RESUMO
The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.
Assuntos
Colágeno/farmacologia , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Velocidade do Fluxo Sanguíneo , Humanos , Fragmentos Fab das Imunoglobulinas , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Trombina/farmacologiaRESUMO
We have demonstrated that the cross-linking of ribosomal protein S19 (RP S19) on platelets by activated factor XIII provides chemotactic potency to monocytes/macrophages for a resolution of coagulum. Factor XIII is activated by an active form of prothrombin, thrombin. We here report that RP S19 is present as a complex with prothrombin in the blood stream. Formation of this complex was blocked by a mutation of the glycosaminoglycan-binding basic cluster (Lys(23) -Lys(29) ) in RP S19. Prothrombin-RP S19 interaction was enhanced by an absence of Ca(2+) and the plasma RP S19 concentration was significantly low in the patient treated with warfarin, indicating participation of the γ-carboxyl glutamic acid domain of prothrombin making a salt bridge with the basic cluster. The complex formation likely explains why a protein as small as RP S19 can prevent from a filtering system of renal glomeruli at a steady state. The translocation of RP S19 from prothrombin to platelets during blood coagulation seems to be also advantageous for RP S19 from the perspective of oligomerisation by activated factor XIII, which should have been activated by thrombin.
Assuntos
Protrombina/metabolismo , Proteínas Ribossômicas/sangue , Sequência de Aminoácidos , Anticoagulantes/farmacologia , Sítios de Ligação , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Cálcio/sangue , Reagentes de Ligações Cruzadas , Fator X/metabolismo , Fator XIIIa/metabolismo , Humanos , Técnicas In Vitro , Complexos Multiproteicos/sangue , Complexos Multiproteicos/química , Estrutura Terciária de Proteína , Protrombina/química , Protrombina/genética , Proteínas Ribossômicas/química , Varfarina/farmacologiaRESUMO
INTRODUCTION: Increased plasma levels of von Willebrand factor (VWF) have been reported in acute myocardial infarction (AMI). Recently, we showed reduced activity of a VWF-cleaving protease (ADAMTS13) in AMI patients. However, there is no information as to whether ADAMTS13 affects the pathogenesis of unstable angina (UA). Thus, the purpose of this study was to examine changes in plasma VWF and ADAMTS13 levels in UA patients. MATERIALS AND METHODS: Plasma VWF and ADAMTS13 levels (mU/ml) were measured in 45 patients with UA, 55 with stable exertional angina (SEA) and 47 with chest pain syndrome (CPS) at the time of coronary angiography. Levels were also measured in 15 UA patients after 6 months of follow-up. RESULTS: VWF antigen levels (mU/ml) increased significantly in UA patients compared with SEA or CPS (2129.3+/-739.5, 1571.8+/-494.2 and 1569.5+/-487.0, respectively; P < 0.0001 in UA vs. SEA or CPS). ADAMTS13 antigen levels (mU/ml) were significantly lower in UA patients than SEA or CPS (737.3+/-149.5, 875.3+/-229.0 and 867.7+/-195.5, respectively; P < 0.01 in UA vs. SEA or CPS). Furthermore, there was a significant inverse correlation between VWF and ADAMTS13 antigen levels (r = -0.302, P = 0.0002). The antigen levels at 6 months of follow-up were not different compared to the acute phase in the 15 UA patients that had repeated blood sampling. CONCLUSIONS: These findings suggest that there is prolonged thrombogenicity in UA patients represented as an imbalance between VWF and ADAMTS13 activity.
Assuntos
Proteínas ADAM/sangue , Angina Instável/sangue , Angina Instável/enzimologia , Fator de von Willebrand/metabolismo , Proteína ADAMTS13 , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Angina Pectoris/sangue , Angina Pectoris/enzimologia , Estudos de Casos e Controles , Dor no Peito/sangue , Dor no Peito/enzimologia , Doença Crônica , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , SíndromeRESUMO
Von Willebrand factor (VWF), a cofactor in platelet adhesion and aggregation, increases hemostasis and thrombosis. Recently, a metalloprotease that cleaves VWF multimers has been identified, namely ADAMTS13. The aim of this study was to investigate the relation between serial changes in plasma VWF and ADAMTS13 and the prognosis after acute myocardial infarction (AMI). We measured serial changes of plasma VWF and ADAMTS13 antigen levels in 92 patients with AMI and 40 control subjects. VWF levels were significantly higher in patients with AMI compared with controls (p <0.01) on admission, peaked 3 days after admission, and remained high for 14 days. In contrast, on admission, ADAMTS13 levels were significantly lower in patients with AMI compared with controls (p <0.0001), with minimum antigen levels reached after 3 days, and remained lower for 14 days. The ratio of VWF/ADAMTS13 antigen levels was higher in patients with AMI compared with controls throughout the time course. Cox hazards analysis revealed that the early increase of VWF and VWF/ADAMTS13 ratio levels and the early decrease of ADAMTS13 levels were significant predictors of future thrombotic events during the 1-year follow-up period. Kaplan-Meier analysis demonstrated that patients with major decreases of ADAMTS13 levels and high increases of VWF/ADAMTS13 levels had significantly greater probabilities for development of thrombotic events (p = 0.0104 and 0.0209, respectively). In conclusion, these findings suggest that monitoring the changes of VWF and ADAMTS13 antigen levels in the early phase might be valuable for predicting and preventing thrombosis during 1-year follow-up in patients with AMI.
Assuntos
Proteínas ADAM/sangue , Infarto do Miocárdio/sangue , Fator de von Willebrand/análise , Proteína ADAMTS13 , Adulto , Idoso , Idoso de 80 Anos ou mais , Angina Instável/etiologia , Anticoagulantes/uso terapêutico , Aspirina/uso terapêutico , Baixo Débito Cardíaco/etiologia , Estudos de Casos e Controles , Feminino , Seguimentos , Previsões , Heparina/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/tratamento farmacológico , Admissão do Paciente , Readmissão do Paciente , Inibidores da Agregação Plaquetária/uso terapêutico , Prognóstico , Trombose/etiologiaRESUMO
ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.
Assuntos
Proteínas ADAM/sangue , Proteínas ADAM/imunologia , Proteína ADAMTS13 , Western Blotting , Meios de Cultivo Condicionados , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Imunoprecipitação , Testes de NeutralizaçãoRESUMO
The presence of unusually large multimers of von Willebrand factor (VWF) is thought to be a major pathogenic factor for thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is a protease that regulates the multimeric size and function of VWF by cleaving VWF. Hence, congenital or acquired deficiency of ADAMTS13 causes life-threatening illness of TTP. Mutations in the ADAMTS13 gene cause inherited TTP, and the development of autoantibodies that inhibit ADAMTS13 activity frequently are associated with acquired TTP. ADAMTS13 consists of 1,427 amino acid residues and is composed of multiple structural and functional domains, containing a signal peptide, a propeptide, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (Tsp1) motif, a cysteine-rich domain, a spacer domain, seven additional Tsp1 repeats, and two CUB domains. In particular, the cysteine-rich/spacer domains are essential for VWF cleavage and are the principal epitopes recognized by autoantibodies in patients with acquired TTP. Therefore, it is likely that these domains are involved in the recognition and binding of ADAMTS13 to VWF. ADAMTS13 circulates in the blood in an active state, and efficiently cleaves unfold form of VWF induced under shear stress caused by blood flow, preventing the accumulation of pathogenic unusually large VWF multimers (ULVWF). Thus, ADAMTS13 helps maintain vascular homeostasis by preventing the excess thrombus formation.
Assuntos
Homeostase/fisiologia , Metaloendopeptidases/metabolismo , Púrpura Trombocitopênica Trombótica/metabolismo , Fator de von Willebrand/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Animais , Homeostase/genética , Humanos , Metaloendopeptidases/genética , Púrpura Trombocitopênica Trombótica/genética , Fator de von Willebrand/genéticaRESUMO
The newly identified platelet collagen receptor glycoprotein VI binds to fibrous collagen, inducing platelet activation. Several antibodies against GPVI have been reported, including a patient's auto-antibodies, that activates platelets through their ability to crosslink this glycoprotein. We have developed a monoclonal antibody (mAb) against GPVI using the recombinant extracellular domain of GPVI as an antigen. This antibody, mAb 204-11, induced platelet aggregation and tyrosine phosphorylation of proteins similar to those induced by GPVI-reactive proteins, collagen and convulxin. Its interaction with GPVI was analyzed by measuring the effect of the antibody on GPVI binding to collagen using a dimeric form of recombinant GPVI, GPVI-Fc2. MAb 204-11 inhibited the binding of GPVI-Fc2 to fibrous collagen particles, but enhanced the GPVI binding to immobilized collagen, suggesting that the antibody binds to a region near the collagen binding site of GPVI. MAb 204-11 also inhibited the GPVI binding to convulxin at a low concentration, but not completely. Since mAb 204-11 reacts specifically with GPVI and is applicable for immunoblotting and immunoprecipitation, this antibody would be useful for studies on GPVI.
Assuntos
Anticorpos Monoclonais/farmacologia , Colágeno/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Reações Antígeno-Anticorpo , Sítios de Ligação , Colágeno/química , Epitopos , Fragmentos Fc das Imunoglobulinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Glicoproteínas da Membrana de Plaquetas/imunologia , Ligação Proteica/efeitos dos fármacos , Proteínas RecombinantesRESUMO
We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.
Assuntos
Endopeptidases/química , Precursores Enzimáticos/metabolismo , Protrombina/metabolismo , Proteínas Recombinantes/química , Actinas/metabolismo , Animais , Biotecnologia/métodos , Western Blotting , Células CHO , Bovinos , Linhagem Celular , Galinhas , Cromatografia de Afinidade , Cricetinae , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Precursores Enzimáticos/isolamento & purificação , Vetores Genéticos , Humanos , Cinética , Metotrexato/farmacologia , Camundongos , Mieloma Múltiplo/metabolismo , Mutação , Plasmídeos/metabolismo , Agregação Plaquetária , Regiões Promotoras Genéticas , Protrombina/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Sefarose/química , Tetra-Hidrofolato Desidrogenase/genética , Trombina/metabolismo , Fatores de Tempo , TransfecçãoAssuntos
Proteínas ADAM/sangue , Proteínas ADAM/genética , Western Blotting/métodos , Púrpura Trombocitopênica Trombótica/sangue , Púrpura Trombocitopênica Trombótica/genética , Proteína ADAMTS13 , Adulto , Estudos de Casos e Controles , Cromossomos Humanos Par 9 , Ensaio de Imunoadsorção Enzimática , Feminino , Genes Recessivos , Triagem de Portadores Genéticos , Heterozigoto , Humanos , Masculino , Mutação , Púrpura Trombocitopênica Trombótica/diagnósticoRESUMO
Antithrombin (AT) inhibits several blood coagulation proteases, including activated factor X (FXa), by forming stable complexes with these proteases. Herein, we demonstrate that AT forms a stable complex with zymogen factor X (FX). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography analyses showed that AT and FX formed an SDS-stable complex, which is distinct in apparent molecular mass from an FXa-AT complex, in the presence of heparin. Amino-terminal sequence analysis of the complex following SDS-PAGE under reducing conditions provided clear evidence that AT forms this complex with the heavy chain of FX, because two sequences, HGSPVDI (residues 1-7 of AT) and SVAQATS (residues 1-7 of the heavy chain of FX), were identified. Furthermore, sequence SLNPNRV, which corresponds to residues 394-400 of AT, was identified in the non-reduced FX-AT complex, indicating that FX cleaved the Arg393-Ser394 bond in a reactive centre loop of AT. Unfractionated heparin induced FX-AT complex formation more effectively than low-molecular weight heparin or AT-binding pentasaccharide, and appeared to promote complex formation mainly via a template effect. These data suggest that AT is capable of forming a stable complex with zymogen FX by acting as an inhibitor in the presence of heparin.
Assuntos
Antitrombinas/química , Precursores Enzimáticos/química , Fator X/metabolismo , Heparina/farmacologia , Inibidores de Serina Proteinase/química , Antitrombinas/farmacologia , Arginina/química , Arginina/metabolismo , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/metabolismo , Fator X/antagonistas & inibidores , Fator X/química , Heparina/metabolismo , Humanos , Serina/química , Serina/metabolismo , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
Plaque disruption with subsequent thrombus formation is a major cause of atherothrombotic diseases and von Willebrand factor (VWF), which is cleaved by ADAMTS-13, plays a critical role in thrombus formation. However, the role of ADAMTS-13 during thrombogenesis on atherosclerotic vessel remains unknown. We examined the localization of ADAMTS-13 in coronary thrombi obtained from patients with acute myocardial infarction. We also investigated the roles of ADAMTS-13 in thrombus formation using type I collagen-coated flow chambers (100S(-1) and 1500S(-1)) and on injured neointima of rabbit femoral arteries. ADAMTS-13 was present in thrombi of human coronary arteries, where it co-localized with VWF. In a flow chamber, both the average of the surface covered by platelet adhesion and the long axes of platelet thrombi were significantly augmented by an antibody to the ADAMTS-13 disintegrin-like domain (WH2-22-1A) at a shear rate of 1500s(-1), but not by an antibody to the ADAMTS-13 thrombospondin 1-3 domain (WH10). WH2-22-1A also reduced the activity of plasma ADAMTS-13 to cleave large VWF multimers during perfusion. Thrombi on injured neointima were induced by repeated balloon injury of rabbit femoral arteries, and were composed of platelet and fibrin, like human coronary thrombi. WH2-22-1A significantly augmented thrombus formation on injured neointima. These results suggest that the disintegrin-like domain of ADAMTS-13 functions in attenuating thrombus growth on diseased arteries exposed to a high shear rate.
Assuntos
Proteínas ADAM/metabolismo , Aterosclerose/patologia , Colágeno Tipo I/metabolismo , Vasos Coronários/patologia , Regulação da Expressão Gênica , Trombose/patologia , Proteína ADAMTS13 , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adesividade Plaquetária , Coelhos , Trombospondinas/química , Fator de von Willebrand/metabolismoRESUMO
Plasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (> 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood ( approximately 5 x 10(4) platelets/microL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)-labeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.
Assuntos
Plaquetas/fisiologia , Adesão Celular/fisiologia , Trombose/fisiopatologia , Fator de von Willebrand/farmacologia , Anticoagulantes/farmacologia , Remoção de Componentes Sanguíneos , Plaquetas/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Colágeno , Fator VIII/farmacologia , Humanos , Contagem de PlaquetasRESUMO
To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. The k(cat)/K(m) value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mm(-)1s(-)1) was 3-fold higher than that of wild-type VIIa (30.3 mm(-)1 s(-)1) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K(m) value but not to an increase in the k(cat) value. On the other hand, the k(cat)/K(m) value for S-2288 hydrolysis by VIIa-39 (17.9 mm(-)1 s(-)1) was 18-fold higher than that of wild-type (1.0 mm(-)1 s(-)1) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K(m) value but also to an increase in the k(cat) value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. (2001) J. Biol. Chem. 276, 17229-17235), the replacement of the 170 loop of VIIa with that of trypsin induced a 10-fold enhancement of the k(cat) value for S-2288 hydrolysis as compared with that of wild-type VIIa in the absence of sTF. These results suggested that the 99 and the 170 loop structures of VIIa independently affect the K(m) and k(cat) values, respectively. Furthermore, we studied the effect of mutations on proteolytic activity toward S-alkylated lysozyme as a macromolecular substrate and the activation of natural macromolecular substrate factor X.
Assuntos
Fator VIIa/metabolismo , Tromboplastina/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Fator VIIa/química , Fator VIIa/genética , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Muramidase/metabolismo , Mutagênese , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
A severe lack of von Willebrand factor-cleaving protease (VWF-CP) activity can cause thrombotic thrombocytopenic purpura (TTP). This protease was recently identified as a member of the ADAMTS family, ADAMTS-13. It consists of a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, additional Tsp1 repeats, and CUB domains. To explore the structural and functional relationships of ADAMTS-13, we prepared here 13 sequential COOH-terminal truncated mutants and a single-point mutant (ArgGlyAsp [RGD] to ArgGlyGlu [RGE] in the cysteine-rich domain) and compared the activity of each mutant with that of the wild-type protein. The results revealed that the truncation of the cysteine-rich/spacer domains caused a remarkable reduction in VWF-CP activity. We also prepared immunoglobulin G (IgG) fractions containing inhibitory autoantibodies against ADAMTS-13 from plasma from 3 patients with acquired TTP, and we performed mapping of their epitopes using the aforementioned mutants. The major epitopes of these antibodies were found to reside within the cysteine-rich/spacer domains. These results suggest that the ADAMTS-13 cysteine-rich/spacer domains are essential for VWF-CP activity.
Assuntos
Metaloendopeptidases/química , Fator de von Willebrand/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Autoanticorpos/sangue , Cisteína , Mapeamento de Epitopos , Humanos , Imunoglobulina G/sangue , Metaloendopeptidases/imunologia , Metaloendopeptidases/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína/fisiologia , Púrpura Trombocitopênica Trombótica/imunologiaRESUMO
We report here 7 new mutations in the ADAMTS13 gene responsible for Upshaw-Schulman syndrome (USS), a catastrophic phenotype of congenital thrombotic thrombocytopenic purpura, by analyzing 5 Japanese families. There were 3 mutations that occurred at exon-intron boundaries: 414+1G>A at intron 4, 686+1G>A at intron 6, and 1244+2T>G at intron 10 (numbered from the A of the initiation Met codon), and we confirmed that 2 of these mutations produced aberrantly spliced messenger RNAs (mRNAs). The remaining 4 mutations were missense mutations: R193W, I673F, C908Y, and R1123C. In expression experiments using HeLa cells, all mutants showed no or a marginal secretion of ADAMTS13. Taken together with the findings in our recent report we determined the responsible mutations in a total of 7 Japanese patients with USS with a uniform clinical picture of severe neonatal hyperbilirubinemia, and in their family members, based on ADAMTS13 gene analysis. Of these patients, 2 were homozygotes and 5 were compound heterozygotes. The parents of one homozygote were related (cousins), while those of the other were not. Molecular models of the metalloprotease, fifth domain of thrombospondin 1 (Tsp1-5), and Tsp1-8 domains of ADAMTS13 suggest that the missense mutations could cause structural defects in the mutants.
Assuntos
Processamento Alternativo , Anemia Hemolítica/genética , Metaloendopeptidases/genética , Mutação de Sentido Incorreto , Trombocitopenia/genética , Proteínas ADAM , Proteína ADAMTS13 , Adulto , Éxons , Saúde da Família , Feminino , Expressão Gênica , Humanos , Recém-Nascido , Íntrons , Japão , Icterícia Neonatal/genética , Masculino , Metaloendopeptidases/química , Linhagem , Estrutura Terciária de Proteína , Trombospondina 1/químicaRESUMO
The natural anticoagulant-activated protein C may inhibit inflammation and fibrosis in the lung. Platelet-derived growth factor is involved in the pathogenesis of lung fibrosis. This study assessed the effect of activated protein C on platelet-derived growth factor expression in human cell lines and in an in vivo model of lung fibrosis. Activated protein C significantly inhibited the secretion and expression of platelet-derived growth factor in human lung cell lines, primary bronchial epithelial cells, and macrophages. In vitro studies also showed that the endothelial activated protein C receptor is expressed by lung epithelial cells and macrophages, and that this receptor and the proteolytic activity of activated protein are implicated in the inhibition of platelet-derived growth factor expression. In the in vivo model of lung fibrosis, intratracheal administration of activated protein C decreased the expression of platelet-derived growth factor and suppressed the development of lung fibrosis. Concomitant intratracheal administration of activated protein C and anti-endothelial activated protein C receptor or anti-platelet-derived growth factor suppressed the inhibitory activity of activated protein C in vivo. In brief, this study describes a novel biological function of activated protein C that may further explain its inhibitory activity on lung inflammation and fibrosis.