RESUMO
The anticoagulant effect of rivaroxaban, a direct inhibitor of activated factor X (FX), might be influenced by its dosing time because the activity of the coagulofibrinolytic system exhibits daily rhythmicity. In rats, FX activity follows a 24-h rhythm with a peak in the middle of the light phase and a trough at the beginning of the dark phase. Consistent with these findings, a single dose of rivaroxaban had a stronger inhibitory effect on FX activity after dosing at the beginning of the light phase than after dosing at the beginning of the dark phase. A similar chronopharmacological effect was seen in a quantitative model of venous stasis thrombosis. In comparison, the dosing time had minimal influence on the pharmacokinetics of rivaroxaban. These data indicate that the anticoagulant effect of rivaroxaban is influenced by the dosing time. Further studies should confirm this finding in a clinical setting.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Cronofarmacocinética , Ritmo Circadiano/fisiologia , Cronofarmacoterapia , Inibidores do Fator Xa/farmacologia , Rivaroxabana/administração & dosagem , Rivaroxabana/farmacologia , Animais , Relação Dose-Resposta a Droga , Fator X/fisiologia , Masculino , Fotoperíodo , Ratos Wistar , Rivaroxabana/farmacocinética , Fatores de TempoRESUMO
Aside from their role in hemostasis, coagulant and fibrinolytic proteases are important mediators of inflammation in diseases such as asthma, atherosclerosis, rheumatoid arthritis, and cancer. The blood circulating zymogens of these proteases enter damaged tissue as a consequence of vascular leak or rupture to become activated and contribute to extravascular coagulation or fibrinolysis. The coagulants, factor Xa (FXa), factor VIIa (FVIIa), tissue factor, and thrombin, also evoke cell-mediated actions on structural cells (e.g., fibroblasts and smooth muscle cells) or inflammatory cells (e.g., macrophages) via the proteolytic activation of protease-activated receptors (PARs). Plasmin, the principle enzymatic mediator of fibrinolysis, also forms toll-like receptor-4 (TLR-4) activating fibrin degradation products (FDPs) and can release latent-matrix bound growth factors such as transforming growth factor-ß (TGF-ß). Furthermore, the proteases that convert plasminogen into plasmin (e.g., urokinase plasminogen activator) evoke plasmin-independent proinflammatory actions involving coreceptor activation. Selectively targeting the receptor-mediated actions of hemostatic proteases is a strategy that may be used to treat inflammatory disease without the bleeding complications of conventional anticoagulant therapies. The mechanisms by which proteases of the coagulant and fibrinolytic systems contribute to extravascular inflammation in disease will be considered in this review.
Assuntos
Coagulação Sanguínea , Fibrinólise , Inflamação/etiologia , Peptídeo Hidrolases/fisiologia , Fator X/fisiologia , Produtos de Degradação da Fibrina e do Fibrinogênio/fisiologia , Fibrinolisina/fisiologia , Humanos , Multimerização Proteica , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/química , Receptor PAR-1/fisiologia , Receptores de Fatores de Crescimento/genética , Ativador de Plasminogênio Tecidual/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaAssuntos
Arginina/fisiologia , Fator X/fisiologia , Mutação , Idoso , Arginina/genética , Fator X/química , Fator X/genética , Humanos , Masculino , Modelos MolecularesRESUMO
INTRODUCTION: Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease. MATERIALS AND METHODS: We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, prothrombin fragment 1+2) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined. RESULTS: Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased prothrombin fragment 1+2 and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P<0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.001). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function. CONCLUSIONS: Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.
Assuntos
Coagulação Sanguínea/fisiologia , Endotélio Vascular/fisiologia , Falência Renal Crônica/fisiopatologia , Trombofilia/fisiopatologia , Idoso , Antígenos/fisiologia , Antitrombina III/fisiologia , Fenômenos Biológicos , Biomarcadores/sangue , Creatinina/sangue , Selectina E/sangue , Fator VII/fisiologia , Fator X/fisiologia , Feminino , Humanos , Interleucina-6/sangue , Falência Renal Crônica/complicações , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/fisiologia , Proteína S/metabolismo , Protrombina/fisiologia , Diálise Renal/efeitos adversos , Solubilidade , Trombomodulina/sangue , Trombofilia/complicações , Tromboplastina/fisiologiaRESUMO
STUDY OBJECTIVE: To validate a Thromboelastograph (Haemoscope Corporation, Niles, IL) assay for functional fibrinogen. DESIGN: Correlation study of the Thromboelastograph assay with two conventional fibrinogen assays by the standard Clauss method. SETTING: Research laboratory of a university medical center. PARTICIPANTS AND INTERVENTIONS: Blood samples were obtained from 19 healthy volunteers. MEASUREMENT AND MAIN RESULTS: Thromboelastograph assays, using heparinized whole blood from 19 healthy donors, indicated that reptilase-XIIIa mixture (Activatorf)-generated clot shear elasticity in dynes per square centimeter (Gf) correlated with fibrinogen (mg/dL). Blood from four donors was used to define the contribution of hematocrit (Hct) to Gf by titration with platelet-rich plasma. The Gf versus Hct gave linear correlations (r2 = 0.746) with Gf = 1258 - 17.8 x % Hct. A commercial collection of 19 normal, 10 borderline, and one deficient for functional fibrinogen-citrated plasmas was assayed for Gf after recalcification using Activatorf. Of the 30 plasma samples, four were from factor X- or factor VII-deficient donors and one was from a coumadin-treated donor. There was a linear correlation of Activatorf Gf with functional fibrinogen (r2 = 0.940) with Gf = -730 + 9.21 x fibrinogen (mg/dL). CONCLUSION: Thrombelastography with Activatorf may be used to determine fibrinogen levels in whole blood.
Assuntos
Fibrinogênio/análise , Tromboelastografia , Transtornos da Coagulação Sanguínea/sangue , Elasticidade , Fator VII/fisiologia , Fator X/fisiologia , Hematócrito , Humanos , Técnicas In Vitro , Modelos Lineares , Ativação Plaquetária/fisiologia , Plasma Rico em Plaquetas/fisiologiaRESUMO
Factor X is a vitamin K-dependent, liver-produced serine protease that serves a pivotal role in coagulation as the first enzyme in the common pathway to fibrin formation. Inherited factor X deficiency is a rare autosomal recessive bleeding disorder that is estimated to occur in 1:1,000,000 individuals up to 1:500 carriers. Several international registries of FX-deficient patients have greatly expanded the knowledge of clinical phenotype. A proposed classification of severity is based on FX:C activity measurements: an FX:C measurement <1% is severe, an FX:C measurement of 1-5% is moderate and an FX:C measurement of 6-10% is mild. Levels above 20% are infrequently associated with bleeding and heterozygotes are usually asymptomatic. Among patients with FX:C levels <10%, unlike moderate or severe haemophilia A and B, mucocutaneous bleeding symptoms such as epistaxis and menorrhagia occur in the majority. In addition, patients with moderate-severe deficiency may have symptoms similar to that of haemophilia A and B, including haemarthrosis, intracranial haemorrhage, and gastrointestinal bleeding. Genotype characterization may offer important clues about clinical prognosis. More than 80 mutations of the F10 gene have been identified, most of which are missense mutations. There is no specific FX replacement product yet readily available, but fresh frozen plasma and prothrombin complex concentrates can be used for treatment of bleeding symptoms and preparation for surgery.
Assuntos
Coagulantes/uso terapêutico , Deficiência do Fator X/genética , Fator X/fisiologia , Hemorragia/genética , Sistema de Registros/estatística & dados numéricos , Adulto , Fatores de Coagulação Sanguínea/uso terapêutico , Testes de Coagulação Sanguínea , Criança , Fator X/uso terapêutico , Deficiência do Fator X/classificação , Deficiência do Fator X/diagnóstico , Deficiência do Fator X/tratamento farmacológico , Feminino , Genótipo , Hemorragia/tratamento farmacológico , Heterozigoto , Homozigoto , Humanos , Lactente , Recém-Nascido , Irã (Geográfico)/epidemiologia , Itália/epidemiologia , Masculino , Mutação de Sentido Incorreto , Plasma , Gravidez , Prevalência , Doenças Raras , Índice de Gravidade de Doença , Reino Unido/epidemiologia , Adulto JovemRESUMO
Heparin inhibits the intrinsic tenase complex (factor IXa-factor VIIIa) via interaction with a factor IXa exosite. To define the role of this exosite, human factor IXa with alanine substituted for conserved surface residues (R126, N129, K132, R165, N178) was characterized. Chromogenic substrate hydrolysis by the mutant proteases was reduced 20-30% relative to factor IXa wild type. Coagulant activity was moderately (N129A, K132A, K126A) or dramatically (R165A) reduced relative to factor IXa wild type. Kinetic analysis demonstrated a marked reduction in apparent cofactor affinity (23-fold) for factor IXa R165, and an inability to stabilize cofactor activity. Factor IXa K126A, N129A, and K132A demonstrated modest reductions ( approximately 2-fold) in apparent cofactor affinity, and accelerated decay of intrinsic tenase activity. In the absence of factor VIIIa, factor IXa N178A and R165A demonstrated a defective Vmax(app) for factor X activation. In the presence of factor VIIIa, Vmax(app) varied in proportion to the predicted factor IXa-factor VIIIa concentration. However, factor IXa R165A had a 65% reduction in the kcat for factor X, suggesting an additional effect on catalysis. The ability of factor IXa to compete for physical assembly into the intrinsic tenase complex was enhanced by EGR-chloromethylketone bound to the factor IXa active site or addition of factor X, and reduced by selected mutations in the heparin-binding exosite (N178A, K126A, R165A). These results suggest that the factor IXa heparin-binding exosite participates in both cofactor binding and protease activation, and cofactor affinity is linked to active site conformation and factor X interaction during enzyme assembly.
Assuntos
Regulação Alostérica/fisiologia , Arginina/fisiologia , Cisteína Endopeptidases/metabolismo , Fator IXa/metabolismo , Fator X/fisiologia , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/fisiologia , Fator IXa/genética , Fator VIIIa/metabolismo , Heparina/metabolismo , Humanos , Cinética , Conformação Proteica , Proteínas Recombinantes/metabolismoRESUMO
Membrane phospholipids are essential in blood coagulation reactions. The importance of negatively changed phosphatidylserine has been shown. The roles of other phospholipids in the blood coagulation system, however, are not clear. This study examined the effects of phosphatidylcholine on the blood coagulation system using liposomes containing varying concentrations of phosphatidylcholine in the presence of phosphatidylserine at a constant concentration. In addition, with phosphatidylserine and phosphatidylcholine at constant concentrations, the effects of phosphatidylethanolamine and lysophosphatidylcholine on the blood coagulation system were examined. Using an in vitro reconstructed system of the activated factor X-prothrombin system, blood coagulation was measured by the rate of thrombin formation after the addition of liposome preparations. The results showed suppression of the system by phosphatidylcholine and phosphatidylethanolamine and acceleration by lysophosphatidylcholine. The results of the present study suggest that the cell membrane, the 'location' of blood coagulation, is one of the regulatory factors, and that changes in phosphatidylcholine content and phospholipid composition of the cell membrane regulate the coagulation reaction.
Assuntos
Fator X/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Fosfatidilcolinas/farmacologia , Fosfatidiletanolaminas/farmacologia , Protrombina/efeitos dos fármacos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Bovinos , Fator V/efeitos dos fármacos , Fator V/fisiologia , Fator X/fisiologia , Humanos , Lipossomos/síntese química , Lipossomos/química , Protrombina/fisiologiaRESUMO
Classical hemophilia results from a defect of the intrinsic tenase complex, the main factor X (FX) activator. Binding of factor VIIa to tissue factor triggers coagulation, but little amplification of thrombin production occurs. Handling of hemophilia by injection of the deficient or missing (thus foreign) factor often causes immunological complications. Several strategies have been designed to bypass intrinsic tenase complex, but none induce true auto-amplification of thrombin production. In an attempt to re-establish a cyclic amplification of prothrombin activation in the absence of tenase, we prepared a chimera of FX having fibrinopeptide A for the activation domain (FX(FpA)). We reasoned that cascade initiation would produce traces of thrombin that would activate FX(FpA) (contrary to its normal homologue). Given that the activation domain of FX is released upon activation, thrombin cleavage would produce authentic FXa that would produce more thrombin, which in turn would activate more chimeras. FX(FpA) was indeed activable by thrombin, albeit at a relatively low rate (5 x 10(3) M(-1) s(-1)). Nevertheless, FX(FpA) allowed in vitro amplification of thrombin production, and 100 nM efficiently corrected thrombin generation in tenase-deficient plasmas. A decisive advantage of FX(FpA) could be that the artificial cascade is self-regulating: FX(FpA) had little influence on the clotting time of normal plasma, yet corrected that of tenase deficiency. Another advantage could be the half-life of FX(FpA) in blood; FX has a half-life of about 30 h (less than 3 h for FVIIa). It is also reasonable to expect little or no immunogenicity, because FX and fibrinopeptide A both circulate normally in the blood of hemophiliacs.
Assuntos
Cisteína Endopeptidases/fisiologia , Fator X/química , Fator X/fisiologia , Proteínas de Neoplasias/fisiologia , Trombina/metabolismo , Coagulação Sanguínea , Coagulantes/química , Cisteína Endopeptidases/metabolismo , DNA Complementar/metabolismo , Dissulfetos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fator X/metabolismo , Fibrinopeptídeo A/química , Fibrinopeptídeo A/metabolismo , Hemofilia A/metabolismo , Humanos , Cinética , Modelos Biológicos , Modelos Químicos , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Trombina/química , Fatores de TempoRESUMO
It is uncommon for similar pathways/systems to be involved in highly divergent functions within single organisms. Earlier, we have shown that trocarin D, a venom prothrombin activator, from the Australian rough-scaled snake Tropidechis carinatus, is structurally and functionally similar to the blood coagulation factor Xa (FXa). The presence of a haemostatic system in these snakes implies that they have two parallel prothrombin activating systems: one in the plasma, that participates in the life saving process of blood clotting and the other in their venom, where it acts as a toxin. Here, we report the complete cDNA sequence encoding the blood coagulation factor X (FX) from the liver of T. carinatus. Deduced T. carinatus FX sequence shows approximately 80% identity with trocarin D but approximately 50% identity with the mammalian FX. Our present study confirms the presence of two separate genes--one each for FX and trocarin D, that code for similar proteins in T. carinatus snake. These two genes have different expression sites and divergent uses suggesting that snake venom prothrombin activators have probably evolved by the duplication of the liver FX gene and subsequently marked for tissue-specific expression in the venom gland.
Assuntos
Fator X/genética , Protrombina/genética , Serpentes/fisiologia , Trombina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fator X/fisiologia , Hemostasia , Fígado , Dados de Sequência Molecular , Protrombina/fisiologia , Análise de Sequência de DNA , Homologia de Sequência , Serpentes/sangue , Distribuição TecidualRESUMO
Treatment of Beas-2B airway epithelial cells with crocidolite asbestos induced tissue factor (TF) mRNA and TF-dependent procoagulant activity. The mitogen-activated protein kinase (MAPK) inhibitors UO126 and SB203850 decreased TF expression in both naive and crocidolite-treated Beas-2B cells to the same extent. Calphostin, an inhibitor of classical and novel protein kinase C (PKC) isotypes, reduced TF mRNA in both intact and crocidolite-treated Beas-2B cells by about 50%. Conversely, the phosphatidylinositol 3-kinase (PI3 kinase) inhibitor LY294002 and a selective PKCzeta inhibitory peptide decreased TF mRNA expression in asbestos-treated cells to a greater extent than in naive cells, suggesting that signaling via this pathway contributes to asbestos-induced TF expression. These results demonstrate that crocidolite asbestos induces TF expression by Beas-2B cells and suggest that the process involves the PI3 kinase-PKCzeta signaling pathway, representing a newly recognized potential mechanism by which asbestos may contribute to lung remodeling.
Assuntos
Asbesto Crocidolita/farmacologia , Carcinógenos/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Quinase C/fisiologia , Tromboplastina/efeitos dos fármacos , Células Cultivadas , Fator X/fisiologia , Humanos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Mucosa Respiratória/fisiologia , Transdução de Sinais , Tromboplastina/fisiologiaRESUMO
Factor X Padua, first described a few years ago, is characterized by a defect only in the extrinsic system. In this present paper, the molecular basis for this peculiar defect is investigated. Polymerase chain reaction amplification and direct sequencing of the entire FX coding sequence and of exon-intron junctions detected in the proposita a C-to-T translocation in exon 8 of nucleotide 875 at the homozygous level. This resulted in the substitution of tryptophan for arginine 251. A niece of the proposita was shown to be heterozygous for the abnormality. Molecular modeling suggested that the mutation does not alter significantly folding and stability of the protein but may be involved in the Ca2+ binding site.
Assuntos
Transtornos da Coagulação Sanguínea/genética , Fator X/genética , Mutação de Sentido Incorreto , Sítios de Ligação/genética , Cálcio/metabolismo , Fator X/química , Fator X/fisiologia , Saúde da Família , Feminino , Humanos , Pessoa de Meia-Idade , Modelos Moleculares , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
A number of studies suggest that blood-clotting factor X (FX) uses secondary site(s) to interact (as a substrate) with its activators. Numerous pieces of evidence also imply that, within prothrombinase (as an enzyme), activated FX (FXa) uses exosite(s) for cofactor Va and/or prothrombin recognition. Similarly, FXa exosite(s) seem to govern interaction with inhibitors. An obvious difference between FXa and thrombin resides within a region called exosite-1: positively charged in thrombin and clearly of opposite polarity in FXa. To investigate the role of this potential cation-binding exosite, we prepared a series of mutants within loops 34-40 and 70-80 of FX. Overall, the mutations induced relatively subtle, non-synergistic modulation. The potential exosite was dispensable for FX activation and is unlikely to constitute a critical region for factor Va binding, albeit it is clearly important for prothrombin activation. Our data also implicate loop 34-40 of FXa in the interaction with the tissue factor pathway inhibitor, in prevention of plasminogen activator inhibitor-1 binding, and in tempering inhibition by heparin-activated antithrombin. Compared with FX, mutants with reduced electrostatic potential potentiated thrombin production in FX-depleted plasma, whereas mutants with inverted electrostatic potential impeded clotting. Despite the definite consequences observed, disruption of the potential cation-binding exosite of FX had rather weak effects, far from what would be expected if this region was as crucial as in thrombin.
Assuntos
Fator X/química , Trombina/química , Sequência de Aminoácidos , Antitrombinas/química , Cátions , DNA Complementar/metabolismo , Ativação Enzimática , Fator Va/química , Fator X/fisiologia , Fator Xa/química , Heparina/química , Humanos , Hidrólise , Cinética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Protrombina/química , Eletricidade Estática , Fatores de TempoRESUMO
Tissue factor (TF) is a transmembrane glycoprotein, currently considered as being the major regulator of the coagulation cascade and the initiator of thrombogenesis in vivo. When TF comes in contact with blood, it forms a high-affinity complex with factors VII/VIIa, activating factors IX and X and thus leading to the formation of an insoluble fibrin clot. The regulation of TF-VIIa activity plays a key role in blood-vessel wall interactions. Selective patterns of cellular expression of TF are observed in tissues. TF is constitutively localized only on the surface of cells anatomically separated from the blood, where it plays an essential role in hemostasis by limiting hemorrhage after vessel wall injury. A number of pathophysiologic stimuli are capable of inducing TF transcription and activity in endothelial cells and monocytes. An aberrant TF expression in contact with blood is implicated in thrombotic complications of atherosclerosis, including acute myocardial infarction. Recent findings have demonstrated cell-derived microparticles containing TF in the circulating blood of patients with acute coronary syndromes, capable of triggering and propagating thrombus growth. This observation suggests a new view of thrombosis that does not necessarily require the exposure of vessel wall-derived TF at the site of vascular injury to initiate and propagate thrombosis.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/etiologia , Fibrinolíticos/uso terapêutico , Tromboplastina/antagonistas & inibidores , Tromboplastina/fisiologia , Angina Instável/sangue , Angina Instável/tratamento farmacológico , Angina Instável/etiologia , Animais , Arteriosclerose/sangue , Arteriosclerose/tratamento farmacológico , Arteriosclerose/etiologia , Coagulação Sanguínea/fisiologia , Doenças Cardiovasculares/sangue , Modelos Animais de Doenças , Fator IX/fisiologia , Fator VII/fisiologia , Fator VIIa/fisiologia , Fator X/fisiologia , Proteínas de Helminto/uso terapêutico , Hemostasia , Humanos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/etiologia , Síndrome , Tromboplastina/genética , Trombose/sangue , Trombose/tratamento farmacológico , Trombose/etiologiaRESUMO
Factor X (FX) has high structure homology with other proteins of blood coagulation such as factor IX (FIX) and factor VII (FVII). These proteins present at their amino-terminal extremity a gamma-carboxyglutamic acid containing domain (Gla domain), followed by two epidermal growth factor-like (EGF1 and EGF2) domains, an activation peptide, and a serine protease domain. After vascular damage, the tissue factor-FVIIa (TF-FVIIa) complex activates both FX and FIX. FXa interacts stoichiometrically with tissue pathway inhibitor (TFPI), regulating TF-FVIIa activity by forming the TF-FVIIa-TFPI-FXa quaternary complex. Conversely, FXa boosts coagulation by its association with its cofactor, factor Va (FVa). To investigate the contribution of the Gla and EGF1 domains of FX in these complexes, FX chimeras were produced in which FIX Gla and EGF1 domains substituted the corresponding domains of FX. The affinity of the two chimeras, FX/FIX(Gla) and FX/FIX(EGF1), for the TF-FVIIa complex was markedly reduced compared with that of wild-type-FX (wt-FX) independently of the presence of phospholipids. Furthermore, the association rate constants of preformed FX/FIX(Gla)-TFPI and FX/FIX(EGF1)-TFPI complexes with TF-FVIIa were, respectively, 10- and 5-fold slower than that of wt-FXa-TFPI complex. Finally, the apparent affinity of FVa was 2-fold higher for the chimeras than for wt-FX in the presence of phospholipids and equal in their absence. These data demonstrate that FX Gla and EGF1 domains contain residues, which interact with TF-FVIIa exosites contributing to the formation of the TF-FVIIa-FX and TF-FVIIa-TFPI-FXa complexes. On the opposite, FXa Gla and EGF1 domains are not directly involved in FVa binding.
Assuntos
Ácido 1-Carboxiglutâmico/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator VIIa/metabolismo , Fator X/química , Tromboplastina/metabolismo , Animais , Fator X/fisiologia , Humanos , Camundongos , Protrombina/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
To understand the role of the factor X (fX) activation peptide (AP), a deletion mutagenesis approach was employed. Two single-chain, variant enzymes were generated in which 41 residues were deleted from the AP: fX (des-137-183) and fX(des-137-183;N191A), which lacks a carbohydrate moiety at Asn191 due to an alanine substitution. Deletion of the fX AP did not impact fXa catalytic activity. Activation of the variant zymogens, however, was altered. Neither mutant enzyme was activated by the fX coagulant protein from Russell's viper venom (RVV-X(1)). Activation by factor VIIa (fVIIa) and fVIIa in the presence of cofactor, lipidated tissue factor (TF), occurred at an accelerated rate for both variants as compared to wild-type fX (WTfX). Similar to fVII, the mutants auto-activated in a cofactor-independent manner, which was characterized by a lag period and accelerated dose-dependently by plasma fXa (kcat/Km, 0.046 +/- 0.004 micro M(-1) s(-1)). Both mutants were also found to be activated by fVIIa (0.31 +/- 0.03 micro M(-1) s(-1)), fIXa (0.30 +/- 0.03 micro M(-1) s(-1)), and thrombin (0.00078 +/- 0.00015 micro M(-1) s(-1)). In all cases, the rate of activation was faster for fX(des-137-183;N191A) as compared to fX(des-137-183). We propose that the fX AP and Asn191 carbohydrate serve primarily as negative autoregulation mechanisms to prevent spurious activation of fX and secondarily in cofactor dependence and activator specificity.
Assuntos
Fator X/fisiologia , Peptídeos/fisiologia , Deleção de Sequência , Sequência de Aminoácidos , Carboidratos , Catálise , Fator VIIa/metabolismo , Fator X/química , Fator X/genética , Fator Xa/metabolismo , Homeostase , Humanos , Cinética , Lipossomos , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/genética , Tromboplastina/metabolismoRESUMO
We investigated the effects of addition of recombinant activated coagulation factor VII (rFVIIa) to coagulation factor-deficient plasma and whole blood, using thrombelastograms (TEGs). The addition of rFVIIa to factor II- or X-deficient plasma did not correct hemostatic parameters, whereas it produced partial responses in factor V-, VIII- or IX-deficient plasma and good responses in factor VII-, XI- or XII-deficient plasma. Furthermore, the addition of rFVIIa and platelets (30-100 x 10(3)/ micro l) to platelet-poor plasma produced marked corrections, producing TEGs similar to those of platelet-rich plasma. These results indicate that factors II and X are essential for the hemostatic effects of rFVIIa, and that factors V and VIII promote these effects. We believe that TEGs are, at present, one of the most useful tools for evaluating in vitro hemostatic effects of rFVIIa.