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...

The Ron/STK receptor tyrosine kinase is a member of the c-Met family of receptors and is activated by hepatocyte growth factor-like protein (HGFL). Ron activation results in a variety of cellular responses in vitro, such as activation of macrophages, proliferation, migration, and invasion, suggesting a broad biologic role in vivo. Nevertheless, HGFL-deficient mice grow to adulthood with few appreciable phenotypic abnormalities. We report here that in striking contrast to the loss of its only known ligand, complete loss of Ron leads to early embryonic death. Embryos that are devoid of Ron (Ron -/-) are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization analysis demonstrates that Ron is expressed in the trophectoderm at embryonic day (E) 3.5 and is maintained in extraembryonic tissue through E7.5, compatible with an essential function at this stage of development. Hemizygous mice (Ron +/-) grow to adulthood; however, these mice are highly susceptible to endotoxic shock and appear to be compromised in their ability to downregulate nitric oxide production. These results demonstrate a novel role for Ron in early mouse development and suggest that Ron plays a limiting role in the inflammatory response. eration and motility of keratinocytes in vitro. The functions of Ron in vivo are not yet understood, but these findings suggest its involvement in a wide spectrum of biologic activities.An equally complex pattern of Ron expression is observed in the developing mouse, implying a functional requirement for Ron during embryogenesis. Ron expression is detected at embryonic day (E) 12.5 in the liver, perhaps reflecting fetal hematopoiesis, and in the central nervous system, primarily in the spinal and dorsal root ganglia. By E14.5, Ron expression has been initiated in the digestive tract epithelium, skin keratinocytes, and bone (22, 23). Information is not available on the expression of Ron prior to E7.5 in the mouse or on its expression in extraembryonic tissue.In spite of the growing body of evidence that Ron and HGFL are involved in embryogenesis and/or inflammation, HGFL-deficient mice grow to adulthood with few ascertainable defects, aside from the accumulation of lipid-filled cytoplasmic vesicles in hepatocytes, which does not perturb synthetic or secretory hepatic function (24). Activation of macrophages is delayed, but nevertheless attained, in the absence of HGFL, and hematopoiesis and wound healing appear to be unaltered in mice that are devoid of HGFL. To directly determine the specific functions of Ron in development and in the adult mouse, we have examined the consequences of Ron gene disruption in mice. In contrast to the loss of the ligand HGFL, we report that the complete loss of the Ron receptor tyrosine kinase leads to death in the periimplantation stage of development. MethodsDisruption of the mouse Ron gene. In construction of the targeting vector, exons 1-14 and part of exon 15 of the mouse Ron gene, isolated from a 129/Ola geno...

Mice carrying a conditional prothrombin knockout allele (fII lox ) were established to develop an experimental setting for exploring the importance of thrombin in the maintenance of vascular integrity, the inflammatory response, and disease processes in adult animals. In the absence of Cre-mediated recombination, homozygous fII lox/lox mice or compound heterozygous mice carrying one fII lox allele and one constitutive-null allele were viable. Young adults exhibited neither spontaneous bleeding events nor diminished reproductive success. However, the induction of Cre recombinase in fII lox mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fII lox allele within the liver, the loss of circulating prothrombin, and profound derangements in coagulation function. Consistent with the notion that thrombin regulates coagulation and inflammatory pathways, an additional early consequence of reducing prothrombin was impaired antimicrobial function in mice challenged with Staphylococcus aureus peritonitis. However, life expectancy in unchallenged adults genetically depleted of prothrombin was very short (~5-7 days). The loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, particularly in the heart and brain.

SummarySeveral members of a family from Scranton, Pennsylvania were identified to have normal levels of prothrombin antigen while their prothrombin clotting activity was approximately 50% of normal. There has been no previous history of bleeding or other clinical manifestations in this family. The genomic DNA from the proband was amplified for all exons in the prothrombin gene and analyzed by single strand conformation polymorphism (SSCP)/heteroduplex analysis followed by DNA sequence analysis and restriction enzyme digestion. A mutation at nucleotide 20040 in exon 14 was identified and confirmed by restriction enzyme digestion. This mutation results in the substitution of Thr for Lys at amino acid 556. Amino acid 556 has been reported as one of the key residues for the binding of Na+ in the thrombin portion of the protein.

In an e ort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and¯anking DNA were identi®ed. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5'¯anking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyltransferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides 7585 to 7465 and from 7465 to 7285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides 7585 to 7508 and nucleotides 7375 to 7285 appear to bind speci®c proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene.