RESUMEN
Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder that may cause life-threatening hemorrhages in patients with plasma cell dyscrasias (PCDs). Early diagnosis and treatment require a thorough understanding of its underlying pathophysiology. Two patients with IgG MGUS presented with dramatically decreased plasma von Willebrand factor (VWF) and a severe type-1 pattern on multimer analysis. A prompt response to intravenous immunoglobulins (IVIG), but not to VWF/FVIII, was consistent with accelerated immunologic clearance of plasma VWF. Another IgG MGUS patient showed a type-2 pattern and a less pronounced response to IVIG, suggesting that additional mechanism(s) contributed to AVWS evolution. In a patient with Waldenström's macroglobulinemia and severe depletion of plasma VWF, multimer analysis indicated association of the IgM paraprotein with VWF before, but not after plasmapheresis, resulting in destruction of the agarose gel and a characteristically distorted band structure of VWF multimers. A type-2 pattern with highly abnormal VWF triplets and laboratory evidence of excessive fibrinolytic activity suggested that plasmin-mediated VWF degradation contributed to AVWS in a patient with multiple myeloma (MM) and AL amyloidosis. Finally, in a patient with IgG MM, maximally prolonged PFA-100® closure times and a specific defect in ristocetin-induced platelet agglutination, both of which resolved after remission induction, indicated interference of the paraprotein with VWF binding to platelet GPIb. Importantly, in none of the six patients, circulating autoantibodies to VWF were detected by a specific in-house ELISA. In summary, when evaluating PCD patients with severe bleeding symptoms, AVWS due to various pathogenic mechanisms should be considered.
Asunto(s)
Paraproteinemias/sangre , Paraproteinemias/diagnóstico , Enfermedades de von Willebrand/sangre , Enfermedades de von Willebrand/diagnóstico , Anciano , Autoanticuerpos/sangre , Femenino , Humanos , Inmunoglobulinas Intravenosas/administración & dosificación , Masculino , Persona de Mediana Edad , Paraproteinemias/tratamiento farmacológico , Enfermedades de von Willebrand/tratamiento farmacológico , Factor de von Willebrand/metabolismoRESUMEN
Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders.
Asunto(s)
Suero Antilinfocítico/farmacología , Complemento C5/fisiología , Monocitos/efectos de los fármacos , Proteína Disulfuro Isomerasas/fisiología , Tromboplastina/metabolismo , Células Cultivadas , Activación de Complemento/efectos de los fármacos , Activación de Complemento/fisiología , Complemento C5/metabolismo , Disulfuros/metabolismo , Disulfuros/farmacología , Evaluación Preclínica de Medicamentos , Humanos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/fisiología , Modelos Biológicos , Monocitos/metabolismo , Oxidación-Reducción/efectos de los fármacos , Proteína Disulfuro Isomerasas/metabolismo , Factores de TiempoRESUMEN
High on-treatment platelet reactivity has been associated with adverse cardiovascular events in patients receiving anti-platelet agents, but the molecular mechanisms underlying this phenomenon remain incompletely understood. Succinate, a citric acid cycle intermediate, is released into the circulation under conditions of mitochondrial dysfunction due to hypoxic organ damage, including sepsis, stroke, and myocardial infarction. Because the G protein-coupled receptor (GPCR) for succinate, SUCNR1 (GPR91), is present on human platelets, we hypothesized that succinate-mediated platelet stimulation may counteract the pharmacological effects of cyclooxygenase-1 and ADP receptor antagonists. To test this hypothesis in a controlled in-vitro study, washed platelets from healthy donors were treated with acetylsalicylic acid (ASA) or small-molecule P2Y(1) or P2Y(12) inhibitors and subsequently analyzed by light transmittance aggregometry using arachidonic acid (AA), ADP and succinate as platelet agonists. Aggregation in response to succinate alone was highly variable with only 29% of donors showing a (mostly delayed) platelet response. In contrast, succinate reproducibly and concentration-dependently (10-1000 µM) enhanced platelet aggregation in response to low concentrations of exogenous ADP. Furthermore, while succinate alone had no effect in the presence of platelet inhibitors, responsiveness of platelets to ADP after pretreatment with P2Y(1) or P2Y(12) antagonists was fully restored, when platelets were co-stimulated with 100 µM succinate. Similarly, succinate completely (at 1000 µM) or partially (at 100 µM) reversed the inhibitory effect of ASA on AA-induced platelet aggregation. In contrast, succinate failed to restore platelet responsiveness in the presence of both ASA and the P2Y(12) antagonist, suggesting that concomitant signaling via different GPCRs was required. Essentially identical results were obtained, when flow cytometric analysis of surface CD62P expression was used as a different readout for platelet activation. In summary, extracellular succinate may have a co-stimulatory role in platelet aggregation and, by (partially) antagonizing the effects of platelet inhibitors, may contribute to the inter-individual variability frequently observed in platelet function testing.
Asunto(s)
Aspirina/farmacología , Plaquetas/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Agonistas del Receptor Purinérgico P2Y/farmacología , Ácido Succínico/farmacología , Plaquetas/citología , Femenino , Humanos , Masculino , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
Tissue factor (TF) plays a critical role in tumour growth and metastasis, and its enhanced release into plasma in association with cellular microparticles (MPs) has recently been associated with pathological cancer progression. We have previously demonstrated significantly elevated levels of plasma TF antigen as well as systemic coagulation and platelet activation in patients with localised prostate cancer. In this prospective study, we used a highly sensitive one-stage clotting assay to measure preoperative TF-specific procoagulant activity (PCA) of plasma MPs in 68 consecutive patients with early-stage prostate cancer to further explore the relevance of circulating TF in this tumour entity. Automated calibrated thrombography was used to monitor thrombin generation in cell-free plasma samples in the absence of exogenous TF or phospholipids. Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer and TF-specific PCA of plasma MPs (p<0.001). Furthermore, MP-associated TF PCA was higher in patients with (n=29) than in those without (n=39) laboratory evidence of an acute-phase reaction (p=0.004) and decreased to normal levels within one week after radical prostatectomy. Overall, we found a significant correlation between TF-specific PCA of plasma MPs and plasma D-dimer (p=0.002), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. Thrombin generation in plasma was also significantly increased in patients compared to controls (p<0.01). Collectively, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent a potential link between hypercoagulability, inflammation, and disease progression.
Asunto(s)
Reacción de Fase Aguda/fisiopatología , Micropartículas Derivadas de Células/metabolismo , Neoplasias de la Próstata/fisiopatología , Trombina/metabolismo , Tromboplastina/metabolismo , Reacción de Fase Aguda/diagnóstico , Reacción de Fase Aguda/etiología , Reacción de Fase Aguda/patología , Anciano , Anticuerpos Monoclonales , Coagulación Sanguínea , Pruebas de Coagulación Sanguínea , Línea Celular Tumoral , Separación Celular , Progresión de la Enfermedad , Citometría de Flujo , Humanos , Lipopolisacáridos/metabolismo , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Prospectivos , Neoplasias de la Próstata/complicaciones , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Sensibilidad y Especificidad , Trombina/genética , Tromboplastina/inmunologíaRESUMEN
Myeloperoxidase (MPO) is a cationic heme enzyme stored in neutrophilic polymorphonuclear leukocytes (PMNs) that has recently been implicated in inflammatory cell signaling and tissue damage. Although PMNs play a critical role in both innate immunity and vascular thrombosis, no previous study has systematically investigated the effect of MPO on blood coagulation. Here, we show that PMN-derived MPO inhibits the procoagulant activity (PCA) of lipidated recombinant human tissue factor (rhTF) in a time- and concentration-dependent manner that involves, but is not entirely dependent on the enzyme's catalytic activity. Similarly, MPO together with its substrate, H2O2, inhibited the PCA of plasma microvesicles isolated from lipopolysaccharide (LPS)-stimulated whole blood, an effect additive to that of a function blocking TF antibody. Treatment of whole blood with LPS or phorbol-myristate-acetate dramatically increased MPO plasma levels, and co-incubation with 4-ABAH, a specific MPO inhibitor, significantly enhanced the PCA in plasma supernatants. MPO and MPO/H2O2 also inhibited the PCA of activated platelets and purified phospholipids (PLs), suggesting that modulation of negatively charged PLs, i.e., phosphatidylserine, rather than direct interference with the TF/FVIIa initiation complex was involved. Consistently, pretreatment of activated platelets with MPO or MPO/H2O2 attenuated the subsequent binding of lactadherin, which specifically recognizes procoagulant PS on cell membranes. Finally, endogenously released MPO regulated the PCA of THP1 cells in an autocrine manner dependent on the binding to CD11b/CD18 integrins. Collectively, these findings indicate that MPO is a negative regulator of PL-dependent coagulation and suggest a more complex role of activated PMNs in haemostasis and thrombosis.
Asunto(s)
Neutrófilos/fisiología , Peroxidasa/metabolismo , Trombosis/metabolismo , Coagulación Sanguínea , Factor VIIa/metabolismo , Células HL-60 , Humanos , Peróxido de Hidrógeno/metabolismo , Lipopolisacáridos/metabolismo , Fosfatidilserinas/metabolismo , Fosfolípidos/metabolismo , Unión Proteica , Vesículas Secretoras/metabolismo , Células THP-1 , Tromboplastina/metabolismoRESUMEN
BACKGROUND: In acute myeloid leukemia (AML), disseminated intravascular coagulation (DIC) contributes to morbidity and mortality, but the underlying pathomechanisms remain incompletely understood. METHODS: We conducted a prospective study on 69 patients with newly diagnosed AML to further define the correlates of systemic coagulation activation in this hematological malignancy. Tissue factor procoagulant activity (TF PCA) of isolated peripheral blood mononuclear cells (PBMCs) and TF expression by circulating microparticles (MPs) were assessed by single-stage clotting and thrombin generation assay, respectively. Soluble plasma TF antigen and secretion of vascular endothelial growth factor (VEGF) by cultured PBMCs were measured by ELISA. Cell-free plasma DNA was quantified by staining with a fluorescent dye. RESULT: TF PCA of PBMCs was significantly increased in AML patients as compared to healthy controls. Furthermore, TF PCA was significantly associated with decompensated DIC at presentation, as defined by a plasma fibrinogen level of ≤1 g/L (n = 11). In addition to TF PCA and circulating blasts, serum lactate dehydrogenase, a surrogate marker for leukemic cell turnover, correlated with plasma D-Dimer in the total patient cohort and was significantly increased in DIC patients, suggesting a role for myeloblast apoptosis/necrosis in activation of the TF-dependent coagulation pathway. Consistently, TF-bearing plasma MPs were more frequently detected and levels of soluble TF antigen were significantly higher in DIC vs. non-DIC patients. No association was found between TF PCA expression and VEGF secretion by isolated PBMCs, but significantly increased levels of cell-free plasma DNA pointed to a contribution of the intrinsic contact pathway to systemic coagulation activation in the total patient cohort and in patients with lower TF PCA expression. While PBMC-associated TF PCA had no effect on long-term survival, DIC occurrence at presentation increased the risk of early mortality. CONCLUSION: In newly diagnosed AML, TF expression by PBMCs and shedding of TF-bearing plasma MPs are central to the pathogenesis of DIC, but additional pathways, such as DNA liberation, may contribute to systemic coagulation activation.
RESUMEN
BACKGROUND: Patients with germ cell tumors (GCTs) receiving cisplatin-based chemotherapy are at increased risk of thrombosis, but the underlying cellular and molecular mechanisms remain obscure. OBJECTIVE: To study baseline tissue factor (TF) expression by GCT cell lines and its modulation by cisplatin treatment. METHODS: TF expression was assessed by single-stage clotting and thrombin generation assay, flow cytometry, ELISA, and Western blot analysis. Cell cycle analysis and detection of phosphatidylserine (PS) membrane exposure were carried out by flow cytometry. TF mRNA was analyzed by quantitative RT-PCR. RESULTS: Significant expression of TF-specific procoagulant activity (PCA) was detected on three non-seminoma (NT2, 2102Ep, NCCIT) and one seminoma cell line (TCam-2). Treatment with 0.4µM cisplatin (corresponding to the IC50) for 48hrs increased TF PCA on NT2 cells 3-fold, an effect that was largely independent of PS exposure and that could not be explained by translocation of active TF from intracellular storage pools. Cisplatin-induced TF PCA expression in NT2 cells did not occur before 12hrs, but was steady thereafter and accompanied by a 2-fold increase in total and surface-located TF antigen. Importantly, increased TF gene transcription or production and release of an intermediate factor were not involved in this process. Cell cycle analysis suggested that cisplatin-induced G2/M arrest resulted in an accumulation of procoagulant TF on the membrane surface of NT2 cells. CONCLUSIONS: In addition to induction of apoptosis/necrosis with PS-mediated activation of preformed TF, cisplatin may alter the procoagulant phenotype of GCT cells through an increase in total cellular TF antigen.
Asunto(s)
Cisplatino/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Neoplasias de Células Germinales y Embrionarias/metabolismo , Tromboplastina/metabolismo , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HL-60 , Humanos , Neoplasias de Células Germinales y Embrionarias/patologíaRESUMEN
The management of patients with acquired factor V inhibitors is challenging, because their bleeding risk is highly variable and only poorly correlated with routine coagulation tests. Furthermore, there is no standardized treatment for bleeding control or inhibitor eradication. An 84-year-old white man underwent uneventful surgery for a ruptured intracerebral haemangioma. There were no perioperative coagulation abnormalities. Eight weeks after surgery, however, the prothrombin and the activated partial thromboplastin times were found to be maximally prolonged without signs of acute haemorrhage. A factor V inhibitor of 212 Bethesda units was diagnosed. We used a fluorogenic real-time thrombin generation assay with low concentrations of tissue factor (TF) to analyse the factor V inhibitor for interference with coagulation in platelet-poor plasma. Compared with three bleeding patients with acquired haemophilia A and severely deficient thrombin generation, total thrombin generation capacity was similar in the patient and healthy controls. However, the lag phase was significantly prolonged, suggesting a defect in the initiation/amplification, but not in the propagation phase of TF-triggered thrombin generation. This defect could be fully reproduced by purified patient IgG and largely corrected by ex-vivo addition of activated prothrombin complex concentrate, but not recombinant human FVIIa. Addition of normal platelets to the patient's plasma resulted in a pronounced shortening of the lag phase, suggesting that platelet-derived factor V can escape the inhibitor. Our findings offer an explanation for the absence of spontaneous bleeding in this patient and support the concept of platelet transfusions for the management of acute haemorrhages in patients with acquired factor V inhibitors.
Asunto(s)
Deficiencia del Factor V/sangre , Factor V/antagonistas & inhibidores , Trombina/biosíntesis , Anciano de 80 o más Años , Humanos , MasculinoAsunto(s)
Plaquetas/patología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Vías Secretoras/genética , Trombocitopenia/genética , Enfermedad de von Willebrand Tipo 2/genética , Factor de von Willebrand/metabolismo , Adulto , Plaquetas/metabolismo , Femenino , Hemorragia/genética , Humanos , Lactante , Mutación/genética , Linaje , Agregación Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Embarazo , Estudios Prospectivos , Unión Proteica/genética , Multimerización de Proteína/genética , Proteolisis , Vesículas Secretoras/metabolismo , Enfermedad de von Willebrand Tipo 2/diagnóstico , Factor de von Willebrand/administración & dosificaciónRESUMEN
Tissue factor (TF) expressed on sub-cellular membrane vesicles, so-called plasma microparticles (MPs), has recently emerged as a potential key player in intravascular coagulation activation in various disease states. In this report, we demonstrate significantly increased levels of TF-specific procoagulant activity (PCA) of plasma MPs in five patients presenting with overt disseminated intravascular coagulation (DIC) due to an underlying malignancy, including non-small-cell lung cancer (n = 1), melanoma (n = 1), prostate cancer (n = 2), and acute promyelocytic leukemia (n = 1). Clotting experiments on available tumor cell samples suggested that cancer cells were a potential source of circulating TF-positive MPs at least in three of the five patients. Furthermore, follow-up plasma samples from two surviving patients revealed that response of their malignancies to specific anti-cancer therapy was paralleled by resolution of overt DIC and a significant decline in MP-associated TF PCA. Levels of plasma TF antigen, as assessed by an enzyme-linked immunosorbent assay, were also increased at presentation albeit to a lesser extent compared to MP-associated TF PCA, likely due to insufficient solubilization of the phospholipid-incorporated full-length TF molecule by the detergent. In summary, our findings suggest that MP-associated TF PCA may play an important pathogenic role in the evolution of overt DIC in various types of malignancy.