RESUMO
Essentials Activated clotting factor X (FXa) acquires fibrinolytic cofactor function after cleavage by plasmin. FXa-mediated plasma fibrinolysis is enabled by active site modification blocking a second cleavage. FXa-directed oral anticoagulants (DOACs) alter FXa cleavage by plasmin. DOACs enhance FX-dependent fibrinolysis and plasmin generation by tissue plasminogen activator. BACKGROUND: When bound to an anionic phospholipid-containing membrane, activated clotting factor X (FXa) is sequentially cleaved by plasmin from the intact form, FXaα, to FXaß and then to Xa33/13. Tissue-type plasminogen activator (t-PA) produces plasmin and is the initiator of fibrinolysis. Both FXaß and Xa33/13 enhance t-PA-mediated plasminogen activation. Although stable in experiments using purified proteins, Xa33/13 rapidly loses t-PA cofactor function in plasma. Bypassing this inhibition, covalent modification of the FXaα active site prevents Xa33/13 formation by plasmin, and the persistent FXaß enhances plasma fibrinolysis. As the direct oral anticoagulants (DOACs) rivaroxaban and apixaban bind to the FXa active site, we hypothesized that they similarly modulate FXa fibrinolytic function. METHODS: DOAC effects on fibrinolysis and the t-PA cofactor function of FXa were studied in patient plasma, normal pooled plasma and purified protein experiments by the use of light scattering, chromogenic assays, and immunoblots. RESULTS: The plasma of patients taking rivaroxaban showed enhanced fibrinolysis correlating with FXaß. In normal pooled plasma, the addition of rivaroxaban or apixaban also shortened fibrinolysis times. This was related to the cleavage product, FXaß, which increased plasmin production by t-PA. It was confirmed that these results were not caused by DOACs affecting activated FXIII-mediated fibrin crosslinking, clot ultrastructure and thrombin-activatable fibrinolysis inhibitor activation in plasma. CONCLUSION: The current study suggests a previously unknown effect of DOACs on FXa in addition to their well-documented anticoagulant role. By enabling the t-PA cofactor function of FXaß in plasma, DOACs also enhance fibrinolysis. This effect may broaden their therapeutic indications.
Assuntos
Fator Xa/química , Pirazóis/farmacologia , Piridonas/farmacologia , Rivaroxabana/farmacologia , Administração Oral , Anticoagulantes/química , Coagulação Sanguínea/efeitos dos fármacos , Domínio Catalítico , Reagentes de Ligações Cruzadas/química , Inibidores do Fator Xa/farmacologia , Fibrina/química , Fibrinolisina/química , Fibrinólise , Humanos , Fosfolipídeos/química , Trombina/química , Terapia Trombolítica , Trombose , Ativador de Plasminogênio Tecidual/químicaAssuntos
Terapia Antirretroviral de Alta Atividade/efeitos adversos , Infecções por HIV/tratamento farmacológico , Doenças Mitocondriais/induzido quimicamente , Doenças Mitocondriais/fisiopatologia , Oftalmoplegia Externa Progressiva Crônica/induzido quimicamente , Oftalmoplegia Externa Progressiva Crônica/fisiopatologia , Fármacos Anti-HIV/efeitos adversos , Blefaroptose/induzido quimicamente , Blefaroptose/metabolismo , Blefaroptose/fisiopatologia , DNA Mitocondrial/efeitos dos fármacos , Didanosina/efeitos adversos , Predisposição Genética para Doença/genética , Humanos , Lipodistrofia/induzido quimicamente , Lipodistrofia/metabolismo , Lipodistrofia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Mutação/genética , Músculos Oculomotores/efeitos dos fármacos , Músculos Oculomotores/patologia , Músculos Oculomotores/fisiopatologia , Oftalmoplegia/induzido quimicamente , Oftalmoplegia/metabolismo , Oftalmoplegia/fisiopatologia , Oftalmoplegia Externa Progressiva Crônica/metabolismo , Recuperação de Função Fisiológica/fisiologia , Fatores de Risco , TempoRESUMO
Statins are generally well tolerated, but can cause myopathy and have been associated with mitochondrial abnormalities. The aim of this study was to determine whether muscle mitochondrial DNA (mtDNA) levels are altered during statin therapy. We retrospectively quantified mtDNA in 86 skeletal muscle biopsy specimens collected as part of a previously published clinical trial of high-dose simvastatin or atorvastatin versus placebo.
Assuntos
DNA Mitocondrial/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Sinvastatina/efeitos adversos , Adulto , Idoso , Atorvastatina , Método Duplo-Cego , Feminino , Ácidos Heptanoicos/efeitos adversos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pirróis/efeitos adversos , Estudos Retrospectivos , Sinvastatina/uso terapêutico , Ubiquinona/metabolismoRESUMO
We examined the prevalence of cleavage site mutations, both within and outside the gag region, in 28 protease inhibitor (PI) cross-resistant patients treated with indinavir, ritonavir, and/or saquinavir compared to control patients treated with reverse transcriptase inhibitors. Three human immunodeficiency virus protease cleavage sites within gag (p2/NC, NC/p1, and NC/TFP) showed considerable in vivo evolution before and after therapy with indinavir, ritonavir, and/or saquinavir. Another gag cleavage site (p1/p6(gag)) showed a trend compared to matched controls. The other eight recognized cleavage sites showed relatively little difference between PI-resistant cases and controls. An A-->V substitution at the P2 position of the NC/p1 and NC/TFP cleavage sites was the most common (29%) change selected by the PIs used in this study.
Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação , Sequência de Aminoácidos , Substituição de Aminoácidos , Estudos de Casos e Controles , Resistência Microbiana a Medicamentos , Quimioterapia Combinada , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/uso terapêutico , HIV-1/enzimologia , Humanos , Indinavir/farmacologia , Indinavir/uso terapêutico , Dados de Sequência Molecular , Polimorfismo Genético , Inibidores da Transcriptase Reversa/uso terapêutico , Ritonavir/farmacologia , Ritonavir/uso terapêutico , Saquinavir/farmacologia , Saquinavir/uso terapêuticoRESUMO
Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.
Assuntos
Fator Xa/metabolismo , Hirudinas/isolamento & purificação , Hirudinas/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Calbindinas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Cricetinae , Ativação Enzimática , Fator Xa/genética , Hirudinas/genética , Humanos , Proteínas Ligantes de Maltose , Fator de Acasalamento , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/genética , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Protrombina/genética , Protrombina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Saccharomyces cerevisiae , TransfecçãoRESUMO
Congenital dysfibrinogenemia is a rare cause of unexplained thrombosis. However, most individuals with dysfibrinogenemia are asymptomatic, suggesting that co-morbid factors contribute to thrombo-embolic events. The potential roles of additional genetic or acquired prothrombotic risk factors are poorly understood because detailed family studies are lacking. Herein, we describe a family whose propositus was a young Caucasian man with recurrent venous thrombo-emboli and dysfibrinogenemia due to heterozygosity for an Arg-->Cys substitution at residue 275 in the gamma-chain. The only additional thrombophilic abnormality found in the proband was heterozygosity for a G/A transition at position -455 in the fibrinogen beta-chain promoter; a genotype associated with high acute phase levels of fibrinogen. The proband's father, who died of a cerebral artery thrombosis, carried the gammaR275C substitution but not the beta-promoter -455 variant. Among 14 living relatives, eight were heterozygous for one or the other mutation and only one, a 21-year-old niece, was dually affected. None had suffered bleeding or thrombosis. In vitro studies of the proband's purified fibrinogen revealed markedly abnormal thrombin-catalyzed polymerization and delayed fibrin clot lysis by tPA-activated plasmin. We hypothesize that the gammaR275C substitution predisposes to thrombosis by generating clots that are relatively resistant to fibrinolysis. The clinical risk is low, however, in the absence of an additional thrombophilic mutation. The beta-promoter variant could, theoretically, contribute to this risk by augmenting expression of the dysfibrinogen under conditions of stress. Like the common hereditary thrombophilias, heterozygous familial dysfibrinogenemia induces thrombosis in the setting of multiple prothrombotic influences.
Assuntos
Fibrinogênio/genética , Mutação Puntual , Trombose/genética , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Trombose/etiologiaRESUMO
Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI activation, and Trp(215) appears to very important for clotting and protein C activation.
Assuntos
Trombina/química , Coagulação Sanguínea , Fluorescência , Humanos , Mutação , Relação Estrutura-Atividade , Trombina/genética , TriptofanoRESUMO
Human immunodeficiency virus (HIV) type 1 drug-resistance testing is quickly moving from the research laboratory to the clinic as data defining its utility as a prognostic indicator of response to therapy become available. In July 1998, a panel of the International AIDS Society-USA did not recommend the widespread application of resistance testing, but by May 2000 this panel endorsed and recommended the incorporation of resistance testing in patient-care management. Considerable data supporting the use of drug-resistance testing have now been published or presented at international conferences. These data strongly suggest that drug-resistance testing is of considerable value in many clinical settings. Prospective trials of resistance testing as a clinical management tool are still ongoing, and the long-term benefits still need to be evaluated. Nevertheless, early results from several studies showed a significantly better virological response when treatment regimens were based on resistance-testing data, rather than on the standard of care. HIV drug-resistance testing is also useful as a tool for new antiretroviral drug design and development, as well as for monitoring the spread of primary HIV drug resistance.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/uso terapêutico , Resistência Microbiana a Medicamentos , Infecções por HIV/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana/métodos , Administração dos Cuidados ao Paciente , Inibidores da Transcriptase Reversa/uso terapêuticoRESUMO
Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.
Assuntos
Pâncreas/enzimologia , Pichia/genética , alfa-Amilases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Glicosilação , Humanos , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , alfa-Amilases/química , alfa-Amilases/metabolismoAssuntos
Afibrinogenemia/genética , Fibrinogênio/genética , Mutação , Adulto , Sequência de Aminoácidos , Sítios de Ligação , Coagulação Sanguínea/fisiologia , Cristalização , Feminino , Fibrinogênio/química , Fibrinogênio/metabolismo , Transtornos Hemorrágicos/genética , Humanos , Lactente , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Relação Estrutura-Atividade , Trombofilia/genéticaRESUMO
Meizothrombin and meizothrombin(desF1) are intermediates formed during the conversion of prothrombin to thrombin by factor Xa, factor Va, phospholipids, and Ca2+ (prothrombinase). These intermediates are active toward synthetic peptide substrates but have limited ability to interact with platelets or macromolecular substrates such as fibrinogen. Meizothrombin and meizothrombin(desF1) activate protein C, however, and may exert primarily an anticoagulant effect. In this study, we investigated the inhibition of meizothrombin and meizothrombin(desF1) by two glycosaminoglycan-dependent protease inhibitors, heparin cofactor II (HCII) and antithrombin (AT). Purified recombinant meizothrombin and meizothrombin(desF1) were inhibited by HCII in the presence of dermatan sulfate with maximal second-order rate constants of 8 x 10(6) M-1.min-1 and 1.8 x 10(7) M-1.min-1, respectively, but were inhibited less than one-tenth as fast by AT in the presence of heparin. Similarly, the products of the prothrombinase reaction were inhibited in situ more effectively by HCII than by AT. When HCII and dermatan sulfate were present continuously during the prothrombinase reaction, meizothrombin was trapped as a sodium dodecyl sulfate-stable complex with HCII and no amidolytic activity could be detected with a thrombin substrate. Our findings indicate that HCII is an effective inhibitor of meizothrombin and meizothrombin(desF1) and, therefore, might regulate the anticoagulant activity of these proteases.
Assuntos
Precursores Enzimáticos/antagonistas & inibidores , Esterases/antagonistas & inibidores , Cofator II da Heparina/farmacologia , Inibidores de Serina Proteinase/farmacologia , Trombina/antagonistas & inibidores , Antitrombina III/farmacologia , Glicosaminoglicanos/metabolismo , Humanos , Modelos Químicos , Estrutura Secundária de Proteína , Protrombina/metabolismo , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
The carboxyl-terminal region of the gamma chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross-linking, and binding to the platelet fibrin(ogen) receptor. Protein fragments encoding amino acids Val143 to Val411 (rFbggammaC30) or Val143 to Leu427 (gamma'C30) from the carboxyl end of the gamma or gamma' chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site. rFbggammaC30 and gamma'C30 were both readily cross-linked by factor XIIIa, but only rFbggammaC30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, gammaC30-Q329R and gammaC30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbggammaC30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbggammaC30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not. Similarly, rFbggammaC30 and gamma'C30 were protected from plasmin degradation by the presence of Ca2+ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca2+-binding site and polymerization pocket are contained within each of these fragments. The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested. These results indicate that the polymerization pocket "a", which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amino acids Gln329 and Asp364 play a crucial role in fibrin polymerization.
Assuntos
Cálcio/metabolismo , Fibrinogênio/química , Sequência de Aminoácidos , Sítios de Ligação , Biopolímeros , Fibrinogênio/metabolismo , Fibrinolisina/metabolismo , Humanos , Hidrólise , Masculino , Agregação Plaquetária , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trombina/metabolismoRESUMO
After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the alpha chain of one fibrin molecule and the C-terminal region of a gamma chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) gamma chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the alpha chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.
Assuntos
Fibrina/química , Oligopeptídeos/química , Dimerização , Fibrina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Conformação ProteicaRESUMO
Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.
Assuntos
Precursores Enzimáticos/fisiologia , Trombina/fisiologia , Antitrombina III/metabolismo , Arginina/análogos & derivados , Arginina/metabolismo , Ligação Competitiva , Coagulação Sanguínea , Compostos de Dansil/metabolismo , Ativação Enzimática , Hirudinas/metabolismo , Humanos , Agregação Plaquetária , Proteína C/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade , Trombomodulina/metabolismoRESUMO
A patient had both lupus anticoagulant hypoprothrombinemia syndrome and celiac disease. The presence of a neutralizing antiprothrombin antibody in the patient's serum was demonstrated by coagulation tests, immunoadsorption, and Western blot analysis. The probable cause for the severe hypoprothrombinemia was clearance of prothrombin-antibody complexes from the circulation. Studies showed the antiprothrombin antibody binding to human prothrombin was phospholipid- and Ca(++)-independent; the antibody did not bind to human thrombin. The target epitope of the antibody was studied by Western blot analysis of mutated recombinant human prothrombin molecules. The antibody reacted with the fragment 2-A region of prothrombin, spanning the second kringle domain and the thrombin A chain within prothrombin. Based on this new method, the proposed mechanism for the neutralizing action of the antibody is impairment of prothrombin activation by the prothrombinase complex, either by steric hindrance of the hydrolysis of prothrombin by factor Xa or by interference of the interaction of prothrombin with factor Va; both reactions are required for efficient conversion of prothrombin to thrombin.
Assuntos
Doença Celíaca/complicações , Epitopos/química , Inibidor de Coagulação do Lúpus/análise , Lúpus Eritematoso Sistêmico/complicações , Western Blotting , Criança , Feminino , Testes Hematológicos , Humanos , Hipoprotrombinemias/etiologia , Protrombina/imunologia , Protrombina/metabolismo , SíndromeRESUMO
BACKGROUND: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. RESULTS: The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. CONCLUSIONS: The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.
Assuntos
Fibrinogênio/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Alinhamento de Sequência , Transglutaminases/farmacologiaRESUMO
Meizothrombin is a transient intermediate produced during the activation of prothrombin by the prothrombinase complex. Because meizothrombin is very sensitive to further activation and autolysis, its isolation is possible only in the presence of active site thrombin inhibitors. This complicates studies of the activities and functions of meizothrombin. As a model, we have expressed a mutant human prothrombin cDNA (R155A, R271A, R284A) with three of the cleavage sites modified so that they are no longer cleaved by factor Xa or thrombin. Several stable baby hamster kidney cell lines were isolated that secreted up to 20 micrograms/ml of carboxylated mutant prothrombin. After purification, the mutant prothrombin was activated by the prothrombinase complex or by ecarin, resulting in the formation of a meizothrombin-like molecule. Electrophoretic analysis and NH2-terminal sequence analysis were consistent with cleavage of a single bond between Arg320-Ile321 and proper processing of the prepropeptide. The meizothrombin was stable for weeks at 4 degrees C. Activation in the presence of dansylarginine N-(3-ethyl-1,5-pentanediyl) amide confirmed the conversion of prothrombin via meizothrombin. Compared with human plasma-derived thrombin, recombinant meizothrombin demonstrated approximately 7% clotting activity, 100% p-toluene-sulfonylarginine methyl ester esterase activity, and approximately 35% S2238 amidolytic activity, and could attenuate fibrinolysis.
Assuntos
Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/química , Mutação Puntual , Protrombina/metabolismo , Trombina/biossíntese , Trombina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Precursores Enzimáticos/isolamento & purificação , Estabilidade Enzimática , Fibrinólise , Humanos , Rim , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Fosfolipídeos/metabolismo , Plasmídeos , Protrombina/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Espectrometria de Fluorescência , Trombina/isolamento & purificação , Tromboplastina/metabolismo , TransfecçãoRESUMO
We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.