RESUMEN
INTRODUCTION: Systemic hypercoagulation is often a severe complication of infective and inflammatory diseases, which overcome the hemostatic balance and lead to multiple thrombotic occlusions in the microvasculature and organ damage and is related to high mortality rates. SATI is a potent dual inhibitor of FXa and thrombin with antithrombotic efficacy in venous and arterial thrombosis models. In this study, the antithrombotic efficacy of SATI was investigated in a microthrombosis model in rats with an induced hypercoagulant state. MATERIALS AND METHODS: The hypercoagulant state was generated by infusion of TF in sixty rats (12 groups, consisting of 5 rats each). SATI was administered in two different doses by constant infusion and its antithrombotic efficacy was investigated using two different approaches: 1) measuring 125I-fibrin deposition in various organs and 2) continuous whole-body imaging of 111In-platelet biodistribution in anesthetized animals. RESULTS: After start of the TF infusion in rats with radioactively-labeled fibrinogen, the radioactivity was accumulated in liver, spleen, kidney, and mostly in the lung as a consequence of fibrin generation. SATI efficiently reduced the pulmonary deposition of fibrin in a dose- and time-dependent manner. In the SATI groups the splenic and renal radioactivity was enhanced at later time points probably as consequence of the clearance of 125I-fibrin(ogen). Imaging of rats that received 111In-platelets prior to systemic TF administration showed retention of the radioactivity mainly in the lungs in the control group. SATI efficiently blocked the platelet accumulation in the lungs and increased platelet recruitment by the spleen. CONCLUSIONS: SATI is a promising candidate for prevention of microcirculatory disturbances by inhibiting fibrin deposition and platelet accumulation in the lungs and thereby conferring organ protection. Both methods used in this study are suitable for investigating the antithrombotic efficacy of new drugs in microthrombosis. Continuous imaging of 111In-platelets allowed for follow-up of thrombus formation in living animals without the need for tissue harvesting.
Asunto(s)
Inhibidores del Factor Xa , Trombina/antagonistas & inhibidores , Animales , Plaquetas/metabolismo , Factor Xa/farmacología , Fibrina/metabolismo , Microcirculación , Ratas , Distribución TisularAsunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antitrombinas/efectos adversos , Dabigatrán/efectos adversos , Inhibidores del Factor Xa/efectos adversos , Factor Xa/uso terapéutico , Hemorragia/tratamiento farmacológico , Pirazoles/efectos adversos , Piridinas/efectos adversos , Piridonas/efectos adversos , Proteínas Recombinantes/uso terapéutico , Rivaroxabán/efectos adversos , Tiazoles/efectos adversos , Administración Oral , Anticuerpos Monoclonales Humanizados/farmacología , Antitrombinas/uso terapéutico , Pérdida de Sangre Quirúrgica/prevención & control , Hemorragia Cerebral/inducido químicamente , Hemorragia Cerebral/terapia , Ensayos Clínicos como Asunto , Dabigatrán/antagonistas & inhibidores , Dabigatrán/inmunología , Dabigatrán/uso terapéutico , Aprobación de Drogas , Urgencias Médicas , Factor Xa/farmacología , Inhibidores del Factor Xa/uso terapéutico , Hemorragia/inducido químicamente , Humanos , Pirazoles/antagonistas & inhibidores , Pirazoles/uso terapéutico , Piridinas/antagonistas & inhibidores , Piridinas/uso terapéutico , Piridonas/antagonistas & inhibidores , Piridonas/uso terapéutico , Proteínas Recombinantes/farmacología , Rivaroxabán/antagonistas & inhibidores , Rivaroxabán/uso terapéutico , Tiazoles/antagonistas & inhibidores , Tiazoles/uso terapéutico , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Factor Xa/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Embrión de Pollo , Factor X/farmacología , Factor X/uso terapéutico , Factor Xa/uso terapéutico , Proteínas del Helminto/farmacología , Proteínas del Helminto/uso terapéutico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Receptor PAR-1/metabolismo , Pez CebraRESUMEN
A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.
Asunto(s)
Cisteína Endopeptidasas/fisiología , Melanoma/metabolismo , Proteínas de Neoplasias/fisiología , Protrombina/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismo , Línea Celular Tumoral/química , Cisteína Endopeptidasas/efectos de los fármacos , Factor V/farmacología , Factor VIIa/farmacología , Factor Xa/farmacología , Citometría de Flujo , Humanos , Melanoma/química , Proteínas de Neoplasias/efectos de los fármacosRESUMEN
A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.