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We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg100, the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII-Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.

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 (© 1998 by The American Society of Hematology.

Abstract Factor VII levels are regulated by environmental and genetic factors. Two polymorphisms, a G-to-A transversion at nucleotide 10976 resulting in Arg 353 Gln 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 Arg 353 Gln almost always segregates on alleles containing the insert in UK and Italian populations, thereby making it impossible to independently evaluate the impact of Arg 353 Gln 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 Arg 353 Gln 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 Arg 353 Gln and the insert compared with 72 normal subjects who had neither Arg 353 Gln 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 Arg 353 Gln 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, Arg 353 Gln but not the insert significantly reduced VII:C and VII:Ag after adjustment for age and plasma triglycerides ( P P =.02, respectively). To evaluate the mechanism responsible for reduced factor VII levels in individuals with Arg 353 Gln, we performed transient transfection assays with factor VII cDNA containing the base substitution resulting in Gln 353 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 Arg 353 Gln polymorphism alone can decrease plasma factor VII levels.

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.

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatasecalcineurin,anditsfunctionisinhibitedbytheimmunosuppressiveagentcyclosporinA(CsA).Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colonystimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response. The nuclear factor of activated T cells (NFAT) was originallyidentifiedinTcellsasanessentialtranscriptionfactorfor

Objective Under physiologic conditions in animals and pathologic conditions in humans, purinergic mechanisms contribute significantly to detrusor contractions. Although the activation of P2X1 receptors (P2X1R) accounts for the largest portion of bladder smooth muscle (BSM) responses to ATP, previous reports have shown that P2X1R antagonists do not completely abolish the purinergic component of neurogenic contractions, suggesting the presence of other P2XR subtypes on BSM. P2X4R has been identified in bladder tissue, however whether this receptor is functionally relevant warrants investigation. The aim of this study was to examine the extent of P2X4R expression in BSM tissue and to investigate its pharmacological contribution to ATP-mediated detrusor contractions. Methods P2X4R mRNA and protein expression was investigated in mouse bladder tissue (without mucosa) and in cultured BSM cells by real-time RT-PCR and western blotting respectively. In vitro isometric tension studies were performed in mouse BSM strips without mucosa. Purinergic detrusor contractions were elicited by administration of α-β-methylene-ATP (αβmATP), and the purinergic component of neurogenic contractions induced by electrical field stimulations (EFS) was isolated by pre-treatment with the muscarinic receptor antagonist atropine. The inhibitory effect of two P2X4R selective antagonists, 5-BDBD and BX430, on the αβmATP- and EFS-induced contractions was investigated in the presence of P2X1R antagonist NF449. In addition, the effect of the P2X4R positive modulator ivermectin (IVC) on αβmATP responses was investigated. Data were analyzed by a repeated measures analysis of variance. Results P2X4R mRNA was detected in smooth muscle from both bladder tissue and cultured BSM cells, although its expression was significantly lower than P2X1R expression. Immunoreactivity for P2X4R was detected in lysates from both mouse BSM tissue and smooth muscle cells. Functional studies indicated that although P2X1R activation is predominantly responsible for purinergic contractions in mouse detrusor, a significant portion of the contractile response to both αβmATP (22.3±7%) and EFS (27.5±4% of purinergic component of EFS) was resistant to P2X1R inhibition. This NF449-resistant component was abolished by administration of P2X4R antagonists 5-BDBD or BX430. In addition, responses to αβmATP increased significantly upon administration of IVC. Conclusions The expression of P2X4R in detrusor smooth muscle together with the identification of a P2X4R-sensitive component of bladder contractions suggest that the activation of this P2X receptor subtype could significantly contribute to ATP-mediated BSM responses. P2X4R may thus potentially represent a novel target for the management of detrusor dysfunctions associated with alterations in purinergic signaling. Funding Source(s) Department of Veterans Affairs, Research Service BX001790; BX002806

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.

Investigation of the molecular basis of a severe factor VII (fVII) deficiency revealed compound heterozygosity in the fVII gene. On the paternal allele the patient had 3 structural gene abnormalities frequently associated with fVII deficiency. A new mutation, a C to T transition at position −55 relative to the translational start site, was found on the maternal allele. The study demonstrates that this mutation partially impeded binding of the transcriptional activator, hepatic nuclear factor 4, to the fVII promoter while greatly reducing reporter gene expression in hepatic cells.

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein essential for normal hemostasis. We have discovered that cultured human umbilical vein endothelial cells incorporate inorganic sulfate into vWF. Following immunoisolation and analysis by polyacrylamide or agarose gel electrophoresis, metabolically labeled vWF was found to have incorporated [35S]-sulfate into all secreted multimer species. The time course of incorporation shows that sulfation occurs late in the biosynthesis of vWF, near the point at which multimerization occurs. Quantitative analysis suggests the presence, on average, of one molecule of sulfate per mature vWF subunit. Virtually all the detectable sulfate is released from the mature vWF subunit by treatment with endoglycosidases that remove asparagine-linked carbohydrates. Sulfated carbohydrate was localized first to the N-terminal half of the mature subunit (amino acids 1 through 1,365) by partial proteolytic digestion with protease V8; and subsequently to a smaller fragment within this region (amino acids 273 through 511) by sequential digestions with protease V8 and trypsin. Thus, the carbohydrate at asparagine 384 and/or 468 appears to be the site of sulfate modification. Sodium chlorate, an inhibitor of adenosine triphosphate- sulfurylase, blocks sulfation of vWF without affecting either the ability of vWF to assemble into high molecular weight multimers or the ability of vWF multimers to enter Weible-Palade bodies. The stability of vWF multimers in the presence of an endothelial cell monolayer also was unaffected by the sulfation state. Additionally, we have found that the cleaved propeptide of vWF is sulfated on asparagine-linked carbohydrate.

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.

Summary. Factor (F)VII plays a critical role in initiation of coagulation. Several segments within the 5′ flanking region of the FVII gene were previously demonstrated to recognize hepatic nuclear proteins, but few have been identified. To identify a regulatory protein binding the nuclear hormone response region (−237 to −200) of the FVII 5′ flanking region and demonstrate that the interaction is functional. Electrophoretic mobility shift assays and mutation analysis showed that ARP1, an orphan nuclear hormone receptor, interacted with two regions of the FVII 5′ flanking region, the hepatic nuclear factor 4 binding region (−77 to −47) and the nuclear hormone response region (−237 to −200). Transfection experiments demonstrated that reporter gene expression was decreased from vectors including the nuclear hormone response segment compared with that containing only the minimal promoter between positions −109 and +1, and that ARP1 also repressed expression through an interaction with the minimal promoter. These data indicate a role for ARP1 in transcriptional modulation of the FVII gene.

SummaryFactor VII (FVII) requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active FVIIa conformation. This event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. The TF-induced allosteric enhancement of FVIIa’s activity contributes to the procoagulant activity of the complex. We have characterized two naturally occurring mutations (S363I W364C) on FVII gene. Both homozygous patients for each mutation have a normal FVII:Ag level associated to an undetectable FVII coagulant activity. The patient carrying the allele 364C had a more severe hemorrhagic diathesis than the S363I mutant. To understand the mechanism of these deficiency, in vitro expression analysis with further biochemical characterization of recombinant proteins of both mutants FVII-363I, FVII-364C and wild type (WTFVII) FVII constructs were done. The results recapitulated the patients’ plasma data with normal Ag level and no detectable coagulant activity. The D-F-Pip-R-pNA and CH3SO2-D-CHA-A-But-R chromogenic substrates were used to evaluate the amidolytic activity of WT and mutant FVII in presence and absence of recombinant tissue factor (rTF). Binding of FVII to rTF by a solid phase binding assay was done using recombinant human rTF. The results of amidolytic assays showed that rTF enhances 28 fold the value of the specificity of constant (kcat/Km) in WT but no activity was detectable in either mutant constructs under any condition. The equilibrium dissociation constant of rTF-FVIIa interaction showed Kd equal to 4.4 ± 0.2nM, 4.9 ± 0.5nM and 6 ± 0.9 of WT, 363I and 364C FVII forms, respectively. The Kd values of the non activated forms were equal to 24.7 ± 3.3, 24.4 ± 3.1 and 20.6 ± 4nM, respectively. These data demonstrate that, compared to the WT form, FVII-363I and FVII-364C have no significant affinity change for TF and that the detrimental effect of these two mutations is attributable to the loss of an efficient catalytic machinery in the FVII molecule causing a severe deficiency of coagulant activities.

A case of a novel mutation in the F7 gene that results in factor VII coagulant activity (VII:c) of less than 1% and VII antigen (VII:Ag) levels of 10% is presented. DNA analysis revealed a homozygous 15–base pair (bp) in-frame insertion-type mutation at nucleotide 10554. This insertion consisted of a duplication of residues leucine (L)213 to aspartic acid (D)217 (leucine, serine, glutamic acid, histidine, and aspartic acid), probably arising by slipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separated by 4 bp. Molecular graphic analyses showed that the insertion is located at the surface of the catalytic domain in an exposed loop stabilized by extensive salt-bridge and hydrogen bond formation at which the calcium binding site is located. The mutation probably interferes with protein folding during VII biosynthesis and/or diminishes functional activity through the loss of calcium binding. In vitro expression studies demonstrated that the levels of VII:Ag in lysates of cells transfected with wild type VII (VIIWT) were equivalent to those with mutant type VII (VIIMT), but the level of secreted VIIMT was 5% to 10% that of VIIWT. Pulse chase studies demonstrated that VIIMT did not accumulate intracellularly, and studies with inhibitors of protein degradation showed that recombinant VIIMT was partially degraded in the pre-Golgi compartment. Accordingly, only small amounts of VIIMT with undetectable procoagulant activity were secreted into conditioned media. These results demonstrate that a combination of secretion and functional defects is the mechanism whereby this insertion causes VII deficiency.

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.

BACKGROUND: Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. METHODOLOGY/PRINCIPAL FINDINGS: Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, p

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 O-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 O-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 1b 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 O-linked carbohydrates in the vWf-glycoprotein 1b interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetin-dependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease.

Sodium bisulfite reacts with cytosine and 5-methylcytosine, forming the 5,6-dihydrosulfonate adducts which deaminate to the uracil and thymine adducts, respectively. At alkaline pH, the sulfonate groups are then released, generating uracil and thymine. In DNA, the resulting G:U and G:T base mismatches generated are potential sites of mutagenesis. Using a human damage-specific DNA binding protein as a probe, we have found protein-recognizable lesions in bisulfite-treated DNA and poly d(I-C), but not in treated poly d(A-T) or poly d(A-U). Although this suggests that the lesion recognized is cytosine-derived, there was no correlation between the number of uracils induced and the number of binding sites, suggesting that the protein-bound damage is not a uracil-containing mismatch. Modification of the treatment protocol to reduce elimination of the bisulfite from the base adducts increased the level of binding, suggesting that the protein recognizes a base-sulfonate adduct.

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 or more intracellular proteases whose functions are essential for the induction and/or expression of the transformed phenotype. We have isolated an endopeptidase activity capable of hydrolyzing the substrate Boc-Val-Pro-Arg-MCA (Boc = butoxycarbonyl; MCA = 7-amino-4-methylcoumarin) from C3H/10T1/2 mouse embryo fibroblast cells. This intracellular protease was inhibited by the soybean-derived Bowman-Birk inhibitor (BBI), chymostatin, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, all of which have anticarcinogenic activity, but was unaffected by soybean trypsin inhibitor, which lacks anticarcinogenic activity. Other protease inhibitors affected the proteolytic activity to an extent that correlates with their relative ability to suppress transformation in vitro. The enzyme has a mass of about 70 kDa, contains a single subunit, and exhibits maximal activity at pH 7.0. Diisopropyl fluorophosphate covalently binds to this enzyme and blocks its activity, indicating that the enzyme is a serine protease. We have previously demonstrated that several protease inhibitors are effective suppressors of radiation-induced transformation of C3H/10T1/2 cells. Since these agents reduce the Boc-Val-Pro-Arg-MCA-hydrolyzing activity to an extent that correlates with their ability to inhibit malignant transformation in vitro, this endopeptidase activity may be a cellular target of the anticarcinogenic protease inhibitors.