RESUMO
OBJECTIVE: To examine whether tissue factor pathway inhibitor or acquired activated protein C (APC) resistance influences the increased risk of coronary heart disease (CHD) due to estrogen plus progestin therapy. APPROACH AND RESULTS: Prospective nested case-control study of 205 cases of CHD and 481 matched controls in the Women's Health Initiative randomized trial of estrogen plus progestin therapy. After multivariable covariate adjustment, both baseline tissue factor pathway activity (P=0.01) and APC resistance (P=0.004) were associated positively with CHD risk. Baseline tissue factor pathway activity and APC resistance singly or jointly did not significantly modify the effect of estrogen plus progestin on CHD risk. Compared with placebo, estrogen plus progestin decreased tissue factor pathway inhibitor activity and increased APC resistance but these changes did not seem to modify or mediate the effect of estrogen plus progestin on CHD risk. CONCLUSIONS: Tissue factor pathway inhibitor activity and APC resistance are related to CHD risk in women, but may not explain the increased CHD risk due to estrogen plus progestin therapy. The data from this study do not support the clinical use of measuring these hemostatic factors to help stratify risk before hormone therapy. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00000611.
Assuntos
Resistência à Proteína C Ativada/complicações , Doença das Coronárias/induzido quimicamente , Terapia de Reposição de Estrogênios/efeitos adversos , Estrogênios/efeitos adversos , Lipoproteínas/metabolismo , Progestinas/efeitos adversos , Resistência à Proteína C Ativada/sangue , Resistência à Proteína C Ativada/diagnóstico , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Doença das Coronárias/sangue , Doença das Coronárias/diagnóstico , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Pós-Menopausa , Estudos Prospectivos , Medição de Risco , Fatores de RiscoRESUMO
Activated protein C (APC) resistance, often associated with the factor V (FV) Leiden mutation, is the most common risk factor for venous thrombosis. We observed increased APC resistance in carriers of fibrinogen γ gene (FGG) haplotype 2, which is associated with reduced levels of the alternatively spliced fibrinogen γ' chain. This finding prompted us to study the effects of fibrinogen and its γ' chain on APC resistance. Fibrinogen, and particularly the γA/γ' isoform, improved the response of plasma to added APC in the thrombin generation-based assay. Similarly, a synthetic peptide mimicking the C-terminus of the fibrinogen γ' chain, which binds thrombin and inhibits its activities, greatly increased the APC sensitivity of normal and FV Leiden plasma, likely due to its ability to inhibit thrombin-mediated activation of FV and FVIII. Although the fibrinogen γ' peptide also inhibited protein C activation by the thrombin/thrombomodulin complex, it still increased the sensitivity of plasma to endogenously formed APC when thrombin generation was measured in the presence of soluble thrombomodulin. We conclude that fibrinogen, and particularly fibrinogen γ', increases plasma APC sensitivity. The fibrinogen γ' peptide might form the basis for pharmacologic interventions to counteract APC resistance.
Assuntos
Fator V/metabolismo , Fibrinogênios Anormais/metabolismo , Proteína C/metabolismo , Resistência à Proteína C Ativada/sangue , Resistência à Proteína C Ativada/complicações , Resistência à Proteína C Ativada/genética , Adulto , Sequência de Aminoácidos , Fator V/genética , Feminino , Fibrinogênios Anormais/genética , Haplótipos , Heterozigoto , Humanos , Masculino , Dados de Sequência Molecular , Fatores de Risco , Trombina/metabolismo , Trombomodulina/sangue , Trombose Venosa/sangue , Trombose Venosa/etiologia , Trombose Venosa/genéticaRESUMO
Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd â¼1 nM). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded ß-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nM). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.
Assuntos
Coagulantes/química , Fator VIIa/química , Fator Xa/química , Lipoproteínas/antagonistas & inibidores , Peptídeos/química , Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/síntese química , Coagulantes/metabolismo , Coagulantes/uso terapêutico , Fator VIIa/metabolismo , Fator Xa/metabolismo , Hemofilia A/tratamento farmacológico , Hemofilia A/metabolismo , Hemorragia/tratamento farmacológico , Hemorragia/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited â¼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.
Assuntos
Domínio Catalítico , Protrombina/metabolismo , Tromboplastina/metabolismo , Trombose/enzimologia , Substituição de Aminoácidos , Animais , Coagulação Sanguínea , Ativação Enzimática/genética , Humanos , Cinética , Camundongos , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Protrombina/química , Protrombina/genética , Tromboplastina/química , Tromboplastina/genética , Trombose/genéticaRESUMO
BACKGROUND AND PURPOSE: To test whether changes in plasma tissue factor pathway inhibitor (TFPI) levels or activated protein C resistance (normalized activated protein C resistance ratio [nAPCsr]) modify the increased risk of ischemic stroke due to postmenopausal hormone therapy. METHODS: Nested case-control study of 455 cases of ischemic stroke and 565 matched control subjects in the Women's Health Initiative trials of postmenopausal hormone therapy. RESULTS: Baseline free TFPI was associated with ischemic stroke risk (OR per SD increase, 1.17; 95% CI, 1.01-1.37; P=0.039), but baseline nAPCsr was not (OR per SD increase, 0.89; 95% CI, 0.75-1.05; P=0.15). Baseline TFPI levels and nAPCsr did not modify the effect of postmenopausal hormone therapy on ischemic stroke. Treatment-induced mean changes of -28% in free TFPI and +65% in nAPCsr did not change the risk of ischemic stroke (interaction P=0.452 and 0.971, respectively). In subgroup analyses, baseline nAPCsr was inversely associated with lacunar strokes (OR per SD increase, 0.74; 95% CI, 0.57-0.96; P=0.025) and baseline free TFPI interacted with treatment to increase large vessel atherosclerotic strokes (P=0.008). CONCLUSIONS: Procoagulant changes in TFPI or nAPCsr do not modify the increased ischemic stroke risk due to postmenopausal hormone therapy. Clinical Trial Registration- URL: www.clinicaltrials.gov. Unique identifier: NCT 00000611.
Assuntos
Resistência à Proteína C Ativada/sangue , Isquemia Encefálica/sangue , Isquemia Encefálica/epidemiologia , Terapia de Reposição Hormonal , Lipoproteínas/sangue , Pós-Menopausa/sangue , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/epidemiologia , Idoso , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Proteína C/metabolismo , Estudos Retrospectivos , Fatores de RiscoRESUMO
Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet FV in 4 patients with severe congenital FV deficiency (3 with plasma FV clotting activity [FV:C] < 1%). When triggered with tissue factor (TF) concentrations up to 50pM, platelet-poor plasma (PPP) from the patients with undetectable plasma FV showed no thrombin generation, whereas platelet-rich plasma (PRP) formed thrombin already at 1 to 5pM of TF. Thrombin generation in PRP from the FV-deficient patients was enhanced to near-normal levels by platelet activators (collagen or Ca(2+)-ionophore) and could be completely suppressed by specific FV inhibitors, suggesting FV dependence. Accordingly, platelet FV antigen and activity were measurable in all FV-deficient patients and platelet FVa could be visualized by Western blotting. Normalization of the tissue factor pathway inhibitor (TFPI) level, which is physiologically low in FV-deficient plasma, almost completely abolished thrombin generation in PRP from the FV-deficient patients. In conclusion, patients with undetectable plasma FV may contain functional FV in their platelets. In combination with low TFPI level, residual platelet FV allows sufficient thrombin generation to rescue these patients from fatal bleeding.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Deficiência do Fator V/sangue , Transtornos Hemorrágicos/sangue , Trombina/biossíntese , Adulto , Fatores de Coagulação Sanguínea/genética , Deficiência do Fator V/genética , Feminino , Transtornos Hemorrágicos/genética , Humanos , Imunoprecipitação , Lipoproteínas/metabolismo , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Ativação Plaquetária , Índice de Gravidade de Doença , Adulto JovemRESUMO
Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K(D) of 0.11 +/- 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.
Assuntos
Domínio Catalítico/genética , Mutação , Proteína C/genética , Proteína C/metabolismo , Trombina/biossíntese , Arginina , Ligação Competitiva , Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/química , Fatores de Coagulação Sanguínea/metabolismo , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Ativação Enzimática , Humanos , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteína C/química , Proteína C/isolamento & purificação , Proteína S/metabolismo , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Trombina/metabolismoRESUMO
BACKGROUND: Coagulation factor V (FV), present in plasma and platelets, has both pro- and anticoagulant functions. OBJECTIVE: We investigated an FV-deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding. METHODS/RESULTS: Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet-poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet-rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC-cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon ) that favors a "closed conformation" of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5-fold) unfavorable kinetic parameters (Km , Vmax ) of prothrombin activation, but also a lower rate of APC-catalyzed inactivation in the presence of protein S. CONCLUSIONS: FVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid-binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.
Assuntos
Fator V , Trombofilia , Testes de Coagulação Sanguínea , Fator V/genética , Homozigoto , Humanos , Mutação , Trombofilia/genéticaRESUMO
Severe factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n = 11; 4.0 +/- 1.0 ng/mL free TFPI) than in controls (n = 20; 11.5 +/- 4.8 ng/mL), while persons with partial FV deficiency had inter-mediate levels (n = 16; 7.9 +/- 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding.
Assuntos
Deficiência do Fator V/sangue , Lipoproteínas/sangue , Adulto , Idoso , Estudos de Casos e Controles , DNA/genética , Fator V/química , Fator V/genética , Fator V/metabolismo , Deficiência do Fator V/complicações , Deficiência do Fator V/congênito , Deficiência do Fator V/genética , Feminino , Hemofilia A/sangue , Transtornos Hemorrágicos/sangue , Transtornos Hemorrágicos/etiologia , Transtornos Hemorrágicos/prevenção & controle , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Masculino , Pessoa de Meia-Idade , Complexos Multiproteicos/sangue , Mutação , Ressonância de Plasmônio de Superfície , Trombina/biossínteseRESUMO
We used the thrombin generation assay to evaluate the hypercoagulable state according to JAK2(V617F) mutational status in essential thrombocythemia (ET) and polycythemia vera (PV) patients. Thrombin generation was determined in the presence and absence of activated protein C (APC), and APC resistance was expressed as normalized APC sensitivity ratio (nAPCsr). Tissue factor pathway inhibitor (TFPI), total and free protein S (PS), prothrombin (FII), factor V (FV), and neutrophil elastase were measured in plasma; CD11b was measured on neutrophils. Compared with normal controls, patients had a lower endogenous thrombin potential in the absence of APC but had a higher endogenous thrombin potential in the presence of APC, showing the occurrence of APC resistance. The nAPCsr increased in JAK2(V617F) carriers compared with noncarriers and was highest in JAK2(V617F) homozygous patients. FII, FV, free PS, and TFPI levels were reduced in patients, mainly in JAK2(V617F) carriers. Multiple regression analysis indicated the low free PS level as major determinant of the increased nAPCsr. Elastase was increased in patients and inversely correlated with free PS. In conclusion, these data indicate the occurrence of acquired APC resistance in ET and PV patients, probably because of a reduction in free PS levels. The APC-resistant phenotype is influenced by the JAK2(V617F) mutational load.
Assuntos
Janus Quinase 2/metabolismo , Mutação de Sentido Incorreto , Policitemia Vera/enzimologia , Proteína C/metabolismo , Trombina/metabolismo , Trombocitemia Essencial/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Antígeno CD11b , Fator V/genética , Fator V/metabolismo , Feminino , Heterozigoto , Homozigoto , Humanos , Janus Quinase 2/genética , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Policitemia Vera/genética , Proteína C/genética , Proteína S/genética , Proteína S/metabolismo , Protrombina/genética , Protrombina/metabolismo , Trombina/genética , Trombocitemia Essencial/genéticaRESUMO
BACKGROUND: Protein S, which circulates in plasma in both free and bound forms, is an anticoagulant protein that stimulates activated protein C and tissue factor pathway inhibitor. Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned. DESIGN AND METHODS: Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, or having type I or type III deficiency according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals). RESULTS: Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, tissue factor pathway inhibitor and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and two large deletions were identified in the genetically characterized families. CONCLUSIONS: Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased risk of thrombosis. These findings may, however, be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.
Assuntos
Deficiência de Proteína S/sangue , Trombofilia/etiologia , Trombose/etiologia , Adolescente , Adulto , Intervalo Livre de Doença , Família , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Deficiência de Proteína S/classificação , Deficiência de Proteína S/genética , Adulto JovemRESUMO
BACKGROUND: Protein S, which circulates in plasma in a free and bound form, is an anticoagulant protein that stimulates both activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned. DESIGN AND METHODS: Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, type I deficient or type III deficient according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals). RESULTS: Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, TFPI and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the APC- and TFPI-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and 2 large deletions were identified in the genetically characterized families. CONCLUSIONS: Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased thrombosis risk. However, these findings may be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.
Assuntos
Deficiência de Proteína S/genética , Proteína S/metabolismo , Trombofilia/sangue , Trombofilia/genética , Trombose Venosa/sangue , Trombose Venosa/genética , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína S/classificação , Proteína S/genética , Deficiência de Proteína S/classificação , Deficiência de Proteína S/complicações , Fatores de Risco , Trombofilia/etiologia , Trombose Venosa/etiologia , Adulto JovemRESUMO
In the last decades evidence was obtained that protein S not only acts as cofactor of activated protein C (APC) in the downregulation of coagulation, but also expresses anticoagulant activity in the absence of APC. The search for the mechanism(s) underlying the APC-independent anticoagulant activity of protein S was hampered by the fact that protein S exhibited 2 seemingly identical anticoagulant activities in model systems and in plasma. Later it was shown that the anticoagulant activity of purified protein S in model systems was dependent on the concentration of phospholipid vesicles and was explained by low amounts of protein S multimers generated during purification that effectively inhibited phospholipid-dependent coagulation reactions via competition for phospholipid binding sites. Plasma does not contain multimers, and the anticoagulant activity of protein S in plasma was not affected by the phospholipid concentration but was dependent on the amount of tissue factor (TF) used for initiation of thrombin generation. This led to the discovery that protein S acts as cofactor of tissue factor pathway inhibitor (TFPI) which stimulates the inhibition of factor Xa by TFPI approximately 10-fold. The current review describes the background of the TFPI-cofactor activity of protein S as well as the rationale for the observation that the TFPI/protein S system particularly inhibits the TF pathway at low procoagulant stimuli.
Assuntos
Lipoproteínas/fisiologia , Proteína S/fisiologia , Anticoagulantes/metabolismo , Coagulação Sanguínea/fisiologia , Fator VIIa/antagonistas & inibidores , Inibidores do Fator Xa , Humanos , Cinética , Modelos Biológicos , Proteína S/química , Estrutura Quaternária de ProteínaRESUMO
BACKGROUND: Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. OBJECTIVE: This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. MATERIALS AND METHODS: FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). RESULTS: The TFPIr (0.25-0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28-0.31) to FVL heterozygotes (0.35, 95% CI 0.34-0.37) and homozygotes (0.39, 95% CI 0.37-0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. CONCLUSION: We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.
Assuntos
Transtornos da Coagulação Sanguínea/diagnóstico , Testes de Coagulação Sanguínea/métodos , Fator V/metabolismo , Fator Va/metabolismo , Lipoproteínas/metabolismo , Fragmentos de Peptídeos/metabolismo , Coagulação Sanguínea , Transtornos da Coagulação Sanguínea/genética , Fator V/genética , Fator Xa/metabolismo , Heterozigoto , Homozigoto , Humanos , Mutação/genética , ProteóliseRESUMO
OBJECTIVE: To assess the effect on hemostasis parameters of a new combined oral contraceptive (COC). STUDY DESIGN: In this randomized, single centre, open-label, exploratory study, healthy women received either 15â¯mg estetrol/3 mg drospirenone (E4/DRSP) (nâ¯=â¯39), 30â¯mcg ethinylestradiol/150â¯mcg levonorgestrel (EE/LNG) (nâ¯=â¯30), or 20â¯mcg ethinylestradiol/3 mg drospirenone (EE/DRSP) (nâ¯=â¯32) for six 28-day cycles. Blood was collected at baseline, cycle 3, and cycle 6. Median change from baseline was evaluated for procoagulant, anticoagulant, and fibrinolytic parameters, and for sex hormone-binding globulin (SHBG). RESULTS: Median change of endogenous thrombin potential (ETP) based activated protein C sensitivity resistance (APCr) at cycle 6 was +30% for E4/DRSP, +165% for EE/LNG (p-value <0.05 vs E4/DRSP), and +219% for EE/DRSP (p-value <0.05 vs E4/DRSP). Changes to prothrombin fragment 1â¯+â¯2 and SHBG for E4/DRSP, EE/LNG, and EE/DRSP were +23%, +71%, and +64% (p-value <0.05 vs E4/DRSP); and +55%, +74% and +251% (p-value <0.05 vs E4/DRSP), respectively. At cycle 6, changes to other hemostasis parameters for E4/DRSP were similar or smaller than for EE/LNG or EE/DRSP. CONCLUSIONS: In this study, changes in hemostasis parameters after treatment with 6 cycles of E4/DRSP were smaller or similar to those observed for EE/LNG. Similar, but more pronounced changes were also observed versus EE/DRSP, which supports the hypothesis that the effect of COCs on hemostasis parameters is mainly mediated by the estrogenic component. Further studies are needed to provide more insight into the venous thromboembolic risk of E4/DRSP. IMPLICATIONS STATEMENT: This study reports that the effects on hemostasis parameters of a COC containing 15â¯mg E4/3 mg DRSP are less or similar to those for EE/LNG or EE/DRSP. It also demonstrates that the choice of estrogen modulates the effects of COCs on hemostasis parameters.
Assuntos
Resistência à Proteína C Ativada/induzido quimicamente , Androstenos/farmacologia , Anticoncepcionais Orais Combinados/farmacologia , Estetrol/farmacologia , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Hemostasia/efeitos dos fármacos , Adolescente , Adulto , Androstenos/administração & dosagem , Androstenos/efeitos adversos , Anticoncepcionais Orais Combinados/administração & dosagem , Anticoncepcionais Orais Combinados/efeitos adversos , Estetrol/administração & dosagem , Estetrol/efeitos adversos , Estrogênios/administração & dosagem , Estrogênios/efeitos adversos , Etinilestradiol/administração & dosagem , Etinilestradiol/efeitos adversos , Feminino , Humanos , Ciclo Menstrual/efeitos dos fármacos , Pessoa de Meia-Idade , Globulina de Ligação a Hormônio Sexual/efeitos dos fármacos , Adulto JovemRESUMO
Coagulation factor V (FV), present in plasma and platelets, is an indispensable clotting factor, as demonstrated by the uniform lethality of FV knock-out mice. Surprisingly, however, severe FV deficiency is rarely fatal in humans. In fact, although several cases of life-threatening intracranial haemorrhage have been reported in FV-deficient newborns, many patients with undetectable FV levels experience only mild to moderate bleeding and do not require routine prophylaxis. While the reasons for this variable phenotypic expression are largely unknown, several observations from different laboratories indicate platelets as crucial players in FV deficiency. Moreover, we have recently shown that plasma levels of tissue factor pathway inhibitor are considerably reduced in FV-deficient plasma, which results in enhanced thrombin generation especially at very low FV levels (<2%). The present review discusses and integrates these findings in the context of the biology of FV and the clinical features of FV deficiency.
Assuntos
Deficiência do Fator V/congênito , Hemorragia/congênito , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Fator V/metabolismo , Deficiência do Fator V/sangue , Hemorragia/sangue , Humanos , Recém-Nascido , Lipoproteínas/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismoRESUMO
Exogenously administered estrogens and progestogens as during combined oral contraceptive use increase the risk of venous thrombosis. The thrombin generation-based APC resistance assay is a global coagulation test that enables quantification of the net prothrombotic effect of combined oral contraceptives and that predicts the risk of thrombosis. The thrombotic risk of the levonorgestrel-releasing intrauterine system is unknown. It was the objective of this study to evaluate the thrombotic risk by comparing the APC resistance before and after insertion of a levonorgestrel-releasing or a copper-containing intrauterine device. We measured normalized APC-sensitivity ratios (nAPCsr) before and three months after insertion of the levonorgestrel-intrauterine system in 56 women and the copper-intrauterine device in 18 women. In women without hormonal contraceptive use or a pregnancy in the three months before collection of the baseline samples, nAPCsr were lower three months after insertion of the levonorgestrel-intrauterine system than at baseline (difference -0.29; 95% CI -0.04 to -0.53) and hardly changed after insertion of the copper-intrauterine device (difference -0.11; 95% CI -1.03 to 0.82). In women who switched from a combined oral contraceptive to the levonorgestrel-system the difference was more pronounced (-1.48; 95% CI -0.85 to -2.11). In this study we observed that the levonorgestrel-intrauterine system decreases the resistance to APC which indicates that the levonorgestrel-intrauterine system does not have a prothrombotic effect.
Assuntos
Resistência à Proteína C Ativada/etiologia , Anticoncepcionais Orais Combinados/efeitos adversos , Dispositivos Intrauterinos de Cobre/efeitos adversos , Dispositivos Intrauterinos Medicados/efeitos adversos , Levanogestrel/efeitos adversos , Trombose Venosa/etiologia , Resistência à Proteína C Ativada/sangue , Resistência à Proteína C Ativada/genética , Adulto , Biomarcadores/sangue , Feminino , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Linhagem , Medição de Risco , Fatores de Risco , Trombina/metabolismo , Fatores de Tempo , Trombose Venosa/sangue , Trombose Venosa/genética , Adulto JovemRESUMO
OBJECTIVE: To compare the effects of oral and transdermal contraceptives containing similar hormone formulations on vascular risk markers. METHODS: We conducted a randomized, investigator-blinded, crossover, clinical trial with 24 healthy women, aged 18-35 years, who received 2 months of transdermal or oral contraceptive, 2 months washout, then 2 months of the alternative medication. The transdermal contraceptive contained 0.75 mg ethinyl estradiol and 6 mg norelgestromin. The oral contraceptive contained 35 mcg ethinyl estradiol and 250 mcg norgestimate. Blood samples taken before and after each treatment were analyzed in batch for D-dimer, von Willebrand factor, factor VIII, total and free protein S, antithrombin, fibrinogen, C-reactive protein, and normalized activated protein C sensitivity ratio (nAPCsr) determined with two thrombin generation-based assays, the alpha2macroglobulin-thrombin end point method (alpha2M-IIa) and calibrated automated thrombinography. Repeated measures analysis of variance was used for analysis. RESULTS: For both contraceptives (transdermal, oral) there were significant declines in free (19%, 11%) and total protein S (19%, 13%) and antithrombin (13%, 10%); increases in fibrinogen (8%, 10%), C-reactive protein (220%, 292%), nAPCsr alpha2M-IIa (81%, 61%), and nAPCsr calibrated automated thrombinography (102%, 68%), all P<.05. Transdermal contraceptives had a greater effect than oral contraceptives on free protein S (P=.07), nAPCsr alpha2M-IIa (P=.06), and nAPCsr calibrated automated thrombinography (P=.03). CONCLUSION: Oral and transdermal contraception with similar hormones had similar adverse effects on vascular risk markers. This suggests that this transdermal contraceptive has at least a similar thrombosis risk as its oral counterpart. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00554632 LEVEL OF EVIDENCE: I.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Anticoncepcionais Orais Hormonais/administração & dosagem , Anticoncepcionais Orais Hormonais/efeitos adversos , Trombose/induzido quimicamente , Administração Cutânea , Adolescente , Adulto , Análise de Variância , Antitrombinas/análise , Antitrombinas/metabolismo , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Proteína C-Reativa , Estudos Cross-Over , Combinação de Medicamentos , Etinilestradiol/administração & dosagem , Etinilestradiol/efeitos adversos , Fator VIII/análise , Fator VIII/metabolismo , Feminino , Fibrinogênio/análise , Fibrinogênio/metabolismo , Humanos , Norgestrel/administração & dosagem , Norgestrel/efeitos adversos , Norgestrel/análogos & derivados , Oximas/administração & dosagem , Oximas/efeitos adversos , Proteína S/análise , Proteína S/metabolismo , Fatores de Risco , Trombose/sangue , Trombose/epidemiologia , Fator de von Willebrand/análise , Fator de von Willebrand/metabolismoRESUMO
INTRODUCTION: Activated protein C (APC) resistance is associated with an increased risk of venous thrombosis. High levels of estradiol and progesterone, e.g. during ovarian stimulation and pregnancy, as well as exogenously administered estrogens and progestagens during oral contraceptive use, induce an acquired form of APC resistance. Several coagulation factors display a cyclic pattern during the menstrual cycle due to the fluctuation of estradiol and progesterone. The aim of the study was to evaluate whether varying levels of estradiol and progesterone during the menstrual cycle are associated with differences in sensitivity to APC. MATERIALS AND METHODS: Normalized APC sensitivity ratios (nAPCsr) were determined with the thrombin generation-based APC-resistance test at six different time points during the menstrual cycle in thirteen wildtype women and six women with factor VLeiden. RESULTS: Mean nAPCsr varied slightly during the menstrual cycle. Women without factor VLeiden were more likely to have lower nAPCsr at the beginning of the cycle than later on in the cycle (1.34 versus 1.54 and 1.58, Friedman ranking test p=0.009). CONCLUSIONS: The sensitivity to APC differs between the different phases of the menstrual cycle. This cyclic variability could be useful in improving studies on APC resistance in women.
Assuntos
Estradiol/sangue , Fator V/análise , Ciclo Menstrual/sangue , Progesterona/sangue , Proteína C , Resistência à Proteína C Ativada/sangue , Adulto , Testes de Coagulação Sanguínea , Fator V/genética , Feminino , Humanos , Mutação , Reação em Cadeia da Polimerase/métodos , Proteína C/farmacologia , Protrombina/genética , Valores de Referência , Sensibilidade e Especificidade , Trombina/química , Trombina/efeitos dos fármacos , Fatores de TempoRESUMO
OBJECTIVE: Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. METHODS AND RESULTS: High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated alphaIIb beta3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active alphaIIb beta3. CONCLUSIONS: These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication.