SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

Abstract

Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbβ3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbβ3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbβ3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbβ3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.

Structure and function of the ubiquitin‐proteasome system in platelets

“…In addition, αIIbβ3 (or GPIIb/IIIA) and C‐type lectin‐like receptor 2 (CLEC‐2) are involved in ubiquitination‐dependent modulation of platelet function. The αIIbβ3‐associated protein SHARPIN induces ubiquitination and activation of NF‐κB proteins via the linear ubiquitin chain assembly complex (LUBAC) and the E3 ligases HOIP and HOIL‐1 after platelet stimulation with thrombin or inflammatory agonists . Another E3 ubiquitin ligase, RN181, was shown to interact with platelet integrin αIIbβ3 suggesting a further link between integrin signal transduction and ubiquitination .…”

“…Here activation is accomplished by ubiquitination of the NF‐κB‐inhibitor IκBα and its subsequent degradation by the proteasome complex (Figure ) . The proteasome is also relevant for modulating the signaling pathways of αIIbβ3, GPVI, and CLEC‐2 (Figure ) and might play a pivotal role in additional signaling pathways in platelets . In this review, we summarize the known fundamental regulatory mechanisms and functional aspects of the proteasome in human platelets.…”

“…[14][15][16] The proteasome is also relevant for modulating the signaling pathways of αIIbβ3, GPVI, and CLEC-2 ( Figure 1) and might play a pivotal role in additional signaling pathways in platelets. [17][18][19][20][21][22] In this review, we summarize the known fundamental regulatory mechanisms and functional aspects of the proteasome in human platelets.…”

Melatonin enhances thrombopoiesis through ERK1/2 and Akt activation orchestrated by dual adaptor for phosphotyrosine and 3‐phosphoinositides

“…HSCs commit and differentiate into MKs, which then mature in a process that is characterized by increased ploidy. Mature MKs release platelets after remarkable morphologic changes, which require a profound rearrangement of the cytoskeleton . Previous studies, including our own work, have revealed that various factors, primarily the specific cytokine thrombopoietin (TPO), can directly promote platelet production by acting at different stages through the extracellular signal‐regulated kinase 1/2 (ERK1/2) and Akt signaling pathways .…”

“…Mature MKs release platelets after remarkable morphologic changes, which require a profound rearrangement of the cytoskeleton. 1,2 Previous studies, including our own work, have revealed that various factors, primarily the specific cytokine thrombopoietin (TPO), can directly promote platelet production by acting at different stages through the extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathways. [3][4][5] In addition, some factors may also indirectly facilitate platelet production through TPO, which is suggested by the data that approximately 60% of TPO in circulation is controlled by the platelet glycoprotein Ibα (GPIbα).…”