NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.

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We show here that NFAT1 is rapidly activated, then slowly deactivated, by stimulation of T cells through their antigen receptor. Within minutes of T-cell receptor stimulation, NFAT1 is dephosphorylated, translocates from the cytoplasm into the nucleus, and shows an increase in its ability to bind to DNA. These changes are dependent on calcium mobilization and calcineurin activation, since they are also elicited by ionomycin and are blocked by the immunosuppressive drug cyclosporin A. After several hours of T-cell receptor stimulation, the majority of the NFAT1 in the cell reverts to its original phosphorylated form, reappears in the cytoplasm, and again displays a low affinity for DNA. Deactivation of NFAT1 is facilitated by phorbol 12-myristate 13-acetate and inhibitors of capacitative calcium entry and most likely reflects the slow return of intracellular free calcium concentrations towards resting levels. Our results suggest that calcineurin-dependent signalling pathways mediate the early activation of NFAT1, while phorbol 12-myristate 13-acetate-dependent feedback pathways contribute to the late deactivation. Persistent NFAT-dependent cytokine gene transcription in activated T cells may be mediated by other NFAT family proteins in addition to NFAT1 during the immune response.

Factor VII levels are regulated by environmental and genetic factors. Two polymorphisms, a G-to-A transversion at nucleotide 10,976 resulting in Arg353Gln and a decanucleotide insert at position -323 in the 5'-flanking region of the factor VII gene, have been associated with a 20% to 25% reduction in plasma factor VII levels. However Arg353Gln almost always segregates on alleles containing the insert in UK and Italian populations, thereby making it impossible to independently evaluate the impact of Arg353Gln on factor VII levels in these ethnic groups. We have evaluated the influence of genotype on factor VII levels in 99 healthy Polish blood donors and observed that Arg353Gln frequently occurs in the absence of the insert. In univariate analysis, the mean levels of factor VII coagulant activity (VII:C) and factor VII antigen (VII:Ag) were significantly lower in 16 people who were heterozygous for Arg353Gln and the insert compared with 72 normal subjects who had neither Arg353Gln nor the insert (88.8% of normal and 83.1% versus 102% and 100%, P = .019 and P = .0003, respectively). In nine subjects heterozygous for Arg353Gln alone, VII:C and VII:Ag were significantly decreased compared with the normal subjects (81.9% and 83%, respectively, P = .007 and P = .004). In multivariate analysis, Arg353Gln but not the insert significantly reduced VII:C and VII:Ag after adjustment for age and plasma triglycerides (P < .05 and P = .02, respectively). To evaluate the mechanism responsible for reduced factor VII levels in individuals with Arg353Gln, we performed transient transfection assays with factor VII cDNA containing the base substitution resulting in Gln353 and wild-type factor VII cDNA in COS-1 cells. The levels of VII:Ag in the cell lysates were similar, but the amino acid substitution significantly reduced factor VII secretion into the media to 74.9% of wild-type (P = .0001). Based on these in vivo and in vitro studies, we conclude that the Arg353Gln polymorphism alone can decrease plasma factor VII levels.

Summary. Aims: The aim of this study was to investigate associations between coronary heart disease risk and polymorphisms in the coagulation factor (F)VII gene in participants of a large prospective study. Methods: One thousand nine hundred and ®fty-seven men were genotyped for four FVII polymorphisms, À670A3C, À402G3A, a 10 base pair insertion at À323 (0 > 10) in the promoter, and R353Q in the structural gene. Associations among genotypes and estimated haplotypes, plasma FVII levels, and coronary heart disease risk were evaluated, and the function of the promoter polymorphisms was assessed in reporter gene assays. Results: The À670A3C and À402G3A polymorphisms were in complete allelic association. The haplotype containing À670C and À402A (frequency 0.23) was associated with signi®cantly increased plasma FVII coagulant activity and increased risk of an initial coronary event, particularly acute myocardial infarction, which remained after correction for conventional risk factors. In contrast, the À323 insertion and Q353 alleles (frequency 0.11 and 0.10, respectively) were associated with decreased plasma FVII levels, but hazard ratios for coronary events in carriers of these alleles were not signi®cantly different from unity. In transiently transfected hepatoma cells, increased basal expression of the reporter gene was directed by a promoter fragment with rare haplotype À670C/À630G/À402A rather than by a promoter fragment with common haplotype À670A/À630A/À402G; À402A was not responsible for this effect. Conclusions: The promoter haplotype, À670C/À630G/ 402A, was associated with signi®cantly increased plasma FVII coagulant activity, risk of an initial coronary event, particularly acute myocardial infarction, and reporter gene expression.

By transfecting the full-length cDNA for human von Willebrand factor (vWf) into a line of Chinese hamster ovary cells with a defect in carbohydrate metabolism, we have prepared recombinant vWf specifically lacking 0-linked carbohydrates. We have compared this under-glycosylated protein to fully glycosylated recombinant vWf with respect to several structural and binding properties. vWf deficient in 0-linked glycans was synthesized, assembled into multimers, and secreted in an apparently normal manner and was not prone to degradation in the extracellular milieu. It did not differ from fully glycosylated vWf in ability to bind to heparin or to collagen type I but did interact less well with glycoprotein lb on formalin-fixed platelets. This decreased interaction was evidenced in both a lessened overall binding to platelets and in diminished capacity to promote platelet agglutination, in the presence of ristocetin. In contrast, no difference was seen in platelet binding in the presence of botrocetin. These data indicate a possible role for 0-linked carbohydrates in the vWf-glycoprotein lb interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetindependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease. (J. Clin. Invest. 1992Invest. . 90:2258Invest. -2267

We have identified a point mutation in the promoter of the factor VII gene responsible for a severe bleeding disorder in a patient from a large French-Canadian family with known consanguinity. The proband has an extremely low plasma level of factor VII antigen and factor VII coagulant activity (<1 percent of normal) and suffers from hemarthroses and chronic arthropathy. Sequencing of the patient’s factor VII 5′ flanking region, intron/exon junctions, and coding regions showed a homozygous point mutation, a C to G transversion at position −94 relative to the translation start site. We show here that this mutation prevented binding of transcription factor Sp1 and of other nuclear proteins to this region of the factor VII promoter and resulted in a 20-fold reduction in reporter gene expression in HepG2 cells. These data underscore the importance of this region of the factor VII promoter for in vivo expression of the factor VII gene.© 1998 by The American Society of Hematology.

Several different protease inhibitors have the ability to suppress transformation in vitro and carcinogenesis in vivo. The mechanism(s) by which protease inhibitors suppress carcinogenesis, however, is not fully understood. Presumably, these agents inhibit one (1, 2, 4).The mechanism by which protease inhibitors suppress malignant transformation is unknown. We believe they act by inhibiting one or more proteases that are critical for the induction and/or expression of the transformed phenotype. One approach to determining how protease inhibitors suppress carcinogenesis involves the identification of the particular cellular protease(s) with which the anticarcinogenic protease inhibitors interact. We have assayed C3H/1OTV2 cell homogenates for protease activity using many different fluorogenic substrates (with different amino acid sequences) and have observed that the cleavage of all but two of these substrates is unaffected by protease inhibitors that suppress malignant transformation. The present report involves a detailed study of a particular cellular proteolytic activity in C3H/10T½ cells that is strongly inhibited by the anticarcinogenic protease inhibitors. The anticarcinogenic protease inhibitors utilized here prevent malignant transformation induced by many different carcinogens in in vitro and in vivo carcinogenesis systems (1-6).