Platelet studies have had a profound impact on our understanding of adhesion receptor function as exemplified by the adhesion and signaling roles of integrin ?IIb?3 in hemostasis and thrombosis. Less is known about whether and how ?IIb?3 signaling regulates platelets in their roles as sentinels and effectors of immunity and inflammation. In this regard, we have discovered that ?IIb?3 interacts through its ?IIb cytoplasmic tail with SHARPIN, an obligate component of the linear ubiquitin chain assembly complex (LUBAC). This complex is the only known enzyme responsible for M1 linear ubiquitination of key proteins involved in immune and inflammatory signaling through the NF-?B pathway in leukocytes. Platelets express many proteins of the canonical NF-?B pathway. Therefore, we will now employ complementary approaches, including studies of peripheral blood platelets, megakaryocytes and platelets derived from human induced pluripotent stem cells, and gene-targeted mice to test the following central hypothesis: Through dynamic and mutually exclusive interactions with ?IIb?3 and LUBAC, SHARPIN regulates platelet function in inflammation and immunity as well as in hemostasis and thrombosis.
Aim 1 will employ biochemical and advanced imaging techniques to test whether physical and functional linkages exist between ?IIb?3, LUBAC and the NF-?B pathway in human and mouse platelets. Attention will be focused on whether SHARPIN functions to maintain ?IIb?3 in a low-affinity state in resting platelets, but dissociates from ?IIb and assembles the LUBAC complex to facilitate ?IIb?3 and NF-?B activation in response to platelet agonists generated during hemostasis and inflammation.
Aim 2 will introduce genetic modifications into human megakaryocytes and platelets derived from induced pluripotent stem cells to model the effects of SHARPIN knockdown or knockout on ?IIb?3 and LUBAC functions in the appropriate primary cells. Using this system, optogenetic techniques new to the platelet field will be used to determine the functional consequences of conditional SHARPIN interactions with ?IIb?3 or with its two LUBAC protein partners.
Aim 3 will address whether SHARPIN is required for ?IIb?3 and platelet functions in response to inflammatory stimuli and vascular injury in vivo. To accomplish this, platelet-specific SHARPIN knockout mice will be generated using the Pf4-Cre system and studied in several mouse models of platelet-dependent inflammation, hemostasis and thrombosis. Altogether, these studies will provide new insights into the role of SHARPIN in platelet pathobiology, with mechanistic and potential therapeutic implications beyond hemostasis and for platelets in inflammation and immunity.

Public Health Relevance

A key platelet adhesion receptor in hemostasis is integrin ?IIb?3, but its role in inflammation is incompletely understood. Here we will investigate newly-identified interactions between ?IIb?3 and SHARPIN, an intracellular protein implicated in immune and inflammatory responses as a member of an enzyme complex that mediates linear ubiquitination of signaling proteins. This idea will be tested using novel techniques in vitro and in vivo, with potential clinical implications for platelets beyond their traditional roles in hemostasis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
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Kindzelski, Andrei L
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University of California San Diego
Internal Medicine/Medicine
Schools of Medicine
La Jolla
United States
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Liao, Zhongji; Kasirer-Friede, Ana; Shattil, Sanford J (2017) Optogenetic interrogation of integrin ?V?3 function in endothelial cells. J Cell Sci 130:3532-3541
Ley, Klaus; Rivera-Nieves, Jesus; Sandborn, William J et al. (2016) Integrin-based therapeutics: biological basis, clinical use and new drugs. Nat Rev Drug Discov 15:173-83
Liao, Zhongji; Kato, Hisashi; Pandey, Manjula et al. (2015) Interaction of kindlin-2 with integrin ?3 promotes outside-in signaling responses by the ?V?3 vitronectin receptor. Blood 125:1995-2004
Desgrosellier, Jay S; Lesperance, Jacqueline; Seguin, Laetitia et al. (2014) Integrin ?v?3 drives slug activation and stemness in the pregnant and neoplastic mammary gland. Dev Cell 30:295-308
van Sorge, Nina M; Cole, Jason N; Kuipers, Kirsten et al. (2014) The classical lancefield antigen of group a Streptococcus is a virulence determinant with implications for vaccine design. Cell Host Microbe 15:729-740
Kasirer-Friede, Ana; Kang, Jian; Kahner, Bryan et al. (2014) ADAP interactions with talin and kindlin promote platelet integrin ?IIb?3 activation and stable fibrinogen binding. Blood 123:3156-65
Casar, B; Rimann, I; Kato, H et al. (2014) In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated ?1 integrin and induction of FAK/PI3K/Akt motility signaling. Oncogene 33:255-68
Ye, Feng; Petrich, Brian G; Anekal, Praju et al. (2013) The mechanism of kindlin-mediated activation of integrin ?IIb?3. Curr Biol 23:2288-95
Kato, Hisashi; Liao, Zhongji; Mitsios, John V et al. (2012) The primacy of ?1 integrin activation in the metastatic cascade. PLoS One 7:e46576
Kahner, Bryan N; Kato, Hisashi; Banno, Asoka et al. (2012) Kindlins, integrin activation and the regulation of talin recruitment to ýýIIbýý3. PLoS One 7:e34056

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