Tesis doctoral de Cristina Puy García
Background: the endothelial cell protein c receptor (epcr) binds to protein c through its γ-carboxyglutamic acid (gla) domain and enhances its thrombin-thrombomodulin complex-dependent activation. Our group has previously shown that factor (f) vii also binds to epcr through its gla domain with high affinity (kd ~ 40 nm). Interestingly, soluble epcr (sepcr) increased dose-dependently the plasma clotting time, suggesting that epcr could play an anticoagulant role upon fviia binding. Hypothesis: the binding of epcr to fvii/viia reduces the anticoagulant activity of the latter, making epcr play a new, unsuspected anticoagulant role. Methods and results: experiments with fviia-cleavable chromogenic substrates showed that fviia undergoes a change in its amidolytic activity upon binding to sepcr. This set of experiments also revealed that fviia interacts simultaneously with soluble tf (stf) and sepcr. Importantly, sepcr decreased the activation of fx and fix by fviia-tf on the surface of tf-expressing cells. Western-blot analyses revealed that the autoactivation of fvii and the tf-dependent activation of fvii by fxa were dose-dependently reduced by sepcr. Flow cytometric analyses showed that sepcr reduced the binding of fviia to tf on the cell surface, which provides an explanation for the previous findings. Importantly, western-blot analyses revealed that the activation of fvii by fxa on the surface of human aortic endothelial cells was favoured about two-fold in the presence of a monoclonal antibody able to block fvii binding to epcr. Looking for an explanation, we demonstrated by surface plasmon resonance that epcr prevented the binding of fvii to anionic phospholipids i.E. Phosphatydilserine (ps). Moreover, immunofluorescence experiments showed that epcr and ps are located at different regions of the cell surface, thus reinforcing the notion that epcr moves fvii away from fxa. Conclusions: sepcr decreases the coagulant activity of fvii/viia. Membrane epcr inhibits fvii activation. Pathological conditions predisposing to low epcr exposure on the endothelial surface may disrupt the haemostatic balance not only by favouring lesser activation of protein c but also by inducing an increase in the circulating levels of fviia.
Datos académicos de la tesis doctoral «Modulación de la actividad coagulante del factor vii/viia por el receptor endotelial de la proteína c«
- Título de la tesis: Modulación de la actividad coagulante del factor vii/viia por el receptor endotelial de la proteína c
- Autor: Cristina Puy García
- Universidad: Navarra
- Fecha de lectura de la tesis: 13/10/2009
Dirección y tribunal
- Director de la tesis
- Ramón Montes Díaz
- Tribunal
- Presidente del tribunal: Francisco España furió
- pascual Marco vera (vocal)
- José ramón González porras (vocal)
- ramón Lecumberri villamediana (vocal)