Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. X Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin Kdependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).Blood coagulation can be initiated by a complex of tissue factor (TF), a membrane-bound glycoprotein, and factor VII, a plasma coagulation factor (for reviews, see refs. 1 and 2). The physiological significance of this extrinsic pathway can be judged by the severe bleeding frequently observed in individuals who are markedly deficient in factor VII (3, 4). In contrast, individuals who have deficiencies or abnormalities in proteins that are involved in the early steps of the intrinsic pathway of coagulation-i.e., high molecular weight kininogen, prekallikrein, and factor XII-are asymptomatic (5). The TF-factor VII complex activates factor IX, a component of the intrinsic pathway, as well as factor X (6). Thus, it is reasonable that the association of TF and factor VII may be the crucial event triggering the initiation of clotting in vivo.The cDNAs for all of the proteins involved in TF-initiated coagulation, with the exception of TF itself, have already been cloned and sequenced (7-13). The TF apoprotein has been purified from both bovine and human sources (14-16). Approximately 50-70% of the amino acid sequence of both species has now been determined, and this has permitted us to select suitable amino acid sequences to serve as a basis for constructing oligonucleotide probes. This in turn has enabled the isolation and characterization of two human placental TF cDNA clones that contain the entire coding region of the mature protein. The nucleotide sequence of these clones, together with amino acid sequence data, has allowed us to formulate a primary structure for the human TF apoprotein §. MATERIALS AND METHODSTF Purification and Sequencing. A monoclonal antibody (17) prepared against human TF that had been purified by using factor VII affinity columns (15) was used for immunoaffinity isolation of TF. Briefly, TF was extracted from human brain or placental tissue acet...

The T235 allele of the angiotensinogen gene (AGT) has been associated with hypertension. Blood pressure increases faster over time in black children than in white children, and in adults hypertension is more prevalent in blacks. We sought evidence for a role for angiotensinogen to contribute to racial differences in blood pressure in a study of 148 white and 62 black normotensive children (mean age, 14.8 yr). The frequency of the T235 allele was 0.81 in blacks and 0.42 in whites (X2 = 77.3, P = 0.0001). The mean angiotensinogen level was 19% higher in blacks than in whites (P = 0.0001 for males, P = 0.004 for females). Genotype was positively related to serum angiotensinogen in white children (P = 0.0001 for males, P = 0.004 for females), but a similar relationship was absent in blacks where the frequency of M235 may have been too low to discern an association. Longitudinal blood pressure (measured twice yearly) adjusted for body mass index showed a marginally significant relationship to the angiotensinogen level (P = 0.07). An independent relationship of serum angiotensinogen with body mass index (P = 0.0001) and race (P = 0.0003) was also observed. In summary, T235 was more frequent, and the level of angiotensinogen was higher in blacks than in whites. Such a racial difference in the renin-angiotensin system may contribute to the disparity in blood pressure levels of white and black young people. (J. Clin. Invest. 1995. 95:948-953.)

The fibroblast growth factor receptors (FGFR) are tyrosine kinases that are present in many types of endothelial and tumor cells and play an important role in tumor cell growth, survival, and migration as well as in maintaining tumor angiogenesis. Overexpression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs represents an attractive strategy for development of cancer treatment options by simultaneously inhibiting tumor cell growth, survival, and migration as well as tumor angiogenesis. Here, we describe a potent, selective, small-molecule FGFR inhibitor, (R)-(E)-2-(4-(2-(5-(1-(3,5-Dichloropyridin-4-yl)ethoxy)-1H-indazol-3yl)vinyl)-1H-pyrazol-1-yl)ethanol, designated as LY2874455. This molecule is active against all 4 FGFRs, with a similar potency in biochemical assays. It exhibits a potent activity against FGF/FGFR-mediated signaling in several cancer cell lines and shows an excellent broad spectrum of antitumor activity in several tumor xenograft models representing the major FGF/FGFR relevant tumor histologies including lung, gastric, and bladder cancers and multiple myeloma, and with a well-defined pharmacokinetic/pharmacodynamic relationship. LY2874455 also exhibits a 6-to 9-fold in vitro and in vivo selectivity on inhibition of FGF-over VEGF-mediated target signaling in mice. Furthermore, LY2874455 did not show VEGF receptor 2-mediated toxicities such as hypertension at efficacious doses. Currently, this molecule is being evaluated for its potential use in the clinic.

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Abstract-Renin and aldosterone secretion is often lower in blacks than in whites, characteristics that resemble a milder form of Liddle syndrome in which a mutation in the amiloride-sensitive epithelial sodium channel (ENaC) of the kidney results in enhanced resorption of sodium. In the present study, we looked for evidence that the intrinsic level of ENaC activity is indeed higher in blacks than in whites. In overnight urine samples collected from young people (249 white and 181 black subjects, mean age 13.4 years), the urinary aldosterone/potassium ratio, which is typically very low in Liddle syndrome, was lower in blacks than in whites: 0.421Ϯ0.024 (meanϮSE) versus 0.582Ϯ0.016 nmol/mmol (PϽ0.0001). In addition, all but 1 of 5 molecular variants in ENaC were much more common in blacks than in whites. G442V in the ␤-subunit, present in 16% of the blacks and in only 1 white, was associated with parameters reflective of a greater Na retention and potentially a higher ENaC activity: a lower plasma aldosterone concentration (Pϭ0.070), a lower urinary aldosterone excretion rate (Pϭ0.052), a higher potassium excretion rate (Pϭ0.048), and a lower urinary aldosterone/potassium ratio (Pϭ0.027). In a second cohort consisting of 126 black and 161 white normotensive subjects and 232 black and 188 white hypertensive subjects, ␤G442V did not show a significant association with hypertension (Pϭ0.089). On the other hand, a variant that was twice as common in whites, ␣T663A, was associated with being normotensive both in blacks (Pϭ0.018) and in whites (Pϭ0.034). Expression of either ␤G442V or ␣T663A in Xenopus oocytes did not result in a change in basal Na current, consistent with the variants being in linkage disequilibrium with alleles at active loci. In conclusion, several lines of evidence are presented to suggest that ENaC activity is higher in blacks than in whites, which could contribute to racial differences in Na retention and the risk for hypertension. (Hypertension. 1999;34:631-637.)

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC 50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1␤ or tumor necrosis factor-␣-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stressactivated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.The mitogen-activated protein kinases (MAPK) 1 are a highly conserved family of signal transduction molecules that transmits extracellular signals from the membrane to the nucleus. There are three major branches of MAPK signaling that include ERK, c-Jun N-terminal kinase (JNK) and p38. The JNK and p38 branches of the MAPK family are activated by stress stimuli including cytokines, osmotic stress, mitogens, UV irradiation, chemotherapeutic agents, and anisomycin (1, 2). There are three kinases that form a MAPK signaling module where a MAPK is activated by a MAPK kinase (MAPKK), which in turn is regulated by a MAPKK kinase (MAPKKK) (Fig. 1). The upstream activation of JNK and p38 is complex, allowing for activation of this pathway in multiple cells and by multiple stimuli. Cellular and receptor specificity of the pathway is conferred by protein-protein interactions where the MAPK and a MAPKK assemble with a specific MAPKKK on scaffold proteins such as JNK-interacting protein (3, 4) or ␤-arrestin (5). The resulting signaling module acts as a bridge joining the appropriate receptor to the downstream effectors, enabling activation of the stress-activated MAPK.The most distal point at which signal and cell specificity for JNK and p38 activation is conferred is at the MAPKKK level. Mixed lineage kinases (MLKs) are a family of MAPKKKs activating JNK and p38. There are currently seven mammalian kinases belonging to the MLK family that have recently been reviewed (6, 7). These kinases can be divided into three subclasses based on sequence similarity and domain structure, and they include MLK1-4, dual leucine zipper kinases, and the zipper sterile ␣-motif kinases (ZAKs). Although much is known about the mechanisms re...

An insertion (I)/deletion (D) polymorphism of the angiotensin I-converting enzyme (ACE) gene that has been associated with certain cardiovascular disorders accounts for nearly half the variation in serum ACE level in white subjects. Whether a similar association of serum ACE with the I/D polymorphism occurs in other racial groups is not known. We studied the I/D polymorphism of ACE in relation to serum ACE activity in 141 white and 62 black healthy, unrelated children and adolescents (mean age, 14.7 years). The mean level of ACE activity in whites homozygous for the D allele was higher than in heterozygotes (P = .002) and in homozygotes for the I allele (P = .0001), consistent with an earlier study. In blacks, on the other hand, no significant difference in serum ACE activity between genotypes was observed. An additional finding was a significantly positive relationship between serum ACE activity and diastolic pressure (P = .009). In children and adolescents, serum ACE activity is related to the ACE gene I/D polymorphism in whites but not in blacks. The results indicate a potentially important ethnic variation in genetic regulation of serum ACE activity and the relationship of the I/D polymorphism to cardiovascular disease.

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