RESUMEN
OBJECTIVE: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. CONCLUSIONS: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.
Asunto(s)
Plaquetas/enzimología , Traumatismos de las Arterias Carótidas/enzimología , Moléculas de Adhesión Celular Neuronal/sangre , Proteínas de la Matriz Extracelular/sangre , Proteínas del Tejido Nervioso/sangre , Neuropéptidos/sangre , Fosfolipasa C gamma/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Serina Endopeptidasas/sangre , Trombosis/enzimología , Proteína de Unión al GTP rac1/sangre , Proteína de Unión al GTP rhoA/sangre , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Coagulación Sanguínea , Traumatismos de las Arterias Carótidas/sangre , Traumatismos de las Arterias Carótidas/etiología , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/genética , Retracción del Coagulo , Citoesqueleto/enzimología , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Ratones de la Cepa 129 , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Activación Plaquetaria , Proteína Reelina , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Transducción de Señal , Trombosis/sangre , Trombosis/etiologíaRESUMEN
High resolution structural information on amyloid fibrils is crucial for the understanding of their formation mechanisms and for the rational design of amyloid inhibitors in the context of protein misfolding diseases. The Src-homology 3 domain of phosphatidyl-inositol-3-kinase (PI3K-SH3) is a model amyloid system that plays a pivotal role in our basic understanding of protein misfolding and aggregation. Here, we present the atomic model of the PI3K-SH3 amyloid fibril with a resolution determined to 3.4 Å by cryo-electron microscopy (cryo-EM). The fibril is composed of two intertwined protofilaments that create an interface spanning 13 residues from each monomer. The model comprises residues 1-77 out of 86 amino acids in total, with the missing residues located in the highly flexible C-terminus. The fibril structure allows us to rationalise the effects of chemically conservative point mutations as well as of the previously reported sequence perturbations on PI3K-SH3 fibril formation and growth.