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1.
J Thromb Haemost ; 17(1): 113-125, 2019 01.
Article in English | MEDLINE | ID: mdl-30415489

ABSTRACT

Essentials Fibrin clots are often implicated in the progression of liver fibrosis. Liver fibrosis was induced in transgenic mice with defects in clot formation or stabilization. Liver fibrosis and fibrin(ogen) deposition do not require fibrin polymerization or factor XIIIa. Fibrin(ogen) is an in vivo substrate of tissue transglutaminase in experimental liver fibrosis. SUMMARY: Background Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis. Methods Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin-mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit-deficient (FXIII-/- ) mice. Female wild-type mice, FXIII-/- mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4 ) twice weekly for 4 weeks or 6 weeks (1 mL kg-1 , intraperitoneal). Results Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin-catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4 -challenged FXIII-/- mice or FibAEK mice as compared with wild-type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase-2 (TGM2)-deficient (TGM2-/- ) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2-/- mice did not affect CCl4 -induced liver fibrosis. Conclusions These results indicate that neither traditional fibrin clots, formed by the thrombin-activated FXIII pathway nor atypical TGM2-crosslinked fibrin(ogen) contribute to experimental CCl4 -induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.


Subject(s)
Chemical and Drug Induced Liver Injury/enzymology , Fibrinogen/metabolism , GTP-Binding Proteins/metabolism , Liver Cirrhosis, Experimental/enzymology , Liver/enzymology , Transglutaminases/metabolism , Animals , Carbon Tetrachloride , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/pathology , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Factor XIII/genetics , Factor XIII/metabolism , Factor XIII Deficiency/enzymology , Factor XIII Deficiency/genetics , Factor XIIIa/genetics , Female , Fibrinogen/genetics , Liver/pathology , Liver Cirrhosis, Experimental/blood , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology , Mice, Inbred C57BL , Mice, Knockout , Protein Glutamine gamma Glutamyltransferase 2 , Substrate Specificity
2.
J Thromb Haemost ; 16(11): 2258-2269, 2018 11.
Article in English | MEDLINE | ID: mdl-30152921

ABSTRACT

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APCMin/+ ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APCMin/+ mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts.


Subject(s)
Disease Progression , Intestinal Neoplasms/metabolism , Prostatic Neoplasms/metabolism , Receptor, PAR-1/metabolism , Animals , Apoptosis , Cell Transformation, Neoplastic , Crosses, Genetic , Gene Deletion , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuroendocrine Tumors/metabolism , Prostatic Neoplasms/genetics , Protein C/metabolism , Thrombin/metabolism
3.
J Thromb Haemost ; 16(7): 1369-1382, 2018 07.
Article in English | MEDLINE | ID: mdl-29758126

ABSTRACT

Essentials The efficacy of systemic antifibrinolytics for hemophilic non-mucosal bleeding is undetermined. The effect of systemically inhibiting fibrinolysis in hemophilic mice and rats was explored. Neither bleeding nor the response to factor treatment was improved after inhibiting fibrinolysis. The non-mucosal bleeding phenotype in hemophilia A appears largely unaffected by fibrinolysis. SUMMARY: Background Fibrinolysis may exacerbate bleeding in patients with hemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain hemostatic control. Although antifibrinolytic drugs have been proven to be effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non-mucosal tissues remain an open question of significant clinical interest. Objective To determine whether inhibiting fibrinolysis improves the outcome in non-mucosal hemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard ex vivo clotting/fibrinolysis assay can be used as a predictive surrogate for in vivo efficacy. Methods A highly sensitive TVT model was employed in hemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in non-mucosal tissue. In mice, induced and congenital hemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid [TXA]). In hemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry evaluation of the same animals to gauge the predictive value of such assays. Results The beneficial effect of systemic TXA therapy observed ex vivo could not be confirmed in vivo in hemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes encoding plasminogen or tissue-type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in hemophilic mice. Conclusions The findings here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of HA rodents in non-mucosal tissues.


Subject(s)
Antifibrinolytic Agents/pharmacology , Coagulants/pharmacology , Factor VIII/pharmacology , Factor VIIa/pharmacology , Fibrinolysis/drug effects , Hemophilia A/drug therapy , Tail/blood supply , Tranexamic Acid/pharmacology , Vascular System Injuries/drug therapy , Animals , Disease Models, Animal , Factor VIII/genetics , Factor VIII/metabolism , Fibrinolysis/genetics , Genetic Predisposition to Disease , Hemophilia A/blood , Hemophilia A/genetics , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Plasminogen/deficiency , Plasminogen/genetics , Rats, Transgenic , Recombinant Proteins/pharmacology , Tissue Plasminogen Activator/deficiency , Tissue Plasminogen Activator/genetics , Vascular System Injuries/blood , Vascular System Injuries/genetics
6.
J Thromb Haemost ; 13(1): 57-71, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25353084

ABSTRACT

BACKGROUND: The coagulation cascade has been shown to participate in chronic liver injury and fibrosis, but the contribution of various thrombin targets, such as protease activated receptors (PARs) and fibrin(ogen), has not been fully described. Emerging evidence suggests that in some experimental settings of chronic liver injury, platelets can promote liver repair and inhibit liver fibrosis. However, the precise mechanisms linking coagulation and platelet function to hepatic tissue changes following injury remain poorly defined. OBJECTIVES: To determine the role of PAR-4, a key thrombin receptor on mouse platelets, and fibrin(ogen) engagement of the platelet αII b ß3 integrin (αIIb ß3 ) in a model of cholestatic liver injury and fibrosis. METHODS: Biliary and hepatic injury was characterized following 4 week administration of the bile duct toxicant α-naphthylisothiocyanate (ANIT) (0.025%) in PAR-4-deficient mice, mice expressing a mutant form of fibrin(ogen) incapable of binding integrin αII b ß3 (Fibγ(Δ5) ), and wild-type mice. RESULTS: Elevated plasma thrombin-antithrombin and serotonin levels, hepatic fibrin deposition, and platelet accumulation in liver accompanied hepatocellular injury and fibrosis in ANIT-treated wild-type mice. PAR-4 deficiency reduced plasma serotonin levels, increased serum bile acid concentration, and exacerbated ANIT-induced hepatocellular injury and peribiliary fibrosis. Compared with PAR-4-deficient mice, ANIT-treated Fibγ(Δ5) mice displayed more widespread hepatocellular necrosis accompanied by marked inflammation, robust fibroblast activation, and extensive liver fibrosis. CONCLUSIONS: Collectively, the results indicate that PAR-4 and fibrin-αII b ß3 integrin engagement, pathways coupling coagulation to platelet activation, each exert hepatoprotective effects during chronic cholestasis.


Subject(s)
Blood Coagulation , Blood Platelets/metabolism , Chemical and Drug Induced Liver Injury/prevention & control , Cholestasis/prevention & control , Liver Cirrhosis, Experimental/prevention & control , Liver/metabolism , Platelet Activation , 1-Naphthylisothiocyanate , Animals , Antithrombin III , Bile Acids and Salts/blood , Blood Coagulation/genetics , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/pathology , Cholestasis/blood , Cholestasis/chemically induced , Cholestasis/genetics , Cholestasis/pathology , Fibrinogens, Abnormal/genetics , Fibrinogens, Abnormal/metabolism , Genotype , Liver/pathology , Liver Cirrhosis, Experimental/blood , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/genetics , Liver Cirrhosis, Experimental/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Mutation , Necrosis , Peptide Hydrolases/blood , Phenotype , Platelet Activation/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Receptors, Thrombin/deficiency , Receptors, Thrombin/genetics , Serotonin/blood , Signal Transduction
7.
J Thromb Haemost ; 6(5): 812-9, 2008 May.
Article in English | MEDLINE | ID: mdl-18315549

ABSTRACT

BACKGROUND: Multiple studies suggest that the hemostatic and innate immune systems functionally cooperate in establishing the fraction of tumor cells that successfully form metastases. In particular, platelets and fibrinogen have been shown to support metastatic potential through a mechanism coupled to natural killer (NK) cell function. As the transglutaminase that ultimately stabilizes platelet/fibrin thrombi through the covalent crosslinking of fibrin, factor (F) XIII is another thrombin substrate that is likely to support hematogenous metastasis. OBJECTIVE: Directly define the role of FXIII in tumor growth, tumor stroma formation, and metastasis. METHODS: Tumor growth and metastatic potential were quantitatively and qualitatively evaluated in wild-type mice and gene-targeted mice lacking the catalytic FXIII-A subunit. RESULTS: Loss of FXIIIa function significantly diminished hematogenous metastatic potential in both experimental and spontaneous metastasis assays in immunocompetent mice. However, FXIII was not required for the growth of established tumors or tumor stroma formation. Rather, detailed analyses of the early fate of circulating tumor cells revealed that FXIII supports the early survival of micrometastases by a mechanism linked to NK cell function. CONCLUSIONS: Factor XIII is a significant determinant of metastatic potential and supports metastasis by impeding NK cell-mediated clearance of tumor cells. Given that these findings parallel previous observations in fibrinogen-deficient mice, an attractive hypothesis is that FXIII-mediated stabilization of fibrin/platelet thrombi associated with newly formed micrometastases increases the fraction of tumor cells capable of evading NK cell-mediated lysis.


Subject(s)
Factor XIII/physiology , Killer Cells, Natural/immunology , Neoplasm Metastasis/pathology , Neoplasms/etiology , Neoplastic Cells, Circulating/pathology , Transglutaminases/metabolism , Animals , Blood Platelets , Factor XIII/metabolism , Fibrin , Mice , Mice, Knockout , Thrombosis , Tumor Escape/physiology
8.
J Cell Sci ; 113 ( Pt 9): 1553-64, 2000 May.
Article in English | MEDLINE | ID: mdl-10751147

ABSTRACT

Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus. The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments. Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined. To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners. From this screen, the cytoskeletal protein betaI-spectrin was isolated. Protein interaction assays demonstrate that MLP and betaI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions. betaI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins. The MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin. Confocal microscopy studies also reveal that MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue. Given that beta-spectrin is a known costamere protein, we propose that sarcolemma-associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the beta-spectrin network to the alpha-actinin crosslinked actin filaments of the myofibril.


Subject(s)
Cytoskeletal Proteins/metabolism , Muscle Proteins/metabolism , Spectrin/metabolism , Animals , Cell Line , Humans , LIM Domain Proteins , Muscle, Skeletal/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley
9.
Oncogene ; 16(24): 3097-104, 1998 Jun 18.
Article in English | MEDLINE | ID: mdl-9671388

ABSTRACT

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogen-deficient mice were crossed with transgenic mice expressing Polyoma middle T antigen under the control of the mouse mammary tumor virus long terminal repeat. Virgin females carrying the Polyoma middle T antigen uniformly developed multiple, bilateral mammary tumors, regardless of the presence or absence of circulating plasminogen. Both the age at which these tumors became palpable and subsequent tumor growth were indistinguishable between plasminogen-deficient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogen-deficient mice was significantly reduced as compared to littermate controls with respect to frequency of occurrence, total number of metastases, and total metastatic tumor burden. Plasminogen activators, as well as other key factors that govern the conversion of plasminogen to plasmin, were expressed within the mammary tumors, suggesting that the plasminogen/plasmin system may promote metastasis by contributing to tumor-associated extracellular proteolysis. The data provide direct evidence that plasmin(ogen) is a tumor progression factor in PymT-induced mammary cancer, and support the hypothesis that hemostatic factors play an important role in tumor biology.


Subject(s)
Antigens, Polyomavirus Transforming/physiology , Lung Neoplasms/pathology , Mammary Neoplasms, Experimental/pathology , Plasminogen/genetics , Animals , Base Sequence , Blotting, Northern , DNA Primers , Female , Immunohistochemistry , In Situ Hybridization , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/etiology , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Transgenic , Plasminogen Activator Inhibitor 1/genetics , Urokinase-Type Plasminogen Activator/genetics
10.
Mol Cell Biol ; 17(8): 4750-60, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9234731

ABSTRACT

The muscle LIM protein (MLP) is a muscle-specific LIM-only factor that exhibits a dual subcellular localization, being present in both the nucleus and in the cytoplasm. Overexpression of MLP in C2C12 myoblasts enhances skeletal myogenesis, whereas inhibition of MLP activity blocks terminal differentiation. Thus, MLP functions as a positive developmental regulator, although the mechanism through which MLP promotes terminal differentiation events remains unknown. While examining the distinct roles associated with the nuclear and cytoplasmic forms of MLP, we found that nuclear MLP functions through a physical interaction with the muscle basic helix-loop-helix (bHLH) transcription factors MyoD, MRF4, and myogenin. This interaction is highly specific since MLP does not associate with nonmuscle bHLH proteins E12 or E47 or with the myocyte enhancer factor-2 (MEF2) protein, which acts cooperatively with the myogenic bHLH proteins to promote myogenesis. The first LIM motif in MLP and the highly conserved bHLH region of MyoD are responsible for mediating the association between these muscle-specific factors. MLP also interacts with MyoD-E47 heterodimers, leading to an increase in the DNA-binding activity associated with this active bHLH complex. Although MLP lacks a functional transcription activation domain, we propose that it serves as a cofactor for the myogenic bHLH proteins by increasing their interaction with specific DNA regulatory elements. Thus, the functional complex of MLP-MyoD-E protein reveals a novel mechanism for both initiating and maintaining the myogenic program and suggests a global strategy for how LIM-only proteins may control a variety of developmental pathways.


Subject(s)
Muscle Proteins/metabolism , Muscle, Skeletal/cytology , MyoD Protein/metabolism , Transcription Factors , Animals , Cell Differentiation , Cell Nucleus/chemistry , Cytoplasm/chemistry , DNA-Binding Proteins/metabolism , Dimerization , Helix-Loop-Helix Motifs , LIM Domain Proteins , Mice , Muscle Development , Muscle Proteins/analysis , Muscle, Skeletal/growth & development , Myogenic Regulatory Factors/metabolism , Myogenin/metabolism , Protein Binding , Rats , Recombinant Fusion Proteins/metabolism , TCF Transcription Factors , Transcription Factor 7-Like 1 Protein
11.
Cell ; 87(4): 709-19, 1996 Nov 15.
Article in English | MEDLINE | ID: mdl-8929539

ABSTRACT

Plasmin(ogen) is an extracellular serine protease implicated in the activation of latent growth factors and procollagenase, degradation of extracellular matrix components, and fibrin clearance. Plasminogen (Plg) deficiency in mice results in high mortality, wasting, spontaneous gastrointestinal ulceration, rectal prolapse, and severe thrombosis. Furthermore, Plg-deficient mice display delayed wound healing following skin injury, a defect partly related to impaired keratinocyte migration. We generated mice deficient in Plg and fibrinogen (Fib) and show that removal of fibrin(ogen) from the extracellular environment alleviates the diverse spontaneous pathologies previously associated with Plg deficiency and corrects healing times. Mice deficient in Plg and Fib are phenotypically indistinguishable from Fib-deficient mice. These data suggest that the fundamental and possibly only essential physiological role of Plg is fibrinolysis.


Subject(s)
Afibrinogenemia/physiopathology , Blood Coagulation Disorders/physiopathology , Plasminogen/deficiency , Wasting Syndrome/etiology , Afibrinogenemia/genetics , Age Factors , Animals , Blood Coagulation Disorders/etiology , Blood Coagulation Disorders/genetics , Blood Coagulation Disorders/mortality , Cell Movement , Digestive System/pathology , Enzyme Activation , Fibrinogen/genetics , Fibrinolysis , Keratinocytes/physiology , Liver/pathology , Mice , Mice, Mutant Strains , Plasminogen/genetics , Skin/cytology , Skin Physiological Phenomena , Wasting Syndrome/mortality , Wound Healing/genetics
13.
Proc Natl Acad Sci U S A ; 93(12): 5899-904, 1996 Jun 11.
Article in English | MEDLINE | ID: mdl-8650190

ABSTRACT

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.


Subject(s)
Fibrin/metabolism , Receptors, Cell Surface/metabolism , Tissue Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/metabolism , Animals , Base Sequence , DNA Primers , Mice , Molecular Sequence Data , Receptors, Cell Surface/deficiency , Receptors, Urokinase Plasminogen Activator , Tissue Plasminogen Activator/deficiency , Wound Healing
14.
Proc Natl Acad Sci U S A ; 93(13): 6258-63, 1996 Jun 25.
Article in English | MEDLINE | ID: mdl-8692802

ABSTRACT

Tissue factor (TF) is the cellular receptor for coagulation factor VI/VIIa and is the membrane-bound glycoprotein that is generally viewed as the primary physiological initiator of blood coagulation. To define in greater detail the physiological role of TF in development and hemostasis, the TF gene was disrupted in mice. Mice heterozygous for the inactivated TF allele expressed approximately half the TF activity of wild-type mice but were phenotypically normal. However, homozygous TF-/- pups were never born in crosses between heterozygous mice. Analysis of mid-gestation embryos showed that TF-/- embryos die in utero between days 8.5 and 10.5. TF-/- embryos were morphologically distinct from their TF+/+ and TF+/- littermates after day 9.5 in that they were pale, edematous, and growth retarded. Histological studies showed that early organogenesis was normal. The initial failure in TF-/- embryos appeared to be hemorrhaging, leading to the leakage of embryonic red cells from both extraembryonic and embryonic vessels. These studies indicate that TF plays an indispensable role in establishing and/or maintaining vascular integrity in the developing embryo at a time when embryonic and extraembryonic vasculatures are fusing and blood circulation begins.


Subject(s)
Blood Coagulation Disorders/complications , Fetal Death/etiology , Hemorrhage , Thromboplastin/deficiency , Animals , Base Sequence , DNA Primers , Genes, Lethal , Heterozygote , Homozygote , Mice , Molecular Sequence Data , Phenotype , Thromboplastin/genetics
15.
Nat Med ; 2(3): 287-92, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8612226

ABSTRACT

Activation of plasminogen (Plg) has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling events, including wound healing. However, there has been no definitive proof of involvement of Plg in such processes. We now report that healing of skin wounds is severely impaired in mice made deficient in Plg by targeted gene disruption. The results demonstrate that Plg is required for normal repair of skin wounds in mice and support the assumption that it also plays a central role in other disease processes involving extracellular matrix degradation, such as cancer invasion.


Subject(s)
Plasminogen/genetics , Plasminogen/physiology , Wound Healing/genetics , Wound Healing/physiology , Animals , Endopeptidases/metabolism , Extracellular Matrix/pathology , Gene Expression Regulation , Gene Targeting , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Mutant Strains , Skin/injuries , Skin/metabolism , Skin/pathology
16.
J Biol Chem ; 270(28): 16886-94, 1995 Jul 14.
Article in English | MEDLINE | ID: mdl-7622505

ABSTRACT

The urokinase-type plasminogen activator receptor (uPAR) gene was disrupted in mice in order to explore the role of cell surface-associated plasminogen activation in development and hemostasis. Homozygous, uPAR-/- mice were born and survived to adulthood with no overt phenotypic abnormalities. There was no indication of loss of fetal animals based on the Mendelian pattern of transmission of the mutant uPAR gene. uPAR-/- mice carried no detectable uPAR in lung, spleen, and other tissues when measured both immunologically by Western blot analysis and functionally by ligand cross-linking analyses. In addition, activated peritoneal macrophages collected from uPAR-/- mice failed to promote plasminogen activation in vitro. The loss of the receptor also resulted in a redistribution of uPA in some tissues but had no impact on pro-uPA activation in the urogenital tract. Thus, in the absence of other challenging factors such as infection, injury, or other functional deficits, uPAR deficiency does not compromise fertility, development, or hemostasis. These mice provide a means to test the proposed function of uPA/uPAR in wound repair, atherogenesis, and tumor cell invasion in vivo.


Subject(s)
Embryonic and Fetal Development , Fertility , Receptors, Cell Surface/physiology , Urokinase-Type Plasminogen Activator/metabolism , Animals , Base Sequence , Enzyme Precursors/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Neoplasm Invasiveness , Plasminogen/metabolism , Pregnancy , Receptors, Cell Surface/analysis , Receptors, Cell Surface/genetics , Receptors, Urokinase Plasminogen Activator , Urogenital System/metabolism
17.
Genes Dev ; 9(7): 794-807, 1995 Apr 01.
Article in English | MEDLINE | ID: mdl-7705657

ABSTRACT

Plasminogen (Plg)-deficient mice were generated to define the physiological roles of this key fibrinolytic protein and its proteolytic derivatives, plasmin and angiostatin, in development, hemostasis, and reproduction. Plg-/- mice complete embryonic development, survive to adulthood, and are fertile. There is no evidence of fetal loss of Plg-/- mice based on the Mendelian pattern of transmission of the mutant Plg allele. Furthermore, embryonic development continues to term in the absence of endogenous, sibling-derived, or maternal Plg. However, Plg-/- mice are predisposed to severe thrombosis, and young animals developed multiple spontaneous thrombotic lesions in liver, stomach, colon, rectum, lung, pancreas, and other tissues. Fibrin deposition in the liver was a uniform finding in 5- to 21-week-old mice, and ulcerated lesions in the gastrointestinal tract and rectal tissue were common. A remarkable finding, considering the well-established linkage between plasmin and the proteolytic activation of plasminogen activators, was that the level of active urokinase-type plasminogen activator in urine was unaffected in Plg-/- mice. Therefore, Plg plays a pivotal role in fibrinolysis and hemostasis but is not essential for urokinase proenzyme activation, development, or growth to sexual maturity.


Subject(s)
Embryonic and Fetal Development/physiology , Plasminogen/deficiency , Reproduction/physiology , Thrombosis/etiology , Angiostatins , Animals , Base Sequence , Fibrin/metabolism , Fibrinolysis/physiology , Hemostasis/physiology , Liver/pathology , Mice , Molecular Sequence Data , Mutagenesis , Oligodeoxyribonucleotides , Peptide Fragments/physiology , Plasminogen/genetics , Plasminogen/physiology , RNA, Messenger/metabolism , Urokinase-Type Plasminogen Activator/metabolism
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