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1.
Circ Res ; 114(3): 444-453, 2014 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-24265393

RESUMO

RATIONALE: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor-bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. OBJECTIVE: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. METHODS AND RESULTS: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI-mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2-mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein-coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2-induced G protein-coupled receptor signaling pathways. CONCLUSIONS: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.


Assuntos
Plaquetas/metabolismo , Proteína Adaptadora GRB2/fisiologia , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/genética , Transdução de Sinais/genética , Motivos de Aminoácidos/genética , Animais , Células Cultivadas , Proteína Adaptadora GRB2/genética , Hemostasia/genética , Motivo de Inibição do Imunorreceptor Baseado em Tirosina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária/genética , Trombose/genética
2.
Bioconjug Chem ; 25(1): 52-62, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24341642

RESUMO

IL-4 signaling into a cell occurs via assembly of a receptor complex that consists of a high-affinity IL-4Rα chain and a low affinity chain, where the low-affinity chain is either γc or IL-13Rα1. It has been previously shown that mutational disruption of the low affinity interface in the IL-4DM (double mutein) yields an antagonist that inhibits IL-4 as well as IL-13-dependent responses. The present study reveals that new types of IL-4 antagonists can be generated by site-specific chemical modification. The chemically modified IL-4 analogues consist of (1) mixed disulfides created by refolding IL-4 cysteine muteins in the presence of different thiol compounds or (2) maleimide conjugates created by modifying cysteine muteins with maleimide derivatives. IL-4 analogues chemically modified at position 121 retain marginal binding affinity to γc or IL-13Rα1 receptor ectodomains during SPR interaction analysis. The biological activity of the analogues is strongly reduced in HEK-Blue IL-4/IL-13 cells as well as in Jurkat cells. Since the IL-4 analogues modified at position 121 have the ability to inhibit γc (IL-4)- and IL13Rα1 (IL-4/IL-13)-dependent responses in Jurkat and HEK-Blue cell lines, they effectively act as IL-4 antagonists. The results of our IL-4 study provide the first example of a cytokine that is transformed into a competitive inhibitor by site-specific chemical modification.


Assuntos
Interleucina-13/antagonistas & inibidores , Interleucina-4/análogos & derivados , Interleucina-4/antagonistas & inibidores , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Interleucina-4/química , Células Jurkat , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
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