RESUMO
Neutrophils and neutrophil extracellular traps (NETs) have been shown to be involved in coagulation. However, the interactions between neutrophils or NETs and fibrin(ogen) in clots, and the mechanisms behind these interactions are not yet fully understood. In this in vitro study, the role of neutrophils or NETs on clot structure, formation and dissolution was studied with a combination of confocal microscopy, turbidity and permeation experiments. Factor (F)XII, FXI and FVII-deficient plasmas were used to investigate which factors may be involved in the procoagulant effects. We found both neutrophils and NETs promote clotting in plasma without the addition of other coagulation triggers, but not in purified fibrinogen, indicating that other factors mediate the interaction. The procoagulant effects of neutrophils and NETs were also observed in FXII- and FVII-deficient plasma. In FXI-deficient plasma, only the procoagulant effects of NETs were observed, but not of neutrophils. NETs increased the density of clots, particularly in the vicinity of the NETs, while neutrophils-induced clots were less stable and more porous. In conclusion, NETs accelerate clotting and contribute to the formation of a denser, more lysis resistant clot architecture. Neutrophils, or their released mediators, may induce clotting in a different manner to NETs, mediated by FXI.
Assuntos
Coagulação Sanguínea , Armadilhas Extracelulares/metabolismo , Fator XI/metabolismo , Neutrófilos/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Meios de Cultura/química , Meios de Cultura/farmacologia , Fibrina/química , Humanos , Neutrófilos/citologia , Receptores de IgG/metabolismo , Trombina/farmacologiaRESUMO
Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.
Assuntos
Fator XIII/metabolismo , Fibrina/metabolismo , Fibrinólise , Domínio Catalítico , Cisteína , Ativação Enzimática , Fator XIII/química , Fator XIII/genética , Humanos , Cinética , Mutação , Domínios Proteicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.
Assuntos
Tromboembolia/terapia , Trombose/sangue , Trombose/terapia , Anticoagulantes/uso terapêutico , Biomarcadores/sangue , Coagulação Sanguínea , Eritrócitos/metabolismo , Fator VIII/metabolismo , Fator XII/metabolismo , Fator XIII/metabolismo , Humanos , Macrófagos/metabolismo , Países Baixos , Fenótipo , Placa Aterosclerótica/sangue , Placa Aterosclerótica/diagnóstico , Placa Aterosclerótica/terapia , Polifosfatos/metabolismo , Fatores de Risco , Transdução de Sinais , Tromboembolia/sangue , Tromboembolia/diagnóstico , Trombose/diagnósticoRESUMO
Essentials Elevated lipoproteinp(a) is an independent and causal risk factor for atherothrombotic diseases. rs3798220 (Ile/Met substitution in apo(a) protease-like domain) is associated with disease risk. Recombinant I4399M apo(a) altered clot structure to accelerate coagulation/delay fibrinolysis. Evidence was found for increased solvent exposure and oxidation of Met residue. SUMMARY: Background Lipoprotein(a) (Lp[a]) is a causal risk factor for a variety of cardiovascular diseases. Apolipoprotein(a) (apo[a]), the distinguishing component of Lp(a), is homologous with plasminogen, suggesting that Lp(a) can interfere with the normal fibrinolytic functions of plasminogen. This has implications for the persistence of fibrin clots in the vasculature and hence for atherothrombotic diseases. A single-nucleotide polymorphism (SNP) (rs3798220) in the gene encoding apo(a) has been reported that results in an IleâMet substitution in the protease-like domain (I4399M variant). In population studies, the I4399M variant has been correlated with elevated plasma Lp(a) levels and higher coronary heart disease risk, and carriers of the SNP had increased cardiovascular benefit from aspirin therapy. In vitro studies suggested an antifibrinolytic role for Lp(a) containing this variant. Objectives We performed a series of experiments to assess the effect of the IleâMet substitution on fibrin clot formation and lysis, and on the architecture of the clots. Results We found that the Met variant decreased coagulation time and increased fibrin clot lysis time as compared with wild-type apo(a). Furthermore, we observed that the presence of the Met variant significantly increased fibrin fiber width in plasma clots formed ex vivo, while having no effect on fiber density. Mass spectrometry analysis of a recombinant apo(a) species containing the Met variant revealed sulfoxide modification of the Met residue. Conclusions Our data suggest that the I4399M variant differs structurally from wild-type apo(a), which may underlie key differences related to its effects on fibrin clot architecture and fibrinolysis.
Assuntos
Apoproteína(a)/sangue , Apoproteína(a)/genética , Coagulação Sanguínea/genética , Fibrinólise/genética , Lipoproteína(a)/sangue , Lipoproteína(a)/genética , Polimorfismo de Nucleotídeo Único , Trombose/sangue , Trombose/genética , Adulto , Apoproteína(a)/química , Feminino , Fibrina/química , Fibrina/metabolismo , Predisposição Genética para Doença , Células HEK293 , Homozigoto , Humanos , Lipoproteína(a)/química , Masculino , Metionina , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Oxirredução , Fenótipo , Conformação Proteica , Proteínas Recombinantes/sangue , Relação Estrutura-Atividade , TransfecçãoRESUMO
UNLABELLED: Essentials Patients with cirrhosis have hemostatic changes, which may contribute to a risk of thrombosis. This in vitro study compares clot formation and structure between patients and healthy subjects. Clot formation is delayed in patients; ultimately, however, clot permeability is decreased. The thrombogenic structure of fibrin clots may contribute to the thrombotic risk in cirrhosis. ABSTRACT: Background and Objectives Patients with cirrhosis can be at risk of thrombotic complications due to an imbalance between hemostatic components. However, little is known on how the disease affects clot generation or how alterations in the structure of fibrin clots may affect the hemostatic function of these patients. Methods We investigated the formation and structure of clots generated with plasma and purified fibrinogen of 42 patients with cirrhosis. Clots generated with plasma and fibrinogen of 29 healthy volunteers were studied for comparison. Clot formation and structure were assessed by turbidity, permeation studies, confocal laser and scanning electron microscopy (SEM). The extent of fibrinogen oxidation was assessed by measuring the carbonyl content of purified fibrinogen samples. Results Tissue factor and thrombin-induced clotting of plasma was delayed in patients. The clotting rate was also decreased, but change in turbidity, fibrin density and fiber thickness were largely comparable to healthy volunteers. Conversely, clot permeability was significantly decreased in patients. When clots were generated with purified fibrinogen, differences in clot formation and structure similar to those in plasma were found. The carbonyl content was increased in patient fibrinogen and correlated with disease severity and clot permeability. Conclusions Delayed clot formation in cirrhosis ultimately results in decreased clot permeability. Similar alterations in clots generated with purified fibrinogen suggest that modifications of the molecule are (partly) responsible. Taken together, these findings are indicative of hypercoagulable features of clots of patients with cirrhosis, which may explain the increased risk of thrombosis associated with this condition.
Assuntos
Coagulantes/química , Fibrinogênio/química , Fibrose/sangue , Adulto , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Fator XIII/química , Feminino , Fibrina/química , Voluntários Saudáveis , Hemostasia , Hemostáticos , Humanos , Masculino , Malondialdeído/sangue , Microscopia Confocal , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Oxigênio/química , Permeabilidade , Trombose/sangueRESUMO
The clinical phenotype of patients with congenital dysfibrinogenaemia is highly heterogeneous, from absence of symptoms to mild bleeding, or thrombosis. A few mutations are associated with a specific phenotype, but generally the clinical course is not predictable. We investigated whether fibrin clot properties are correlated with the patient's phenotype and/or genotype. Ex vivo plasma fibrin clot characteristics, including turbidity, fibrinolysis, clot permeability and fibrin fibre density assessed by laser scanner confocal microscopy were investigated in 24 genotyped patients with congenital dysfibrinogenaemia compared to normal pool plasma. Compared to normal pool plasma, the patients were characterised by slower fibrin polymerisation (lag time, 345.10 ± 22.98 vs. 166.00s), thinner fibrin fibres (maximum absorbance, 0.15 ± 0.01 vs. 0.31), prolonged clot lysis time (23.72 ± 0.97 vs. 20.32 min) and larger clot pore size (21.5×10(-9) ± 4.48×10(-9) vs. 7.96×10(-9)cm(2)). Laser scanning confocal microscopy images confirmed disorganised fibrin networks in all patients. Patients with tendency to bleed showed an increased permeability compared to asymptomatic patients (p=0.01) and to patients with a thrombotic history (p=0.02) while patients with thrombotic history had a tendency to have a prolonged clot lysis time. Fibrin clot properties were similar among hotspot mutations. Further studies including a larger number of patients are needed to evaluate whether analysis of permeability and clot lysis time may help to distinguish the clinical phenotype in these patients and to assess differences according to the genotype.
Assuntos
Anemia Diseritropoética Congênita/sangue , Anemia Diseritropoética Congênita/patologia , Tempo de Lise do Coágulo de Fibrina/métodos , Fibrina/metabolismo , Fibrina/ultraestrutura , Adulto , Idoso , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/ultraestrutura , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
INTRODUCTION: Post-thrombotic syndrome (PTS) is a chronic sequel of deep vein thrombosis (DVT). The clot structure and fibrinolytic potential in PTS is currently unknown. OBJECTIVE: To assess the fibrinolytic potential and clot structure in patients with PTS. MATERIALS AND METHODS: Patients with a history of DVT were included in a case-control study: patients with PTS (cases n=30) and without PTS (controls n=30), and 30 apparently healthy individuals (HI) without venous thromboembolism (VTE) or venous insufficiency were enrolled. Fibrinolysis and clot structure were assessed by turbidimetric assays, permeation, and confocal microscopy. Fibrinogen was measured by Clauss and fibrinogen γ' by ELISA. RESULTS: We observed a significant trend of decreasing maximum turbidity from HI (median 0.52 [IQR 0.46-0.62]), to controls (0.49 [IQR 0.41-0.55]), to cases (0.46 [IQR 0.39-0.49]) p=0.020. Fibrinogen was lower in patients (cases and controls) (3.69g/L [IQR 3.31-4.26]) compared to HI (4.17 [IQR 3.69-4.65]) p=0.041. Patients with recurrent VTE had lower maximum turbidity and lower permeation than patients with one episode of VTE; (0.31 [IQR 0.25-0.39] versus 0.38 [IQR 0.34-0.44] p=0.008) and (6.0×10(-9)/cm(2) [IQR 5.1-7.9] versus 7.7×10(-9)/cm(2) [IQR 6.0-10.0] p=0.047) respectively, at equal fibrinogen levels. There were no differences in lysis time, confocal microscopy, or fibrinogen γ'. CONCLUSIONS: Lower maximum turbidity, indicating a tendency towards thinner fibres and denser clots, was found in patients with PTS as well as in patients with recurrent VTE. Fibrinogen levels did not explain these differences in clot structure. The abnormal clot structure may contribute to the increased thrombotic risk profile in patients with PTS.
Assuntos
Coagulação Sanguínea , Fibrinogênio/análise , Síndrome Pós-Trombótica/sangue , Síndrome Pós-Trombótica/patologia , Trombose Venosa/sangue , Trombose Venosa/patologia , Idoso , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Optimal hemostatic management during orthotopic liver transplantation (OLT) remains a challenge. The cause of bleeding during OLT is multifactorial, and may include hemostatic imbalance. Fibrinogen concentrates are increasingly being used to control perioperative bleeding during OLT. However, administration is based on arbitrary thresholds of fibrinogen levels. Importantly, studies on fibrin clot structure during OLT are lacking. OBJECTIVE: We determined the hemostatic efficacy of fibrinogen concentrate in correcting the fibrin structure. METHODS: Plasma samples taken at various times during OLT from 15 patients and 15 healthy controls were spiked with 1 g L(-1) fibrinogen concentrate or saline. Turbidity, fibrin fiber density and permeability of the fibrin clots were assessed. RESULTS: Clotting rate and turbidity were significantly decreased at the start of surgery, and decreased even further during surgery. Addition of fibrinogen significantly increased the clotting rate and turbidity at all time points, but did not normalize it. Fibrin density was significantly reduced after reperfusion as compared with the density at the start of surgery and in healthy controls. Fibrin density improved significantly after addition of fibrinogen in samples taken at the start of surgery and after reperfusion. The severely impaired polymerization and decreased density after reperfusion were accompanied by significantly increased permeability of the clot as compared with the start of surgery and in controls, which was completely restored after addition of fibrinogen. CONCLUSIONS: Ex vivo addition of fibrinogen concentrate during OLT substantially improves the structural properties of the fibrin clot, which, particularly after reperfusion, shows hypocoagulable features. These data support the use of fibrinogen concentrate to control bleeding complications during OLT.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Perda Sanguínea Cirúrgica/prevenção & controle , Fibrina/metabolismo , Fibrinogênio/farmacologia , Hemostáticos/farmacologia , Transplante de Fígado/efeitos adversos , Adulto , Idoso , Testes de Coagulação Sanguínea , Estudos de Casos e Controles , Fator XIII/metabolismo , Feminino , Fibrina/química , Humanos , Masculino , Pessoa de Meia-Idade , Permeabilidade , Polimerização , Porosidade , Fatores de TempoRESUMO
Congenital dysfibrinogenemia is a qualitative congenital fibrinogen disorder characterized by normal antigen levels of a dysfunctional fibrinogen. The diagnosis is usually based on discrepancies between fibrinogen activity and antigen levels, but could require more specialized techniques for the assessment of fibrinogen function, owing to some limitations in routine assays. Molecular abnormalities, which are frequently heterozygous missense mutations localized in exon 2 of FGA and exon 8 of FGG, lead to defects in one or more phases of fibrinogen to fibrin conversion, fibrin network formation, and other important functions of fibrinogen. The clinical phenotype is highly heterogeneous, from no manifestations to bleeding and/or thrombotic events. Asymptomatic propositi and relatives with the predisposing genotype are at risk of developing adverse outcomes during the natural course of the disease. Correlations between genotype and phenotype have not yet been clearly established, with the exception of some abnormal fibrinogens that severely increase the risk of thrombosis. Functional analysis of polymerization and fibrinolysis, structural studies of the fibrin network and the viscoelastic properties of fibrin clot could help to predict the phenotype of congenital dysfibrinogenemia, but have not yet been evaluated in detail. The management is essentially based on personal and family history; however, even individuals who are still asymptomatic and without a family history should be carefully assessed and monitored. Particular situations, such as pregnancy, delivery, and surgery, require a multidisciplinary approach.
Assuntos
Afibrinogenemia , Coagulação Sanguínea , Fibrinogênios Anormais/genética , Mutação , Afibrinogenemia/sangue , Afibrinogenemia/diagnóstico , Afibrinogenemia/genética , Afibrinogenemia/terapia , Animais , Coagulação Sanguínea/genética , Testes de Coagulação Sanguínea , Análise Mutacional de DNA , Feminino , Fibrinogênios Anormais/metabolismo , Predisposição Genética para Doença , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Fenótipo , Valor Preditivo dos Testes , Gravidez , Complicações Hematológicas na Gravidez/sangue , Complicações Hematológicas na Gravidez/genética , Complicações Hematológicas na Gravidez/terapia , Prognóstico , Fatores de Risco , Trombose/sangue , Trombose/genética , Trombose/terapiaRESUMO
OBJECTIVE: Thrombotic changes in fibrin networks contribute to increased cardiovascular risk in patients with abdominal aortic aneurysm (AAA). Given that aspirin modulates the fibrin network, we aimed to determine if aspirin therapy is associated with changes in ex-vivo fibrin clot characteristics in AAA patients and also conducted an exploratory analysis of 5-year mortality in these individuals. METHODS: We recruited 145 male patients, divided into controls (aortic diameter < 3 cm, n = 49), AAA not taking aspirin (AAA-Asp, n = 50) and AAA on 75 mg day(-1) aspirin (AAA+Asp, n = 46), matched for aneurysm size. Characteristics of clots made from plasma and plasma-purified fibrinogen were investigated using turbidimetric analysis, permeation studies, and confocal and electron microscopy. Plasma fibrinogen, D-dimer and inflammatory marker levels were also measured. RESULTS: Maximum absorbance (MA) of plasma clots from controls was lower than that of AAA patients not on aspirin (AAA-Asp) at 0.30 ± 0.01 and 0.38 ± 0.02 au, respectively (P = 0.002), whereas aspirin-treated subjects had MA similar to controls (0.31 ± 0.02 P = 0.9). Plasma clot lysis time displayed an identical pattern at 482 ± 15, 597 ± 24 and 517 ± 27 s for control, AAA-Asp and AAA+Asp (P = 0.001 and P = 0.8). The lysis time of clots made from purified fibrinogen of AAA-Asp was longer than that of AAA+Asp patients (756 ± 47 and 592 ± 52 s, respectively; P = 0.041). Permeation studies and confocal and electron microscopy showed increased clot density in AAA-Asp compared with the AAA+Asp group. Mortality in AAA-Asp and AAA+Asp was similar, despite increased cardiovascular risk in the latter group, and both exhibited higher mortality than controls. CONCLUSION: Aspirin improves fibrin clot characteristics in patients with AAA, which may have important clinical implications.
Assuntos
Aneurisma da Aorta Abdominal/complicações , Aspirina/uso terapêutico , Fibrina/metabolismo , Fibrinólise , Idoso , Estudos de Casos e Controles , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Although both coagulation and fibrinolysis are associated with cardiovascular disease (CVD) the underlying nature and pathways of many of these associations are still unclear. Our aim was to determine which of the current or 5-year prior levels of total fibrinogen, fibrinogen γ', plasminogen activator inhibitor-1 (PAI-1act) and global fibrinolytic potential were the stronger determinant of arterial structure and function. MATERIALS AND METHODS: This prospective study consisted of 2010 Africans over the age of 35 years with 5-year follow-up data available for 1288 participants. Cardiovascular measurements included arterial stiffness, blood pressure and carotid intima media thickness. RESULTS: Fibrinogen γ' showed stronger associations with blood pressure than total fibrinogen also in the presence of other CVD risk factors. PAI-1act was positively associated with blood pressure both cross-sectionally and prospectively, with the longitudinal association being the stronger determinant, also after adjustment for known CVD risk factors. Clot lysis time (CLT) was positively associated, both prospectively and cross-sectionally, with intima media thickness and negatively with markers of arterial stiffness but not after adjustment for known CVD risk factors. CONCLUSIONS: Fibrinogen γ' was more strongly associated with CVD function than total fibrinogen. PAI-1act was significantly associated with blood pressure with changes in PAI-1 levels preceding changes in blood pressure. Different mechanisms may be at play determining arterial wall stiffness/thickening and blood pressure as observed from the opposing associations with PAI-1act and CLT. CLT was not independently related to cardiovascular measures as its associations were weakened in the presence of other known CVD risk factors.
Assuntos
População Negra , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etnologia , Fibrinogênio/metabolismo , Fibrinólise/fisiologia , África/epidemiologia , Coagulação Sanguínea , Doenças Cardiovasculares/epidemiologia , Estudos de Coortes , Progressão da Doença , Feminino , Tempo de Lise do Coágulo de Fibrina , Humanos , Masculino , Estudos Prospectivos , Fatores de RiscoRESUMO
BACKGROUND: Factor XIII is a 320 kDa tetramer, comprising two enzymatic A-subunits and two carrier B-subunits (FXIII A2 B2). Activated FXIII (FXIIIa) catalyses the formation of ε-(γ-glutamyl)lysyl covalent bonds between γ-γ, γ-α and α-α chains of adjacent fibrin molecules and also cross-links the major plasmin inhibitor, α2-antiplasmin, to fibrin. OBJECTIVES: We investigated the role of FXIII cross-linking of fibrin directly in clot morphology and its functional effect on clot formation and lysis, in the absence of α2-antiplasmin. RESULTS AND CONCLUSIONS: Our data show that the presence of FXIII during clot formation results in fibrin clots that have a significant 2.1-fold reduction in pore size, as determined by the Darcy constant, Ks, and formed thinner fibers (74.7 ± 1.5 nm) and higher density of fibers compared with those without FXIII (86.0 ± 1.7 nm, P < 0.001), as determined by scanning electron microscopy. Additionally, fibrinolysis showed a significant increase in the time to lysis for clots formed in the presence of FXIII in both static and flow systems. These data demonstrate that independent of α2-antiplasmin, FXIII activity plays a role in increasing the stability of the fibrin clot by altering its structure and increasing the resistance to fibrinolysis.
Assuntos
Fator XIII/fisiologia , Fibrina/fisiologia , Cromatografia em Gel , Fibrina/química , Fibrinólise , Humanos , Microscopia ConfocalRESUMO
BACKGROUND: Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the γA/γ' fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl-terminal end of the γ' chain. This interaction down-regulates thrombin-mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown. OBJECTIVES: To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen γ' in plasma, and to verify whether the fibrinogen γ' carboxyl-terminal peptide affects thrombin-mediated FV activation. METHODS: Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen γ' carboxyl-terminal peptide (0-500 µmol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma. RESULTS: Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen γ' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma. CONCLUSIONS: Total fibrinogen and the fibrinogen γ' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen γ' peptide is a novel mechanism of the anticoagulant activity of fibrinogen γ'.
Assuntos
Anticoagulantes/farmacologia , Fator V/agonistas , Fibrinogênios Anormais/farmacologia , Sequência de Aminoácidos , Anticoagulantes/uso terapêutico , Fator V/uso terapêutico , Fibrinogênios Anormais/química , Fibrinogênios Anormais/uso terapêutico , Humanos , Dados de Sequência MolecularRESUMO
Fibrinogen is an abundant plasma protein that, when converted to fibrin by thrombin, provides the main building blocks for the clot. Dys-, a-, and hypo-fibrinogenemias have been variably linked to a normal phenotype, bleeding or even thrombosis. Meanwhile, increased fibrinogen concentrations in the blood have been associated with risk for thrombosis. More recently, studies have focussed on abnormal fibrin structure as a cause for thrombosis. Fibrin clots that have high fiber density and increased resistance to fibrinolysis have been consistently associated with risk for thrombosis. Fibrin structure measurements can (i) provide an overall assessment of hemostatic capacity of a sample, (ii) include effects of thrombin generation and fibrinogen concentrations, (iii) include effects of fibrinogen mutations, polymorphisms, and modifications, and (iv) give an indication of clot mechanical strength and resistance to fibrinolysis. A fibrinogen splice variation of the γ-chain (γ') is discussed as a model for changes in fibrin structure in relation to thrombosis. Results from prospective studies on fibrin structure are awaited. Studies of fibrin formation under flow, interactions of fibrin with blood cells, the mechanical properties of the fibrin clot, and nanoscale/molecular characterization of fibrin formation are likely to expose new causal mechanisms for the role of fibrin in thrombotic disease. Future studies into the causality and mechanisms may lead to new opportunities using fibrin structure in the diagnosis or treatment of thrombosis.
Assuntos
Fibrina/fisiologia , Fibrinogênio/fisiologia , Trombose/fisiopatologia , Causalidade , HumanosAssuntos
Testes de Coagulação Sanguínea/normas , Coagulação Sanguínea , Doenças Cardiovasculares/diagnóstico , Fibrina/metabolismo , Análise de Variância , Doenças Cardiovasculares/sangue , Europa (Continente) , Humanos , Variações Dependentes do Observador , Permeabilidade , Projetos Piloto , Valor Preditivo dos Testes , Valores de Referência , Reprodutibilidade dos TestesRESUMO
CONTEXT: Diabetes is associated with increased incidence of atherothrombotic disease. The fibrin network forms the backbone of the arterial thrombus, and fibrin clot structure determines predisposition to cardiovascular events. OBJECTIVES: The aim of the study was to investigate fibrin clot structure/fibrinolysis in the largest type 2 diabetes cohort and analyze associations with cardiometabolic risk factors and vascular pathology. DESIGN: Clot structure/fibrinolysis was assessed in 875 participants of the Edinburgh Type 2 Diabetes Study [age, 68 (range, 60-75) yr; 450 males] by turbidimetric assays, and clots were visualized by confocal microscopy. Four parameters of clot structure/fibrinolysis were analyzed, and plasma levels of fibrinogen and plasminogen activator inhibitor-1 were studied by Clauss assay and ELISA, respectively. RESULTS: Clot maximum absorbance was increased in females compared with males (0.37 ± 0.005 and 0.34 ± 0.005 arbitrary unit, respectively; P < 0.001), and took longer to lyse (803 ± 20 and 665 ± 12 sec, respectively; P < 0.001). These gender differences in clot structure and fibrinolysis were still evident after correcting for fibrinogen and plasminogen activator inhibitor-1 plasma levels. A prothrombotic fibrin structure profile was associated with increased body mass index and low levels of high-density lipoprotein in women and with inadequate diabetes control in men. Clot formation time was related to previous cardiac ischemic events in both men and women after adjusting for traditional risk factors [odds ratio, 1.22 (95% confidence interval, 1.07, 1.38); and 1.33 (1.15, 1.50), respectively], and prothrombotic clots were associated with low ankle brachial index, renal impairment, and smoking, regardless of gender. CONCLUSIONS: Women with type 2 diabetes have compact clots with compromised fibrinolysis compared with men. There are gender-specific associations between clotting parameters and cardiometabolic risk factors in this population, whereas vascular abnormalities, impaired renal function, and smoking are associated with prothrombotic clot structure profile regardless of gender.
Assuntos
Biomarcadores , Coagulação Sanguínea/fisiologia , Doenças Cardiovasculares/etiologia , Diabetes Mellitus Tipo 2/sangue , Fibrina/química , Fibrina/fisiologia , Doenças Metabólicas/etiologia , Idoso , Biomarcadores/análise , Biomarcadores/sangue , Testes de Coagulação Sanguínea , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Estudos de Coortes , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Fibrina/metabolismo , Fibrinólise/fisiologia , Humanos , Masculino , Doenças Metabólicas/sangue , Doenças Metabólicas/diagnóstico , Pessoa de Meia-Idade , Fatores de Risco , Fatores Sexuais , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Fibrinogen contains an alternatively spliced γ-chain (γ'), which mainly exists as a heterodimer with the common γA-chain (γA/γ'). Fibrinogen γ' has been reported to inhibit thrombin and modulate fibrin structure, but the underlying mechanisms are unknown. OBJECTIVE: We aimed to investigate the molecular mechanism underpinning the influence of γ' on fibrin polymerization, structure and viscoelasticity. METHODS: γA/γA and γA/γ' fibrinogens were separated using anion exchange chromatography. Cross-linking was controlled with purified FXIIIa and a synthetic inhibitor. Fibrin polymerization was analyzed by turbidity and gel-point time was measured using a coagulometer. We used atomic force microscopy (AFM) to image protofibril formation while final clot structure was assessed by confocal and scanning electron microscopy. Clot viscoelasticity was measured using a magnetic microrheometer. RESULTS: γA/γ' fibrin formed shorter oligomers by AFM than γA/γA, which in addition gelled earlier. γA/γ' clots displayed a non-homogenous arrangement of thin fibers compared with the uniform arrangements of thick fibers for γA/γA clots. These differences in clot structure were not due to thrombin inhibition as demonstrated in clots made with reptilase. Non-cross-linked γA/γA fibrin was approximately 2.7 × stiffer than γA/γ'. Cross-linking by FXIIIa increased the stiffness of both fibrin variants; however, the difference in stiffness increased to approximately 4.6 × (γA/γA vs. γA/γ'). CONCLUSIONS: Fibrinogen γ' is associated with the formation of mechanically weaker, non-uniform clots composed of thin fibers. This is caused by direct disruption of protofibril formation by γ'.
Assuntos
Coagulação Sanguínea , Fibrina/metabolismo , Fibrinogênios Anormais/metabolismo , Processamento Alternativo , Testes de Coagulação Sanguínea , Cromatografia por Troca Iônica , Elasticidade , Fator XIIIa/metabolismo , Fibrina/ultraestrutura , Fibrinogênios Anormais/genética , Humanos , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Nefelometria e Turbidimetria , Polimerização , Conformação Proteica , Relação Estrutura-Atividade , Fatores de Tempo , ViscosidadeAssuntos
Coagulação Sanguínea , Deficiência do Fator XIII/diagnóstico , Fator XIII/análise , Algoritmos , Autoanticorpos/sangue , Biomarcadores/sangue , Coagulação Sanguínea/genética , Testes de Coagulação Sanguínea/normas , Análise Mutacional de DNA/normas , Eletroforese em Gel de Poliacrilamida/normas , Fator XIII/genética , Fator XIII/imunologia , Deficiência do Fator XIII/sangue , Deficiência do Fator XIII/classificação , Deficiência do Fator XIII/genética , Deficiência do Fator XIII/imunologia , Fibrina/metabolismo , Predisposição Genética para Doença , Humanos , Valor Preditivo dos TestesRESUMO
BACKGROUND: : The presence of an abdominal aortic aneurysm (AAA) independently predicts cardiovascular disease (CVD) and its complications. Levels of plasma markers of fibrin turnover are raised in men with a large AAA (at least 5.5 cm) and predict CVD risk in healthy subjects. This study examined fibrin turnover in men with a small AAA. METHODS: : Seventy-five men with a small AAA (30-55 mm) were compared with 90 controls matched for age, sex and race. Haemostatic and fibrinolytic parameters were assessed. RESULTS: : Men with a small AAA had higher mean levels of fibrinogen (2.92 versus 2.59 g/l; P = 0.019), thrombin-antithrombin (TAT) complex (4.57 versus 1.89 ng/ml; P < 0.001), prothrombin F1 + 2 (1.13 versus 0.82 ng/ml; P = 0.004) and D-dimer (346.7 versus 120.2 ng/ml; P < 0.001). All markers correlated with maximum aortic diameter determined by ultrasonography. On multivariable regression the association between presence of an AAA and fibrinogen, TAT complex, prothrombin F1 + 2 and D-dimer levels remained significant after adjustment for confounding influences. CONCLUSION: : Fibrin turnover was increased in these men with a small AAA, independently of concomitant CVD, conventional risk factors and inflammatory markers. Enhanced fibrin turnover may contribute to the risk of cardiac complications in this group.