RESUMEN
Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands.
Asunto(s)
Caveolas/metabolismo , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , Receptores de Superficie Celular/metabolismo , Reología , Estrés Mecánico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Bovinos , Caveolas/efectos de los fármacos , Caveolina 1/metabolismo , Membrana Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Fosforilación/efectos de los fármacos , Presión , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The glycoprotein (GP) IIb-IIIa complex is an attractive anti-platelet target for the prevention of thrombotic events associated with coronary artery disease. Although GPIIb-IIIa antagonists inhibit GPIIb-IIIa binding to its ligands, the interactions have not been fully clarified, particularly with respect to their ability to induce structural changes in the complex that lead to exposure of neoantigenic epitopes or ligand-induced binding sites (LIBS). In this study we used the anti-LIBS monoclonal antibody (mAb) D3 to further define the activation states of purified active and inactive GPIIb-IIIa. We also compared the data obtained in the purified system to that observed with intact human platelets. Active GPIIb-IIIa expressed significantly greater high-affinity D3 LIBS sites compared to the inactive form. In addition, the ligand recognition peptides RGDS and H12 caused increased expression of the D3 epitope, with RGDS eliciting a much more potent response. The response of the purified GPIIb-IIIa to these peptides paralleled that observed with human platelets. To explore whether the platelet antagonists abciximab, eptifibatide and tirofiban induced expression of the D3 LIBS site, a modified competitive ELISA was developed. Our data indicate that the use of purified GPIIb-IIIa with this ELISA system provides a reproducible approach for exploring the interactions between GPIIb-IIIa and its antagonists. Whereas abciximab caused no detectable increase in the expression of the D3 epitope on purified GPIIb-IIIa, eptifibatide, tirofiban, RGDS, and H12 induced differential expression of the high-affinity LIBS. Studies with intact platelets suggested that abciximab blocked the binding of the D3 and LIBS6 mAbs, and that the pre bound anti-LIBS D3 sterically hindered abciximab binding.
Asunto(s)
Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Anticuerpos Monoclonales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/inmunología , Unión Competitiva , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Ligandos , Oligopéptidos/farmacología , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Conformación Proteica/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Sudden coronary artery occlusion is one of the leading causes of death. Several in vitro models have been used to study the relationship between hemodynamic forces and platelet function. However, very few in vivo studies exist that fully explore this relationship due to the lack of rheologic data for the platelet. For this purpose, micropipette aspiration techniques were used in the present study to determine the mechanical properties of platelets. The data were analyzed by two mathematical models: (1) an erythrocyte-type membrane model which yielded a platelet shear modulus of 0.03+/-0.01 dyn cm[-1] (mean+/-SD) and a viscous modulus of 0.12+/-0.04 dyn s cm[-1]. (2) An endothelial-type cell model which approximated the platelet Young's modulus to be 1.7+/-0.6 x 10(3) dyn cm(-2) with a viscous modulus of 1.0+/-0.5 x 10(4) dyn s cm(-2). The endothelial-type cell model more accurately describes the mechanics occurring at the micropipette tip and permits more appropriate assumptions to be made in quantifying the rheologic properties of a platelet. Results from this study can be integrated into numerical models of blood flow in stenosed coronary arteries to elucidate the impact of local hemodynamics on platelets and thrombus formation in coronary artery disease.