RESUMEN
Primary pulmonary hypertension (PPH), an often fatal disease, is characterized by elevated pulmonary artery pressures in the absence of a secondary cause. Endovascular occlusion in the smallest pulmonary arteries occurs by proliferation of cells and matrix, with thrombus and vasospasm. Diagnosis is often delayed because the initial symptoms of fatigue and dyspnea on exertion are nonspecific and definitive diagnosis requires invasive procedures. The average life expectancy after diagnosis is two to three years with death usually due to progressive right heart failure. The aetiology of the disease is unknown. Although most cases appear to be sporadic, approximately 6% of cases recorded in the NIH Primary Pulmonary Hypertension Registry are inherited in an autosomal dominant manner with reduced penetrance. Following a genome-wide search using a set of highly polymorphic short tandem repeat (STR) markers and 19 affected individuals from six families, initial evidence for linkage was obtained with two chromosome 2q markers. We subsequently genotyped patients and all available family members for 19 additional markers spanning approximately 40 centiMorgans (cM) on the long arm of chromosome 2. We obtained a maximum two-point lod score of 6.97 at theta = 0 with the marker D2S389; multipoint linkage analysis yielded a maximum lod score of 7.86 with the marker D2S311. Haplotype analysis established a minimum candidate interval of approximately 25 cM.
Asunto(s)
Cromosomas Humanos Par 2 , Hipertensión Pulmonar/genética , Centrómero , Mapeo Cromosómico , Femenino , Ligamiento Genético , Haplotipos , Humanos , Masculino , National Institutes of Health (U.S.) , Linaje , Sistema de Registros , Estados UnidosRESUMEN
Four patients who received bone marrow transplants were studied sequentially during the posttransplant period to define the pattern of recovering lymphoid cell types. Three patients received T cell-depleted, HLA-matched marrow, and one received untreated marrow from an identical twin. Blood lymphoid cells were labeled with 25 different pairs of monoclonal antibodies. In each sample, one antibody was conjugated to fluorescein and one to phycoerythrin, thus allowing simultaneous assessment of the expression of the two markers using the fluorescence activated cell sorter. A total of 14 antibodies were used, routinely including HLE, Leu-M3, Leu-4, Leu-1, Leu-5, Leu-9, Leu-6, Leu-2, Leu-3, HLA-DR, Leu-7, Leu-11, Leu-15, and Leu-12. Other antibodies were used to further define some populations. This study has allowed us to define six distinct cell types that have appeared in all four patients by day 90 posttransplantation, and which account for 90-100% of all circulating lymphoid cells. These cell types are (a) T helper cells expressing Leu-1, Leu-4, Leu-9, Leu-5, Leu-3, and variable amounts of HLA-DR; (b) T suppressor cells expressing Leu-1, Leu-4, Leu-9, Leu-5, Leu-2, and variable amounts of HLA-DR; (c) B cells expressing Leu-12, B1, HLA-DR, IgD, and IgM, but none of the T cell antigens; (d) an unusual B cell phenotype (Leu-1 B) expressing all of the B cell markers, and also having low amounts of Leu-1, but none of the other T cell antigens; (e) natural killer (NK) cells expressing Leu-11, Leu-15, Leu-5 but none of the other T cell or B cell markers; (f) NK cells expressing Leu-11, Leu-15, Leu-5, and low levels of Leu-2. Both NK types also express Leu-7 on some, but not all cells. The relative frequencies of these cell types varied among the patients and with time, but the striking findings were the presence of relatively few mature T cells, large numbers of NK cells, and the preponderance of the unusual Leu-1 B cell over conventional B cells in all three patients who developed B cells. Sorting experiments confirmed the NK activity of the major NK cell phenotypes, and DNA analysis confirmed that all of the cells studied were of donor origin. In addition, analysis of Ig genes in one patient showed that the Leu-1 B cells were not clonally rearranged.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Trasplante de Médula Ósea , Linfocitos/inmunología , Adolescente , Adulto , Antígenos de Diferenciación de Linfocitos T , Antígenos de Superficie/inmunología , Linfocitos B/inmunología , Recuento de Células , Preescolar , ADN/genética , Femenino , Humanos , Células Asesinas Naturales/inmunología , Leucemia/terapia , Linfocitos/clasificación , Masculino , Fenotipo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Various immune responses have been described in epileptic patients and animal models of epilepsy, but immune responses in brain after a single seizure are poorly understood. We studied immune responses in brain after a single brief generalized tonic-clonic seizure in mice. C57bl/6 mice, either unanesthetized or anesthetized (pentobarbital, ethyl chloride) received either electrical (15-30 mA, 100 Hz, 1s) or sham stimulation (subcutaneous electrodes over frontal lobe, no current). Electrical stimulation of unanesthetized mice resulted in tonic-clonic convulsions with hind-limb extension (maximal seizure), tonic-clonic convulsions without hind-limb extension (submaximal seizure), or no seizure. In contrast, such stimulation of anesthetized mice did not result in seizure. Mice were killed at 1h-7 days after seizure. Brains or regions dissected from brain (neocortex, hippocampus, midbrain, cerebellum) of each group were pooled, single cell suspensions prepared, and cells separated according to density. CD4(+) (CD3(+)CD45(Hi)) and CD8(+) (CD3(+)CD45(Hi)) T cell and CD45R(+) (CD45(Hi)) B cell numbers were determined by flow cytometry. At 24h after a maximal seizure, CD4(+) and CD8(+) T cells and CD45R(+) B cells appeared in brain, reaching peak numbers at 48 h, but were no longer detected at 7days. CD4(+) T cells and CD45R(+) B cells were preferentially found in neocortex compared with hippocampus, whereas CD8(+) T cells were preferentially found in hippocampus at 24h after a maximal seizure. In contrast, virtually no lymphocytes were detected in brains of unstimulated or sham stimulated mice, unanesthetized stimulated mice after submaximal or no seizure, and anesthetized stimulated mice at 1 h-7 day. Neither Ly6-G+ neutrophils nor erythrocytes were detected in brains of any animals, nor was there any detectable increase of blood-brain barrier permeability by uptake of Evans Blue dye. The results indicate that lymphocyte entry into brain after a single brief seizure is due to a selective process of recruitment into cortical regions.
Asunto(s)
Hipocampo/patología , Linfocitos/fisiología , Neocórtex/patología , Infiltración Neutrófila/fisiología , Convulsiones/patología , Anestesia , Animales , Anticuerpos Monoclonales , Linfocitos B/fisiología , Relación CD4-CD8 , Movimiento Celular , Cerebelo/patología , Colorantes , Electrodos Implantados , Electrochoque , Eritrocitos/fisiología , Azul de Evans , Citometría de Flujo , Hipocampo/inmunología , Masculino , Mesencéfalo/patología , Ratones , Ratones Endogámicos C57BL , Neocórtex/inmunología , Convulsiones/inmunologíaRESUMEN
The effect of previous irradiation on the sensitivity of the glow peaks of LiF:Mg,Ti (TLD-100) is investigated up to levels of dose of 400 Gy in both slow-cooled and naturally cooled materials following the 400°C/1 hour pre-irradiation anneal. It is demonstrated that the naturally cooled samples can be re-used up to accumulated levels of dose of 50 Gy without recalibration. At 400 Gy a significant decrease in sensitivity of approximately 25% is observed for all the glow peaks (excluding peak 3). In slow-cooled materials even 100 Gy does not alter the sensitivity of the material.
Asunto(s)
Dosimetría Termoluminiscente , Titanio , Diseño de Equipo , Fluoruros , Compuestos de Litio , Dosis de RadiaciónRESUMEN
Human factor VIII--von Willebrand factor (vWF) is a large, multimeric glycoprotein that plays a central role in the blood coagulation system, serving both as a carrier for factor VIIIC (antihemophilic factor) and as a major mediator of platelet-vessel wall interaction. Diminished or abnormal vWF activity results in von Willebrand's disease (vWD), a common and complex hereditary bleeding disorder. Overlapping vWF cDNA clones that span 8.2 kilobases of the vWF messenger RNA have been obtained. vWF accounts for approximately 0.3 percent of endothelial cell messenger RNA and was undetectable in several other tissues examined. A large single copy gene for vWF is located on the short arm of chromosome 12 (12p12----12pter). No gross gene rearrangement or deletion was detected in the DNA of two patients with severe vWD.
Asunto(s)
Factores de Coagulación Sanguínea/genética , Cromosomas Humanos 6-12 y X , ADN/aislamiento & purificación , Factor de von Willebrand/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Humanos , ARN MensajeroRESUMEN
The generally mild bleeding disorder of von Willebrand disease is associated with abnormalities of two distinct plasma proteins, the large multimeric von Willebrand factor (vWF), which mediates platelet adhesion, and von Willebrand antigen II (vW AgII), which is of unknown function. The two proteins were found to have a common biosynthetic origin in endothelial cells and megakaryocytes, which explains their simultaneous absence in the severe form of this hereditary disease. Shared amino acid sequences from a 100-kilodalton plasma glycoprotein and from vW AgII are identical to amino acid sequences predicted from a complementary DNA clone encoding the 5' end of vWF. In addition, these proteins have identical molecular weights and immunologic cross reactivities. Monoclonal antibodies prepared against both proteins recognize epitopes on the pro-vWF subunit and on a 100-kilodalton protein that are not present on the mature vWF subunit in endothelial cell lysates. In contrast, polyclonal antibodies against vWF recognize both pro-vWF and vWF subunits. Thus, the 100-kilodalton plasma glycoprotein and vW AgII are identical proteins and represent an extremely large propolypeptide that is first cleaved from pro-vWF during intracellular processing and then released into plasma.
Asunto(s)
Antígenos/metabolismo , Factor de von Willebrand/metabolismo , Secuencia de Aminoácidos , Antígenos/inmunología , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/metabolismo , Endotelio/metabolismo , Humanos , Peso Molecular , Fragmentos de Péptidos/análisis , Precursores de Proteínas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid inhibitor of tissue plasminogen activator (tPA) and urokinase. Clinical studies suggest that PAI-1 may play a crucial role in the regulation of fibrinolysis. A number of factors modulate PAI-1 activity in endothelial cell culture, and the isolation of PAI-1 cDNA now allows study of PAI-1 regulation at the mRNA level. We examined the effect of endothelial cell growth factor (ECGF) and heparin on PAI-1 expression in human umbilical vein endothelial cell (HUVEC) culture. The addition of ECGF and heparin to HUVEC cultures results in a 3-10-fold decrease in the PAI-1 activity secreted into the conditioned media. This effect is mediated at the mRNA level. A decrease in PAI-1 is also seen with higher concentrations of ECGF alone, but is greatly enhanced by the addition of heparin. No significant change in tPA antigen or mRNA levels was observed.
Asunto(s)
Endotelio Vascular/metabolismo , Glicoproteínas/biosíntesis , Sustancias de Crecimiento/farmacología , Heparina/farmacología , Activadores Plasminogénicos/antagonistas & inhibidores , Inactivadores Plasminogénicos , Células Cultivadas , Medios de Cultivo , Sinergismo Farmacológico , Factores de Crecimiento Endotelial , Endotelio Vascular/citología , Humanos , ARN Mensajero/aislamiento & purificación , Activador de Tejido Plasminógeno/metabolismo , Venas Umbilicales/citologíaRESUMEN
Deficiency of adenosine deaminase (ADA) is the cause of an autosomal recessive form of immunodeficiency. We sought to define, at a molecular level, the mutations responsible for ADA deficiency in the cell line GM-1715, derived from an immunodeficient patient. Full-length complementary DNA (cDNA) for ADA was synthesized and cloned from the cell line. Sequence analysis of the clones revealed a point mutation in codon 101 (CGG to CAG) that predicts an amino acid change from arginine to glutamine. Southern blot analysis, based on silent polymorphisms in the cDNA sequence, indicated that only one of the defective alleles of the GM-1715 line had been sequenced. The mutation that was identified appears to be responsible for the loss of function in this allele, since the predicted primary structure of the enzyme is otherwise entirely normal.
Asunto(s)
Adenosina Desaminasa/genética , Síndromes de Inmunodeficiencia/genética , Mutación , Nucleósido Desaminasas/genética , Alelos , Secuencia de Bases , Línea Celular , ADN/análisis , Humanos , Síndromes de Inmunodeficiencia/enzimología , Polimorfismo GenéticoRESUMEN
vWF is a multimeric glycoprotein that serves as the major carrier in plasma of Factor VIII (FVIII). We have used an anti-human vWF MAb W5-6A to investigate the FVIII binding site on vWF. W5-6A inhibited FVIII binding to vWF-coated polystyrene tubes in a concentration-dependent manner with 90% inhibition of FVIII binding at a concentration of 10 micrograms/ml. The W5-6A epitope was identified by screening a vWF fragment library using the bacteriophage expression vector lambda gt11. DNA sequence analysis of 29 immunoreactive phage clones localized the W5-6A epitope to a nonadecapeptide spanning amino acid residues threonine 78 to threonine 96 at the amino-terminus of the mature vWF polypeptide. Purified beta-galactosidase/vWF fusion protein from one of these clones, vWF9, was incubated with radiolabeled W5-6A and caused near complete inhibition of W5-6A binding to vWF. Inhibitory activity was lost after vWF9 trypsinization or reduction and alkylation. These data indicate that (a) the antigenic determinant recognized by W5-6A localizes to a nonadecapeptide at the NH2 terminus of the mature vWF polypeptide, (b) disulfide bonds within vWF9 may be necessary to maintain the structure required for immunoreactivity with W5-6A, and (c) W5-6A recognizes an immunogenic region on vWF that may be at (or near) the major FVIII binding domain.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos , Factor VIII/antagonistas & inhibidores , Factor de von Willebrand/inmunología , Bacteriófago lambda/genética , Unión Competitiva , Western Blotting , ADN Viral/análisis , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Factor VIII/metabolismo , Vectores Genéticos , Humanos , Péptidos/inmunologíaRESUMEN
After successful bone marrow transplantation, patient hematopoietic and lymphoid cells are replaced by cells derived from the donor marrow. To document and characterize successful engraftment, host and donor cells must be distinguished from each other. We have used DNA sequence polymorphism analysis to determine reliably the host or donor origin of posttransplant cell populations. Using a selected panel of six cloned DNA probes and associated sequence polymorphisms, at least one marker capable of distinguishing between a patient and his sibling donor can be detected in over 95% of cases. Posttransplant patient peripheral leukocytes were examined by DNA restriction enzyme digestion and blot hybridization analysis. We have studied 18 patients at times varying from 13 to 1,365 d after marrow transplantation. Mixed lymphohematopoietic chimerism was detected in 3 patients, with full engraftment documented in 15. One patient with severe combined immunodeficiency syndrome was demonstrated to have T cells of purely donor origin, with granulocytes and B cells remaining of host origin. Posttransplant leukemic relapse was studied in one patient and shown to be of host origin. DNA analysis was of particular clinical value in three cases where failure of engraftment or graft loss was suspected. In two of the three cases, full engraftment was demonstrated and in the third mixed lymphohematopoietic chimerism was detected. DNA sequence polymorphism analysis provides a powerful tool for the documentation of engraftment after bone marrow transplantation, for the evaluation of posttransplant lymphoma or leukemic relapse, and for the comprehensive study of mixed hematopoietic and lymphoid chimeric states.
Asunto(s)
Trasplante de Médula Ósea , ADN/genética , Adulto , Secuencia de Bases , Niño , Quimera , Clonación Molecular , Femenino , Rechazo de Injerto , Sistema Hematopoyético/metabolismo , Humanos , Lactante , Tejido Linfoide/metabolismo , Masculino , Polimorfismo GenéticoRESUMEN
Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 +/- 8 micrograms) than littermate controls (70.5 +/- 8 micrograms, P < 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 +/- 8 micrograms) was not significantly different (P = 0.63) than that of control animals receiving saline (37 +/- 1 micrograms), while hydroxyproline content was significantly increased in heterozygote (77 +/- 12 micrograms, P = 0.06) and wild-type (124 +/- 19 micrograms, P < 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.
Asunto(s)
Bleomicina/farmacología , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/genética , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Animales , Femenino , Fibrinólisis/genética , Hidroxiprolina/análisis , Pulmón/química , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Inhibidor 1 de Activador Plasminogénico/fisiologíaRESUMEN
Type IIB von Willebrand Disease (vWD) is characterized by the selective loss of large von Willebrand Factor (vWF) multimers from plasma, presumably due to their increased reactivity with platelets and subsequent clearance from the circulation. Using the PCR, one of a panel of four potential missense mutations was identified in each of the 14 patients studied from 11 unrelated families. None of these substitutions was encountered in a large panel of normal DNAs. These changes all represent C----T transitions at CpG dinucleotides, proposed "hot spots" for mutation in the human genome. The four resulting amino acid substitutions, Arg543----Trp, Arg545----Cys, Val553----Met, and Arg578----Gln, are all clustered within the GpIb binding domain of vWF. Disruption of this latter functional domain may explain the pathogenesis of Type IIB vWD. By sequence polymorphism analysis, the Arg543----Trp substitution was shown to have occurred as at least two independent mutational events. This latter observation, along with the identification of mutations in all 14 patients studied and their localization to the GpIb binding domain, all strongly suggest that these substitutions represent the authentic defects responsible for Type IIB vWD. This panel of mutations may provide a useful diagnostic tool for the majority of patients with Type IIB vWD.
Asunto(s)
Glicoproteínas de Membrana Plaquetaria/metabolismo , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética , Alelos , Secuencia de Bases , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Mutación , Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Unión Proteica , Factor de von Willebrand/metabolismoRESUMEN
Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.
Asunto(s)
Clonación Molecular , ADN/análisis , Endotelio/análisis , Glicoproteínas/genética , Activadores Plasminogénicos/antagonistas & inhibidores , Inactivadores Plasminogénicos , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Codón , Glicoproteínas/análisis , Glicoproteínas/inmunología , Humanos , ARN Mensajero/análisisRESUMEN
Combined Factors V and VIII deficiency is an autosomal recessive bleeding disorder identified in at least 58 families comprising a number of different ethnic groups. Affected patients present with a moderate bleeding tendency and have Factor V and Factor VIII levels in the range of 5-30% of normal. The highest frequency of the mutant gene is found in Jews of Sephardic and Middle Eastern origin living in Israel with an estimated disease frequency of 1:100,000. We sought to identify the gene responsible for combined Factors V and VIII deficiency using a positional cloning approach. Of 14 affected individuals from 8 unrelated Jewish families, 12 were the offspring of first-cousin marriages. After a genome-wide search using 241 highly polymorphic short tandem repeat (STR) markers, 13 of the 14 affected patients were homozygous for two closely linked 18q markers. Patients and all available family members were genotyped for 11 additional STRs spanning approximately 11 cM on the long arm of chromosome 18. Multipoint linkage analysis yielded a maximal log of the odds (LOD) score of 13.22. Haplotype analysis identified a number of recombinant individuals and established a minimum candidate interval of 2.5 cM for the gene responsible for combined Factors V and VIII deficiency. The product of this locus is likely to operate at a common step in the biosynthetic pathway for these two functionally and structurally homologous coagulation proteins. Identification of this gene should provide new insight into the biology of Factor V and Factor VIII production.
Asunto(s)
Cromosomas Humanos Par 18 , Deficiencia del Factor V/genética , Ligamiento Genético , Hemofilia A/genética , Homocigoto , Mapeo Cromosómico/métodos , Deficiencia del Factor V/complicaciones , Marcadores Genéticos , Haplotipos , Hemofilia A/complicaciones , Humanos , Linaje , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator, is an important regulator of the blood fibrinolytic system. Elevated plasma levels of PAI-1 are associated with thrombosis, and high levels of PAI-1 within platelet-rich clots contribute to their resistance to lysis by t-PA. Consequently, strategies aimed at inhibition of PAI-1 may prove clinically useful. This study was designed to test the hypothesis that a 14-amino acid peptide, corresponding to the PAI-1 reactive center loop (residues 333-346), can rapidly inhibit PAI-1 function. PAI-1 (0.7 microM) was incubated with peptide (55 microM) at 37 degrees C. At timed intervals, residual PAI-1 activity was determined by addition of reaction mixture samples to t-PA and chromogenic substrate. The T1/2 of PAI-1 activity in the presence of peptide was 4 +/- 3 min compared to a control T1/2 of 98 +/- 18 min. The peptide also inhibited complex formation between PAI-1 and t-PA as demonstrated by SDS-PAGE analysis. However, the capacity of the peptide to inhibit PAI-1 bound to vitronectin, a plasma protein that stabilizes PAI-1 activity, was markedly attenuated. Finally, the peptide significantly enhanced in vitro lysis of platelet-rich clots and platelet-poor clots containing recombinant PAI-1. These results indicate that a 14-amino acid peptide can rapidly inactivate PAI-1 and accelerate fibrinolysis in vitro. These studies also demonstrate that PAI-1 function can be directly attenuated in a physiologic setting and suggest a novel approach for augmenting fibrinolysis in vivo.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Plaquetas/fisiología , Fragmentos de Péptidos/farmacología , Inhibidor 1 de Activador Plasminogénico/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/fisiología , Unión Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Factores de TiempoRESUMEN
BACKGROUND: The genetic factors responsible for the wide variation in plasma von Willebrand factor (VWF) levels observed among individuals are largely unknown, although these genes are also likely to contribute to variability in the severity of von Willebrand disease (VWD) and other bleeding and thrombotic disorders. We have previously mapped two genes contributing to the regulation of plasma VWF levels in mice (Mvwf1 on chromosome 11 and Mvwf2 on chromosome 6). OBJECTIVE: To identify additional quantitative trait loci (QTL) contributing to the genetic regulation of murine plasma VWF levels. METHODS: To map genetic loci contributing to the > 7-fold difference in plasma VWF levels between two mouse strains (A/J and CASA/RkJ), high-density individual genotyping and R/qtl analyses were applied to a previously generated set of approximately 200 F2 mice obtained from an intercross of these two inbred lines. RESULTS: Genomic loci for two additional candidate VWF modifier genes were identified: Mvwf3 on chromosome 4 and Mvwf4 on chromosome 13. These loci demonstrate primarily epistatic effects when co-inherited with two CASA/RkJ Vwf alleles, although Mvwf4 may also exert a small, independent, additive effect. CONCLUSIONS: Mvwf3 and Mvwf4, combined with the effect of Mvwf2, explain approximately 45% of the genetic variation in plasma VWF level among the A/J and CASA/RkJ strains. Mvwf3 and Mvwf4 exhibit homology of synteny to three human chromosomal segments (on chromosomes 1, 5 and 6) previously reported by the Genetic Analysis of Idiopathic Thrombophilia (GAIT) study, suggesting that orthologs of Mvwf3 and Mvwf4 may also encode important VWF modifier genes in humans.
Asunto(s)
Regulación de la Expresión Génica/genética , Sitios de Carácter Cuantitativo , Factor de von Willebrand/genética , Animales , Cromosomas de los Mamíferos , Patrón de Herencia/genética , Ratones , Ratones Mutantes , Modelos Animales , Factor de von Willebrand/análisisRESUMEN
Thrombus formation is initiated by platelets and leads to cardiovascular, cerebrovascular, and peripheral vascular disease, the leading causes of morbidity and mortality in the Western world. A number of antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use, especially in surgery, is limited by hemorrhage. Here, we describe an antiplatelet agent that can have its activity controlled by a matched antidote. We demonstrate that an RNA aptamer targeting von Willebrand factor (VWF) can potently inhibit VWF-mediated platelet adhesion and aggregation. By targeting this important adhesion step, we show that the aptamer molecule can inhibit platelet aggregation in PFA-100 and ristocetin-induced platelet aggregation assays. Furthermore, we show that a rationally designed antidote molecule can reverse the effects of the aptamer molecule, restoring platelet function quickly and effectively over a clinically relevant period. This aptamer-antidote pair represents a reversible antiplatelet agent inhibiting a platelet specific pathway. Furthermore, it is an important step towards creating safer drugs in clinics through the utilization of an antidote molecule.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Oligonucleótidos/metabolismo , Inhibidores de Agregación Plaquetaria/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Factor de von Willebrand/metabolismo , Aptámeros de Nucleótidos/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Humanos , Oligonucleótidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Pruebas de Función Plaquetaria , Ristocetina/farmacología , Técnica SELEX de Producción de Aptámeros , Trombosis , Factor de von Willebrand/químicaRESUMEN
We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.
Asunto(s)
Bacteriófagos/enzimología , Bacteriófagos/genética , Integrasas/fisiología , Integración Viral/genética , Integración Viral/fisiología , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , ADN/genética , Cartilla de ADN/genética , Expresión Génica , Genes Reporteros , Genoma , Humanos , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Recombinación Genética , Selección Genética , Homología de Secuencia de Ácido NucleicoRESUMEN
BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) is a serpin that has a key role in the control of fibrinolysis through proteinase inhibition. PAI-1 also has a role in regulating cell adhesion processes relevant to tissue remodeling and metastasis; this role is mediated by its binding to the adhesive glycoprotein vitronectin rather than by proteinase inhibition. Active PAI-1 is metastable and spontaneously transforms to an inactive latent conformation. Previous attempts to crystallize the active conformation of PAI-1 have failed. RESULTS: The crystal structure of a stable quadruple mutant of PAI-1(Asn150-->His, Lys154-->Thr, Gln319-->Leu, Met354-->Ile) in its active conformation has been solved at a nominal 3 A resolution. In two of four independent molecules within the crystal, the flexible reactive center loop is unconstrained by crystal-packing contacts and is disordered. In the other two molecules, the reactive center loop forms intimate loop-sheet interactions with neighboring molecules, generating an infinite chain within the crystal. The overall conformation resembles that seen for other active inhibitory serpins. CONCLUSIONS: The structure clarifies the molecular basis of the stabilizing mutations and the reduced affinity of PAI-1, on cleavage or in the latent form, for vitronectin. The infinite chain of linked molecules also suggests a new mechanism for the serpin polymerization associated with certain diseases. The results support the concept that the reactive center loop of an active serpin is flexible and has no defined conformation in the absence of intermolecular contacts. The determination of the structure of the active form constitutes an essential step for the rational design of PAI-1 inhibitors.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Fibrinólisis/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/química , Conformación Proteica , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Mutación/genética , Inhibidor 1 de Activador Plasminogénico/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Inhibidores de Serina Proteinasa/química , Serpinas/química , Vitronectina/metabolismoRESUMEN
UNLABELLED: Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome-wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates. SUMMARY: Background Previous studies identified common variants at the ABO and VWF loci and unknown variants in a chromosome 2q12 linkage interval that contributed to the variation in plasma von Willebrand factor (VWF) levels. Whereas the association with ABO haplotypes can be explained by differential VWF clearance, little is known about the mechanisms underlying the association with VWF single-nucleotide polymorphisms (SNPs) or with variants in the chromosome 2 linkage interval. VWF propeptide (VWFpp) and mature VWF are encoded by the VWF gene and secreted at the same rate, but have different plasma half-lives. Therefore, comparison of VWFpp and VWF association signals can be used to assess whether the variants are primarily affecting synthesis/secretion or clearance. Methods We measured plasma VWFpp levels and performed genome-wide linkage and association studies in 3238 young and healthy individuals for whom VWF levels had been analyzed previously. Results and conclusions Common variants in an intergenic region on chromosome 7q11 were associated with VWFpp levels. We found that ABO serotype-specific SNPs were associated with VWFpp levels in the same direction as for VWF, but with a much lower effect size. Neither the association at VWF nor the linkage on chromosome 2 previously reported for VWF was observed for VWFpp. Taken together, these results suggest that the major genetic factors affecting plasma VWF levels, i.e. variants at ABO, VWF and a locus on chromosome 2, operate primarily through their effects on VWF clearance.