RESUMEN
Platelet integrin αIIbß3 possesses a Leu/Pro polymorphism at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b). The Pro33 isoform has been suggested to exhibit prothrombotic features. αIIbß3-expressing CHO (Chinese hamster ovary) cells on immobilized fibrinogen show activation of the MAP kinase family member ERK2, with an enhanced ERK2 activity in Pro33 cells compared to Leu33 cells. In our present work, we examined how the Leu/Pro polymorphism modulates the ERK2 activation stimulated by 2 differently triggered outside-in signalings. We either treated the CHO cells with Mn2+ or allowed them to adhere to fibrinogen. Moreover, we studied which signaling cascades are involved in ERK2 activation. In contrast to immobilized fibrinogen, Mn2+ did not significantly increase ERK2 activation. However, Mn2+ had a synergistic effect on ERK2 phosphorylation when combined with immobilized fibrinogen. Pro33 cells adherent to fibrinogen exhibited a significantly greater ERK2 activity than Leu33 cells in the presence of Mn2+, which peaked after 10 min of adhesion. Our data showed that Src family and rho kinases play a crucial role in the integrin αIIbß3-dependent outside-in signaling to ERK2.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Manganeso/farmacología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Quinasas Asociadas a rho/genética , Familia-src Quinasas/genética , Animales , Plaquetas/metabolismo , Células CHO , Cationes Bivalentes , Adhesión Celular/efectos de los fármacos , Cricetulus , Fibrinógeno/química , Fibrinógeno/metabolismo , Regulación de la Expresión Génica , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación , Fosforilación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Quinasas Asociadas a rho/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbß3, αvß3, and α5ß1 are known to assemble FN fibrils on platelets. In our study, we examined the contribution of these integrins to FN binding, unfolding, and assembly on platelets in suspension and adherent platelets in the presence or absence of agonists. Phorbol 12-myristate 13-acetate (PMA), but not adenosine diphosphate (ADP), induced binding of FN to platelets in suspension. In contrast, adherent platelets were able to deposit FN on their surfaces in the absence of agonists. ß3 integrins had a major impact on the interaction of FN on platelets. αvß3 showed a similar contribution to the binding of FN as αIIbß3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5ß1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than ß3 integrins. None of the distinct antibodies directed against one of the three integrins caused a complete inhibition of binding, unfolding, and assembly of FN by platelets. Thus, it is likely that αIIbß3, αvß3, and α5ß1 or another still unknown receptor can be substituted.
Asunto(s)
Plaquetas/química , Fibronectinas/química , Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Plaquetas/metabolismo , Adhesión Celular , Fibronectinas/sangre , Transferencia Resonante de Energía de Fluorescencia , Humanos , Integrina alfa5beta1/agonistas , Integrina alfa5beta1/sangre , Integrina alfaVbeta3/agonistas , Integrina alfaVbeta3/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/agonistas , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Mapeo de Interacción de Proteínas , Desplegamiento ProteicoRESUMEN
Biomechanical forces can induce the transformation of fibronectin (Fn) from its compact structure to an extended fibrillar state. Adsorption of plasma proteins onto metallic surfaces may also influence their conformation. We used a cone-plate rheometer to investigate the effect of shear and stainless steel on conformational changes of Fn. In control experiments, cones grafted once or twice with polyethylene glycol were used. Plasma Fn was added at concentrations of 50 or 100 µg/ml to bovine serum albumin (BSA)- or Fn-coated plates and subsequently exposed to dynamic shear rates stepwise increasing from 50 to 5000 s-1 within 5 min and subsequently decreasing from 5000 to 50 s-1 within 5 min. The viscosity (mPa s) of Fn solutions was recorded over 10 min. Upon exposure to shear, the viscosity in the sample increased, suggesting conformational changes in Fn. Western blotting and densitometric analyses demonstrated that conformational changes of plasma Fn depended both on shear and protein concentration. However, there was no significant difference in fibril formation between BSA- or Fn-coated plates, suggesting that physical properties of stainless steel and biomechanical forces such as shear can affect the molecular structure of Fn. Our model may provide useful information of surface- and flow-induced alterations of plasma proteins.
Asunto(s)
Fibronectinas/sangre , Fibronectinas/química , Microfibrillas/química , Resistencia al Corte/fisiología , Adulto , Velocidad del Flujo Sanguíneo/fisiología , Adhesión Celular/fisiología , Fibronectinas/aislamiento & purificación , Humanos , Microfibrillas/fisiología , Conformación Proteica , Albúmina Sérica Bovina/química , Acero InoxidableRESUMEN
: Shear stress alone can activate platelets resulting in a subsequent platelet aggregation, so-called 'shear-induced platelet aggregation'. In our work, we analyzed how differently elevated shear stress impacts the Src and focal adhesion kinase (FAK) activation in fibrinogen-adherent human platelets. We detected the extents of Src pY418 and FAK pY397 activations in platelets on immobilized fibrinogen and over BSA under shear conditions. Moreover, we analyzed the role of αIIbß3 in the shear-induced platelet signaling by performing our experiments in the presence of the αIIbß3-antagonist Abciximab. Abnormally high shear rates (5000âs) significantly increased the extent of phosphorylation of both tyrosine kinases after short (2âmin) incubation time independently of the presence or absence of the integrin αIIbß3 ligand, fibrinogen. We could see considerably greater Src activation on immobilized fibrinogen than on BSA, but the extent of FAK Y397 phosphorylation was independent on the matrix. Abciximab not only reduced the Src and FAK signaling in platelets exposed to 5000âs on immobilized fibrinogen, but in platelets exposed to 5000âs over BSA as well. Our data indicate that whereas Src activation under shear stress is dominantly ligand-dependent, FAK signaling seems to be mostly shear induced.