Integrin αIIbß3 outside-in signaling.

Integrin αIIbß3 is a highly abundant heterodimeric platelet receptor that can transmit information bidirectionally across the plasma membrane, and plays a critical role in hemostasis and thrombosis. Upon platelet activation, inside-out signaling pathways increase the affinity of αIIbß3 for fibrinogen and other ligands. Ligand binding and integrin clustering subsequently stimulate outside-in signaling, which initiates and amplifies a range of cellular events driving essential platelet processes such as spreading, thrombus consolidation, and clot retraction. Integrin αIIbß3 has served as an excellent model for the study of integrin biology, and it has become clear that integrin outside-in signaling is highly complex and involves a vast array of enzymes, signaling adaptors, and cytoskeletal components. In this review, we provide a concise but comprehensive overview of αIIbß3 outside-in signaling, focusing on the key players involved, and how they cooperate to orchestrate this critical aspect of platelet biology. We also discuss gaps in the current understanding of αIIbß3 outside-in signaling and highlight avenues for future investigation.

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbß3 Outside-in Signaling.

The class I PI3K family of lipid kinases plays an important role in integrin αIIbß3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbß3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbß3-expressing CHO cells blocked Rap1 activity and integrin αIIbß3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbß3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbß3-mediated outside-in signaling and cell spreading.

Hepatocyte mARC1 promotes fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)-siRNA to understand the in vivo impacts of MTARC1. Results: We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and in vivo GalNAc-siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD. Impact and implications: We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 for NAFLD.

PI3K inhibitors in thrombosis and cardiovascular disease.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate important intracellular signalling and vesicle trafficking events via the generation of 3-phosphoinositides. Comprising eight core isoforms across three classes, the PI3K family displays broad expression and function throughout mammalian tissues, and the (patho)physiological roles of these enzymes in the cardiovascular system present the PI3Ks as potential therapeutic targets in settings such as thrombosis, atherosclerosis and heart failure. This review will discuss the PI3K enzymes and their roles in cardiovascular physiology and disease, with a particular focus on platelet function and thrombosis. The current progress and future potential of targeting the PI3K enzymes for therapeutic benefit in cardiovascular disease will be considered, while the challenges of developing drugs against these master cellular regulators will be discussed.

Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity.

BACKGROUND AND PURPOSE: Rapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. EXPERIMENTAL APPROACH: The effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. KEY RESULTS: Rapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. CONCLUSIONS AND IMPLICATIONS: Rapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.

Identification of PtdIns(3,4)P2 effectors in human platelets using quantitative proteomics.

After decades in PtdIns(3,4,5)P3's shadow, PtdIns(3,4)P2 has now emerged as a bona fide regulator of important cellular events, including endocytosis and cell migration. New understanding of PtdIns(3,4)P2's cellular roles has been possible via novel approaches to observe and quantify cellular PtdIns(3,4)P2 dynamics, alongside methods to target the kinases and phosphatases governing phosphoinositide turnover. Despite this, the mechanisms by which PtdIns(3,4)P2 orchestrates its cellular roles remain more poorly understood, most notably because, to date, few PtdIns(3,4)P2 effectors have been identified. Here, we develop and apply an affinity-proteomics strategy to conduct a global screen for PtdIns(3,4)P2 interactors in human platelets; a primary cell type with striking PtdIns(3,4)P2 accumulation. Through an integrated approach, coupling affinity capture of PtdIns(3,4)P2-binding proteins to both label-free and isobaric tag-based quantitative proteomics, we identify a diverse PtdIns(3,4)P2 interactome. Included are long-established PtdIns(3,4)P2-binding proteins such as PLEKHA1, PLEKHA2, AKT and DAPP1, and a host of potentially novel effectors, including MTMR5, PNKD, RASA3 and GAB3. The PtdIns(3,4)P2 interactome shows an enrichment of pleckstrin homology (PH) domain-containing proteins, and through bioinformatics and array analyses we characterise the PH domain of MTMR5 and define its phosphoinositide selectivity. The interactome is also diverse in function, including several proteins known to support protein trafficking and cytoskeletal mobilisation. Such proteins have the ability to drive key platelet events, and to fulfil recently-defined roles for PtdIns(3,4)P2 in a wider range of cell types. Moreover, this study will serve as a valuable resource for the future characterisation of effector-driven PtdIns(3,4)P2 function.

Critical roles for the phosphatidylinositide 3-kinase isoforms p110ß and p110γ in thrombopoietin-mediated priming of platelet function.

Thrombopoietin (TPO) enhances platelet activation through activation of the tyrosine kinase; JAK2 and the lipid kinase phosphatidylinositide 3-kinase (PI3K). The aim of our study was to identify the PI3K isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism. We found that p110ß plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin αIIbß3 activation and α-granule secretion, (ii) synergistic enhancement of PAR-mediated activation of the small GTPase RAP1, a regulator of integrin activation and (iii) phosphorylation of the PI3K effector Akt. More importantly, the synergistic effect of TPO on phosphorylation of extracellular-regulated kinase (ERK1/2) and thromboxane (TxA2) synthesis was dependent on both p110ß and p110γ. p110ß inhibition/deletion, or inhibition of p110γ, resulted in a partial reduction, whereas inhibiting both p110ß and p110γ completely prevented the synergistic effect of TPO on ERK1/2 phosphorylation and TxA2 synthesis. The latter was ablated by inhibition of MEK, but not p38, confirming a role for ERK1/2 in regulating TPO-mediated increases in TxA2 synthesis. In conclusion, the synergistic effect of TPO on RAP1 and integrin activation is largely mediated by p110ß, whereas p110ß and p110γ contribute to the effect of TPO on ERK1/2 phosphorylation and TxA2 formation.

In-depth PtdIns(3,4,5)P3 signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function.

The class I phosphoinositide 3-kinase (PI3K) isoforms play important roles in platelet priming, activation, and stable thrombus formation. Class I PI3Ks predominantly regulate cell function through their catalytic product, the signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], which coordinates the localization and/or activity of a diverse range of binding proteins. Notably, the complete repertoire of these class I PI3K effectors in platelets remains unknown, limiting mechanistic understanding of class I PI3K-mediated control of platelet function. We measured robust agonist-driven PtdIns (3,4,5)P3 generation in human platelets by lipidomic mass spectrometry (MS), and then used affinity-capture coupled to high-resolution proteomic MS to identify the targets of PtdIns (3,4,5)P3 in these cells. We reveal for the first time a diverse platelet PtdIns(3,4,5)P3 interactome, including kinases, signaling adaptors, and regulators of small GTPases, many of which are previously uncharacterized in this cell type. Of these, we show dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1) to be regulated by Src-family kinases and PI3K, while platelets from DAPP1-deficient mice display enhanced thrombus formation on collagen in vitro. This was associated with enhanced platelet α/δ granule secretion and αIIbß3 integrin activation downstream of the collagen receptor glycoprotein VI. Thus, we present the first comprehensive analysis of the PtdIns(3,4,5)P3 signalosome of human platelets and identify DAPP1 as a novel negative regulator of platelet function. This work provides important new insights into how class I PI3Ks shape platelet function.

A new approach to measuring phosphoinositides in cells by mass spectrometry.

The phosphoinositide family of phospholipids, defined here as PtdIns, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, play pivotal roles in organising the location and activity of many different proteins acting on biological membranes, including those involved in vesicle and protein trafficking through the endolysosomal system and receptor signal transduction at the plasma membrane. Accurate measurement of the cellular levels of these lipids, particularly the more highly phosphorylated species, is hampered by their high polarity and low cellular concentrations. Recently, much progress has been made in using mass spectrometry to measure many different lipid classes in parallel, an approach generally referred to as 'lipidomics'. Unfortunately, the acidic nature of highly phosphorylated phosphoinositides makes them difficult to measure using these methods, because they yield low levels of useful ions; this is particularly the case with PtdIns(3,4,5)P3. We have solved some of these problems by methylating the phosphate groups of these lipids with TMS-diazomethane and describe a simple, integrated approach to measuring PtdIns, PtdInsP, PtdInsP2 and PtdInsP3 classes of lipids, in parallel with other phospholipid species, in cell and tissue extracts. This methodology is sensitive, accurate and robust, and also yields fatty-acyl compositions, suggesting it can be used to further our understanding of both the normal and pathophysiological roles of these important lipids.

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) is required to maintain physiological levels of PtdIns and PtdInsP(2) in the mouse.

We disrupted the gene encoding lysophosphatidylinositol-acyltransferase-1 (LPIAT1) in the mouse with the aim of understanding its role in determining cellular phosphoinositide content. LPIAT1(-/-) mice were born at lower than Mendelian ratios and exhibited a severe developmental brain defect. We compared the phospholipid content of livers and brains from LPIAT1(-/-) and LPIAT1(+/+) littermates by LC-ESI/MS. In accord with previous studies, the most abundant molecular species of each phosphoinositide class (PtdIns, PtdInsP, PtdInsP2 and PtdInsP3) possessed a C38∶4 complement of fatty-acyl esters (C18∶0 and C20∶4 are usually assigned to the sn-1 and sn-2 positions, respectively). LPIAT1(-/-) liver and brain contained relatively less of the C38∶4 species of PtdIns, PtdInsP and PtdInsP2 (dropping from 95-97% to 75-85% of the total species measured for each lipid class) and relatively more of the less abundant species (PtdInsP3 less abundant species were below our quantification levels). The increases in the less abundant PtdIns and PtdInsP2 species did not compensate for the loss in C38∶4 species, resulting in a 26-44% reduction in total PtdIns and PtdInsP2 levels in both brain and liver. LPIAT1(-/-) brain and liver also contained increased levels of C18∶0 lyso-PtdIns (300% and 525% respectively) indicating a defect in the reacylation of this molecule. LPIAT1(-/-) brain additionally contained significantly reduced C38∶4 PC and PE levels (by 47% and 55% respectively), possibly contributing to the phenotype in this organ. The levels of all other molecular species of PC, PE, PS and PA measured in the brain and liver were very similar between LPIAT1(-/-) and LPIAT1(+/+) samples. These results suggest LPIAT1 activity plays a non-redundant role in maintaining physiological levels of PtdIns within an active deacylation/reacylation cycle in mouse tissues. They also suggest that this pathway must act in concert with other, as yet unidentified, mechanisms to achieve the enrichment observed in C38∶4 molecular species of phosphoinositides.