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Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell–specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1–mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell–mediated maintenance of immune homeostasis.

Neutrophils are vital for inflammation and immune defense. Dependent on β2 integrins, spherical neutrophils spread on vascular endothelium after arrest, which is critical for their recruitment from circulation to resist high shear flow and to initiate intravascular crawling. Here, we use high-resolution quantitative dynamic footprinting microscopy to monitor neutrophil spreading on a substrate of recombinant ICAM-1 and P-selectin under flow. A homogenous binding assay using the conformation-reporter antibodies mAb24 (reporting high-affinity β2, H+) and KIM127 (reporting extended β2, E+) showed three conformations of activated β2 integrins. E−H+ β2 integrins increased before E+H− and E+H+ conformations at the beginning of neutrophil spreading. Integrin extension depended on Syk-mediated integrin outside-in signaling. The ring of E−H+ and E+H+, but not E+H− β2 integrins was fully formed during late neutrophil spreading just before migration. Using kindlin-3-GFP fusion proteins, a ring of kindlin-3 was observed before the ring of H+ integrins appeared. These findings show spatially coordinated integrin activation during spreading. The previously unrecognized E−H+ conformation is the pioneer integrin for neutrophil spreading and appears to be organized by kindlin-3.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact–mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell–specific loss of talin, a β integrin–binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4β1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway-dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell-induced thrombosis.

Background: Animal models of hemostasis play a crucial role in understanding hemostasis and developing novel therapeutic treatments. Hemostasis is a mechanical phenomenon and recent studies have demonstrated that biophysical parameters, such as platelet margination (Walton et al. Blood 2017), thrombus porosity (Welsh et al. Blood 2014), shear forces (Nesbitt et al. Nat Med 2009), compression forces (Ju et al. Nat Comm 2018), and single platelet forces, as measured by platelet contraction cytometry, developed by our laboratory, (Myers et al. Nat Mat. 2017) affect hemostatic processes and may be pathologically altered in disease states. However, little is known about the most basic biophysical interspecies differences between platelets of various animal hemostasis models, which could provide critical insight into interpreting data and results. Here, we report the results of our initial study on platelet biophysics in humans (n=5), mice (n=8), pigs (n=5), and dogs (n=6), which are commonly used for hemostasis models due to anatomical similarities to human vessels, organs, and cell function (Siller-Matula et al. Thromb & Haem 2008) (Jagadeeswaran et al. Circ Res 2015). We focus on understanding differences in platelet adhesion on collagen and fibrinogen, spreading area, singe cell contraction forces, and bulk contraction, which all may influence the initiation and stability of blood clots (Fig. 1). Our data demonstrates striking differences between each species, especially concerning adhesion, spreading area, and single platelet force. Surprisingly, measurements of volumetric bulk contraction, the most commonly used biophysical assay in hemostasis research, appear to be quite consistent between species. Methods: We performed a comprehensive study of platelet biophysics using adhesion assays, bulk contraction, and single platelet contractile forces on humans, mice, pigs, and dogs' platelets. For the adhesion assays, spreading area, and platelet contractile forces, platelets were gel-filtered as described previously (Myers et al. 2017). Cell Adhesion & Spreading Assay: Gel filtered platelets were diluted to 20M/mL, incubated on coverslips that had been incubated with type 1 collagen (100 µg/mL) or fibrinogen (100 µg/mL), allowed to adhere for 2 hours, counted, and measured. Platelet Contraction Cytometry: Polyacrylamide hydrogels with pairs of fibrinogen microdots are fabricated at physiologically relevant stiffnesses. Gel-filtered thrombin-activated platelets (1 U/mL) are then plated to attach, spread, and contract the microdots together. Individual platelet force is directly proportional to the microdot displacement measured with fluorescent imaging. Bulk Clot Contraction Assay: Platelets are washed as described previously (Lam et al. Nat Mat 2011) and then diluted to 200M/mL in a solution of fibrinogen (2 mg/mL), CaCl2 (5 mM), and thrombin (1 U/mL). Clots are left to form in cuvettes lined with gridded paper and measured every 30 mins for 2.5 hours. All assays were performed on at least 3 subjects from each species and in triplicate when possible. Results: We found both significant biophysical differences and similarities between platelets of different species (Fig. 2). Overall, murine platelets have the highest biophysical similarity to human platelets regarding adhesion, volumetric contraction, and single platelet contractile force despite substantial size differences. Porcine platelets exhibited enhanced adhesion on collagen with little adhesion to fibrinogen; while canine platelets showed the opposite. Such differences could have significant implications on the biophysical initiation of blood hemostasis and thrombosis, as well as the thrombogenicity tests of new materials. In bulk and single platelet contraction, we noted that similarities between most species except for canine platelets which produced forces nearly triple that of humans, the highest contractile forces of any platelets measured to date. As recent work has shown that forces are linked to clot porosity and therefore activation (Mirramezani et al. JTH 2018), such high forces could influence the clot hierarchical structure and subsequent lysis. Taken together, these results demonstrate how the biophysical properties of platelets differ across species and may be another reason why animal studies of hemostasis do not always translate to humans. Disclosures No relevant conflicts of interest to declare.

Extracellular antagonists of alpha 4 integrin are an effective therapy for several autoimmune and inflammatory diseases; however, these agents that directly block ligand binding may exhibit mechanism-based toxicities. Inhibition of alpha 4 integrin signaling by mutations of alpha 4 that block paxillin binding inhibits inflammation while limiting mechanism-based toxicities. Here, we test a pharmacological approach by identifying small molecules that inhibit the alpha 4 integrin-paxillin interaction. By screening a large (approximately 40,000-compound) chemical library, we identified a noncytotoxic inhibitor of this interaction that impaired integrin alpha 4-mediated but not alpha L beta 2-mediated Jurkat T cell migration. The identified compound had no effect on alpha 4-mediated migration in cells bearing the alpha 4(Y991A) mutation that disrupts the alpha 4-paxillin interaction, establishing the specificity of its action. Administration of this compound to mice led to impaired recruitment of mononuclear leukocytes to a site of inflammation in vivo, whereas an isomer that does not inhibit the alpha 4-paxillin interaction had no effect on alpha 4-mediated cell migration, cell spreading, or recruitment of leukocytes to an inflammatory site. Thus, a small molecule inhibitor that interferes with alpha 4 integrin signaling reduces alpha 4-mediated T cell migration in vivo, thus providing proof of principle for inhibition of alpha 4 integrin signaling as a target for the pharmacological reduction of inflammation.

SummaryIntegrins are heterodimeric adhesion receptors essential for metazoan life. In addition to mediating cell-extracellular matrix and cell-cell interactions, integrins are bona fide signalling receptors in that they transmit information in both directions across the plasma membrane. The affinity of integrins for extracellular ligands is regulated through a process termed integrin activation or “inside-out signalling”. On the other hand, ligand binding to integrins can induce the recruitment and activation of a number of enzymes and adaptors such as pp125FAK and Src family kinases, to initiate “outside-in signalling”. Intensive investigation into the mechanisms of integrin signalling has revealed many of the key players; amongst these, one of the most important is talin. Our understanding of how many of these molecules interact is now understood at the atomic level thanks to detailed structural studies. Indeed structural information and model cell systems have provided unique opportunities to dissect the molecular mechanisms of many aspects of integrin signalling. Recent studies have begun testing the biological significance of these mechanisms using in-vivo models, particular genetically modified mice. The generation and characterisation of in-vivo models to study integrin signalling has provided valuable information into the functional significance of integrin signalling in fundamental physiological processes as well as within the context of human disease. Here, I will review recent insights that have been gained into integrin signalling through the use of genetically modified mice focusing on integrin αIIbβ3 (GPIIb-IIIa) and the regulation of its function in haemostasis and thrombosis.

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin alphaIIbbeta3-mediated platelet aggregation and beta1 integrin-mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet beta1 and beta3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian alpha2beta1 and alphaIIbbeta3 integrins in vivo.

Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia–reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin’s capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia–reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin–mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin–mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia–reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable.

Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the ‘niche’. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvβ3 on HSCs. The resultant β3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvβ3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvβ3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvβ3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.

Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin β cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the β3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of αIIbβ3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to β3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of αIIbβ3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-β3 integrin binding affinity results in decelerated αIIbβ3 integrin activation and protection from arterial thrombosis without pathological bleeding.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-cell and cell-extracellular matrix interactions, have been shown to play an important role in facilitating cell contact-mediated suppression by Treg cells. Here we show that integrin activation plays an essential, previously unappreciated role in maintaining the stability of the Treg cell pool. Treg cell-specific loss of talin1, a β integrin-binding protein, or expression of talin1(L325R), a mutant that selectively abrogates integrin activation, resulted in dysregulation of Treg cell identity and lethal systemic autoimmunity. Moreover, the absence of sustained interactions between the integrin LFA-1 on Treg cells and its ligand ICAM-1 on dendritic cells reduced the expression of Foxp3 and caused Treg cells to adopt an effector CD4+ T cell-like phenotype. Taken together, these results reveal a critical role for tonic, integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell identity and stability.

In inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1–deficient neutrophils, whereas only arrest is defective in kindlin-3–deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.

Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin “inside-out” signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent “outside-in” signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.

alphaIIbbeta3 interaction with fibrinogen promotes Src-dependent platelet spreading in vitro. To determine the consequences of this outside-in signaling pathway in vivo, a "beta3(Delta760-762)" knockin mouse was generated that lacked the 3 C-terminal beta3 residues (arginine-glycine-threonine [RGT]) necessary for alphaIIbbeta3 interaction with c-Src, but retained beta3 residues necessary for talin-dependent fibrinogen binding. beta3(Delta760-762) mice were compared with wild-type beta3(+/+) littermates, beta3(+/-) heterozygotes, and knockin mice where beta3 RGT was replaced by beta1 C-terminal cysteine-glycine-lysine (EGK) to potentially enable signaling by Src kinases other than c-Src. Whereas beta3(+/+), beta3(+/-) and beta3/beta1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, beta3(Delta760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including beta3 (Tyr(747)). Unlike control mice, beta3(Delta760-762) mice were protected from carotid artery thrombosis after vessel injury with FeCl(3). Some beta3(Delta760-762) mice exhibited prolonged tail bleeding times; however, none demonstrated spontaneous bleeding, excess bleeding after surgery, fecal blood loss, or anemia. Fibrinogen binding to beta3(Delta760-762) platelets was normal in response to saturating concentrations of protease-activated receptor 4 or glycoprotein VI agonists, but responses to adenosine diphosphate were impaired. Thus, deletion of beta3 RGT disrupts c-Src-mediated alphaIIbbeta3 signaling and confers protection from arterial thrombosis. Consequently, targeting alphaIIbbeta3 signaling may represent a feasible antithrombotic strategy.

Adhesion of platelets to blood vessel walls is a shear stress dependent process that promotes arrest of bleeding and is mediated by the interaction of receptors expressed on platelets with various extracellular matrix (ECM) proteins that may become exposed upon vascular injury. Studies of dynamic platelet adhesion to ECM-coated substrates in conventional flow chambers require substantial fluid volumes and are difficult to perform with blood samples from a single laboratory mouse. Here we report dynamic platelet adhesion assays in two new microfluidic devices made of PDMS. Small cross-sections of the flow chambers in the devices reduce the blood volume requirements to

Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell injury signals trigger the macrophages P2X7 receptor and thereby induce the release both proinflammatory IL-1β and prothrombotic MP. Prothrombotic MP carry tissue factor (TF) and high content of phosphatidylserine (PS), and can induce thrombosis causing major clinical complications in patients. We previously identified the P2X7 receptor as a crucial component of thrombosis in mice, but the mechanistic details of macrophage MP release in this thrombo-inflammatory pathway remain incompletely understood. The the generation of these MP requires thiol-disulfide exchange-dependent activation of the inflammasome and is accompanied by the release of various soluble proteins into the extracellular space. We hypothesized that the released proteome presents regulators and structural components of the MP generation pathway and employed proteomics to unveil their identity. Amongst the most abundant proteins were γ-actin and actin cytoskeleton associated proteins, including PS-binding proteins annexin 1 and annexin 5. Cytoskeletal remodeling processes leading to formation of filopodia downstream of P2X7 receptor activation were crucial for the biogenesis of thrombo-inflammatory MP, since pharmacological inhibitors of inflammasome activation, cytoskeletal remodeling and the thioredoxin system attenuated both, filopodia formation and the release of highly procoagulant MP. Confocal microscopy demonstrated raft dependent translocation of TF onto filopodia that was prevented by the same inhibitory strategies. Surprisingly, phalloidin-staining of non-permeabilized macrophages revealed that F-actin is exposed to the cell surface decorating the base of filopodia. Positive phalloidin-staining of thrombo-inflammatory MP further demonstrated that F-actin remained associated with the MP surface. Strikingly, blocking surface actin by incubation with high concentration of phalloidin prevented the release of PS-rich MP, demonstrating that exposure of F-actin during filopodia formation is functionally linked to the biogenesis of thrombo-inflammatory MP. As the underlying common mechanism for thiol-disulfide exchange-dependent cell surface actin exposure and MP release, we showed that blockade of the cysteine protease caspase 1, which mediates processing and release of IL-1β downstream of inflammasome activation is also required for the release of thrombo-inflammatory MP. Although caspase 1-mediated activation of caplain was required for the release of filamin A localized TF to the cell cortex, calpain was not involved in the release of thrombo-inflammatory MP release. The N-terminus of γ-actin harbors a recognition and cleavage motif for caspase 1. Residual γ-actin released from caspase 1-blocked macrophages showed decreased electrophoretic mobility, indicating prior cleavage of actin that becomes exposed on the cell surface. We show here that the proteome released during thrombo-inflammatory activation of macrophages includes critical players in the biogenesis of MP and may provide diagnostic fingerprints in complex biological samples. Our data demonstrate an entirely unexpected role for caspase 1 and surface exposure of polymerized actin in the severing of prothrombotic MP from filopodia and thus position this protease upstream of both IL-1β processing and thrombo-inflammatory MP in cardiovascular diseases. Disclosures No relevant conflicts of interest to declare.

Introduction: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiological conditions. However, measuring hemostasis in mice is quite variable and current methods do not quantify platelet adhesion or fibrin formation at the site of injury. Methods: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation, and time to hemostatic plug formation in real-time. Repeated vessel injuries of ~50-100µm in diameter were induced using laser ablation technology in the saphenous vein of mice. Results: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein (GP)Ibα (IL4R/GPIb-tg) or talin-1 (talin1f/f PF4-Cre), important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice defective in the extrinsic (tissue factor (TF)low) or the intrinsic (FIX-/-) coagulation pathways, even though platelet adhesion was significantly reduced. Interestingly, fibrin accumulation was markedly increased in lesions of talin1f/f PF4-Cre, IL4R/GPIb-tg and FIX-/- mice, while it was decreased in TFlow mice. These findings suggest that prolonged plasma exposure to TF leads to increased thrombin and fibrin generation in the surrounding tissue. A partial reduction of platelet adhesiveness using clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TFlow mice. Conclusions: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. This new model has several defining characteristics, including the use of intravital fluorescence microscopy to monitor the hemostatic process, the novel use of laser ablation technology to generate vascular lesions with a defined diameter, the ability to repeatedly disrupt the hemostatic process at the same site of injury, and the possibility to perform multiple injuries along the exposed saphenous vein. Based on its sensitivity towards platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool to study the efficacy and safety of antiplatelet agents. Disclosures No relevant conflicts of interest to declare.

Abstract 2218 The purinergic P2X7 receptor contributes to thrombosis by promoting tissue factor (TF) activation and the release of prothrombotic microparticles (MP). On primed macrophages, P2X7 stimulation induces the procoagulant activity of cell surface TF and the release of MP that carry TF, integrin β1 and protein disulfide isomerase (PDI). The generation of TF+ MP is dependent on extracellular thiol-disulfide exchange, but intermediates leading to MP generation downstream of P2X7 signaling are incompletely defined. Tracking of cell surface TF by confocal microscopy shows that constitutive internalization of TF is prevented by P2X7 activation. In non-stimulated cells, inhibition of dynamin-dependent endocytosis retains TF on the cell surface, rapidly upregulates TF activity, and releases procoagulant MP carrying TF and integrin β1. Integrin recycling is dependent on the small GTPase ARF6 that is found incorporated into MP released when internalization is blocked. In contrast, activation of P2X7 releases ARF6 into the MP-depleted supernatant rather than associated with MP. Decreased ARF6 GTP loading in P2X7-stimulated cells and additional data in integrin α4 S988A mutant mice with reduced ARF6 activity support the conclusion that P2X7 interrupts constitutive ARF6 recycling to increase cell surface availability of TF-integrin complexes for incorporation into MP. Confocal imaging shows that P2X7 stimulation promotes filopodia formation and the peripheral transport of TF to the tips of filipodia. Proteomics identifies γ-actin as a thiol-regulated MP protein that is released by P2X7 signaling. We show that specific inhibition of thioredoxin reductase attenuates P2X7-induced actin remodeling and filopodia formation, the generation of highly procoagulant MP carrying γ-actin and PDI, and the increased availability of extracellular thioredoxin and free thiols. Importantly, inhibition of thioredoxin reductase also prevented the processing and release of the pro-inflammatory cytokine IL1β. Thus, the thioredoxin reductase system is a crucial intermediate downstream of P2X7 activation that may couple coagulation and inflammation in cardiovascular diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 862 Hematopoietic stem cells (HSCs) govern hematopoiesis by giving rise to lymphoid, myeloid, and erythroid cells throughout adult life. HSCs reside in a specialized microenvironment termed “niche,” where their functions are regulated by several factors such as cytokines and extracellular matrix (ECM). Nov (CCN3), a member of the CCN family, is a well-known soluble factor that regulates several biological events by binding to integrin receptors, growth factors, and ECM. Recently, Nov has been reported to function as a positive regulator of HSCs, as evidenced by enhanced HSC activity in response to enforced Nov expression in HSCs (Gupta et al., Science, 2007). Furthermore, Nov has been identified as a target gene for Hoxb4, a transcription factor that governs the self-renewal capacity of HSCs (Ohshima et al., Blood, 2011). In addition, Nov expression in HSCs is upregulated by IL-3 via STAT5 activation (Kimura et al., J. Biol. Chem., 2010). Thus, models for the regulation of Nov expression in HSCs have been proposed; however, the mechanisms underlying the regulation of HSC functions by Nov remain unclear. Here, we present a novel mechanism for the enhancement of HSC activity by Nov. We first suspected that thrombopoietin (TPO), an essential cytokine for HSC maintenance, may promote Nov expression, given that TPO not only induces STAT5 activation in HSCs (Seita et al., PNAS, 2007) but also stimulates Hoxb4 expression (Kirito et al., Blood, 2003). Therefore, we examined the expression of Nov by real-time quantitative RT-PCR in CD150+ CD34− c-kit+ Sca-1+ lineage− (CD150+ CD34− KSL) HSCs that were treated with TPO. Similar to IL-3, TPO significantly enhanced Nov expression, compared to that in fresh unstimulated HSCs (p < 0.05). In contrast, stem cell factor (SCF), a critical cytokine for the maintenance of HSC functions, completely lost Nov expression. This strong link between Nov and TPO in HSCs suggests that TPO may play a key role in the regulation of HSCs by Nov. Therefore, we examined the long-term repopulating (LTR) activity of HSCs in transplantation assays following treatment with exogenous Nov in the presence of TPO or SCF. Interestingly, TPO stimulation supported the Nov-induced enhancement of HSC LTR activity (p < 0.05), whereas this positive effect was completely abolished in the presence of SCF. Furthermore, treatment with TPO, but not with SCF, increased the capture of Nov by HSCs, as measured by flow cytometry analyses using Alexa647-labeled Nov (p < 0.001), which strongly suggests that the positive effect of exogenous Nov on the LTR activity of HSCs is specifically dependent on TPO. More importantly, this TPO-mediated promotion of Nov binding to HSCs was blocked by antibodies against integrin αv or β3, indicating that integrin avβ3 is the primary receptor for Nov on HSCs. Previously, we demonstrated that outside-in signaling via phosphorylated Tyr747 of integrin 3 (β3PY747) is indispensable for the TPO-dependent maintenance of mouse HSCs, which requires the activation (conformational change for high affinity ligand binding) of αvβ3 integrin via TPO-induced inside-out signaling (Umemoto et al., Blood ASH abstract, 2009). Given our previous data, the results from the present study suggest that Nov regulates the LTR activity of HSCs through outside-in signaling especially via β3PY747, following its ligation to integrin αvβ3 that has been activated by TPO-induced inside-out signaling. Finally, we confirmed our hypothesis by using β3 integrin mutant mice that harbor an alanine substitution of tyrosine 747 in the cytoplasmic tail of β3 integrin (Y747A), which impairs integrin inside-out and outside-in signaling. Transplantation assays using Y747A-expressing HSCs revealed that inhibition of bidirectional integrin signaling by the Y747A mutation completely abolished the positive TPO-dependent effect of Nov, even when αvβ3 integrin activation was rescued by Mn2+, an external inducer of integrin activation that acts independently of inside-out signaling. Taken together, our findings demonstrate that Nov positively regulates HSC activity through outside-in signaling via β3PY747, following its TPO-dependent ligation to integrin αvβ3. Thus, we present a novel mechanistic link between Nov, β3 integrin, and TPO in HSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 383 Hematopoietic stem cells (HSCs) reside in a specialized microenvironment, bone marrow (BM) ‘niche' for maintaining self-renewal and multi-lineage differentiation capacities through the life. We recently reported that murine HSCs (CD34- c-Kit+ Sca-1+Lin-: CD34-KSL) but not CD34+KSL hematopoietic progenitors highly express CD61 (integrin β3), correlating well with long-term repopulating activity (LTRA) of HSCs (Umemoto et al., J. Immunol, 2006). However, the roles of CD61 on HSC regulation and underlying molecular mechanisms in the BM niche are to date unknown. We herein demonstrate a novel role of CD61 using CD61-deficient (CD61-/-) and mutant CD61 mice that exhibit 747 tyrosine residue substituted with alanine in the cytoplasmic tail of β3 integrin subunit (CD61Y747A KI) whereby the integrin inside-out and outside-in signaling are impaired. We found that the CD61-deficiency influences the sensitivity to 5-fluorouracil (5-FU) administration (0.25 mg/g i.v. one shot), increasing the mortality of CD61-/- mice (CD61-/- mice died within 14 days, n=20, vs. WT mice survive over 90 days, n=20, P Disclosures: No relevant conflicts of interest to declare.

Platelet activation by agonists stimulates high affinity binding of adhesive ligands, such as fibrinogen, to integrin αIIbβ3. In turn, ligand binding initiates Src kinase-dependent outside-in signaling, platelet spreading on sub-endothelial matrices, and platelet aggregation. Short-term integrin blockade by parenteral αIIbβ3 antagonists is effectively anti-thrombotic in certain clinical situations, but long-term blockade by oral αIIbβ3 antagonists has been problematic. As an alternative approach, we evaluated the feasibility of disrupting outside-in αIIbβ3 signaling in vivo. A “β3(Δ760-762)” knock-in mouse was generated that lacked the three C-terminal β3 amino acid residues (RGT) necessary for αIIbβ3-dependent c-Src signaling, but retained β3 residues necessary for agonist- and talin-dependent fibrinogen binding. β3(Δ760-762) mice were compared with wild-type β3+/+ littermates, β3+/− heterozygotes, and “β3/β1(EGK)” knock-in mice where β3 RGT was replaced by three C-terminal residues in β1 (EGK) to potentially enable outside-in integrin signaling by certain Src kinases other than c-Src. Whereas β3+/+, β3+/− and β3/β1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, β3(Δ760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including the β3 cytoplasmic domain itself (Tyr-747). Furthermore, unlike the three groups of control mice, all of the β3(Δ760-762) mice tested were protected from carotid artery occlusive thrombosis following vessel injury with FeCl3. The median tail bleeding time for the β3(Δ760-762) mice was approximately two-fold longer than that of the control mice. However, β3(Δ760-762) mice did not exhibit spontaneous bleeding, excess bleeding after surgical exposure of the carotid artery, fecal blood loss or anemia. Fibrinogen binding to β3(Δ760-762) platelets was normal in response to saturating concentrations of agonists to the PAR4 thrombin receptor or the GP VI collagen receptor; however, responses to ADP were impaired. These studies establish that deletion of β3 RGT disrupts outside-in αIIbβ3 signaling and confers protection from arterial thrombosis in vivo. Currently available anti-platelet drugs either inhibit agonist-induced activation of αIIbβ3 or block fibrinogen binding to the integrin. The studies reported here suggest that targeting outside-in αIIbβ3 signaling may represent an alternative anti-thrombotic strategy.

Interactions of platelets and their adhesion receptors with extracellular matrices are essential for hemostasis. Platelets experience different shear rates as they circulate through the vasculature. Conventional studies of platelets in shear flow are performed in simple flow chambers with relatively large volumes of cells and reagents, limiting testing when these are in short supply. Microfluidics technology should enable the concurrent study of multiple small volume samples across a wide range of shear rates, thereby allowing trends to emerge that might be difficult to detect otherwise. To achieve this goal, we fabricated PDMS microfluidic devices that permit testing of dynamic platelet adhesion over a 100-fold span of shear rates from a single 200μl blood sample. Alternate device design permits synchronous monitoring of platelet adhesion from two genetically distinct blood samples or treatment groups. We have used this technology to gain new insights into integrin αIIbβ3 function in mepacrine-labeled platelets under shear flow. In whole blood, the adhesion of wild-type mouse platelets to a fibrinogen-coated substrate was shear rate-dependent, similarly to human platelets. In contrast, adhesion of αIIbβ3-deficient (β3−/−) platelets was virtually absent above a shear rate of 100 s−1. To distinguish between requirements for the presence of an intact extracellular αIIbβ3 ligand binding domain versus an intact integrin activation process, we used mice with a Y/A mutation at residue 747 in the β3 cytoplasmic tail (β3Y747A), that selectively blocks talin interaction with β3, agonist-induced αIIbβ3 activation, and platelet thrombus formation in vivo. When compared to wild-type and β3−/− platelets, a normal αIIbβ3 extracellular domain on β3Y747A platelets partially rescued dynamic adhesion to fibrinogen by 50–80% at ≤130 s−1, but by only 25% at 250 s−1. Treatment of wild-type platelets with PGE1 to inhibit platelet activation similarly reduced adhesion to fibrinogen at higher shear rates. On fibrillar type I collagen, wild-type platelets formed an initial monolayer and progressively larger thrombi over time. In addition, platelets supported rolling and firm adhesion of granulocytes in a manner dependent on shear rate, platelet P-selectin and granulocyte PSGL1. In contrast, no platelet thrombus growth on collagen was observed with αIIbβ3-deficient or β3Y747A platelets, or with wild-type platelets treated with PGE1. Furthermore, even the initial adhesion of αIIbβ3-deficient and β3Y747A platelets to collagen rapidly declined at increasing shear rates (120%, 30% and 7% of wild-type platelets at 70, 1000 and 4000 s−1, respectively (p < 0.01)). Taken together, these studies establish that microfluidics provides an efficient and high-throughput platform to study mechanisms of dynamic platelet adhesion, activation and thrombus formation on extracellular matrices. Furthermore, they demonstrate a role for talin-dependent αIIbβ3 activation in all of these processes. This platform will be particularly useful under conditions where blood sample volumes or reagents are limiting, such as in neonates, and genetically-modified model organisms.