Integrin Activation Research Articles

Accelerated Platelet Clearance in Mice with Constitutive Store-Operated Calcium Entry Is Independent of Caldag-GEFI/Rap1 Activation

Calcium (Ca2+) is critical for numerous functions of platelets including integrin activation, granule secretion and phosphatidylserine exposure. Circulating platelets maintain low cytosolic Ca2+ concentrations which rapidly rise upon cellular activation. Ca2+ release from intracellular stores triggers influx of extracellular Ca2+, a process known as store-operated Ca2+ entry (SOCE). Critical for SOCE is Stromal interaction molecule 1 (Stim1), a protein expressed in the dense tubular system membrane. Loss of Stim1 in platelets partially impairs platelet activation and procoagulant function. On the other hand, gain-of-function mutations in Stim1 are associated with thrombocytopenia and bleeding. In platelets, Ca2+ also activates CalDAG-GEFI and in turn Rap1 GTPase, the principal pathway mediating integrin activation. We recently demonstrated that unrestricted activation of Rap1 due to loss of the Rap1 GAP Rasa3 causes severe thrombocytopenia due to CalDAG-GEFI/integrin-dependent platelet clearance. To investigate whether CalDAG-GEFI activation mediates thrombocytopenia seen with constitutive Stim1 activation, we crossed Stim1 gain-of-function mice (Stim1Sax/+) with CalDAG-GEFI-/- mice to obtain Stim1Sax/+ x CalDAG-GEFI+/- or -/-mice. We observed no improvement in platelet count or platelet lifespan in Stim1Sax/+ mice with 1 or 0 copies of CalDAG-GEFI. While Rasa3 mutant platelets are predominantly cleared in the spleen, Stim1Sax/+ platelets accumulated to a greater extent in the lung. These results suggest that reduced platelet survival due to constitutive SOCE is not the result of increased CalDAG-GEFI/Rap1 signaling in circulating platelets.

Loss of the Nuclear Receptor Pparγ Primes T Effector (TEFF) Differentiation of Antigen-Specific CD8+ T Cells

Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to the nuclear hormone superfamily, with three distinct types identified: PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). PPARs possess a critical role in the regulation of lipid metabolism, and thus dictate the differentiation and fate of immune cells. PPARγ is a lipid-activated transcription factor mainly known to mediate anti-inflammatory effects on both innate and adaptive immune system. Synthetic ligands for activating PPARγ ameliorate disease outcomes in various models of inflammation and autoimmunity. The role of PPARγ in the immune system has been mainly studied in macrophages, where it is known to mediate M2 polarization. In the present study we investigated how PPARγ affects the differentiation and function of antigen-specific effector (TEFF) and memory (TM) CD8+ T cells using OTI TCR transgenic mice and OTI mice with T cell-specific PPARγ deletion (OTI/PPARγf/f-CD4-cre; thereafter named OTI/PPARγ-KO). Antigen-specific TEFF were generated in vitro by stimulation of OTI and OTI/PPARγ-KO T cells with Ova257-264 followed by culture with IL-2, whereas TM were generated by stimulation with Ova257-264 followed by culture with IL-15. Under both TEFF and TM conditions in vitro, antigen-specific stimulation of OTI/PPARγ-KO cells resulted in significantly greater proliferation and expansion relative to OTI control. Additionally, OTI/PPARγ-KO TM cells cultured with IL-15 co-expressed significantly higher CD44+/CD62L+ fractions indicating a greater expansion of central memory cells. Consistent with these, metabolomics analysis showed that under both culture conditions, PPARγ-deficient OTI cells had increased levels of glycolysis intermediates but also TCA metabolites consistent with a program of metabolically active cells with high levels of glycolysis and OXPHOS. RNA-sequencing studies showed that upon TEFF differentiation in vitro, OTI/PPARγ-KO T cells displayed a differential gene expression program compared to OTI controls. Pathway enrichment analysis revealed that PPARγ-deficient OTI cells were significantly enriched for genes related to T cell activation and proliferation, integrin activation, cytokine production, ribosomal function and biogenesis, RNA processing and protein translation. During antigen-specific stimulation in vivo performed after adoptive transfer of OTI/PPARγ-KO and OTI cells into syngeneic recipients followed by immunization with Ova-expressing Listeria monocytogenes (Lm-OVA), PPARγ deletion resulted in a rapid and enhanced TEFF cell activation and expansion as determined by the higher mitotic index, higher enrichment of Dextramer-positive antigen-specific T cells, and the higher expression of activation markers including KLRG1, CD44 and CD69, but also CTLA-4, PD-1, Lag-3 and Tim-3. These cells also developed higher numbers of TNF-α and IFN-γ-producing T cells, as well as ROR-γt expressing T cells consistent with Tc17 differentiation. In vivo stimulated antigen-specific PPARγ-deficient and control OTI T cells showed no numerical differences in TM differentiation or expansion. However, upon rechallenge in vitro, PPARγ-deficient TM cells exhibited improved capacity for TEFF differentiation with higher release of inflammatory cytokines and expression of CD69, PD-1 and CTLA-4. Our findings uncover a new role of PPARγ ablation in programming CD8+ T responses by regulating metabolism-mediated cues, which imprint T cell differentiation and long-term effector capacity.

Identification of Receptors and Signaling Pathways Mediating the Platelet Contribution to Clotting Parameters in Thromboelastography (TEG)

Viscoelastic testing is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Clot formation and contraction in thromboelastography (TEG) are assumed to be driven by thrombin through its two main actions, the conversion of fibrinogen to fibrin and activation of platelets through protease activated receptors (PARs). Platelet stimulation leads to inside-out activation of αIIbβ3 integrin, a process regulated by the small GTPase Rap1, and fibrin binding to activated αIIbβ3 triggers outside-in signaling and platelet contraction. However, studies have shown no impact of PAR inhibitors on viscoelastic clotting parameters, and the impact of defects in Rap1 signaling on TEG have not been determined. Furthermore, the role of novel ligand-receptor interactions such as fibrin-GPVI on TEG parameters have not been studied. Here we investigated the specific receptors and signaling pathways required for platelet function in standard (kaolin-triggered) TEG using genetic or pharmacological inhibition of platelet proteins in murine and human blood samples. We confirmed the requirement of platelets and αIIbβ3 integrin for mouse whole blood clot formation/contraction in TEG. Loss of the integrin adaptor Talin1 in mouse platelets impaired clotting parameters, but to a lesser extent than αIIbβ3 blockade. We observed no changes in clotting parameters using blood from mice lacking Rap1b, or with combined deficiency of CalDAG-GEFI and P2Y12, the two main pathways mediating Rap1 activation. Deficiency of PAR4, which fully mediates the platelet response to thrombin in mice, only partially reduced the speed of clot formation (α-angle) with no impact on maximal clot strength (maximal amplitude, MA). However, TEG parameters similar to samples lacking platelets were observed for blood from PAR4-deficient mice treated with inhibitors of the GPVI receptor or a key downstream signaling protein Syk. These results were reproduced in human blood samples, where inhibition of both PAR1 and PAR4 combined with Syk inhibition robustly reduced α-angle and MA. In summary, our results suggest that global hemostasis assays such as TEG, which are driven by high concentrations of thrombin, are not sensitive to platelet signaling pathways critical for integrin inside-out activation. Furthermore, we provide evidence that platelet-mediated clot contraction in TEG is dependent on both the thrombin-PAR interaction and GPVI, likely interacting with fibrin. Our findings suggest that current interpretation of platelet function in TEG may not reflect platelet function in vivo, and that novel interactions need to be integrated into clinical assays.

Deletion of 12-Lipoxygenase Improves Platelet Function after Storage and Transfusion in Thrombocytopenic Mice

Introduction Human platelets are stored for up to 7 days for transfusion. During storage, platelets undergo numerous detrimental functional changes. Results from two recent randomized clinical trials challenge the hemostatic efficacy of stored platelets for transfusion. In a recent study in healthy humans, increased 12-HETE levels were associated with reduced platelet performance in vivo. In the current study, we sought to understand how genetic deletion of 12-LOX (the enzyme that generates 12-HETE) affects platelets during storage, before, and after transfusion in vitro and in mouse models. Methods and Results: We obtained platelets from wild-type (WT) and 12-LOX-/- mice and performed storage studies for 24 and 48 hours. Using LC-MS/MS-MRM, we showed that ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) increased significantly in stored platelets from 12-LOX-/- mice, while oxylipins were significantly lower in 12-LOX-/- platelets than in WT platelets. The ω-6/ω-3 PUFA ratio decreased significantly in stored 12-LOX-/- compared to stored WT platelets, highlighting a pronounced increase in ω-3 PUFAs. The circulation time of stored 12-LOX-/- platelets did not differ from stored WT platelets. Baseline, pre-transfusion αIIbβ3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/- platelets than in WT platelets. Surprisingly, once stored platelets were transfused, we observed significantly increased baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. To test whether this translates into better in vivo function, we exposed 24-hour stored platelets to an in vivo model of thrombosis and hemostasis. In brief, hIL4R-TG mice express the receptor for human interleukin 4 instead of GPIbα on the platelet surface. hIL4R-TG mice were rendered thrombocytopenic with an anti-human IL4R antibody. We transfused 24-hour stored WT, or 12-LOX-/- platelets, to thrombocytopenic hIL4R-TG mice, injured the carotid artery by FeCl3, and recorded the arterial blood flow. Indeed, stored 12-LOX-/- platelets had better in vivo function, as evidenced by more frequent and significantly faster vessel occlusions with stored and transfused 12-LOX-/- platelets than transfusion of stored WT platelets. Importantly, stored and transfused 12-LOX-/- occluded vessels in a similar time as normocytic WT mice. Untransfused, thrombocytopenic hIL4R-TG mice and untransfused, normocytic hIL4R-TG mice did not show vessel occlusion. Conclusion Deleting 12-LOX improves the post-transfusion function of stored murine platelets. Pharmacologic inhibition of 12-LOX or dietary alterations of ω-3 and ω-6 PUFAs could significantly enhance human platelet quality and function after storage. Future studies must determine the feasibility and safety of 12-LOX inhibition in stored and transfused human platelets.

Actin Bundling Protein L-Plastin Regulates Megakaryocyte Membrane Rigidity and Platelet Spreading

An adequate number of normally functioning platelets is required for normal hemostasis, while thrombocytopenia and platelet dysfunction are common causes of pathologic bleeding. Platelets are released into the blood from proplatelets (PPs) that extend from megakaryocyte (MK) intracellular invaginated membrane system (IMS) that is continuous with the plasma membrane. Tubulin provides the motor for PP extension, and actin reorganization is believed to be critical for MK migration and PP branching. However, the role for actin in PP formation (PPF) is less well understood. The initiation of PPF requires podosomes - actin-based structures with an F-actin core surrounded by a ring of adhesion molecules (e.g., integrins) and many actin-binding proteins. We have been intrigued by how podosomes form and how the IMS is able to penetrate the dense cortical actin cytoskeleton to form PPs. We recently showed that human MKs and platelets express a novel actin bundling protein, L-plastin. L-plastin levels were inversely correlated with platelet number, and its expression was down-regulated during MK maturation. L-plastin knockdown in cultured human CD34+ derived MKs caused increased PP, IMS, and podosome formation. These data indicate L-plastin is a negative regulator of MK platelet production. To assess the in vivo effects of L-plastin, we have investigated platelet production and function in vivo using L-plastin knockout (KO) mice (Lcp1-/-). Compared to wildtype (WT) littermate controls, Lcp1 KO mice showed both significantly increased platelet counts and increased PPF from cultured murine bone marrow MKs. Elegant work in both HUVECs and epithelial cells has shown high membrane proximal F-actin (MPA) prohibits membrane protrusions, and lowering MPA density is required to sensitize areas for membrane protrusion (Bisaria, Science 2020). We hypothesize that normal MK levels of L-plastin inhibits PPF in less mature MKs by F-actin bundling, which raises the MPA. Conversely, lower L-plastin levels result in lower MPA and enabling PP protrusion. Enhanced L-plastin-mediated F-actin bundling is expected to increase membrane stiffness, so we measured MK membrane stiffness at single cell level using a state-of-the-art Biomembrane Force Probe (BFP). MKs from KO mice showed 68% reduction in membrane stiffness as compared to WT controls (p<0.0001) (Fig 1). Ultrastructure of MKs also revealed increased IMS in Lcp1-/- MKs. Preliminary data suggests that L-plastin deficiency also associated with increased MK podosome formation in vitro, which is consistent with our findings in human CD34+ derived MKs. Although L-plastin levels decrease during terminal MK differentiation, they persist at moderate levels in platelets. Platelet integrin activation, granule release and clot retraction also depend on actin reorganization, but L-plastin function in platelets has not been studied. We observed no difference between WT and Lcp1 KO platelets for measures of inside-out integrin signaling (activation-induced JON/A binding or P-selectin expression). However, when plated on fibrinogen and stimulated with thrombin, platelets from KO mice showed significantly increased spreading compared to WT mice (Fig 2). A similar trend was observed for platelet clot retraction. Taken together these results show that L-plastin regulates MK membrane stiffness, which likely inhibits PPF. L-plastin also appears to participate in platelet outside-in, but not inside-out, integrin signaling. On-going studies are determining whether integrin beta3 is the molecular link between these two findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Platelet ERO1α Is Crucial for Ca2+ Mobilization and Platelet Activation in Atherothrombosis

Endoplasmic reticulum oxidoreductase 1α (ERO1α) is a key oxidase of protein disulfide isomerase (PDI) during protein folding in the ER. Platelet ERO1α was reported to interact with PDI and αIIbβ3 integrin and regulate platelet function. However, the regulatory mechanism and pathophysiological role remain unknown. In the present study, we demonstrate that platelet ERO1α orchestrates intracellular Ca2+ signaling during platelet activation and contributes to thrombogenesis. Intravital microscopy using megakaryocyte-specific Ero1α conditional knockout (CKO) and KO mice reveals that platelet ERO1α is crucial for platelet thrombus formation in mouse models of laser-induced cremaster arteriolar thrombosis and FeCl3-induced carotid arterial thrombosis. Deletion of Ero1α does not affect bleeding times and blood loss at the site of tail amputation, implying that ERO1α is not required for hemostasis. Studies using Ero1α-null platelets, novel function-blocking antibodies, and small-molecule inhibitors reveal that intracellular, but not extracellular, ERO1α promotes P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by multiple agonists, such as thrombin and collagen-related peptide. Importantly, the regulatory function of ERO1α is independent of PDI activity. Mass spectrometry and immunoprecipitation assays identify stromal interaction molecule 1 (STIM1) and sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) as ERO1α binding partners. We have found that ERO1α-STIM1 binding is enhanced during platelet activation, whereas ERO1α-SERCA2 binding is reduced. Intriguingly, platelet ERO1α interacts with STIM1 through Cys49 and Cys56 residues and promotes its function by oxidizing the Cys49-Cys56 disulfide bond, contributing to Ca2+ store content and enhancing Ca2+ mobilization during platelet activation. Using a high throughput screen, we identify a small-molecule ERO1α inhibitor, B20, which inhibits ERO1α activity with an IC50 value of 10.0 μM but does not affect the activities of H2O2 and PDI. Treatment of platelets with B20 recapitulates the functional defects as seen in Ero1α-null platelets. Intravenous injection of B20 and eptifibatide, an αIIbβ3 integrin antagonist, into WT mice significantly inhibits platelet thrombus formation following laser- or FeCl3-induced vascular injury. While treatment of mice with eptifibatide markedly prolongs tail bleeding times and causes blood loss, B20 does not impair hemostatic function. Furthermore, treatment with B20 reduces the infarct volume in ischemic stroke. These results indicate that platelet ERO1α is a novel regulator of Ca2+ signaling and promotes platelet activation and aggregation in a PDI-independent manner, contributing to the pathogenesis of arterial thrombosis and thromboinflammation. Targeting ERO1α may be an attractive therapeutic strategy for treating patients with thrombotic disease.

Spatiotemporal characteristics of P-selectin-induced β2 integrin activation of human neutrophils under flow.

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that β2-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1min in an extracellular Ca2+ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.

β1 integrin monoclonal antibody treatment ameliorates cerebral cavernous malformations.

β1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active β1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of β1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting β1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.

Abstract 14919: EN-RAGE is Associated With Improved Outcomes in Hospitalized COVID-19 Patients

Background: Inflammation has been implicated in the pathogenesis, severity, and outcomes of COVID-19. Extracellular newly identified receptor for advanced glycation end-product (EN-RAGE), a soluble EF-hand calcium-binding protein, causes integrin activation, activates inflammation, and upregulates the synthesis of vascular cell adhesion molecule-1 and intracellular adhesion molecule-1. While it is positively associated with atherosclerosis, high EN-RAGE levels have been shown to predict remission of auto-immune diseases. The role of EN-RAGE as a biomarker in COVID-19 is unknown. Methods: Serum EN-RAGE levels were measured from blood samples of 146 COVID-19 in-patients using Myriad® Cytokine MAP and compared between the subgroups based on race and outcomes. T-test, Chi-square, and multivariable regression analysis were performed to identify the correlation of EN-RAGE with race and outcomes. Results: Of 146 patients (mean age=59 years), 60% were female, 35% were White, and 60% were African Americans. 21 of the 146 (14%) patients died. Mortality was higher in Whites compared to African Americans (21% vs 10%) consistent with CDC data showing higher mortality in Whites compared to non-Whites. Surprisingly, African Americans had significantly higher EN-RAGE levels compared to Whites. (574.6 vs 360.9 respectively, p=0.002). Congruently, EN-RAGE levels were significantly higher in the patients who survived compared to the patients who died. (514.82 vs 336.29, p=0.039) suggesting that increased serum EN-RAGE levels may be associated with improved outcomes in hospitalized COVID-19 patients. Conclusion: Our study reveals a negative association of EN-RAGE levels with in-patient mortality associated with COVID-19 and could possibly be used as a biomarker of survival in hospitalized COVID-19 patients. EN-RAGE levels could partly explain mortality differences based on race. Further studies are required to confirm this association.

Grim-19 plays a key role in mitochondrial steroidogenic acute regulatory protein stability and ligand-binding properties in Leydig cells

Grim-19 (gene associated with retinoid-IFN-induced mortality 19), the essential component of complex I of mitochondrial respiratory chain, functions as a noncanonical tumor suppressor by controlling apoptosis and energy metabolism. However, additional biological actions of Grim-19 have been recently suggested in male reproduction. We investigated here the expression and functional role of Grim-19 in murine testis. Testicular Grim-19 expression was detected from mouse puberty and increased progressively thereafter, and GRIM-19 protein was observed to be expressed exclusively in interstitial Leydig cells (LCs), with a prominent mitochondrial localization. Invivo lentiviral vector-mediated knockdown of Grim-19 resulted in a significant decrease in testosterone production and triggered aberrant oxidative stress in testis, thus impairing male fertility by inducing germ cell apoptosis and oligozoospermia. The control of testicular steroidogenesis by GRIM-19 was validated using the invivo knockdown model with isolated primary LCs and invitro experiments with MA-10 mouse Leydig tumor cells. Mechanistically, we suggest that the negative regulation exerted by GRIM-19 deficiency-induced oxidative stress on steroidogenesis may be the result of two phenomena: a direct effect through inhibition of phosphorylation of steroidogenic acute regulatory protein (StAR) and subsequent impediment to StAR localization in mitochondria and an indirect pathway that is to facilitate the inhibiting role exerted by the extracellular matrix on the steroidogenic capacity of LCs via promotion of integrin activation. Altogether, our observations suggest that Grim-19 plays a potent role in testicular steroidogenesis and that its alterations may contribute to testosterone deficiency-related disorders linked to metabolic stress and male infertility.

Integrin Activation as a Novel Therapeutic Strategy for Podocytopathies

Integrin Activation as a Novel Therapeutic Strategy for Podocytopathies

Tension Enhances the Binding Affinity of β1 Integrin by Clamping Talin Tightly: An Insight from Steered Molecular Dynamics Simulations.

Integrin activation is a predominant step for cell-cell and cell-ECM interactions. Talin and Kindlin are mechanosensitive adaptor proteins that bind to the integrin cytoplasmic tail and mediate integrin activation, cytoskeleton rearrangement, and focal adhesion assembly. However, knowledge about how Talin and Kindlin synergistically assist integrin activation remains unclear. Here, we performed so-called "ramp-clamp" SMD simulations, which modeled the mechanosignaling from Kindlin, to investigate the effect of tension on the interaction of the β1 integrin cytoplasmic tail with the Talin-F3 domain. The present results showed that mild but not excessive stretching enhanced the binding of integrin with Talin. This mechanical regulation on integrin affinity to Talin referred to an event cascade, in which under stretching, the integrin cytoplasmic tail adopted allostery in response to the mechanical stimulus, remodeling of integrin in favor of Talin-association ensued, and finally, a stable, close-knit complex was formed. In the cascade, the torsion angle transition of integrin was the cue for the stable interaction of the complex under tensile force. The present work suggested a model for Talin and Kindlin to synergistically activate integrin. It should help understand integrin activation and its mechanochemical regulation mechanism, integrin-related innate cellular immune responses, cell adhesion, cell-cell interaction, and integrin-related drug development.

Caveolae couple mechanical stress to integrin recycling and activation.

Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active β1-integrin content on the surface of Cav1KO fibroblasts. Florescence recovery after photobleaching analysis and endocytosis/recycling assays revealed that active β1-integrin is mostly endocytosed through the clathrin independent carrier/glycosylphosphatidyl inositol (GPI)-enriched endocytic compartment pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4 and PM tension-dependent manner. Moreover, the threshold for PM tension-driven β1-integrin activation is lower in Cav1KO mouse embryonic fibroblasts (MEFs) than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response.

Novel insights into mouse models of ectopic proplatelet release

AbstractMature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott–Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Reversible Platelet Integrin αIIbβ3 Activation and Thrombus Instability.

Integrin αIIbβ3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbβ3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbβ3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbβ3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.

Asebogenin suppresses thrombus formation via inhibition of Syk phosphorylation.

Thrombosis is a major cause of morbidity and mortality worldwide. Platelet activation by exposed collagen through glycoprotein VI (GPVI) and formation of neutrophil extracellular traps (NETs) are critical pathogenic factors for arterial and venous thrombosis. Both events are regulated by spleen tyrosine kinase (Syk)-mediated signalling events. Asebogenin is a dihydrochalcone whose pharmacological effects remain largely unknown. This study aims to investigate the antithrombotic effects of asebogenin and the underlying molecular mechanisms. Platelet aggregation was assessed using an aggregometer. Platelet P-selectin exposure, integrin activation and calcium mobilization were determined by flow cytometry. NETs formation was assessed by SYTOX Green staining and immunohistochemistry. Quantitative phosphoproteomics, microscale thermophoresis, in vitro kinase assay and molecular docking combined with dynamics simulation were performed to characterize the targets of asebogenin. The in vivo effects of asebogenin on arterial thrombosis were investigated using FeCl3 -induced and laser-induced injury models, whereas those of venous thrombosis were induced by stenosis of the inferior vena cava. Asebogenin inhibited a series of GPVI-induced platelet responses and suppressed NETs formation induced by proinflammatory stimuli. Mechanistically, asebogenin directly interfered with the phosphorylation of Syk at Tyr525/526, which is important for its activation. Further, asebogenin suppressed arterial thrombosis demonstrated by decreased platelet accumulation and fibrin generation and attenuated venous thrombosis determined by reduced neutrophil accumulation and NETs formation, without increasing bleeding risk. Asebogenin exhibits potent antithrombotic effects by targeting Syk and is a potential lead compound for the development of efficient and safe antithrombotic agents.

Force tuning through regulation of clathrin-dependent integrin endocytosis.

Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables β3-integrin-mediated force generation independently of β1 integrin. β3-integrin-mediated forces were associated with a decrease in β3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in β3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.

Nexinhib20 Inhibits Neutrophil Adhesion and β2 Integrin Activation by Antagonizing Rac-1-Guanosine 5'-Triphosphate Interaction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting β2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced β2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

C-Met-integrin cooperation: Mechanisms, tumorigenic effects, and therapeutic relevance.

c-Met is a receptor tyrosine kinase which upon activation by its ligand, the hepatocyte growth factor, mediates many important signalling pathways that regulate cellular functions such as survival, proliferation, and migration. Its oncogenic and tumorigenic signalling mechanisms, greatly contributing to cancer development and progression, are well documented. Integrins, heterogeneous adhesion receptors which facilitate cell-extracellular matrix interactions, are important in biomechanically sensitive cell adhesion and motility but also modulate diverse cell behaviour. Here we review the studies which reported cooperation between c-Met and several integrins, particularly β1 and β4, in various cell models including many tumour cell types. From the various experimental models and results analysed, we propose that c-Met-integrin cooperation occurs via inside-out or outside-in signalling. Thus, either c-Met activation triggers integrin activation and cell adhesion or integrin adhesion to its extracellular ligand triggers c-Met activation. These two modes of cooperation require the adhesive function of integrins and mostly lead to cell migration and invasion. In a third, less conventional, mode of cooperation, the integrin plays the role of a signalling adaptor for c-Met, independently from its adhesive property, leading to anchorage independent survival. Recent studies have revealed the influence of endocytic trafficking in c-Met-integrin cooperation including the adaptor function of integrin occurring on endomembranes, triggering an inside-in signalling, believed to promote survival of metastatic cells. We present the evidence of the cooperation in vivo and in human tissues and highlight its therapeutic relevance. A better understanding of the mechanisms regulating c-Met-integrin cooperation in cancer progression could lead to the design of new therapies targeting this cooperation, providing more effective therapeutic approaches than c-Met or integrin inhibitors as monotherapies used in the clinic.

A Novel GATA1 Variant in the C-Terminal Zinc Finger Compared with the Platelet Phenotype of Patients with A Likely Pathogenic Variant in the N-Terminal Zinc Finger.

The GATA1 transcription factor is essential for normal erythropoiesis and megakaryocytic differentiation. Germline GATA1 pathogenic variants in the N-terminal zinc finger (N-ZF) are typically associated with X-linked thrombocytopenia, platelet dysfunction, and dyserythropoietic anemia. A few variants in the C-terminal ZF (C-ZF) domain are described with normal platelet count but altered platelet function as the main characteristic. Independently performed molecular genetic analysis identified a novel hemizygous variant (c.865C>T, p.H289Y) in the C-ZF region of GATA1 in a German patient and in a Spanish patient. We characterized the bleeding and platelet phenotype of these patients and compared these findings with the parameters of two German siblings carrying the likely pathogenic variant p.D218N in the GATA1 N-ZF domain. The main difference was profound thrombocytopenia in the brothers carrying the p.D218N variant compared to a normal platelet count in patients carrying the p.H289Y variant; only the Spanish patient occasionally developed mild thrombocytopenia. A functional platelet defect affecting αIIbβ3 integrin activation and α-granule secretion was present in all patients. Additionally, mild anemia, anisocytosis, and poikilocytosis were observed in the patients with the C-ZF variant. Our data support the concept that GATA1 variants located in the different ZF regions can lead to clinically diverse manifestations.