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