Plasminogen-deficient Mice Research Articles

Mechanisms of TGF-β 1 –Induced Intimal Growth

Transforming growth factor (TGF)-beta(1) is a potent stimulator of intimal growth. We showed previously that TGF-beta(1) stimulates intimal growth through early upregulation of plasminogen activator inhibitor-1 (PAI-1) and, subsequently, PAI-1-dependent increases in cell migration and matrix accumulation. We also showed that PAI-1 negatively regulates TGF-beta(1) expression in the artery wall. Here we use plasminogen-deficient mice to test whether TGF-beta(1)-stimulated, PAI-1-dependent intimal growth and PAI-1 suppression of TGF-beta(1) expression are mediated through inhibition of plasminogen activation by PAI-1. We also use lineage tracing to investigate the origin of cells in TGF-beta(1)-induced intimas. Surprisingly, both TGF-beta(1)-induced, PAI-1-dependent intimal growth and PAI-1 suppression of TGF-beta(1) expression are independent of plasminogen. Moreover, approximately 50% of cells that migrate into the intima of TGF-beta(1)-overexpressing arteries carry a smooth muscle lineage marker, <1% carry a bone marrow lineage marker, and the remaining cells carry neither marker. Therefore, PAI-1 stimulates intimal growth and suppresses TGF-beta(1) expression through activities other than inhibition of plasminogen activation. In addition, contrary to widely held models, our results do not support a role for plasmin (or thrombospondin) in TGF-beta(1) activation in the artery wall. Further identification of the molecular targets through which PAI-1 stimulates intimal formation and suppresses TGF-beta(1) expression in the artery wall may reveal new approaches for inhibiting intimal formation. Our studies also discount bone marrow as an important source from which TGF-beta(1) recruits intimal cells and suggest instead that TGF-beta(1) induces substantial cell migration either from the adventitia or from an extravascular, but nonhematopoietic source.

Proteolytic Activation of Monocyte Chemoattractant Protein-1 by Plasmin Underlies Excitotoxic Neurodegeneration in Mice

Exposure of neurons to high concentrations of excitatory neurotransmitters causes them to undergo excitotoxic death via multiple synergistic injury mechanisms. One of these mechanisms involves actions undertaken locally by microglia, the CNS-resident macrophages. Mice deficient in the serine protease plasmin exhibit decreased microglial migration to the site of excitatory neurotransmitter release and are resistant to excitotoxic neurodegeneration. Microglial chemotaxis can be signaled by the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 (CC chemokine ligand 2). We show here that mice genetically deficient for MCP-1 phenocopy plasminogen deficiency by displaying decreased microglial recruitment and resisting excitotoxic neurodegeneration. Connecting these pathways, we demonstrate that MCP-1 undergoes a proteolytic processing step mediated by plasmin. The processing, which consists of removal of the C terminus of MCP-1, enhances the potency of MCP-1 in in vitro migration assays. Finally, we show that infusion of the cleaved form of MCP-1 into the CNS restores microglial recruitment and excitotoxicity in plasminogen-deficient mice. These findings identify MCP-1 as a key downstream effector in the excitotoxic pathway triggered by plasmin and identify plasmin as an extracellular chemokine activator. Finally, our results provide a mechanism that explains the resistance of plasminogen-deficient mice to excitotoxicity.

Functional Corpora Lutea Are Formed in Matrix Metalloproteinase Inhibitor-Treated Plasminogen-Deficient Mice

Corpus luteum (CL) formation involves dramatic tissue remodeling and angiogenesis. To determine the functional roles of the plasminogen activator and matrix metalloproteinase (MMP) systems in these processes, we have studied CL formation and function in plasminogen (plg)-deficient mice, with or without treatment with the broad-spectrum synthetic MMP inhibitor galardin. Both the adult pseudopregnant CL model and the gonadotropin-primed immature mouse model were used. We found that CL formed normally not only in plasminogen-deficient mice and in galardin-treated wild-type mice, but also in galardin-treated plg-deficient mice, suggesting that neither of the plasminogen activator and MMP systems is essential for CL formation. Nevertheless, in plg-deficient mice, serum progesterone levels were reduced by approximately 50%, and the progesterone levels were not reduced further by galardin treatment. When CL from plg-deficient mice were stained for several molecular markers for CL development and regression, they appeared healthy and vascularized, and were indistinguishable from CL from wild-type mice. This implies that the reduced progesterone levels were not caused by impaired CL formation. Taken together, our data suggest that neither plasmin nor MMPs, alone or in combination, are required for CL formation. Therefore, the tissue remodeling and angiogenesis processes during CL formation may be mediated by redundant protease systems. However, the reduced serum progesterone levels in plg-deficient mice suggest that plasmin, but not MMPs, plays a role in maintenance of luteal function. This role may be performed through proteolytic activation of growth factors and other paracrine factors.

2006 Sol Sherry Distinguished Lecture in Thrombosis—Crosstalk Between the Hemostatic and Inflammatory Systems

Essentially all of the major coagulation and fibrinolytic system components (including proteases, co-factors, receptors, inhibitors, and substrates) have been experimentally eliminated or functionally altered in gene-targeted mice. Detailed studies of these mutant animals have greatly improved the understanding of the role of these proteins in complex physiological and pathological processes in vivo. Hemostatic derangements were the most predictable consequence of genetically imposed alterations in key coagulation and fibrinolytic factors, but more detailed studies have illuminated another less obvious aspect of the hemostatic system—namely, that coagulation and fibrinolytic factors appear to figure prominently into a wide spectrum of biological processes in vivo that have little or nothing to do with traditional hemostasis. Studies of mice with hemostatic deficits compatible with survival to adulthood (eg, fibrinogen-, plasminogen activator–, and plasminogen-deficient mice) have revealed an important role of these factors in phenomenally diverse biological settings (eg, immune surveillance, neurodegeneration and repair, tumor metastasis, and inflammatory diseases). Regulatory crosstalk between the hemostatic and inflammatory systems has become an increasingly frequent biological theme. The precise mechanisms by which thrombin, fibrinogen, and other hemostatic factors contribute to the inflammatory response remain to be fully defined, but leukocyte receptor interactions with fibrin(ogen) and other hemostatic factors are likely to be one important means of locally regulating leukocyte function. A comprehensive understanding of the role of hemostatic factors in inflammatory processes may suggest new strategies for the treatment of a wide array of inflammatory diseases.

Plasminogen Activator Inhibitor-1 in Chronic Kidney Disease

In 1984, Loskutoff et al. ([1][1]) purified plasminogen activator inhibitor-1 (PAI-1) from conditioned media of cultured endothelial cells. This 50-kd glycoprotein is the primary physiologic inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activators (tPA and uPA,

Successful ovulation in plasminogen-deficient mice treated with the broad-spectrum matrix metalloproteinase inhibitor galardin

Many studies have suggested the hypothesis that the plasminogen activator (PA) system and the matrix metalloproteinase (MMP) system, either separately or in combination, may provide the proteolytic activity that is required for rupture of the follicular wall at the time of ovulation. Our recent studies on ovulation in plasminogen (plg)-deficient mice have, however, shown that plasmin is not required for normal ovulation, leading us to the hypothesis that MMPs may be a more important source of proteolysis for this process. To investigate the role of MMPs and also the possibility of a functional overlap or synergy between the MMP and PA systems during ovulation, we have studied ovulation efficiency in wild-type and plg-deficient mice treated with the broad-spectrum MMP inhibitor galardin. We found that in both wild-type mice and heterozygous plg-deficient (plg +/−) mice that had been treated with galardin prior to ovulation, there was a mild (18–20%) reduction in ovulation efficiency. Surprisingly, galardin treatment of plg-deficient (plg −/−) mice only caused an additional 14% reduction in ovulation efficiency as compared to vehicle-treated plg −/− mice. Our data therefore suggest that although MMPs may play a role in degradation of the follicular wall, they may not be obligatory for ovulation. In contrast to previous studies on tissue remodeling during wound healing and placental development, we have demonstrated that there is no obvious functional overlap or synergy between the PA and MMP systems, which has previously been thought to be essential for the ovulatory process.

Unaltered prion protein cleavage in plasminogen-deficient mice

In normal brains and cultured cells, cellular prion protein (PrP) is partially found as N-terminally truncated fragments, designated C1 and C2. The cleavage of recombinant PrP to a fragment corresponding to C1 can be mediated by the protease plasmin (Pln) in vitro, suggesting that plasmin might be responsible for the generation of the C1 fragment in vivo as well. The cleavage pattern of PrP found in both brain lysates and other tissues of plasminogen knock-out mice, however, is unaltered. The presence of C1 fragment in homogenates from plasminogen-deficient mice in a comparable ratio with full-length PrP as can be found in wild-type animals indicates that other proteases in addition to plasmin are responsible for PrP cleavage in vivo.

Plasminogen Activator System and Vascular Disease

Vascular diseases, such as atherosclerosis, thromboembolic disorders and stroke, in addition to surgical procedures such as restenosis, all share the plasminogen activator system as a central component in the pathogenesis of vascular injury. Since the development of plasminogen deficient mice our knowledge of the effects of this proteolytic system in cardiovascular disease has vastly increased. The plasminogen activator system plays a key role in vascular homeostasis and constitutes a critical response mechanism to cardiovascular injury. The central components of the PA system are the proteolytic activators, urokinase-plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), plasminogen (plg) and its degradation product, plasmin, together with the major inhibitors of this system, plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2). An extensive network of additional proteases, inhibitors, receptors and modulators directly associate and are influenced by the PA system, the largest group being the Matrix Metalloproteinases (MMPs) and their respective inhibitors the Tissue inhibitors of MMPs (TIMPS).

Plasmin/plasminogen is essential for the healing of tympanic membrane perforations

Plasminogen has been proposed to play an important role in different tissue remodeling processes such as wound healing and tissue regeneration after injuries. The healing of tympanic membrane perforations is a well-organized chain of inflammatory events, with an initial invasion of inflammatory cells followed by reparative and restoration phases. Here we show that the healing of tympanic membrane perforations is completely arrested in plasminogen-deficient mice, with no signs of any healing even 143 days after perforation. Inflammatory cells were recruited to the wounded area, but there were no signs of tissue debridement. In addition, removal of fibrin, keratinocyte migration and in-growth of connective tissue were impaired. This contrasts with skin wound healing, where studies have shown that, although the healing process is delayed, it reaches completion in all plasminogen-deficient mice. Our finding that keratinocyte migration and re-epithelialization were completely arrested in plasminogen-deficient mice indicates that plasminogen/plasmin plays a more profound role in the healing of tympanic membrane perforations than in the healing of other epithelial wounds.

Role of Plasminogen in Propagation of Scrapie

To investigate whether plasminogen may feature in scrapie infection, we inoculated plasminogen-deficient (Plg(-/-)), heterozygous plasminogen-deficient (Plg(+/-)), and wild-type (Plg(+/+)) mice by the intracerebral or intraperitoneal (i.p.) route with the RML scrapie strain and monitored the onset of neurological signs of disease, survival time, brain, and accumulation of scrapie disease-associated forms of the prion protein (PrP(Sc)). Only after i.p. inoculation, a slight, although significant, difference in survival (P < 0.05) between Plg(-/-) and Plg(+/+) mice was observed. Neuropathological examination and Western blot analysis were carried out when the first signs of disease appeared in Plg(+/+) animals (175 days after i.p. inoculation) and when mice reached the terminal stage of illness. At the onset of symptoms, PrP(Sc) accumulation was higher in the brain and spleen of Plg(+/+) and Plg(+/-) mice than in those of Plg(-/-) mice, and these differences were paralleled by differences in the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures. Immunohistochemical analysis of the spleens before inoculation did not show any impairment of the immune system affecting follicular dendritic or lymphoid cells in Plg(-/-) mice. Once the disease progressed and mice began to die of infection, differences were no longer apparent in either brains or spleens. In conclusion, our data indicate that plasminogen has no major effect on the survival of scrapie agent-infected mice.

A study of cutaneous lesions caused by Leishmania mexicana in plasminogen-deficient mice

The role of plasminogen, the zymogenic form of the serine protease plasmin, was investigated in the infection of Leishmania mexicana in plasminogen-deficient ( plg −/−) and Plg wild-type ( plg +/+) mice. Differences in the lesion size were observed between male plg +/+ and plg −/− mice. However, these differences were not observed in female mice. In both genders, examination of the lesion tissues at 8 weeks post-infection showed differences in the immunoreactivity pattern with anti- Leishmania antibodies. The parasites were limited to isolated foci in the plg −/− mice lesion, in contrast to the scattered pattern observed in plg +/+ mice. These results support the hypothesis that the interaction of the parasite with the host plasminogen–plasmin system might contribute to the virulence of L. mexicana.

Contrasting roles of plasminogen deficiency in different rheumatoid arthritis models

To investigate the contrasting roles of plasminogen deficiency between models of collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA). We developed a new animal model of arthritis, which we have called local injection-induced arthritis (LIA). In this model, we replaced methylated bovine serum albumin, which is normally used as an immunogen and is injected intraarticularly into the knee joint, with type II collagen (CII) to induce AIA. The severity of CIA, LIA, and AIA in wild-type and plasminogen-deficient mice was evaluated by clinical scoring or histologic grading. Necrosis was determined by histology and immunohistochemistry. After CII immunization alone, wild-type mice developed arthritis in most of the paws as well as in the knee joints, whereas plasminogen-deficient mice were totally resistant to the disease. Local knee injections of CII or saline slightly enhanced the severity of the knee arthritis in wild-type mice during a 60-day experimental period. Unexpectedly, the plasminogen-deficient mice also developed arthritis in joints that were injected with CII or saline. However, the arthritis was milder than that in their wild-type littermates. Sustained tissue necrosis was found only in the plasminogen-deficient mice after the local injection. Our data show that both the antigen and the joint trauma caused by the local injection are critical to explaining the contrasting roles of plasminogen deficiency in CIA and AIA. This further indicates that CIA and AIA have distinct pathogenic mechanisms. The data also suggest that plasmin may be required for the induction of these arthritis models that are critically dependent on complement activation.

The Plasminogen Activator/Plasmin System Is Essential for Development of the Joint Inflammatory Phase of Collagen Type II-Induced Arthritis

The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA, we found that none of the 50 plasminogen-deficient littermates that were tested developed CIA within a 40-day period. Antibody generation after CII immunization as well as the binding of labeled anti-CII antibodies to the surface of cartilage were similar in wild-type and plasminogen-deficient mice. No sign of inflammation was seen when plasminogen-deficient mice were injected with a mixture of monoclonal antibodies against CII. However, after daily injections of human plasminogen, these mice developed arthritis within 5 days. Our finding that infiltration of inflammatory cells into the synovial joints was impaired in plasminogen-deficient mice suggests that uPA and plasminogen are important mediators of joint inflammation. Active plasmin is therefore essential for the induction of pathological inflammatory joint destruction in CIA.

Tissue plasminogen activator promotes the effects of corticotropin-releasing factor on the amygdala and anxiety-like behavior

Stress-induced plasticity in the brain requires a precisely orchestrated sequence of cellular events involving novel as well as well known mediators. We have previously demonstrated that tissue plasminogen activator (tPA) in the amygdala promotes stress-induced synaptic plasticity and anxiety-like behavior. Here, we show that tPA activity in the amygdala is up-regulated by a major stress neuromodulator, corticotropin-releasing factor (CRF), acting on CRF type-1 receptors. Compared with WT, tPA-deficient mice responded to CRF treatment with attenuated expression of c-fos (an indicator of neuronal activation) in the central and medial amygdala but had normal c-fos responses in paraventricular nuclei. They exhibited reduced anxiety-like behavior to CRF but had a sustained corticosterone response after CRF administration. This effect of tPA deficiency was not mediated by plasminogen, because plasminogen-deficient mice demonstrated normal behavioral and hormonal changes to CRF. These studies establish tPA as an important mediator of cellular, behavioral, and hormonal responses to CRF.

Plasminogen regulates pro‐opiomelanocortin processing

Plasminogen-deficient mice exhibit behavioral differences in response to stress, including a markedly reduced acoustic startle reflex response compared with wild-type (WT) littermates. The acoustic startle reflex activates the hypothalamic-pituitary axis and is modulated by these hormones. The purpose of this study was to investigate whether plasminogen plays a role in the processing of hormones in the hypothalamic-pituitary axis. In this study the concentration of plasma, pituitary, and brain hypothalamic-pituitary axis hormones and precursor processing was examined in WT and plasminogen deficient (Plg-/-) mice before and after acoustic startle reflex testing. Plasma adrenocorticotropic hormone (ACTH), beta-endorphin and alpha-melanocyte stimulating hormone were elevated after acoustic startle reflex testing in both WT and (Plg-/-) mice. However, in the Plg-/- mice, beta-endorphin values were 43, 35, and 45% lower in the plasma, pituitary, and whole brain, respectively, compared with the WT mice. Plasmin readily degraded precursor peptides, the 23-kDa precursor, beta-lipotropin, and ACTH, when presented as purified proteins or as the secretory products of mouse pituitary cells (AtT-20). The precursor peptide, 23 kDa, for beta-endorphin and alpha-melanocyte stimulating hormone was reduced in the pituitaries from the Plg-/- mice, and the mRNA for Plg was found in pituitaries from WT mice. Infusion of beta-endorphin and alpha-melanocyte stimulating hormone into the brain of Plg-/- mice increased acoustic startle reflex. The results of this study show that plasmin is involved in the processing of hormones derived from the pro-opiomelanocortin precursor in the intermediate pituitary. A deficiency of plasminogen reduces processing of beta-endorphin and alpha-melanocyte stimulating hormone, and interferes with normal brain function.

The effects of the plasminogen pathway on scar tissue formation.

The authors sought to determine the role of the plasminogen pathway in wound healing. They hypothesized that decreased fibrin degradation may lead to increased collagen deposition. Presuming that the degree of histopathological abnormality correlates with the aesthetic appearance of the scar, we conducted a study that attempted to determine the histopathological appearance of scar tissue in mice with and without impaired function of the plasminogen pathway. Mice with and without deficiencies in the plasminogen pathway underwent surgery. The role of the plasminogen pathway in wound healing was studied by analysis of scar tissue formation using the methods described. A 2-cm incision was made on the dorsum of mice with and without specified genetic deficiencies in the plasminogen pathway. After the animals were killed, the tissue was harvested, fixed, and prepared using hematoxylin and eosin as well as trichrome stains. Histopathological analysis and scoring were performed by two separate investigators in a blinded manner. Student's t test was used to determine statistical significance between groups. A statistically significant difference in collagen orientation was noted between mice with impaired plasminogen pathway function and the wild-type (control) group (P =.0163). A statistical trend toward improved wound healing for plasminogen-deficient mice was found for overall histomorphological score (P =.0706). The role of the plasminogen pathway in wound healing is one that should be noted and may lead to the development of new therapies that reduce scar tissue formation. Hence, the role of other thrombolytic and anti-thrombolytic agents in wound healing should be further investigated to precisely identify agents that play the most significant role in scar tissue formation.

Plasminogen supports tumor growth through a fibrinogen-dependent mechanism linked to vascular patency

AbstractThe growth of Lewis lung carcinoma (LLC) was sustained in plasminogen-deficient mice when transplanted into the dorsal skin but was dramatically suppressed in another anatomic location, the footpad. This unanticipated negative effect of plasminogen deficiency on footpad tumor growth was entirely relieved by superimposing a deficit in fibrinogen. This finding was not simply an unusual feature of LLC tumors—T241 fibrosarcoma growth in the footpad was also restricted by plasminogen deficiency in a fibrinogen-dependent manner. The probable mechanistic basis for suppression of tumor growth was revealed through transmission electron microscopy studies of tumor tissues. Occlusive microvascular thrombi were commonplace within footpad tumors from plasminogen-deficient mice, whereas no such lesions were observed within either dorsal skin tumors from plasminogen-deficient mice or footpad tumors from mice that also lacked fibrinogen. The data infer that tumor growth in the footpad of plasminogen-deficient mice is compromised as a function of the formation and persistence of vaso-occlusive thrombi that limit tumor blood supply. These studies indicate that plasminogen and fibrinogen can serve as critical determinants of tumor growth, but their relative importance is dependent on the tumor microenvironment. Furthermore, these studies suggest that one target of plasmin(ogen) relevant to tumor progression in vivo is intravascular fibrin.

Plasminogen mediates liver regeneration and angiogenesis after experimental partial hepatectomy

Plasmin system components are upregulated after partial hepatectomy, but their contribution to surgery-induced hepatic angiogenesis and regeneration is unclear. Liver regeneration and angiogenesis after partial hepatectomy were examined in mice lacking plasminogen or urokinase plasminogen activator (uPA). Mice with a single-gene deletion of plasminogen or uPA were subjected to 70 per cent partial hepatectomy. Liver regeneration was measured as relative liver weight and cell proliferation index. Angiogenesis was quantified by determining hepatic microvessel density after staining for sinusoidal endothelial cells. The liver remnant weight was significantly reduced in mice lacking plasminogen or uPA compared with that in wild-type mice on days 2 and 7 after partial hepatectomy. This correlated with impaired cell proliferation. In wild-type mice, regeneration was accompanied by a significant increase in microvessel density after hepatectomy; this increase was impaired in plasminogen-deficient mice. Plasminogen and uPA are essential for optimal liver regeneration. In addition, plasminogen appears to be a major determinant in regeneration-associated hepatic angiogenesis.

A functional overlap of plasminogen and MMPs regulates vascularization during placental development.

Both plasminogen activators and matrix metalloproteinases (MMPs) have been implicated in a variety of developmental processes in the mouse during embryo implantation and placentation. We show here that pharmacological treatment of plasminogen-deficient mice with the broad spectrum MMP inhibitor galardin leads to a high rate of embryonic lethality. Implantation sites from plasminogen-deficient galardin-treated mice at 7.5 days post coitus (dpc) showed delay in both decidualization and invasion of maternal vessels into the decidua. At 8.5 dpc, half of the embryos were runted and still at the developmental stage of a 7.5 dpc embryo. Most embryos that escaped these initial defects eventually died, probably from defective vascularization and development of the labyrinth layer of the placenta, although a direct role on embryo development cannot be ruled out. These results demonstrate that the combination of MMPs and plasminogen is essential for the proper development of the placenta. Plasminogen deficiency alone and galardin treatment alone had much less effect and there was a pronounced synergism on both placental vascularization and embryonic lethality, indicating a functional overlap between plasminogen and MMPs.

Mechanisms Linking Hemostatic Factors to Tumor Growth in Mice

A persuasive link between hemostatic factors and cancer has been developed through extensive studies of both human and animal malignancies. Coagulation-, platelet- and fibrinolytic-factors all have been tightly associatedwith tumor growth and/or dissemination. Nevertheless, the mechanisms by which these factors influence tumor growth and progression remain to be fully defined. Recent studies of mice with selected deficits in hemostatic factors have generally supported the hypothesis that hemostatic factors contribute significantly to tumor biology. However, the findings with gene-targeted mice have often been unexpected or tumor model-dependent. Particularly enigmatic was the dual finding that neither fibrin(ogen) nor plasmin(ogen) were significant determinants of Lewis lung carcinoma (LLC) or T241 fibrosarcoma tumor growth following transplantation into the dorsal subcutis of immune-compatible mice. However, follow up studies have revealed that tumor growth is critically dependent on plasminogen when LLC and T241 tumors cells are transplanted to another anatomical location, the footpad. Furthermore, the negative impact of plasminogen deficiency on footpad tumor growth was effectively relieved by genetically superimposing a deficit in fibrinogen. The likely mechanistic basis for the suppression of tumor growth in plasminogen-deficient mice was revealed through transmission electron microscopy studies of tumor tissues. Widespread microvascular thrombi were found within footpad tumors of plasminogen-deficient mice, whereas no such lesions were observed within tumors from mice that also lacked fibrinogen. These occlusive events were shown to result in a substantial lengthening of tumor cell doubling time in vivo, but increased tumor cell dropout is also likely to contribute to the diminution of footpad tumor growth in plasminogen-deficient mice. Taken together, these studies show that both plasminogen and fibrinogen can serve as critical determinants of tumor growth, but their importance is dependent on anatomical location/microenvironment. Furthermore, one target of plasmin(ogen) relevant to tumor success in vivo is intravascular fibrin.