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.

Tissue factor (TF) is the cellular receptor for coagulation factor VII/VIla 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.Tissue factor (TF) is a 47-kDa membrane-bound glycoprotein that functions as the cellular receptor for coagulation factor VII/VIIa. TF is thought to be the primary physiological initiator of blood coagulation following vascular damage (1). Unlike other coagulation factors, TF need not be activated and is delivered to the cell surface as a functional VII/VIla receptor. Factor VIla bound to TF at the cell surface efficiently catalyzes the proteolytic activation of coagulation factors IX and X. This ultimately leads to local thrombin generation and thrombin-catalyzed events such as fibrin formation and the activation of factors V, VIII, XIII, thrombin receptor, and protein C (1).The central role of TF in blood coagulation in vivo is supported by several compelling, but indirect, observations.

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FIIdeficient (FII ؊/؊ ) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII ؊/؊ embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII ؊/؊ embryos within this gestational time frame. However, at least one-quarter of the FII ؊/؊ mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.Prothrombin (FII), a vitamin K-dependent zymogen synthesized by hepatocytes, is activated to form thrombin (FIIa) by factor Xa in the presence of factor Va (FVa), calcium, and a phospholipid surface. FIIa plays a central role in the blood coagulation system by triggering the activation of platelets, converting soluble fibrinogen into insoluble fibrin polymer, and activating regulatory pathways that control the rate of further thrombin formation (1). In the presence of thrombomodulin, FIIa functions as an anticoagulant by activating protein C and protein S, which in turn inactivates factors Va and VIIIa. FIIa is thought to serve a broader biological role than merely controlling blood loss, based on the fact that there are at least two G protein-coupled receptors (i.e., PAR-1 and PAR-3) that are proteolytically activated by thrombin, and these receptors are present on a variety of cell types (2-4). FIIa has been proposed to influence a variety of physiological and pathological processes, including inflammation, tissue repair, neurite outgrowth, atherosclerosis, and tumor cell metastasis (5-9). The expression of both thrombin receptor and FII during organogenesis in the mouse suggests that FIIa may play an important role in development (3), a hypothesis that is supported further by the finding of partial embryonic lethality in mice deficient in tissue factor (TF), factor V (FV), and PAR-1 (10-16). To understand in greater detail the diverse biological roles of FII in vivo, and to directly establish the importance of FII in development, the FII gene was disrupted in mice. We report that FII deficiency results in a loss of vascular integrity and death around the tenth day of gestation in a high percentage of FII Ϫ/Ϫ embryos. Partial embryonic lethali...

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogende®cient 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-de®cient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogende®cient mice was signi®cantly 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.

To understand the biologic function of TIMP-2, a member of the tissue inhibitors of metalloproteinases family, an inactivating mutation was introduced in the mouse Timp-2 gene by homologous recombination. Outbred homozygous mutants developed and procreated indistinguishably from wild type littermates, suggesting that fertility, development, and growth are not critically dependent on TIMP-2. Lack of functional TIMP-2, however, dramatically altered the activation of proMMP-2 both in vivo and in vitro. Fully functional TIMP-2 is essential for efficient activation of proMMP-2 in vivo. No evidence of successful functional compensation was observed. The results illustrate the duality of TIMP-2 function, i.e. at low concentrations, TIMP-2 exerts a "catalytic" or enhancing effect on cell-mediated proMMP-2 activation, whereas at higher concentrations, TIMP-2 inhibits the activation and/or activity of MMP-2.