RESUMO
The molecular defect responsible for a dramatic prolongation of all standard clotting tests discovered in a 15-yr-old boy has been identified. Initial investigations revealed the presence of an activated Factor X (Factor Xa) and thrombin inhibitor which copurified with alpha 1-antitrypsin (alpha 1-AT), thereby suggesting the occurrence of an alpha 1-AT variant similar to alpha 1-AT Pittsburgh. This was confirmed by dot-blot analysis and direct sequencing after amplification by the polymerase chain reaction. A G to T transition at nucleotide 10038 results in the substitution of Met to an Arg, converting alpha 1-AT into an Arg-Ser protease inhibitor (serpin) that inhibited thrombin and Factor Xa more effectively than antithrombin III. Surprisingly, there was no bleeding history in the proband. The common mutation Z, which may explain a reduced expression of the allele bearing the Arg 358 Met mutation, was not observed in the propositus' DNA. To exclude the presence of another mutation, the coding regions and intron/exon junctions were sequenced. No other mutation was found. Recently, the patient experienced his first hemorrhagic episode at the age of 17. The level of the abnormal inhibitor had increased twofold 2 mo before. The large decrease in protein C concentration may account for the mild bleeding tendency in this case, despite the presence of the alpha 1-AT Pittsburgh mutation. An abnormal protein C pattern was observed in patient's plasma, suggesting that the circulating deficiency might be due to a deleterious effect of the abnormal inhibitor on both intracellular processing and catabolism of protein C.
Assuntos
Transtornos da Coagulação Sanguínea/genética , Deficiência de Proteína C , alfa 1-Antitripsina/genética , Sequência de Bases , Análise Mutacional de DNA , Inibidores do Fator Xa , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Oligodesoxirribonucleotídeos/química , Sondas de Oligonucleotídeos , Linhagem , Trombina/antagonistas & inibidoresRESUMO
The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.
Assuntos
Alanina/genética , Antitrombina III/genética , Mutação , Adulto , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA/análise , DNA Polimerase I/metabolismo , Eletroforese em Gel de Ágar , Feminino , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVES: The importance of protease-activated receptor-1 (PAR-1) in blood vessel development has been shown in knock-out mice. As endothelial progenitor cells (EPCs) express functional PAR-1, we examined whether PAR-1 stimulation by the peptide SFLLRN interfered with the angiopoietin pathway, that is EPC commitment, proliferation and migration. METHODS AND RESULTS: Given the strong PAR-1 expression on CD34+ cells, we tested the effect of SFLLRN 75 micromol L(-1) on the emergence of EPCs from cord blood. PAR-1 activation did not modify the number of colonies or the day of emergence, in keeping with the lack of induction of angiopoietin 1 gene expression. Conversely, SFLLRN treatment of EPCs induced angiopoietin 2 gene expression and protein synthesis. Experiments with polyclonal blocking antibodies showed that angiopoietin 2 was involved in the proliferative effect of PAR-1 activation. PAR-1 activation also enhanced migration toward angiopoietin 1 in a Boyden chamber assay. CONCLUSIONS: Our study demonstrates that PAR-1-induced proliferation of EPCs involves angiopoietin 2. PAR-1 also enhances EPC migration toward angiopoietin 1. These findings might explain the role of thrombin in neovascularization via the angiopoietin pathway.
Assuntos
Angiopoietina-1/metabolismo , Angiopoietina-2/fisiologia , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptor PAR-1/metabolismo , Angiopoietina-1/fisiologia , Antígenos CD34 , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Endoteliais/citologia , Sangue Fetal/citologia , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Neovascularização Fisiológica , Fragmentos de Peptídeos/farmacologia , Receptor PAR-1/genéticaRESUMO
On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin.
Assuntos
Núcleo Celular/metabolismo , Endotélio Vascular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Trombina/metabolismo , Trombomodulina/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Catálise , Células Cultivadas , DNA/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas/farmacologia , Receptores de Trombina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trombina/genética , Trombina/farmacologia , Trombomodulina/antagonistas & inibidores , Timidina/metabolismoRESUMO
Small amounts of a variant antithrombin (AT) bearing an Arg-129 to Gln mutation were purified from plasma by means of affinity chromatography on insolubilized heparin at very low ionic strength. As a control, two variant antithrombins, one bearing a Pro-41 to Leu mutation and the other an Arg-47 to His mutation, were purified in the same way. The biochemical characterization of the variants and the kinetic study of thrombin and activated factor X (F Xa) inhibition in the presence of heparin and heparin derivatives suggest that Arg-129 plays a specific role in AT conformation and F Xa inhibition enhancement. Indeed, the purified variant adopted the locked conformation described for AT submitted to mild denaturing conditions (Carrell, R.W., Evans, D.Li. and Stein, P.E. (1991) Nature 353, 576-578) and resembling the latent form of plasminogen activator inhibitor (PAI) (Mottonen, J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geoghegan, K.F., Gerard, R.D. and Goldsmith, E.J. (1992) Nature 355, 270-273). Moreover, the mutant AT was partially reactivated by heparin for thrombin inhibition, but did not respond to the specific pentasaccharide domain of heparin for F Xa inhibition.
Assuntos
Antitrombinas/química , Arginina/química , Inibidores do Fator Xa , Heparina/farmacologia , Antitrombinas/genética , Antitrombinas/isolamento & purificação , Heparina/química , Humanos , Cinética , Mutação , Oligossacarídeos/química , Conformação ProteicaRESUMO
We analyzed the distal promoter region of the thrombomodulin (TM) gene (nucleotides -300 to -2052) in subjects from the Paris Thrombosis Study (PATHROS), a French case-control study of venous thrombosis, to identify polymorphisms that might modify TM gene expression. Eight novel mutations were found in the 40 DNA samples initially screened. Two of these mutations (-1748G/C and -1208/-1209 del TT) were frequent. One rare transition (-1166G/A) might have functional consequences owing to its position. These 3 mutations were screened for in the entire study population of 327 patients and 398 controls. None of the 3 was significantly associated with thrombosis. Interestingly, the -1208/-1209 TT deletion was associated with varicose veins in the patients. This mutation was in tight linkage disequilibrium with the +1418 C/T change in the coding sequence, a known polymorphism that predicts an Ala 455 Val substitution in the sixth epidermal growth factor-like TM module, a domain previously implicated in the proliferative functions of TM. This linkage suggests that the Ala 455 Val mutation may promote changes in these functions and thus be involved in varicose vein formation.
Assuntos
Regiões Promotoras Genéticas/genética , Trombomodulina/genética , Varizes/genética , Trombose Venosa/genética , Adulto , Alelos , DNA/química , DNA/genética , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Protrombina/metabolismo , Trombomodulina/metabolismoRESUMO
"Ce qui a peut-être transformé le plus profondément l'étude desêtres vivants, c'est l'accsà l'analyse d'objets nouveaux," or in other words, "That which had the greatest impact on the study of living subjects is access to the analysis of new objects" (François Jacob, 1965 Nobel Prize of Medicine). In the 20th century, it is recombinant-DNA technology that has opened a new chapter in the history of medicine, genetic medicine, because it has rendered the human genome accessible to analysis. The cardiovascular field has been rather slow to integrate the concepts and the tools of molecular biology and molecular genetics; however, this barrier has now been overcome, and it is an exciting time for the field. This article focuses on studies that are conducted in France in the context of national multidisciplinary INSERM networks and that deal with two types of hereditary forms of cardiovascular diseases, cardiomyopathies, and hereditary thrombophilia. © 1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:207-210).
RESUMO
The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Both PC and PS deficiencies have been implicated in thrombophilia. The molecular basis for hereditary PC and PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele. A small subset of patients who are homozygous or compound heterozygous for a PC gene mutation have severe thrombotic complications at birth, whereas onset occurs later in the other cases. Patients heterozygous for a PC or PS gene abnormality may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. A PC or PS gene defect is associated with the factor V Arg 506 to Gln mutation in 10% to 30% of symptomatic patients, suggesting that clinical expression is controlled by several genes in heterozygous patients.
Assuntos
Deficiência de Proteína C , Proteína C/genética , Deficiência de Proteína S/genética , Mapeamento Cromossômico , Genes/genética , Genes/fisiologia , Humanos , Mutação Puntual/genética , Mutação Puntual/fisiologia , Proteína C/metabolismo , Deficiência de Proteína S/metabolismo , Trombose/genética , Trombose/fisiopatologiaRESUMO
Protein C (PC) is a vitamin K-dependent zymogen that inactivates factors Va and VIIIa after its activation by thrombin complexed to thrombomodulin. We characterized a monoclonal antibody (mAb) against PC, whose only influence on PC functions was to inhibit PC activation by the thrombin-thrombomodulin complex. It recognized an epitope in the PC heavy chain, the conformation of which is calcium-dependent. The mAb did not recognize a natural PC variant that was not activated by the thrombin-thrombomodulin complex (mutation R229Q) and did recognize a synthetic peptide corresponding to PC amino acids 225-235 in an Elisa assay. The peptide inhibited PC activation by the thrombin-thrombomodulin complex. These data confirm that the calcium-binding loop of the serine-protease domain is involved in the interaction of PC with the thrombin-thrombomodulin complex.
Assuntos
Proteína C/química , Trombina/metabolismo , Trombomodulina/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Cálcio/metabolismo , Ativação Enzimática , Epitopos/imunologia , Heterozigoto , Humanos , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Proteína C/genética , Proteína C/imunologia , Proteína C/metabolismo , Conformação ProteicaRESUMO
We used an antithrombin autoantibody (IgG D), the epitope of which encompasses ABE1 and amino acids located within variable region 1, to study thrombin interactions with R358 alpha 1-AT and protein C. IgG D inhibited the thrombin interaction with R358 alpha 1-AT, while hirugen had no effect, indicating that the interaction of R358 alpha 1-AT with thrombin may involve the VR1 subsite. We also obtained evidence that VR1 may be involved in the activation of protein C by thrombin in the absence of thrombomodulin. Moreover, IgG D attenuated the inhibitory effect of calcium ions during protein C activation by thrombin, probably by masking E39 within the VR1 site.
Assuntos
Variação Genética , Mutação Puntual , Proteína C/metabolismo , Trombina/genética , Trombina/metabolismo , alfa 1-Antitripsina/metabolismo , Sequência de Aminoácidos , Hirudinas/química , Hirudinas/farmacologia , Humanos , Imunoglobulina G/farmacologia , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Especificidade por SubstratoRESUMO
A monoclonal antibody (mAb) binding to protein C (PC) heavy chain but not to activated PC was found to inhibit PC activation by free thrombin, suggesting that epitope involved the activation site. Using a set of overlapping synthetic peptides, we confirmed that this mAb recognizes the sequence encompassing the thrombin cleavage site (165QVDPRLI(171)). Surprisingly, epitope was only accessible in the absence of calcium, half-maximal inhibition of mAb binding occurring at 100 microM Ca2+. Thus, our antibody provides direct evidence that conformation and/or accessibility of the activation site differ between the apo and Ca2+-stabilized conformers of PC.
Assuntos
Especificidade de Anticorpos/efeitos dos fármacos , Cálcio/farmacologia , Epitopos , Proteína C/imunologia , Animais , Anticorpos Monoclonais , Ativação Enzimática , Mapeamento de Epitopos , Humanos , Camundongos , Conformação ProteicaRESUMO
BACKGROUND: The FIIG20210A polymorphism has been associated with arterial wall thickness and atherothrombotic diseases in selected subgroups. The FVArg506Gln polymorphism does not seem to be associated with arterial diseases. Few data are available on these polymorphisms and the risk of peripheral arterial disease (PAD). OBJECTIVES: To study the association between the FIIG20210A and FVArg506Gln polymorphisms and PAD and its clinical severity. To examine the potential interactions with traditional vascular risk factors. PATIENTS AND METHODS: We studied 184 consecutive male patients under 70 years of age with symptomatic PAD and 330 age-matched male controls free of symptomatic PAD and with no cardiovascular history. We evaluated the FIIG20210A and FVArg506Gln polymorphisms in all subjects. RESULTS: Mean age was 57.1 +/- 7.2 years (cases) and 56.7 +/- 7.6 years (controls). The FII20210A allele was more frequent in PAD patients with odds ratios (OR) of 3.77 (1.39-10.2) in univariate analysis and 4.30 (1.3-14.7) after adjustment for diabetes, smoking, hypertension and hypercholesterolemia. In smokers or past smokers the magnitude of the association was markedly increased but there was no evidence of an interaction between tobacco exposure and FIIG20210A. In case subjects, the FII20210A allele was also associated with critical ischemia [OR = 4.1 (1.1-15.7), P = 0.039 in multivariate analysis]. FVArg506Gln was not associated with PAD [OR = 0.65 (0.27-1.54) and 0.77 (0.28-2.1) in univariate and multivariate analyses, respectively]. CONCLUSIONS: The FIIG20210A gene polymorphism may be a risk factor for PAD and its severity. In contrast, the FVArg506Gln polymorphism is not associated with PAD.
Assuntos
Artérias/patologia , Fator V/genética , Doenças Vasculares Periféricas/genética , Polimorfismo Genético , Protrombina/genética , Idoso , Alelos , Estudos de Casos e Controles , Diabetes Mellitus/patologia , Genótipo , Humanos , Hipercolesterolemia/genética , Hipertensão/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Fatores de Risco , FumarRESUMO
Oral contraceptive (OC) use is associated with an increased risk of venous thromboembolism. Previous data reported higher thrombotic risk in women using third-generation combined OC than in those using second generation OC. The difference could be explained by differential effects of progestagens on plasma sensitivity to activated protein C (APC). The main purpose of this cross-sectional study was to assess the influence of a progestagen-only OC (chlormadinone acetate) as well as the effect of several combined OC with different progestagen components on APC resistance. The effect of APC on endogenous thrombin potential (ETP) was investigated in the plasma of healthy women using either combined OC (n=82) or progestagen-only OC (n=28), and in non-users (n=64). Carriers of factor V Leiden were excluded. Compared with non-users, there was no significant change in APC resistance in women using progestagen-only OC. Women who used combined OC were less sensitive to APC than non-users (P < 0.001) and the difference was significantly more pronounced in women using third-generation OC (n=41) than in those who used second-generation OC containing levonorgestrel (n=22) (P < 0.05). Compared with OC containing levonorgestrel, use of norethisterone-containing OC (n = 9) was associated with an increased resistance to APC (P < 0.05). Women who used cyproterone-containing OC (n = 10) were less sensitive to APC than those using third-generation OC (P < 0.05) or second-generation OC containing levonorgestrel (P < 0.05). Protein S, factor II and FVIII levels explained in part the OC-related changes in APC sensitivity variations. ETP-based APC resistance may contribute to explain why different brands of OC can be associated with different levels of thrombogenicity.
Assuntos
Resistência à Proteína C Ativada/induzido quimicamente , Anticoncepcionais Orais/efeitos adversos , Congêneres da Progesterona/efeitos adversos , Resistência à Proteína C Ativada/sangue , Adulto , Acetato de Clormadinona/efeitos adversos , Anticoncepcionais Orais Combinados/efeitos adversos , Anticoncepcionais Orais Sintéticos/efeitos adversos , Estudos Transversais , Terapia de Reposição de Estrogênios/efeitos adversos , Feminino , Humanos , Pessoa de Meia-Idade , Tromboembolia/induzido quimicamente , Trombose Venosa/induzido quimicamenteRESUMO
Heparin-induced thrombocytopenia (HIT) occurs in nearly 3% of patients treated with heparin after cardiopulmonary bypass (CPB). HIT carries a risk of severe thrombotic complications, and must be diagnosed rapidly. To identify simple criteria for estimating the probability of HIT after CPB, we retrospectively analyzed the files of 84 patients with suspected HIT after CPB and we analyzed the usefulness of several variables collected at the time of HIT suspicion to estimate HIT probability. HIT was confirmed in 35 cases and ruled out in 49 cases, on the basis of a platelet increment after heparin withdrawal, detection of heparin-dependent antibodies, and absence of other clear cause of thrombocytopenia. A biphasic platelet count from CPB to the first day of suspected HIT, an interval of >/= 5 days from CPB to the first day of suspected HIT, and a CPB duration of = 118 min were independent risk factors for HIT. These variables were combined to create a post-CPB HIT probability score. The score correctly identified 34/35 HIT patients and 28/49-non-HIT patients. This score, which can be applied as soon as HIT is suspected after CPB, has very good negative predictive value (97%). Prospective studies are required to confirm these findings.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Heparina/efeitos adversos , Valor Preditivo dos Testes , Probabilidade , Trombocitopenia/induzido quimicamente , Trombocitopenia/diagnóstico , Idoso , Anticorpos/sangue , Diagnóstico Diferencial , Feminino , Heparina/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Estudos Retrospectivos , Fatores de RiscoRESUMO
PURPOSE: Factor V Leiden and factor II 20210A are inherited disorders of the clotting system that occur frequently in patients with deep vein thrombosis. We conducted this study to determine whether these factors are also common in patients with pulmonary embolism. SUBJECTS AND METHODS: We determined the prevalence of factor V Leiden and factor II 20210A in 773 consecutive patients with objectively documented symptomatic deep vein thrombosis or symptomatic pulmonary embolism, or with a combination of these disorders. RESULTS: Isolated symptomatic deep vein thrombosis occurred in 345 patients; isolated symptomatic pulmonary embolism occurred in 236; and both anomalies occurred in 192. Factor V Leiden was present in 21 (9%) of the patients with isolated symptomatic pulmonary embolism, in 30 (16%) with both manifestations, and in 63 (18%) with isolated symptomatic deep vein thrombosis (P = 0.007). Factor V Leiden was more common among patients with deep vein thrombosis (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.2 to 3.7; P = 0.006) or both pulmonary embolism and deep vein thrombosis (OR = 1.8; 95% CI: 1.0 to 3.3; P = 0.07) than among patients with isolated pulmonary embolism. Factor V Leiden was less common in massive pulmonary embolism (5% [7 of 127]) than in submassive pulmonary embolism (13% [21 of 155], P = 0.03). We found no significant difference in the prevalence of factor II 20210A among the three groups. CONCLUSION: Factors V Leiden and II 20210A vary in prevalence among patients with pulmonary embolism and deep vein thrombosis, suggesting that the risk of pulmonary embolization may vary among patients who have different causes of venous thromboses.
Assuntos
Fator V/metabolismo , Protrombina/metabolismo , Embolia Pulmonar/sangue , Trombose Venosa/sangue , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Embolia Pulmonar/complicações , Trombose Venosa/complicaçõesRESUMO
The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Patients with phenotypic PS deficiency may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. The molecular basis for hereditary PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele.
Assuntos
Deficiência de Proteína S/fisiopatologia , Proteína S/fisiologia , Anticoagulantes/metabolismo , Humanos , Fenótipo , Proteína C/fisiologia , Deficiência de Proteína S/genética , Trombose/genéticaRESUMO
The mutations observed in patients with antithrombin and protein C deficiencies are mostly substitutions of one nucleotide, or deletions/insertions of fewer than 10 nucleotides in the exons and intron-exon junctions. These genomic abnormalities result in missense changes (involving aminoacids important for protein folding), aberrant polypeptide chains and/or premature termination codons, or abnormal splicing precluding DNA transcription. The number of mutations so far identified is such that it is difficult to use genomic DNA analysis for diagnostic purpose. However, identification of the gene defect can be useful in well-defined situations, such as the risk of homozygosity, and complex or ambiguous plasma phenotypes, which frequently occur in protein C deficiency. Protein S deficiency, the molecular bases of which have been less extensively studied, is due to micromodifications of the coding sequence in only half the cases investigated so far. The mechanisms involved in the remaining cases remain to be identified.
Assuntos
Antitrombina III/genética , Mutação , Proteína C/genética , Deficiência de Proteína S/genética , Proteína S/genética , Alelos , Deficiência de Antitrombina III , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 3 , Bases de Dados Factuais , Genes , Humanos , Mutação Puntual , Deficiência de Proteína C , Deleção de SequênciaRESUMO
In a series of 16 propositi with symptomatic protein S deficiency and a protein S gene mutation, we identified a sporadic case of a novel mutation that probably affects gene expression. The mutation, a G to C transversion leading to the substitution of Ala 484 by Pro, was not found in the protein S gene of the patient's parents. Transmission of the paternal and maternal protein S alleles was apparently normal, on the basis of the frequent polymorphism in exon XV. We also checked the transmission of chromosomal material by analysing protein C gene polymorphisms, beta-globin gene frameworks and four variable number of tandem repeats (VNTRs). By combining the results of these analyses, we were able to rule out nonpaternity and to confirm the de novo nature of the mutation.
Assuntos
Deficiência de Proteína S/genética , Proteína S/genética , Alanina/genética , Alelos , Animais , Bovinos , Humanos , Camundongos , Mutação Puntual , Polimorfismo Genético , Prolina/genética , Proteína C/genética , RatosRESUMO
Immunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 10(9) platelets with a mean value of 70.3 +/- 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.
Assuntos
Antitrombina III/sangue , Plaquetas/metabolismo , Antitrombina III/imunologia , Antitrombina III/metabolismo , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Plaquetas/ultraestrutura , Fibrinogênio/sangue , Fibrinogênio/metabolismo , Humanos , Técnicas Imunoenzimáticas , Microscopia Eletrônica , Taxa Secretória/efeitos dos fármacosRESUMO
The association of two missense mutations, a Leu 223 to Phe and an Ile 403 to Met, is described in a family presenting with various protein C deficiency phenotypes. In this family, two subjects were compound heterozygotes with protein C levels of about 25%, the other members being heterozygous for only one of the mutations. The Leu 223 to Phe mutation was also found in 9 members of 3 other families and, in all cases but one, resulted in protein C levels below 60% associated with a high incidence of thrombotic complications. The other mutation, an Ile 403 to Met, was identified in those of the family' members who presented with borderline protein C concentrations. In such a family, the genomic DNA analysis represents the only way to differentiate between the genetic status of each family member. The results highlight the importance of the genotype determination and the poor discriminative power of the plasma assays currently used.