RESUMEN
Direct oral anticoagulants (DOACs), including the direct thrombin inhibitor, dabigatran, and the direct factor Xa (FXa) inhibitors, rivaroxaban, apixaban and edoxaban, are approved for thromboembolism prevention and treatment. These drugs do not require routine coagulation monitoring but, in some circumstances, measurement of drug level or anticoagulant effect may be necessary. Although traditional coagulation tests lack analytical sensitivity and specificity, they are widely available and inexpensive, and can provide useful information regarding the residual anticoagulant effect of DOACs. Hemoclot® and ecarin-based assays can be used to quantify dabigatran level and calibrated chromogenic anti-FXa assays are suitable for measuring rivaroxaban, apixaban and edoxaban levels, but these tests are not yet widely available.
Asunto(s)
Anticoagulantes/farmacología , Pruebas de Coagulación Sanguínea/métodos , Coagulación Sanguínea/efectos de los fármacos , Administración Oral , Anticoagulantes/administración & dosificación , Cromatografía Líquida de Alta Presión/métodos , Dabigatrán/administración & dosificación , Dabigatrán/farmacología , Monitoreo de Drogas/métodos , Humanos , Pirazoles/administración & dosificación , Pirazoles/farmacología , Piridinas/administración & dosificación , Piridinas/farmacología , Piridonas/administración & dosificación , Piridonas/farmacología , Rivaroxabán/administración & dosificación , Rivaroxabán/farmacología , Tiazoles/administración & dosificación , Tiazoles/farmacologíaRESUMEN
Thrombosis is one of the cardinal manifestations of myeloproliferative neoplasms (MPN). The mechanisms leading to a prothrombotic state in MPN are complex and remain poorly understood. Platelet mitochondria play a role in platelet activation, but their number and function have not been extensively explored in MPN to date. We observed an increased number of mitochondria in platelets from MPN patients compared with healthy donors. MPN patients had an increased proportion of dysfunctional platelet mitochondria. The fraction of platelets with depolarized mitochondria at rest was increased in essential thrombocythemia (ET) patients and the mitochondria were hypersensitive to depolarization following thrombin agonist stimulation. Live microscopy showed a stochastic process in which a higher proportion of individual ET platelets underwent mitochondrial depolarization and after a shorter agonist exposure compared to healthy donors. Depolarization was immediately followed by ballooning of the platelet membrane, which is a feature of procoagulant platelets. We also noted that the mitochondria of MPN patients were on average located nearer the platelet surface and we observed extrusion of mitochondria from the platelet surface as microparticles. These data implicate platelet mitochondria in a number of prothrombotic phenomena. Further studies are warranted to assess whether these findings correlate with clinical thrombotic events.
Asunto(s)
Trastornos Mieloproliferativos , Neoplasias , Trombocitemia Esencial , Trombosis , Humanos , Plaquetas/metabolismo , Trombina/metabolismo , Trastornos Mieloproliferativos/metabolismo , Trombocitemia Esencial/metabolismo , Trombosis/metabolismo , Activación Plaquetaria , Neoplasias/metabolismo , MitocondriasRESUMEN
Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.
Asunto(s)
Plaquetas/patología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/patología , Animales , Apoptosis/efectos de los fármacos , Aspirina/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias/patologíaRESUMEN
Rivaroxaban and apixaban are both small molecules that reversibly inhibit factor Xa. Compared with rivaroxaban, apixaban has minimal effects on the prothrombin time and activated partial thromboplastin time. To investigate this phenomenon, we used a factor Xa-directed substrate in a buffer system. Although rivaroxaban and apixaban inhibited factor Xa with similar K i values at equilibrium, kinetic measurements revealed that rivaroxaban inhibited factor Xa up to 4-fold faster than apixaban ( p < 0.001). Using a discontinuous chromogenic assay to monitor thrombin production by prothrombinase in a purified system, rivaroxaban was 4-fold more potent than apixaban (K i values of 0.7 ± 0.3 and 2.9 ± 0.5 nM, respectively; p = 0.02). Likewise, in thrombin generation assays in plasma, rivaroxaban prolonged the lag time and suppressed endogenous thrombin potential to a greater extent than apixaban. To characterize how the two inhibitors differ in recognizing factor Xa, inhibition of prothrombinase was monitored in real-time using a fluorescent probe for thrombin. The data were fit using a mixed-inhibition model and the individual association and dissociation rate constants were determined. The association rates for the binding of rivaroxaban to either free factor Xa or factor Xa incorporated into the prothrombinase complex were 10- and 1,193-fold faster than those for apixaban, respectively, whereas dissociation rates were about 3-fold faster. Collectively, these findings suggest that rivaroxaban and apixaban differ in their capacity to inhibit factor Xa and provide a plausible explanation for the observation that rivaroxaban has a greater effect on global tests of coagulation than apixaban.
RESUMEN
Four direct oral anticoagulants (dabigatran, rivaroxaban, apixaban, edoxaban) have been shown to be at least as effective and safe as warfarin for the prevention of stroke in atrial fibrillation and the prevention and treatment of venous thromboembolism. Although they are administered in fixed doses without routine coagulation monitoring, measurement of anticoagulant effect or drug levels may be useful to determine if: anticoagulant effect is present in patients who are bleeding or require an urgent procedure or thrombolysis; levels are within usual on-therapy range in patients with recurrent thromboembolism during treatment; and levels are outside of the usual on-therapy range in patients with overdose or with extreme clinical characteristics. Traditional coagulation assays are widely available but lack sensitivity to detect clinically relevant anticoagulant effects, and lack accuracy in quantitating drug levels. Specific drug assays are less widely available but can accurately measure drug levels and should be preferred.
Asunto(s)
Anticoagulantes/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Accidente Cerebrovascular/prevención & control , Anticoagulantes/efectos adversos , Coagulación Sanguínea/efectos de los fármacos , Hemorragia/inducido químicamente , Humanos , Tromboembolia Venosa/tratamiento farmacológico , Tromboembolia Venosa/prevención & controlRESUMEN
The in-vitro lysis of plasma clots in acetic acid generally indicates a Factor XIII deficiency that is confirmed by quantitative assay. However, there are two rare, poorly understood circumstances whereby clot lysis in acid occurs when factor XIII activity levels are normal: the presence of either an atypical antifactor XIII antibody, or an unknown acid-activated protease. Our centre has identified four patients with in-vitro clot lysis in acetic acid and normal FXIII levels by activity assay. Our aim was to determine whether the cause of this unusual result was an inhibitory antibody or an aspartic acid protease. In each case, we found an inhibitor that was not an IgG but showed characteristics of an acid protease, including that it was neutralized by pepstatin. The four patients had median pepsinogen I levels five-fold to 10-fold higher than the normal median of 89âµg/l. Pepsinogen II was increased by three-fold to six-fold, but from a lower baseline median of 6.5âµg/l. Cathepsin D levels were normal. Clot lysis in the acid test was observed when recombinant human pepsinogen I was added to normal plasma at similarly high concentrations as in patient samples, consistent with a role of an acid protease. Clot lysis also occurred with addition of pepsinogen II, but required four-fold to seven-fold more than in patient samples. Laboratories should be aware that a positive acid clot lysis test can be misleading if pepsinogen levels are raised and should not use this alone to diagnose FXIII deficiency.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor XIII/análisis , Fibrinólisis/efectos de los fármacos , Pepsinógeno A/sangre , Pepsinógeno C/sangre , Ácido Acético/química , Anciano , Proteasas de Ácido Aspártico/sangre , Factor XIII/antagonistas & inhibidores , Factor XIII/inmunología , Factor XIII/metabolismo , Deficiencia del Factor XIII/sangre , Deficiencia del Factor XIII/diagnóstico , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Técnicas In Vitro , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: There is minimal data on the influence of pre-analytical variables on the use of calibrated automated thrombography (CAT), to measure thrombin generation. OBJECTIVES: To evaluate the impact of centrifugation methods, time after collection, and contact activation inhibition on the CAT assay performed using two commercial reagents. METHODS AND RESULTS: Six different methods of plasma separation were examined. Thrombin generation triggered by a 5 pM tissue factor reagent was not greatly affected by plasma separation method, with similar results obtained with all methods apart from single centrifugation and membrane filtration. Membrane filtration increased APTT and is not recommended. Extended double centrifugation at higher speed was required to minimise the impact of residual phospholipid with 1 pM tissue factor trigger, particularly with inhibition of contact activation. The effect of a delay of up to 24 hours in preparing plasma was assessed. No significant difference in results was observed among samples processed between 0.5 and 6 hours after blood collection into plastic Vacuette® tubes. The presence or absence of corn trypsin inhibitor had a significant impact on all parameters with 1 pM tissue factor trigger, with minor differences seen on Peak and ttPeak results using 5 pM tissue factor. CONCLUSIONS: The impact of pre-analytical variables on thrombin generation results is dependent on the concentration of tissue factor in the trigger reagent used. Results with 1 pM tissue factor are particularly sensitive to centrifugation method and contact activation, and standardisation is required to allow large collaborative studies to be performed.