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Store-operated Ca(2+) entry (SOCE) is the major route of Ca(2+) influx in platelets. The Ca(2+) sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca(2+) channel Orai1 and the inositol trisphosphate receptor (IP(3)R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP(3)R in platelets. Deletion of platelet BIN2 (Bin2(fl/fl,Pf4-Cre) mice) resulted in reduced Ca(2+) store release and Ca(2+) influx in response to all tested platelet agonists. These defects were a consequence of impaired IP(3)R function in combination with defective STIM1-mediated SOC channel activation, while Ca(2+) store content and agonist-induced IP(3) production were unaltered. This severely defective Ca(2+) signaling translated into impaired thrombus formation under flow and a protection of Bin2(fl/fl,Pf4-Cre) mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

Rationale: Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown. Objective: We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)–driven arterial occlusion, tissue infarction, and organ failure. Methods and Results: C57BL/6J mice were injected with CC into the left kidney artery. Primary end point was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, that is, acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain–like deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA. Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase, or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was Conclusions: CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.

Background and Purpose— Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca 2+ entry, which is triggered by the depletion of intracellular calcium stores, contributes to ischemia-induced calcium influx in neurons, but the responsible Ca 2+ channel is not known. Methods— Here, we have generated mice lacking the calcium channel subunit Orai2 and analyzed them in experimental stroke. Results— Orai2-deficient mice were protected from ischemic neuronal death both during acute ischemia under vessel occlusion and during ischemia/reperfusion upon successful recanalization. Calcium signals induced by calcium store depletion or oxygen/glucose deprivation were significantly diminished in Orai2-deficient neurons demonstrating that Orai2 is a central mediator of neuronal capacitative Ca 2+ entry and is involved in calcium overload during ischemia. Conclusions— Our experimental data identify Orai2 as an attractive target for pharmaceutical intervention in acute stroke.

Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1−/−) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1−/−platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1−/− platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis.

Background and Aims Numerous observational studies have reported an association between hyperuricemia (HU) and cardiovascular disease where atherosclerosis is a leading cause. In advanced atherosclerosis, cholesterol crystal (CC) embolism is a life-threatening complication with an average mortality of 62.8%. Clinical manifestations include skin necrosis, intestinal injury and acute kidney injury (AKI). Autopsies and tissue biopsies reveal CC inside the arterial lumen. In a new model of CC-induced AKI, we recently found that fibrin clots formed around CC obstruct peripheral arteries and cause tissue infarction and organ failure. However, the role of asymptomatic HU in CC-induced AKI is currently unknown. Thus, we hypothesized that asymptomatic HU improves the outcomes after CC-induced AKI. Method In vivo, 6 weeks old Alb-creERT2;Glut9lox/lox and Glut9lox/lox control mice were intraperitoneally injected with tamoxifen to deplete hepatic Glut9 expression. Both groups of mice were placed on a standard chow diet enriched with inosine for two weeks. Afterward, a solution of CCs was injected into the left kidney arteries of mice to induce AKI. Serum and kidneys were collected 24 hours later, and kidney function, infarct size and kidney injury evaluated using GFR measurement, colorimetric assays, ELISA and immunohistochemistry of kidney sections. For in vitro studies, we isolated platelets from healthy mice and cultured them in the presence or absence of soluble uric acid (sUA) prior to CC activation. After stimulation, platelet aggregation assays and flow cytometry were performed. Results Our in vivo data showed that Alb-creERT2;Glut9lox/lox mice developed asymptomatic HU without kidney impairment (serum UA levels 9-15 mg/dL), while Glut9lox/lox control mice remained healthy. HU mice displayed a rapid decline in GFR of approx. 80% as compared to only 30% in healthy mice after CC-induced AKI, which was in line with increased serum BUN and IL-6 levels. The rapid GFR decline was due to vasoconstriction in arteries of the contralateral kidney as determined by αSMA/fibrin staining and a reduced ratio of lumen versus artery area in mice with HU after AKI. HU mice also had significantly less kidney infarct size as well as reduced kidney injury (necrosis and edema) and arterial occlusion. The in vitro data showed that sUA had no impact on platelet aggregation, activation and degranulation as compared to medium control in response to CCs. Conclusion We now show that HU has vasoconstrictive effects in the contralateral kidney by reducing GFR, while at the same time protects mice from CC-induced AKI suggesting a compensatory physiological mechanism of autoregulation to protect nephrons. On the other hand, in patients with chronic kidney disease, HU-related vasoconstriction might lead to increased arterial blood pressure; thus, increasing the risk for cardiovascular complications due to platelet activation and aggregation.

Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as calcium, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet zinc transporter, transmembrane protein 163 (TMEM163), which was significantly reduced in BLOC-1 (Dtnbp1(sdy) and Pldn(pa))–, BLOC-2 (Hps6(ru))–, or AP-3 (Ap3b1(pe))–deficient mice and HPS patients (HPS2, HPS3, HPS5, HPS6, or HPS9). We observed similar platelet DG defects and higher intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome marker–positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and that BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.

Zn2+ deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn2+-deficient diets, accounting for 1−4% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn2+ deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn2+ status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn2+ uptake in the gut using different nutritional supplementation of Zn2+ could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn2+ diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn2+ in hemostasis. Storage protein metallothionein maintains or releases Zn2+ in the cytoplasm, and the dynamic change of this cytoplasmic Zn2+ pool is regulated by the redox status of the cell. An increase of labile Zn2+ pool can be toxic for the cells, and therefore cytoplasmic Zn2+ levels are tightly regulated by several Zn2+ transporters located on the cell surface and also on the intracellular membrane of Zn2+ storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn2+ is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn2+ transport and the physiological role of Zn2+ store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn2+ to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn2+ homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.

Objective— TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg 2+ and Ca 2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. Approach and Results— Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice ( Trpm7 R/R ) causes a marked signaling defect in platelets. Trpm7 R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca 2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and β3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca 2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. Conclusions— Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca 2+ entry in platelets. The protection of Trpm7 R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.

Objective— It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain. Approach and Results— We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice. Conclusions— Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes.

Background and Purpose- Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca

Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM)-signaling and thereby regulates diverse functions including platelet adhesion, aggregation and procoagulant activity. GPVI has been proposed as a safe antithrombotic target as its inhibition is protective in models of arterial thrombosis with only minor effects on hemostasis. Here, we demonstrate that genetic deficiency of platelet GPVI in mice decreases experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses show that mouse, as well as human GPVI, supports platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knock-out approach, we identified Galectin-3 as the major counter-receptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed Galectin-3 utilizes ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab)2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study reveals a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression.

Significance Zn2+, Mg2+, and Ca2+ are the most abundant divalent metals in mammals. Different categories of cation-selective channels and transporters are thought to control the levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these minerals are poorly understood. Using genetic mouse models together with biophysical and biochemical analysis, we show that the channel-kinase TRPM7 is a master regulator of the organismal balance of divalent cations. TRPM7 activity is primarily required in the intestine, while TRPM7 function in the kidney—commonly thought to be essential—is expendable. Hence, against current thinking, organismal balance of multiple divalent cations predominantly relies on a common gatekeeper, TRPM7, rather than on individual specialized channels/transporters., Zn2+, Mg2+, and Ca2+ are essential minerals required for a plethora of metabolic processes and signaling pathways. Different categories of cation-selective channels and transporters are therefore required to tightly control the cellular levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these essential minerals are poorly understood. Herein, we identify a central and indispensable role of the channel-kinase TRPM7 for organismal mineral homeostasis. The function of TRPM7 was assessed by single-channel analysis of TRPM7, phenotyping of TRPM7-deficient cells in conjunction with metabolic profiling of mice carrying kidney- and intestine-restricted null mutations in Trpm7 and animals with a global “kinase-dead” point mutation in the gene. The TRPM7 channel reconstituted in lipid bilayers displayed a similar permeability to Zn2+ and Mg2+. Consistently, we found that endogenous TRPM7 regulates the total content of Zn2+ and Mg2+ in cultured cells. Unexpectedly, genetic inactivation of intestinal rather than kidney TRPM7 caused profound deficiencies specifically of Zn2+, Mg2+, and Ca2+ at the organismal level, a scenario incompatible with early postnatal growth and survival. In contrast, global ablation of TRPM7 kinase activity did not affect mineral homeostasis, reinforcing the importance of the channel activity of TRPM7. Finally, dietary Zn2+ and Mg2+ fortifications significantly extended the survival of offspring lacking intestinal TRPM7. Hence, the organismal balance of divalent cations critically relies on one common gatekeeper, the intestinal TRPM7 channel.

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Scott syndrome is a rare bleeding disorder, characterized by altered Ca2+-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano) 6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca2+-dependent K(Ca)3.1Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6min (control 6.4min, P 0.05) with reduced PS exposure (265 to 90%); 2) lowered Ca2+-dependent swelling (280%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.

Cation homeostasis, in relation to various immune-suppressive diseases, is a novel field of investigation. Recently, patients with a loss-of-function mutation in magnesium transporter 1 (MAGT1) were reported to present a dysregulated Mg2+ homeostasis in T lymphocytes. Using Magt1-knockout mice (Magt1−/y), we show that Mg2+ homeostasis was impaired in Magt1−/y B cells and Ca2+ influx was increased after BCR stimulation, whereas T and NK cell function was unaffected. Consequently, mutant B cells displayed an increased phosphorylation of BCR-related proteins differentially affecting protein kinase C activation. These in vitro findings translated into increased frequencies of CD19+ B cells and marginal zone B cells and decreased frequencies of plasma cells among CD45+ splenocytes in vivo. Altogether, our study demonstrates for the first time, to our knowledge, that abolished MAGT1 function causes imbalanced cation homeostasis and developmental responses in B cells. Therefore, this study might contribute to a further understanding of B cell–related pathologies.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice (Twf2a-/-) display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a-/- platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of Twf2a-/- platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune-mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1-deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)35-55 -induced EAE, a model of human MS. The lack of PLD1 reduced chemokine-mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1-deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1-dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.

Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actin-binding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrin-linked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviral-mediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed, SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin β1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation.

A key response of the myocardium to stress is the secretion of factors with paracrine or endocrine function. Intriguing in this respect is peptidase inhibitor 16 (PI16), a member of the CAP family of proteins which we found to be highly upregulated in cardiac disease. Up to this point, the mechanism of action and physiological function of PI16 remained elusive. Here, we show that PI16 is predominantly expressed by cardiac fibroblasts, which expose PI16 to the interstitium via a glycophosphatidylinositol (-GPI) membrane anchor. Based on a reported genetic association of PI16 and plasma levels of the chemokine chemerin, we investigated whether PI16 regulates post-translational processing of its precursor pro-chemerin. PI16-deficient mice were engineered and found to generate higher levels of processed chemerin than wildtype mice. Purified recombinant PI16 efficiently inhibited cathepsin K, a chemerin-activating protease, in vitro. Moreover, we show that conditioned medium from PI16-overexpressing cells impaired the activation of pro-chemerin. Together, our data indicate that PI16 suppresses chemerin activation in the myocardium and suggest that this circuit may be part of the cardiac stress response.

SummaryFour molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

Objective— Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif–bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. Approach and Results— We investigated whether both (hem)immunoreceptor tyrosine activation motif–bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2–depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. Conclusions— These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti–CLEC-2–based agents in the prevention of thrombotic diseases.

Rationale: At sites of vascular injury, exposed subendothelial collagens not only trigger sudden platelet adhesion and aggregation, thereby initiating normal hemostasis, but also can lead to acute ischemic diseases, such as myocardial infarction or stroke. The glycoprotein (GP) VI/Fc receptor γ-chain complex is a central regulator of these processes because it mediates platelet activation on collagens through a series of tyrosine phosphorylation events downstream of the Fc receptor γ-chain–associated immunoreceptor tyrosine-based activation motif. GPVI signaling has to be tightly regulated to prevent uncontrolled intravascular platelet activation, but the underlying mechanisms are not fully understood. Objective: We studied the role of PDZ and LIM domain family member CLP36 in platelet physiology in vitro and in vivo. Methods and Results: We report that CLP36 acts as a major inhibitor of GPVI immunoreceptor tyrosine-based activation motif signaling in platelets. Platelets from mice either expressing a low amount of a truncated form of CLP36 lacking the LIM domain ( Clp36 ΔLIM ) or lacking the whole protein ( Clp36 −/− ) displayed profound hyperactivation in response to GPVI agonists, whereas other signaling pathways were unaffected. This was associated with hyperphosphorylation of signaling proteins and enhanced Ca 2+ mobilization, granule secretion, and integrin activation downstream of GPVI. The lack of functional CLP36 translated into accelerated thrombus formation and enhanced procoagulant activity, assembling a prothrombotic phenotype in vivo. Conclusions: These data reveal an inhibitory function of CLP36 in GPVI immunoreceptor tyrosine-based activation motif signaling and establish it as a key regulator of arterial thrombosis.

Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood. DOI: http://dx.doi.org/10.7554/eLife.20914.001, eLife digest A balanced diet contains a variety of minerals such as magnesium ions, which are required for many chemical reactions in our body. A shortage of magnesium ions is linked to many diseases and is thought to be especially harmful to babies in the womb and shortly after birth. Magnesium ion deficiency is widespread in human populations and in the US is thought to affect up to 68% of people. Despite its prominent role in human health, our understanding of how the body maintains the right balance of magnesium ions remains extremely vague. Magnesium ions can enter and leave a cell by passing through specific types of proteins that form channels in the membrane surrounding the cell. There are thought to be around ten types of these magnesium ion channels in human cells, but we do not know what roles any of them perform in the body. One such channel called TRPM6 may be particularly important because mutations in the gene that encodes this channel can cause magnesium ion deficiency in human infants. However, the loss of TRPM6 in mice disrupts how mouse embryos develop, suggesting that our current view on the role that TRPM6 plays in regulating the magnesium ion balance in humans may be too simplistic. To address this question, Chubanov et al. studied mice with mutations that disrupted the production of TRPM6 in specific tissues only. The experiments show that TRPM6 primarily operates in the placenta and intestine to regulate the balance of magnesium ions in the body. Further experiments show that the loss of TRPM6 in adult mice leads to reduced lifespan, growth defects and poor health by disrupting important biochemical reactions. Supplying the mutant mice with magnesium ion supplements improved their health and could extend lifespans of normal animals. The findings of Chubanov et al. demonstrate that TRPM6 plays a crucial role in regulating the levels of magnesium ions in mice before birth and into adulthood. The next step is to carry out large-scale experiments to investigate the effects of altering the levels of magnesium ions in human diets. DOI: http://dx.doi.org/10.7554/eLife.20914.002