Abstract
The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, mediates platelet adhesion at sites of vascular injury and transmits signals leading to platelet activation. von Willebrand factor/GPIb-IX interaction sequentially activates the Src family kinase Lyn (SFK), phosphoinositide 3-kinase (PI3K), and Akt, leading to activation of integrin α(IIb)β(3) and integrin-dependent stable platelet adhesion and aggregation. It remains unclear how Lyn activates the PI3K/Akt pathway after ligand binding to GPIb-IX. Using platelet-specific Rac1(-/-) mice and the Rac1 inhibitor NSC23766, we examined the role of Rac1 in GPIb-IX-dependent platelet activation. Rac1(-/-) mouse platelets and NSC23766-treated human platelets were defective in GPIb-dependent stable adhesion to von Willebrand factor under shear stress, integrin activation, thromboxane A(2) synthesis, and platelet aggregation. Interestingly, GPIb-induced activation of Rac1 and the guanine nucleotide exchange factor for Rac1, Vav, was abolished in both Lyn(-/-) and SFK inhibitor-treated platelets but was unaffected by the PI3K inhibitor LY294002, indicating that Lyn mediates activation of Vav and Rac1 independently of PI3K. Furthermore, GPIb-induced activation of Akt was abolished in Rac1-deficient platelets, suggesting that Rac1 is upstream of the PI3K/Akt pathway. A Lyn-Vav-Rac1-PI3K-Akt pathway mediates von Willebrand factor-induced activation of integrin α(IIb)β(3) to promote GPIb-IX-dependent platelet activation.
Highlights
Under the high shear-rate flow conditions present in arteries and capillaries, platelet adhesion to the site of vascular injury is mediated by the interaction between subendothelial-bound von Willebrand factor (VWF) and its platelet receptor, the glycoprotein Ib-IX (GPIb-IX) complex.[1,2,3]
The interaction between VWF and GPIb-IX mediates transient platelet adhesion to the injured vessel wall and initiates a signal transduction cascade culminating in the activation of integrin αIIbβ[3], leading to stable platelet adhesion, spreading, and aggregation.[2,4,5,6]
The identified most proximal step to GPIb-IX that propagates platelet activation signals is the activation of Lyn and Lyn-dependent activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway
Summary
Mice containing the Rac[1] conditional allele (Rac1loxP/loxP)[26] were crossed with mice carrying the Pf4-Cre transgene (Pf4-Cre+).[27] Pf4Cre+/ Rac1loxP/loxP mice are notated as Rac1−/− and Pf4-Cre−/ Rac1loxP/loxP. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org. 2762 Arterioscler Thromb Vasc Biol November 2012. Mice were maintained on a mixed SV/129/ C57/Bl-6 background, and littermates were used as control. Human and murine platelets were prepared, as previously described, and used at 3×108/mL.[28,29] Analysis of platelet adhesion under flow was performed as described previously.[11,12] Data are representative of ≥3 experiments, and statistical significance was determined via ANOVA and posttest
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