontaneous fatal hemorrhages. All low-TF mice exhibited a selective heart defect that consisted of hemosiderin deposition and fibrosis. Direct intracardiac measurement demonstrated a 30% reduction (P < 0.001) in left ventricular function in 8-month-old low-TF mice compared with age-matched wild-type mice. Mice expressing low levels of murine FVII (Ϸ1% of wild-type levels) exhibited a similar pattern of hemosiderin deposition and fibrosis in their hearts. In contrast, FIX ؊/؊ mice, a model of hemophilia B, had normal hearts. Cardiac fibrosis in low-TF and low-FVII mice appears to be caused by hemorrhage from cardiac vessels due to impaired hemostasis. We propose that TF expression by cardiac myocytes provides a secondary hemostatic barrier to protect the heart from hemorrhage. E xpression of tissue factor (TF) by adventitial fibroblasts and vascular smooth muscle cells surrounding blood vessels provides a hemostatic barrier that activates coagulation when vascular integrity is disrupted (1). TF is also expressed by cardiac muscle but not by skeletal muscle (1). TF functions as the high-affinity cellular receptor for FVII͞VIIa (2). The coagulation protease cascades are comprised of the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways, which together maintain hemostasis (3).Many murine models of coagulation have been generated that provide new insights into the role of the various procoagulant and anticoagulant proteins in hemostasis (4). For instance, FV Leiden/Leiden mice, which express an FV variant that is resistant to inactivation by activated protein C, and TM Pro/Pro mice, which express a mutated version of thrombomodulin (TM) with reduced thrombin binding, both exhibit prothrombotic phenotypes with increased fibrin deposition in select tissues (5-7). Mice with prohemorrhage phenotypes include models of hemophilia A (FVIII Ϫ/Ϫ ) and B (FIX Ϫ/Ϫ ), as well as fibrinogen-deficient mice (Fbg Ϫ/Ϫ ) and thrombocytopenic mice (NF-E2 Ϫ/Ϫ ) (8-12). Mice with complete deficiencies in TF, FVII, FX, FV, and prothrombin die in utero or shortly after birth (4). We and others have generated mice expressing low levels (Ͻ0.1-1% of wild-type levels) of human TF, murine FVII, and murine FV (13-15). We have shown that low-TF mice have impaired uterine hemostasis (16). A similar phenotype is observed with low-FVII mice.In this study, we performed a detailed characterization of low-TF mice. These mice exhibited shorter lifespans than wildtype mice. Histological analysis of various tissues of low-TF mice revealed hemosiderin deposition and fibrosis selectively in their hearts. Our data suggest that cardiac fibrosis in low-TF mice is caused by hemorrhage from cardiac vessels due to impaired hemostasis. M...