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. Pig -/-mice complete embryonic development, survive to adulthood, and are fertile. There is no evidence of fetal loss of Pig -/-mice based on the Mendelian pattern of transmission of the mutant Pig allele. Furthermore, embryonic development continues to term in the absence of endogenous, sibling-derived, or maternal Pig. However, Pig -/-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 Pig -/-mice. Therefore, Pig plays a pivotal role in fibrinolysis and hemostasis but is not essential for urokinase proenzyme activation, development, or growth to sexual maturity.

To explore the role of the key coagulation factor, fibrinogen, in development, hemostasis, wound repair, and disease pathogenesis, we disrupted the fibrinogen As chain gene in mice. Homozygous, As chain-deficient (As-/-) mice are born normal in appearance, and there is no evidence of fetal loss of these animals based on the Mendelian pattern of transmission of the mutant Ac~ chain allele. All of the component chains of fibrinogen (A¢~, B~, and y) are immunologically undetectable in the circulation of both neonatal and adult As -/-mice, and blood samples fail to either clot or support platelet aggregation in vitro. Overt bleeding events develop shortly after birth in --~30% of Ac~-/-mice, most frequently in the peritoneal cavity, skin, and soft tissues around joints. Remarkably, most newborns displaying signs of bleeding ultimately control the loss of blood, clear the affected tissues, and survive the neonatal period. Juveniles and young adult As-/-mice are predisposed to spontaneous fatal abdominal hemorrhage, but long-term survival is variable and highly dependent on genetic background. The periodic rupture of ovarian follicles in breeding-age As -/-females does not appear to significantly diminish life expectancy relative to males; however, pregnancy uniformly results in fatal uterine bleeding around the tenth day of gestation. Microscopic analysis of spontaneous lesions found in As -/-mice suggests that fibrin(ogen) plays a fundamental role in the organization of cells at sites of injury.[Key Words" Fibrinogen-deficient mice; coagulation; hemostasis; afibrinogenemia; platelet aggregation; wound healing; development] Received May 15, 1995; revised version accepted June 28, 1995.Fibrin(ogen) is the ultimate target of two sophisticated and opposing regulatory systems, the coagulation and fibrinolytic cascades, that together preserve vascular integrity and maintain hemostatic balance (Davie et al. 1991;Esmon 1993;Collen and Lijnen 1994). The coagulation system includes more than a dozen soluble and cell-associated factors that initiate, promote, and ultimately limit the formation of insoluble fibrin polymer (Davie et al. 1991}. A key step in coagulation is the generation of the serine protease, thrombin, which triggers platelet activation (Majerus 1994), converts fibrinogen into a spontaneously polymerizing fibrin monomer (Doolittle 1994), activates the transglutaminase (factor XIII) that covalently cross-links fibrin matrices (Chung and Ichinose 1995), and activates regulatory pathways that both promote and suppress coagulation (Davie et al.

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.

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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

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.

Blood loss at sites of vascular rupture is controlled by the adhesion and aggregation of platelets and the formation of an insoluble fibrin matrix. Fibrinogen is considered to be critical in these processes by both providing an abundant dimeric ligand for alpha IIb beta 3‐mediated platelet aggregation, and serving as the fundamental building block of the fibrin polymer. To establish an in vivo model system to examine in detail the importance of alpha IIb beta 3‐fibrinogen interactions in platelet function, hemostasis, response to injury and vasoocclusive disease, and to test the prevailing hypothesis that the C‐terminal segment of the fibrinogen gamma chain is essential for alpha IIb beta 3 binding, we have used gene‐targeting technology in mice to eliminate the last five residues (QAGDV) from the gamma chain. Mice homozygous for the modified gamma chain gene (gamma delta 5/gamma delta 5) displayed a generally normal hematological profile, including normal platelet count, plasma fibrinogen level, clotting time and fibrin crosslinking. However, both gamma delta 5‐fibrinogen binding to alpha IIb beta 3 and platelet aggregation were highly defective. Remarkably, another alpha IIb beta 3‐dependent process, clot retraction, was unaffected by the gamma delta 5 mutation. Despite the preservation of clotting function, gamma delta 5/gamma delta 5 mice were unable to control blood loss following a surgical challenge and occasionally developed fatal neonatal bleeding events.

Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5'-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.