RESUMEN
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.
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Tromboembolia/terapia , Trombosis/sangre , Trombosis/terapia , Anticoagulantes/uso terapéutico , Biomarcadores/sangre , Coagulación Sanguínea , Eritrocitos/metabolismo , Factor VIII/metabolismo , Factor XII/metabolismo , Factor XIII/metabolismo , Humanos , Macrófagos/metabolismo , Países Bajos , Fenotipo , Placa Aterosclerótica/sangre , Placa Aterosclerótica/diagnóstico , Placa Aterosclerótica/terapia , Polifosfatos/metabolismo , Factores de Riesgo , Transducción de Señal , Tromboembolia/sangre , Tromboembolia/diagnóstico , Trombosis/diagnósticoRESUMEN
Essentials Factor XIII (FXIII)-mediated fibrin crosslinking is delayed in hemophilia. We determined effects of FXIII cotreatment with hemostatic agents on clot parameters. FXIII cotreatment accelerated FXIII activation and crosslinking of fibrin and α2 -antiplasmin. These data provide biochemical rationale for FXIII cotreatment in hemophilia. SUMMARY: Background Hemophilia A results from the absence, deficiency or inhibition of factor VIII. Bleeding is treated with hemostatic agents (FVIII, recombinant activated FVII [rFVIIa], anti-inhibitor coagulation complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment, some patients have prolonged bleeding. FXIII-A2 B2 (FXIII) is a protransglutaminase. During clot contraction, thrombin-activated FXIII (FXIIIa) crosslinks fibrin and α2 -antiplasmin, which promotes red blood cell retention and increases clot stability and weight. We hypothesized that FXIII cotreatment in hemophilia would accelerate FXIII activation, leading to increased fibrin crosslinking. Methods FVIII-deficient plasma and whole blood were clotted with or without hemostatic agents (FVIII, rFVIIa, FEIBA, or recombinant B-domain-deleted porcine FVIII [rpFVIII]) and/or FXIII. The effects on FXIII activation, thrombin generation, fibrin and α2 -antiplasmin crosslinking, clot formation and clot weight were measured by western blotting, calibrated automated thrombography, thromboelastography, and clot contraction assays. Results As compared with FVIII-treated hemophilic plasma, FVIII + FXIII cotreatment accelerated FXIIIa formation without increasing thrombin generation. As compared with buffer-treated or FXIII-treated hemophilic plasma, FVIII treatment and FVIII + FXIII cotreatment increased the generation and amount of crosslinked fibrin, including α-chain-rich high molecular weight species and crosslinked α2 -antiplasmin. In the presence of FVIII inhibitors, as compared with hemostatic treatments (rFVIIa, FEIBA, or rpFVIII) alone, FXIII cotreatment increased whole blood clot weight. Conclusion In hemophilia A plasma and whole blood, FXIII cotreatment with hemostatic agents accelerated FXIIIa formation, increased the generation and amount of fibrin α-chain crosslinked species, accelerated α2 -antiplasmin crosslinking, and increased clot weight. FXIII cotreatment with hemostatic therapy may augment hemostasis through increased crosslinking of fibrin and α2 -antiplasmin.
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Factores de Coagulación Sanguínea/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Coagulantes/uso terapéutico , Factor VIII/uso terapéutico , Factor VIIa/uso terapéutico , Factor XIII/uso terapéutico , Fibrina/metabolismo , Hemofilia A/tratamiento farmacológico , Anciano , Factores de Coagulación Sanguínea/efectos adversos , Coagulantes/efectos adversos , Factor VIII/efectos adversos , Factor VIIa/efectos adversos , Factor XIII/efectos adversos , Femenino , Fibrina/química , Hemofilia A/sangre , Hemofilia A/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , alfa 2-Antiplasmina/metabolismoAsunto(s)
Pruebas de Coagulación Sanguínea/normas , Coagulación Sanguínea , Hemofilia A/diagnóstico , Trombina/metabolismo , Automatización de Laboratorios/normas , Biomarcadores/sangre , Coagulación Sanguínea/efectos de los fármacos , Calibración , Fluorometría/normas , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Humanos , Variaciones Dependientes del Observador , Valor Predictivo de las Pruebas , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
INTRODUCTION: High-titre factor VIII (FVIII) inhibitors complicate peri-operative haemostasis. Recombinant porcine FVIII (r-pFVIII) may provide an alternative haemostatic agent for high-risk procedures and allow FVIII activity monitoring. AIM: Devise an effective haemostatic plan for repair of a progressively symptomatic aortic coarctation in a 5-year-old male with immune tolerance induction (ITI) refractory high-titre FVIII inhibitors. METHODS: Preprocedure human FVIII inhibitor titre was 58 Bethesda Units mL-1 (BU) and cross-reacted to neutralize porcine FVIII at 30 BU. Daily ITI with plasma-derived FVIII concentrate was supplemented with anti-B-cell and anti-plasma cell immunotherapy to reduce FVIII inhibitor titres. Potential haemostatic agents were evaluated in comparative ex vivo thrombin generation assays (TGA). RESULTS: Four weeks after immunosuppression, human and porcine inhibitor titres declined to 16 and 2 BU respectively. TGA with r-pFVIII was less robust than with activated prothrombin complex concentrate (aPCC); however, r-pFVIII was selected for cardiac surgery to secure the ability to assay FVIII levels throughout this high-bleeding risk procedure. Haemostasis with r-pFVIII was excellent; initial trough FVIII activity levels ranged from 0.81-1.17 IU mL-1 . On postoperative day 3, peak and trough levels markedly declined suggesting a rising porcine inhibitor titre. Postprocedure prophylaxis was transitioned to aPCC, informed by TGA. CONCLUSIONS: R-pFVIII provided effective peri-procedural haemostasis with no adverse events. Rapid neutralization of r-pFVIII after the first 60 hours, despite intensive immune suppression, accentuates the importance of careful monitoring. Use of TGA can support bypassing agent selection for convalescence. The comparative cost of r-pFVIII may limit its use to high morbidity clinical scenarios.
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Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Animales , Preescolar , Humanos , Masculino , Proteínas Recombinantes/administración & dosificación , PorcinosRESUMEN
INTRODUCTION: Cancer patients have a 4- to 7- fold increased risk of venous thromboembolism (VTE) compared with general population. Most tumor cells express tissue factor (TF) and constitutively release small membrane microvesicles called tumor microvesicles (TMVs). Clinical studies have shown that circulating MP-TF activity is associated with VTE in pancreatic cancer but not in other types of cancer. Thrombin is a potent platelet agonist and activates platelets via protease activated receptors (PARs). AIM: To determine the contribution of the TF+ TMV-thrombin-platelet pathway to cancer-associated thrombosis. MATERIALS AND METHODS: A human pancreatic adenocarcinoma cell line expressing high levels of TF (BxPc-3) was selected to study the effect of TF+ TMVs on platelet activation and thrombosis. RESULTS: TF+ TMVs induced platelet activation in vitro in a thrombin-dependent manner. The presence of orthotopically grown BxPc-3 tumors in mice was associated with increased levels of thrombin-antithrombin III complexes (TATc) and larger thrombi in an inferior vena cava stenosis model compared with control mice. Furthermore, injection of BxPc-3 TF+ TMVs into mice triggered platelet activation and enhanced venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and wild-type mice treated with the platelet inhibitor clopidogrel, suggesting that platelet activation was required for the enhanced thrombosis. CONCLUSIONS: These studies suggest that platelet inhibitors may reduce thrombosis in cancer patients with elevated levels of TF+ TMVs.
RESUMEN
UNLABELLED: ESSENTIALS: Cancer patients have a high rate of venous thrombosis (VT) but the underlying mechanisms are unknown. Tumor-derived, tissue factor-positive microvesicles in platelet activation in vitro and in vivo were studied. Tumor-derived, tissue factor-positive microvesicles enhanced VT in mice. Platelets may contribute to VT in some cancer patients, and this could be prevented with antiplatelet drugs. BACKGROUND: Cancer patients have an approximately 4-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor-derived, tissue factor-positive (TF(+) ) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF(+) tumor-derived MVs (TMVs) activate platelets in vitro and in mice. MATERIALS AND METHODS: We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF(+) TMVs on platelets and thrombosis. RESULTS AND CONCLUSIONS: We found that both types of TF(+) TMVs activated human platelets and induced aggregation in vitro in a TF and thrombin-dependent manner. Further, injection of BxPc-3 TF(+) TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF(+) TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF(+) TMV-induced platelet activation contributes to thrombosis in cancer patients.
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Micropartículas Derivadas de Células , Tromboplastina/fisiología , Trombosis/tratamiento farmacológico , Adenocarcinoma/fisiopatología , Animales , Plaquetas/citología , Línea Celular Tumoral , Clopidogrel , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/fisiopatología , Neoplasias Pancreáticas/fisiopatología , Activación Plaquetaria , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Embolia Pulmonar/tratamiento farmacológico , Trombina/metabolismo , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
Cardiovascular disease is the leading cause of death and disability worldwide. Among cardiovascular causes of death, venous thrombosis (VT) is ranked third most common in the world. Venous thrombi have high red blood cell and fibrin content; however, the pathophysiologic mechanisms that contribute to venous thrombus composition and stability are still poorly understood. This article reviews biological, biochemical, and biophysical contributions of fibrinogen, factor XIII, and red blood cells to VT, and new evidence suggesting interactions between these components mediate venous thrombus composition and size.
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Coagulación Sanguínea , Eritrocitos/metabolismo , Factor XIII/metabolismo , Fibrinógeno/metabolismo , Trombosis de la Vena/sangre , Animales , Factor XIII/química , Fibrina/metabolismo , Fibrinógeno/química , Humanos , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad , Trombosis de la Vena/fisiopatologíaRESUMEN
In a large proportion of patients with mild bleeding disorders (MBDs) no diagnosis can be established by routine coagulation tests. We investigated whether alterations in plasma clot properties account for MBDs of unknown cause. Ninety-five patients with MBDs of unknown origin and 98 age- and sex-matched healthy controls were investigated. Furthermore, data of 25 patients with a deficiency of factor VIII were analyzed. Plasma clot characteristics in the absence and presence of recombinant tissue plasminogen activator (rtPA) represented by the lag phase, rate of protofibril formation (Vmax), fibrin structure (ΔAbs), time to peak (TTP), half lysis time (t50 and area under the curve (AUC) were measured in turbidometric clot formation and lysis assays. In the fibrinolysis assay, Vmax was lower in patients than in healthy controls. No differences in the other parameters of clot formation and lysis were detected between the groups. There was no clear association of plasma clot properties with the clinical severity of bleeding in patients with MBDs. Patients with known decreased factor VIII levels also showed a lower Vmax. Fibrinogen levels were positively associated with each of the assessed parameters in both groups, with the strongest association with ΔAbs, indicating altered fibrin structure. Factor VIII activity correlated with altered clot characteristics similar to fibrinogen, especially in patients, with the strongest positive correlation to Vmax. This cohort of patients with MBDs of unknown origin showed a lower rate of fibrin formation in the fibrinolysis assay, but otherwise similar plasma clot properties compared to healthy controls.
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Coagulación Sanguínea/fisiología , Tiempo de Lisis del Coágulo de Fibrina/métodos , Hemorragia/sangre , Hemorragia/diagnóstico , Adulto , Pruebas de Coagulación Sanguínea/métodos , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Elevated plasma fibrinogen is associated with arterial thrombosis in humans and promotes thrombosis in mice by increasing fibrin formation and thrombus fibrin content. Fibrinogen is composed of six polypeptide chains: (Aα, Bß, and γ)2. Alternative splicing of the γ chain leads to a dominant form (γA/γA) and a minor species (γA/γ'). Epidemiological studies have detected elevated γA/γ' fibrinogen in patients with arterial thrombosis, suggesting that this isoform promotes thrombosis. However, in vitro data show that γA/γ' is anticoagulant due to its ability to sequester thrombin and suggest its expression is upregulated in response to inflammatory processes. OBJECTIVE: To determine whether γA/γ' fibrinogen is prothrombotic in vivo. METHODS: We separated γA/γA and γA/γ' fibrinogen from human plasma-purified fibrinogen and determined the effects on in vitro plasma clot formation and on in vivo thrombus formation and circulating thrombin-antithrombin complexes in mice. RESULTS AND CONCLUSIONS: Both γA/γA and γA/γ' fibrinogen were cleaved by murine and human thrombin and were incorporated into murine and human clots. When γA/γA or γA/γ' was spiked into plasma, γA/γA increased the fibrin formation rate to a greater extent than γA/γ'. In mice, compared to controls, γA/γA infusion shortened the time to carotid artery occlusion, whereas γA/γ' infusion did not. Additionally, γA/γ' infusion led to lower levels of plasma thrombin-antithrombin complexes following arterial injury, whereas γA/γA infusion did not. These data suggest that γA/γ' binds thrombin in vivo and decreases prothrombotic activity. Together, these findings indicate that elevated levels of γA/γA fibrinogen promote arterial thrombosis in vivo, whereas γA/γ' does not.
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Arterias/patología , Coagulación Sanguínea , Fibrinógeno/química , Fibrinógenos Anormales/química , Trombosis/metabolismo , Animales , Antitrombinas/química , Pruebas de Coagulación Sanguínea , Femenino , Fibrinógeno/genética , Fibrinógenos Anormales/genética , Humanos , Inflamación , Masculino , Ratones , Persona de Mediana Edad , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Trombina/químicaRESUMEN
BACKGROUND: Factor XI (FXI) deficiency is a rare autosomal recessive disorder. Many patients with even very low FXI levels (< 20 IU dL(-1) ) are asymptomatic or exhibit only mild bleeding, whereas others experience severe bleeding, usually following trauma. Neither FXI antigen nor activity predicts the risk of bleeding in FXI-deficient patients. OBJECTIVES: (i) Characterize the formation, structure and stability of plasma clots from patients with severe FXI deficiency and (ii) determine whether these assays can distinguish asymptomatic patients ('non-bleeders') from those with a history of bleeding ('bleeders'). METHODS: Platelet-poor plasmas were prepared from 16 severe FXI-deficient patients who were divided into bleeders or non-bleeders, based on bleeding associated with at least two tooth extractions without prophylaxis. Clot formation was triggered by recalcification and addition of tissue factor and phospholipids in the absence or presence of tissue plasminogen activator and/or thrombomodulin. Clot formation and fibrinolysis were measured by turbidity and fibrin network structure by laser scanning confocal microscopy. RESULTS: Non-bleeders and bleeders had similarly low FXI levels, normal prothrombin times, normal levels of fibrinogen, factor VIII, von Willebrand factor and factor XIII, and normal platelet number and function. Compared with non-bleeders, bleeders exhibited lower fibrin network density and lower clot stability in the presence of tissue plasminogen activator. In the presence of thrombomodulin, seven of eight bleeders failed to form a clot, whereas only three of eight non-bleeders did not clot. CONCLUSIONS: Plasma clot structure and stability assays distinguished non-bleeders from bleeders. These assays may reveal hemostatic mechanisms in FXI-deficient patients and have clinical utility for assessing the risk of bleeding.
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Coagulación Sanguínea , Deficiencia del Factor XI/diagnóstico , Hemorragia/diagnóstico , Adulto , Anciano , Estudios de Cohortes , Factor XI/metabolismo , Deficiencia del Factor XI/sangre , Factor XIII/metabolismo , Femenino , Fibrinólisis , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Fosfolípidos/química , Plasma/metabolismo , Factores de Riesgo , Trombina/química , Factor de von Willebrand/metabolismoRESUMEN
Air pollution exposure is associated with cardiovascular events triggered by clot formation. Endothelial activation and initiation of coagulation are pathophysiological mechanisms that could link inhaled air pollutants to vascular events. Here we investigated the underlying mechanisms of increased endothelial cell procoagulant activity following exposure to soluble components of ultrafine particles (soluble UF). Human coronary artery endothelial cells (HCAEC) were exposed to soluble UF and assessed for their ability to trigger procoagulant activity in platelet-free plasma. Exposed HCAEC triggered earlier thrombin generation and faster fibrin clot formation, which was abolished by an anti-tissue factor (TF) antibody, indicating TF-dependent effects. Soluble UF exposure increased TF mRNA expression without compensatory increases in key anticoagulant proteins. To identify early events that regulate TF expression, we measured endothelial H2O2 production following soluble UF exposure and identified the enzymatic source. Soluble UF exposure increased endothelial H2O2 production, and antioxidants attenuated UF-induced upregulation of TF, linking the procoagulant responses to reactive oxygen species (ROS) formation. Chemical inhibitors and RNA silencing showed that NOX-4, an important endothelial source of H2O2, was involved in UF-induced upregulation of TF mRNA. These data indicate that soluble UF exposure induces endothelial cell procoagulant activity, which involves de novo TF synthesis, ROS production, and the NOX-4 enzyme. These findings provide mechanistic insight into the adverse cardiovascular effects associated with air pollution exposure.
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Contaminantes Atmosféricos/toxicidad , Células Endoteliales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Material Particulado/toxicidad , Trombina/metabolismo , Tromboplastina/metabolismo , Contaminantes Atmosféricos/química , Técnicas de Cultivo de Célula , Línea Celular , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Fibrina/metabolismo , Humanos , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Tamaño de la Partícula , Material Particulado/química , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , SolubilidadAsunto(s)
Factores de Edad , Modelos Animales , Trombosis de la Vena/fisiopatología , Animales , Humanos , RatonesRESUMEN
BACKGROUND: Microparticles (MPs) are sub-micron vesicles shed by activated or apoptotic cells, including platelets and monocytes. Increased circulating MPs are associated with thrombosis; however, their role in thrombogenesis is poorly understood. OBJECTIVE: To determine how MPs promote thrombin generation and modulate fibrin density and stability. METHODS: Platelets and monocytes were isolated from healthy donors. Platelets were stimulated with calcium ionophore, thrombin receptor agonist peptide (TRAP) or TRAP/convulxin. Monocytes and human monocytic THP-1 cells were stimulated with lipopolysaccharide (LPS). MPs were isolated, washed by high-speed centrifugation and assessed using the following: transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA), flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density and stability. RESULTS: MPs from monocytes (M-MPs) and platelets (PMPs) had similar shapes and diameters (100-300 nm). M-MPs had TF activity (16.7 ± 2.4 pm TF per 10(6) MP), supported prothrombinase activity and triggered shorter thrombin generation lag times than buffer controls (5.4 ± 0.5 vs. 84.2 ± 4.8 min, respectively). Compared with controls, M-MPs supported faster fibrin formation (0.24 ± 0.24 vs. 76.7 ± 15.1 mOD min(-1) , respectively), 38% higher fibrin network density and higher clot stability (3.8-fold higher turbidity in the presence of tissue plasminogen activator). In contrast, PMPs did not have TF activity and supported 2.8-fold lower prothrombinase activity than M-MPs. PMPs supported contact-dependent thrombin generation, but did not independently increase fibrin network density or stability. Interestingly, PMPs increased rates of thrombin generation and fibrin formation (1.7- and 1.3-fold, respectively) when mixed with THP-1-derived MPs. CONCLUSION: MPs from platelets and monocytes differentially modulate clot formation, structure and stability, suggesting unique contributions to thrombosis.
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Plaquetas/patología , Micropartículas Derivadas de Células/patología , Fibrina/biosíntesis , Monocitos/patología , Trombina/biosíntesis , Coagulación Sanguínea , Plaquetas/ultraestructura , Micropartículas Derivadas de Células/fisiología , Fibrina/química , Humanos , Monocitos/ultraestructura , Tromboplastina/análisis , Tromboplastina/metabolismo , Trombosis/etiologíaRESUMEN
BACKGROUND: Human eNOS (NOS3) polymorphisms that lower its expression are associated with advanced diabetic nephropathy (DN), and the lack of eNOS accelerates DN in diabetic mice. Diabetes is associated with fibrin deposition. Lack of nitric oxide and fatty acids stimulates the NF-kB pathway, which increases tissue factor (TF). OBJECTIVES: To test the hypothesis that TF contributes to the severity of DN in the diabetic eNOS(-/-) mice fed a high-fat diet (HF). METHODS: We made eNOS(-/-) and wild-type mice diabetic with streptozotocin. Half of them were placed on HF. RESULTS: Blood glucose levels were not affected by either the diet or eNOS genotype. Lack of eNOS in the diabetic mice increased urinary albumin excretion, glomerulosclerosis, interstitial fibrosis, and glomerular basement membrane thickness. HF by itself did not affect DN in the wild-type mice, but significantly enhanced DN in eNOS(-/-) mice. More than half of diabetic eNOS(-/-) mice on HF died prematurely with signs of thrombotic complications. Diabetic kidneys contained fibrin and TF, and their levels were increased by the lack of eNOS and by HF in an additive fashion. The HF diet increased the kidney expression of inflammatory genes. The increase in TF preceded DN, and administration of an anti-mouse TF antibody to diabetic mice reduced the expression of inflammatory genes. CONCLUSION: Together, these data indicate a causal link between TF and the exacerbation of DN in eNOS(-/-) mice. The condition is significantly worsened by enhanced inflammatory responses to an HF diet via TF.
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Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/genética , Óxido Nítrico Sintasa de Tipo III/genética , Tromboplastina/biosíntesis , Alimentación Animal , Animales , Glucemia/metabolismo , Presión Sanguínea , Grasas de la Dieta , Tasa de Filtración Glomerular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente/métodos , Tromboplastina/fisiologíaRESUMEN
Growing evidence suggests that fibrin network structure and stability are important determinants of haemostasis and thrombosis, with alterations in fibrin structure implicated as a causative mechanism in various haemostatic and thrombotic disorders. In haemophilia, for example, deficiency of factor VIII or IX reduces the rate and peak of thrombin generation and produces coarse fibrin clots that show increased susceptibility to fibrinolysis. More recently, studies have shown significant effects of cellular activity and integrin composition on fibrin network and stability. Platelets support the formation of a dense, stable fibrin network via interactions between the alphaIIbbeta3 integrin and the fibrin network, whereas tissue factor-bearing cells regulate fibrin structure and stability predominantly via procoagulant activity. Highly procoagulant extravascular cells (e.g. fibroblasts and smooth muscle cells) support the formation of dense fibrin networks that resist fibrinolysis, whereas unstimulated intravascular cells (e.g. endothelial cells) produce coarser networks that are susceptible to fibrinolysis. Moreover, cellular contributions produce heterogeneous clots in which fibrin network density and stability decrease with increasing distance from the cell surface. Together, these findings suggest that specific plasma and cellular mechanisms link thrombin generation, clot stability and haemostatic or thrombotic outcome. Understanding these mechanisms may provide new therapeutic targets in the management of bleeding and thrombotic disorders.
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Coagulación Sanguínea/fisiología , Fibrina/biosíntesis , Factores de Coagulación Sanguínea/fisiología , Fibrina/química , Fibrinógeno/biosíntesis , Fibrinógeno/química , Fibrinólisis/fisiología , Hemostasis/fisiología , Humanos , Proteínas Recombinantes , Trombina/biosíntesisRESUMEN
BACKGROUND: Activation of tumor cell-associated coagulation and plasminogen activator pathways occurs in malignant disease processes, including breast cancer, and may promote metastatic activity. OBJECTIVES/METHODS: To compare the coagulation and plasminogen activator pathways of normal and metastatic cells, we examined two cell lines from the MCF-10 family of breast cells: near-normal immortalized MCF-10A cells, and metastatic MCF-10CA1 cells. RESULTS: MCF-10CA1 cell motility was significantly increased as compared with that of MCF-10A cells. The two cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. MCF-10A cells produced a stable fibrin network, whereas MCF-10CA1 cells lysed the surrounding fibrin network within 24 h of network formation. Importantly, fibrin located proximal to (within 10 microm) the MCF-10CA1 cell surface lysed substantially faster than fibrin located 100 microm from the surface. MCF-10CA1 cells supported significantly increased plasmin generation rates as compared with MCF-10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF-10CA1 cells had increased expression (mRNA and protein) levels of urokinase plasminogen activator (u-PA) and decreased levels of plasminogen activator inhibitor-1 as compared with MCF-10A cells. Blocking u-PA activity with the active site-directed protease inhibitor amiloride substantially decreased MCF-10CA1 cell motility. Phosphorylated Akt levels were elevated in MCF-10CA1 cells, which partially explains the increased u-PA expression. CONCLUSIONS: These results suggest that the tumor-associated plasminogen activator pathway, not the coagulation pathway, is a key distinguishing feature between metastatic MCF10-CA1 cells and normal MCF-10A cells.
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Coagulación Sanguínea , Neoplasias de la Mama/metabolismo , Activadores Plasminogénicos/metabolismo , Secuencia de Bases , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cartilla de ADN , Femenino , Humanos , Metástasis de la Neoplasia , EmbarazoRESUMEN
Using a cell-based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0-200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV-deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell-based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin.
Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Trombina/metabolismo , Humanos , Modelos Biológicos , Monocitos/fisiología , Activación Plaquetaria/fisiologíaRESUMEN
Elevated plasma homocysteine is associated with an increased risk of atherosclerosis and thrombosis. However, the mechanisms by which homocysteine might cause these events are not understood. We hypothesized that hyperhomocysteinemia might lead to modification of fibrinogen in vivo, thereby causing altered fibrin clot structure. New Zealand White rabbits were injected intraperitoneally (i.p.) every 12 h through an indwelling catheter with homocysteine or buffer for 8 weeks. This treatment raised the plasma homocysteine levels to about 30 micro mol L(-1) compared with 13.5 micro mol L(-1) in control rabbits by the end of the treatment period. The fibrinogen levels were 3.2 +/- 0.6 in homocysteine-treated and 2.5 +/- 1.1 mg mL(-1) in control rabbits. The reptilase time was prolonged to 363 +/- 88 for plasma from homocysteine-treated rabbits compared with 194 +/- 48 s for controls (P < 0.01). The thrombin clotting time (TCT) for the homocysteine-treated rabbits was significantly shorter, 7.5 +/- 1.7 compared with 28.6 +/- 18 s for the controls (P < 0.05). The calcium dependence of the thrombin clotting time was also different in homocysteinemic and control plasmas. Clots from plasma or fibrinogen of homocysteinemic rabbits were composed of thinner fibers than control clots. The clots formed from purified fibrinogen from homocysteine-treated rabbits were lyzed more slowly by plasmin than comparable clots from control fibrinogen. Congenital dysfibrinogenemias have been described that are associated with fibrin clots composed of thin, tightly packed fibers that are abnormally resistant to fibrinolysis, and recurrent thrombosis. Our results suggest that elevated plasma homocysteine leads to a similar acquired dysfibrinogenemia. The formation of clots that are abnormally resistant to fibrinolysis could directly contribute to the increased risk of thrombosis in hyperhomocysteinemia.
Asunto(s)
Fibrina/metabolismo , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/complicaciones , Trombosis/etiología , Animales , Coagulación Sanguínea , Fibrina/ultraestructura , Fibrinógeno/aislamiento & purificación , Fibrinógeno/metabolismo , Fibrinolisina/metabolismo , Homocisteína/sangre , Técnicas In Vitro , Microscopía Electrónica de Rastreo , Conejos , Trombosis/sangreRESUMEN
Fibrin clot structure studies are often performed using optical methods. For example, the clot's fiber structure can be assessed by measuring light scattering as a function of wavelength. From these measurements, one can calculate the mass/length ratio (mu), a relative measure of fibrin thickness. Fiber thickness has important functional correlates in terms of clot stability and resistance to fibrinolysis. Typically, measurements to calculate mass/length ratios are carried out on high-end spectrophotometers. However, limitations of this instrument include the large sample volume required and the inability to read multiple samples at one time. To circumvent these limitations, a plate-reading spectrophotometer is more commonly used to monitor clot formation; increases in absorbance indicate clot formation, while decreases indicate clot lysis. However, it is unclear whether plate-reading spectrophotometers can be used to quantitatively evaluate fibrin fiber structure. In the current study, we compared spectrophotometric analysis of fibrin gels on single-sample and plate-reading spectrophotometers. Results show that a plate-reading spectrophotometer does not give accurate measurements of the fiber mass/length ratio. However, the plate-reading spectrophotometer can provide a qualitative measure of fiber structure for both purified fibrinogen and plasma. We suggest that plate-reading spectrophotometers can provide a convenient, rapid, and inexpensive means of analyzing fibrin clot structure.
Asunto(s)
Fibrina/análisis , Fibrina/ultraestructura , Calcio/farmacología , Dextranos/farmacología , Relación Dosis-Respuesta a Droga , Fibrina/metabolismo , Fibrinólisis , Humanos , Cinética , Reproducibilidad de los Resultados , Análisis Espectral/instrumentación , Análisis Espectral/normas , Trombina/farmacologíaRESUMEN
A small number of thromboembolic events, including deep venous thrombosis and myocardial infarction, have been reported in patients receiving IVIG. These events have primarily occurred in patients receiving high-dose IVIG and have been attributed to an increase in blood viscosity. To test the hypothesis that a procoagulant might be present in IgG preparations, twenty-nine samples of intravenous immunoglobulin (IVIG) from eight different manufacturers were assayed for procoagulant activity. Twenty-six of these samples shortened the clotting time of factor XI-deficient plasma. Of these, fourteen samples had factor XI activities greater than 0.001 U/ml of normal pooled plasma. The remaining samples possessed less than 0. 001 U/ml of normal plasma activity. The procoagulant activity in these samples could be inhibited by an anti-factor XI polyclonal antibody, suggesting that the procoagulant activity was factor XI. The procoagulant activity increased in two samples after storage at 4 degrees C for 4 weeks, likely as a result of factor XIa autoactivation. Additionally, activity in some IVIG samples was able to directly activate factor IX, indicating that activated factor XI was present in these samples. Finally, the degree of factor XI(a) contamination in the samples was correlated with the manufacturer, suggesting that variations in the manufacturing process or source plasma affect the level of factor XI in the IVIG product. Because addition of small amounts of factor XIa to plasma can lead to production of significant amounts of thrombin, we suggest that factor XIa present in some IVIG preparations could contribute to the in vivo risk of thrombosis after IVIG therapy.