Abstract
Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.
Highlights
Integrins play a key role in regulating platelet functions, including platelet adhesion, spreading, clot retraction, and platelet pro-coagulant activity
We found that Protein kinase Cδ (PKCδ) is phosphorylated by integrin αIIbβ3-mediated outside-in signaling in platelets
We further found that integrin αIIbβ3-mediated PKCδ phosphorylation is regulated by Src and proline-rich tyrosine kinase 2 (Pyk2), and αIIbβ3-mediated phosphorylation of Akt is regulated by PKCδ
Summary
Integrins play a key role in regulating platelet functions, including platelet adhesion, spreading, clot retraction, and platelet pro-coagulant activity. PKCδ is phosphorylated on Thr505 and has been implicated in negatively regulating glycoprotein (GP) VI-mediated platelet functional responses, whereas it positively regulated protease-activated receptor (PAR)-mediated platelet responses including granule secretion and thromboxane generation [16]. A previous study implicated PKC isoforms in the regulation of integrin properties in many cell types [22], but the role of specific PKC isoforms in platelet outside-in signaling is limited. PKCδ was reported to be involved in thromboxane A2 (TxA2) generation, and thrombin- and collagen-induced PKCδ phosphorylation is regulated by αIIbβ outside-in signaling, raising the potential role of PKCδ in αIIbβ3-mediated signaling [25]. We found that PKCδ is phosphorylated by integrin αIIbβ3-mediated outside-in signaling in platelets. We concluded that PKCδ plays an important role in integrin αIIbβ3-mediated outside-in signaling in platelets
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