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Abstract: JAK2 V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre-mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet activation and accelerated arterial thrombosis. In Jak2VF CH, both Jak2VF and wild-type (WT) platelets showed increased activation, suggesting cross talk between mutant and WT platelets. Jak2VF platelets showed twofold to threefold upregulation of COX-1 and COX-2, particularly in young platelets, with elevated cPLA2 activation and thromboxane A2 production. Compared with controls, conditioned media from activated Jak2VF platelets induced greater activation of WT platelets that was reversed by a thromboxane receptor antagonist. Low-dose aspirin ameliorated carotid artery thrombosis in VFGp1ba and Jak2VF CH mice but not in WT control mice. This study shows accelerated arterial thrombosis and platelet activation in Jak2VF CH with a major role of increased reticulated Jak2VF platelets, which mediate thromboxane cross talk with WT platelets and suggests a potential beneficial effect of aspirin in JAK2VF CH., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Objectives: To explore effectiveness and sustainability of guideline adherence and antibiotic consumption after establishing treatment guidelines and initiating antimicrobial stewardship (AMS) ward rounds in a university hospital emergency department (ED)., Methods: Data were gathered retrospectively from 2017 to 2021 in the LMU University Hospital in Munich, Germany. Four time periods were compared: P1 (pre-intervention period); P2 (distribution of guideline pocket cards); P3 (reassessment after 3 years); and P4 (refresher of guideline pocket cards and additional daily AMS ward rounds for different medical disciplines). Primary outcome was adherence to guideline pocket cards for community-acquired pneumonia, cystitis, pyelonephritis and COVID-19-associated bacterial pneumonia. Secondary outcomes were reduction in antibiotic consumption and adherence to AMS specialist recommendations., Results: The study included 1324 patients. Guideline adherence increased in P2 for each of the infectious diseases entities. After 3 years (P3), guideline adherence decreased again, but was mostly on a higher level than in P1. AMS ward rounds resulted in an additional increase in guideline adherence (P1/P2: 47% versus 58.6%, P  = 0.005; P2/P3: 58.6% versus 57.3%, P  = 0.750; P3/P4: 57.3% versus 72.5%, P  < 0.001). Adherence increased significantly, not only during workdays but also on weekends/nightshifts. Adherence to AMS specialist recommendations was excellent (91.3%). We observed an increase in use of narrow-spectrum antibiotics and a decrease in the application of fluoroquinolones and cephalosporins., Conclusions: Establishing treatment guidelines in the ED is effective. However, positive effects can be diminished over time. Daily AMS ward rounds are useful, not only to restore but to further increase guideline adherence significantly., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Thrombosis is a frequent cause of cardiovascular mortality and hospitalization. Current antithrombotic strategies, however, target both thrombosis and physiological hemostasis and thereby increase bleeding risk. In recent years the pathophysiological understanding of thrombus formation has significantly advanced and inflammation has become a crucial element. Neutrophils as most frequent immune cells in the blood and their released mediators play a key role herein. Neutrophil-derived cathelicidin next to its strong antimicrobial properties has also shown to modulates thrombosis and thus presents a potential therapeutic target. In this article we review direct and indirect (immune- and endothelial cell-mediated) effects of cathelicidin on platelets and the coagulation system. Further we discuss its implications for large vessel thrombosis and consecutive thromboinflammation as well as immunothrombosis in sepsis and COVID-19 and give an outlook for potential therapeutic prospects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Ul Ain, Schulz and Pircher.)

Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

Desmosomes are important epidermal adhesion units and signalling hubs, which play an important role in pemphigus pathogenesis. Different expression patterns of the pemphigus autoantigens desmoglein (Dsg)1 and Dsg3 across different epidermal layers have been demonstrated. However, little is known about changes in desmosome composition in different epidermal layers or in patient skin. The aim of this study was thus to characterize desmosome composition in healthy and pemphigus skin using super-resolution microscopy. An increasing Dsg1/Dsg3 ratio from lower basal (BL) to uppermost granular layer (GL) was observed. Within BL desmosomes, Dsg1 and Dsg3 were more homogeneously distributed whereas superficial desmosomes mostly comprised one of the two molecules or domains containing either one but not both. Extradesmosomal, desmoplakin (Dp)-independent, co-localization of Dsg3 with plakoglobin (Pg) was found mostly in BL and extradesmosomal Dsg1 co-localization with Pg in all layers. In contrast, in the spinous layer (SL) most Dsg1 and Dsg3 staining was confined to desmosomes, as revealed by the co-localization with Dp. In pemphigus patient skin, Dsg1 and Dsg3 immunostaining was altered especially along blister edges. The number of desmosomes in patient skin was reduced significantly in basal and spinous layer keratinocytes with only few split desmosomes found. In addition, Dsg1-Pg co-localization at the apical BL and Dsg3-Pg co-localization in SL were significantly reduced in patients, suggesting that that extradesmosomal Dsg molecules were affected. These results support the hypothesis that pemphigus is a desmosome assembly disease and may help to explain histopathologic differences between pemphigus phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schmitt, Pircher, Steinert, Meier, Ghoreschi, Vielmuth, Kugelmann and Waschke.)

Background: The chemokine receptor CXCR4 and its ligand CXCL12 have been shown to be a possible imaging and therapeutic target after myocardial infarction (MI). The murine-based and mouse-specific 68 Ga-mCXCL12 PET tracer could be suitable for serial in vivo quantification of cardiac CXCR4 expression in a murine model of MI., Methods and Results: At days 1-6 after MI, mice were intravenously injected with 68 Ga-mCXCL12. Autoradiography was performed and the infarct-to-remote ratio (I/R) was determined. In vivo PET imaging with 68 Ga-mCXCL12 was conducted on days 1-6 after MI and the percentage of the injected dose (%ID/g) of the tracer uptake in the infarct area was calculated. 18 F-FDG-PET was performed for anatomical landmarking. Ex vivo autoradiography identified CXCR4 upregulation in the infarct region with an increasing I/R after 12 hours (1.4 ± 0.3), showing a significant increase until day 2 (4.5 ± 0.6), followed by a plateau phase (day 4) and decrease after 10 days (1.3 ± 1.0). In vivo PET imaging identified similar CXCR4 upregulation in the infarct region which peaked around day 3 post MI (9.7 ± 5.0 %ID/g) and then subsequently decreased by day 6 (2.8 ± 1.0 %ID/g)., Conclusion: Noninvasive molecular imaging of cardiac CXCR4 expression using a novel, murine-based, and specific 68 Ga-mCXCL12 tracer is feasible both ex vivo and in vivo., (© 2020. The Author(s).)

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors 1 . Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2 V617F (JAK2 VF ) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease 1,2 . Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2 VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2 VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2 VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1β reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2 VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1β or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.

Objects: Abnormal use of muscles of tongue and lips is called orofacial myofunctional disorders (OMD). In this study, we aimed to investigate the interrelation between occlusal findings and OMD in primary and mixed dentition and to find out the reasons for malocclusions, and the effect modification of these associations by the number of sources that parents used to receive the information on child oral health issues (NSI)., Setting and Sample Population: Cross-sectional study was performed in one kindergarten in Latvia (cluster sampling). The study sample comprised 141 children-mothers' pairs of pre-school children aged 4-7 years., Materials and Methods: Children' myofunctional situation, occlusion and speech defects were assessed during an examination. Children's mothers were interviewed on their socio-demographic situation, feeding and habits of the child, history of diseases and NSI. Multiple logistic regression models adjusted for lip power, swallowing, breathing and speech cases of long respiratory diseases, age and gender were built to investigate associations between the types of occlusion and OMD/health characteristics. Effect modification of these associations by NSI was analysed., Results: Types of occlusion associated with OMD/health characteristics were as follows: normal occlusion with prolonged respiratory diseases (odds ratio, OR = 0.345 [95% confidence interval, CI 0.16; 0.75]); combined type with tongue thrust habits (OR = 3.11 [0.99; 9.90]), lip-closing strength (OR = 0.99 [0.98; 0.99]); and cross-bite with speech (OR = 3.55 [1.07; 11.78]). These associations remained stable when parents used two or less sources of information on children's oral health., Conclusions: To facilitate occlusal stability, early interdisciplinary examinations should be performed to prevent time-consuming and cost-consuming treatment., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To investigate whether immune cell composition and content of neutrophil extracellular traps (NETs) in relation to clinical outcome are different between acute ischemic stroke (AIS) and acute myocardial infarction (AMI), we performed histologic analysis and correlated results with clinical and procedural parameters., Methods: We retrieved thrombi from patients with AIS (n = 71) and AMI (n = 72) during endovascular arterial recanalization and analyzed their immune cell composition and NET content by immunohistology. We then associated thrombus composition with procedural parameters and outcome in AIS and with cardiac function in patients with AMI. Furthermore, we compared AIS thrombi with AMI thrombi and differentiated Trial of Org 10172 in Acute Stroke Treatment classifications to address potential differences in thrombus pathogenesis., Results: Amounts of leukocytes ( p = 0.133) and neutrophils ( p = 0.56) were similar between AIS and AMI thrombi. Monocytes ( p = 0.0052), eosinophils ( p < 0.0001), B cells ( p < 0.0001), and T cells ( p < 0.0001) were more abundant in stroke compared with AMI thrombi. NETs were present in 100% of patients with AIS and 20.8% of patients with AMI. Their abundance in thrombi was associated with poor outcome scores in patients with AIS and with reduced ejection fraction in patients with AMI., Conclusion: In our detailed histologic analysis of arterial thrombi, thrombus composition and especially abundance of leukocyte subsets differed between patients with AIS and AMI. The presence and amount of NETs were associated with patients' outcome after AIS and AMI, supporting a critical impact of NETs on thrombus stability in both conditions., (© 2020 American Academy of Neurology.)

Background:  von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo , whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes., Methods and Results:  The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo . Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas., Conclusion:  CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Rationale: A reduced rate of myocardial infarction has been reported in patients with atrial fibrillation treated with FXa (factor Xa) inhibitors including rivaroxaban compared with vitamin K antagonists. At the same time, low-dose rivaroxaban has been shown to reduce mortality and atherothrombotic events in patients with coronary artery disease. Yet, the mechanisms underlying this reduction remain unknown., Objective: In this study, we hypothesized that rivaroxaban's antithrombotic potential is linked to a hitherto unknown rivaroxaban effect that impacts on platelet reactivity and arterial thrombosis., Methods and Results: In this study, we identified FXa as potent, direct agonist of the PAR-1 (protease-activated receptor 1), leading to platelet activation and thrombus formation, which can be inhibited by rivaroxaban. We found that rivaroxaban reduced arterial thrombus stability in a mouse model of arterial thrombosis using intravital microscopy. For in vitro studies, atrial fibrillation patients on permanent rivaroxaban treatment for stroke prevention, respective controls, and patients with new-onset atrial fibrillation before and after first intake of rivaroxaban (time series analysis) were recruited. Platelet aggregation responses, as well as thrombus formation under arterial flow conditions on collagen and atherosclerotic plaque material, were attenuated by rivaroxaban. We show that rivaroxaban's antiplatelet effect is plasma dependent but independent of thrombin and rivaroxaban's anticoagulatory capacity., Conclusions: Here, we identified FXa as potent platelet agonist that acts through PAR-1. Therefore, rivaroxaban exerts an antiplatelet effect that together with its well-known potent anticoagulatory capacity might lead to reduced frequency of atherothrombotic events and improved outcome in patients.

Dysregulation of platelet function can contribute to the disease progression in sepsis. The proteasome represents a critical and vital element of cellular protein metabolism in platelets and its proteolytic activity has been associated with platelet function. However, the role of the platelet proteasome as well as its response to infection under conditions of sepsis have not been studied so far. We measured platelet proteasome activity by fluorescent substrates, degradation of poly-ubiquitinated proteins and cleavage of the proteasome substrate Talin-1 in the presence of living E. coli strains and in platelets isolated from sepsis patients. Upregulation of the proteasome activator PA28 (PSME1) was assessed by quantitative real-time PCR in platelets from sepsis patients. We show that co-incubation of platelets with living E. coli (UTI89) results in increased degradation of poly-ubiquitinated proteins and cleavage of Talin-1 by the proteasome. Proteasome activity and cleavage of Talin-1 was significantly increased in α-hemolysin (HlyA)-positive E. coli strains. Supporting these findings, proteasome activity was also increased in platelets of patients with sepsis. Finally, the proteasome activator PA28 (PSME1) was upregulated in this group of patients. In this study we demonstrate for the first time that the proteasome in platelets is activated in the septic milieu.

Clinical observations implicate a role of eosinophils in cardiovascular diseases because markers of eosinophil activation are elevated in atherosclerosis and thrombosis. However, their contribution to atherosclerotic plaque formation and arterial thrombosis remains unclear. In these settings, we investigated how eosinophils are recruited and activated through an interplay with platelets. Here, we provide evidence for a central importance of eosinophil-platelet interactions in atherosclerosis and thrombosis. We show that eosinophils support atherosclerotic plaque formation involving enhanced von Willebrand factor exposure on endothelial cells and augmented platelet adhesion. During arterial thrombosis, eosinophils are quickly recruited in an integrin-dependent manner and engage in interactions with platelets leading to eosinophil activation as we show by intravital calcium imaging. These direct interactions induce the formation of eosinophil extracellular traps (EETs), which are present in human thrombi and constitute a substantial part of extracellular traps in murine thrombi. EETs are decorated with the granule protein major basic protein, which causes platelet activation by eosinophils. Consequently, targeting of EETs diminished thrombus formation in vivo, which identifies this approach as a novel antithrombotic concept. Finally, in our clinical analysis of coronary artery thrombi, we identified female patients with stent thrombosis as the population that might derive the greatest benefit from an eosinophil-inhibiting strategy. In summary, eosinophils contribute to atherosclerotic plaque formation and thrombosis through an interplay with platelets, resulting in mutual activation. Therefore, eosinophils are a promising new target in the prevention and therapy of atherosclerosis and thrombosis., (© 2019 by The American Society of Hematology.)

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIbα-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies.

Objective: Retinoid X receptors (RXR) are a family of nuclear receptors that play critical roles in the regulation of numerous fundamental biological processes including cell proliferation, differentiation, and death. Earlier studies suggested that treatment with RXR agonists attenuates platelet activation in all adults (male and femal) and mice; however, the underlying molecular mechanisms have remained insufficiently understood. To elaborate further on this issue, we characterized megakaryocyte and platelet-specific RXR knockout mice to study platelet function in vitro and arterial thrombosis in vivo., Approach and Results: First, we identified RXRβ as the dominant RXR receptor in mouse platelets, prompting us to generate a megakaryocyte and platelet-specific PF4 Cre ;RXRβ flox/flox mouse. Second, we studied activation, spreading, and aggregation of platelets from C57Bl/6 wild-type mice (WT), PF4 Cre+ ;RXRβ flox/flox mice, and PF4 Cre- ;RXRβ flox/flox littermate controls in the presence or absence of RXR ligands, that is, 9-cis-retinoic acid (9cRA) and methoprene acid (MA). We found that in vitro treatment with RXR ligands attenuates spreading and aggregation of platelets and increases proplatelet particle formation from megakaryocytes (MK). However, these effects are also observed in RXRβ-deficient platelets and MKs and are thus independent of RXRβ. Third, we investigated arterial thrombus formation in an iron chloride (FeCl3)-induced vascular injury model in vivo, which is also not affected by the absence of RXRβ in platelets., Conclusions: Absence of the most abundant RXR receptor in mouse platelets, RXRβ, does not affect platelet function in vitro and thrombus formation in vivo. Furthermore, RXR agonists' mediated effects on platelet function are independent of RXRβ expression. Hence, our data do not support a significant contribution of RXRβ to arterial thrombosis in mice., (© 2019 International Society on Thrombosis and Haemostasis.)

Atherothrombosis is a frequent cause of cardiovascular mortality. It is mostly triggered by plaque rupture and exposure of the thrombogenic subendothelial matrix, which initiates platelet aggregation and clot formation. Current antithrombotic strategies, however, target both thrombosis and physiological hemostasis and thereby increase bleeding risk. Thus, there is an unmet clinical need for optimized therapies. Neutrophil activation and consecutive interactions of neutrophils and platelets contribute mechanistically to thromboinflammation and arterial thrombosis, and thus present a potential therapeutic target. Platelet-neutrophil interactions are mediated through adhesion molecules such as P-selectin and P-selectin glycoprotein ligand 1 as well as glycoprotein Ib and macrophage-1 antigen, which mediate physical cell interactions and intracellular signaling. Release of soluble mediators as well as direct signaling between platelets and neutrophils lead to their reciprocal activation and neutrophil release of extracellular traps, scaffolds of condensed chromatin that play a prothrombotic role in atherothrombosis. In this article, we review the role of neutrophils and neutrophil-derived prothrombotic molecules in platelet activation and atherothrombosis, and highlight potential therapeutic targets., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α 6 β 1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca 2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro . Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo , this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3 -deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell-specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a ( Regnase-1 ), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages., (© 2019 von Gamm et al.)

Background: Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence., Methods: Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay., Findings: We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1β as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1β-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88., Interpretation: Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. FUND: German Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation., (© 2019 Weckbach et al.)

Coronin-1A (Coro1A) belongs to a family of highly conserved actin-binding proteins that regulate cytoskeletal re-arrangement. In mammalians, Coro1A expression is most abundant in the haematopoietic lineage, where it regulates various cellular processes. The role of Coro1A in platelets has been previously unknown. Here, we identified Coro1A in human and mouse platelets. Genetic absence of Coro1A in mouse platelets inhibited agonist-induced actin polymerization and altered cofilin phosphoregulation, leading to a reduction in spreading and low-dose collagen induced aggregation. Furthermore, Coro1A-deficient mice displayed a defect in ferric chloride-induced arterial thrombosis with prolonged thrombus formation and reduced thrombus size. Immunofluorescence analysis revealed a less compact thrombus structure with reduced density of platelets and fibrinogen. In summary, Coro1A has a role in platelet biology with impact on spreading, aggregation and thrombosis., Competing Interests: None., (Georg Thieme Verlag KG Stuttgart · New York.)

A core research area in the Department of Cardiology at Ludwig-Maximilians-University (LMU) Munich focuses on antiplatelet therapy, its translational aspects, and its underlying mechanism with respect to platelet physiology. We are conducting a broad range of investigator-initiated clinical trials (phase II-IV) and preclinical studies on the topic of antithrombotic therapy for percutaneous coronary intervention patients, platelet activation, and reactivity as well as on novel inhibitors of platelet adhesion. Just recently, we completed the large multi-centre investigator-initiated TROPICAL-ACS trial on guided early de-escalation of antiplatelet treatment in acute coronary syndrome (ACS) patients (Sibbing et al. in Lancet 390:1747-1757, 2017; Sibbing et al. in Thromb Haemost 117:1240-1248), done at 33 sites in Europe. Furthermore, besides other ongoing clinical studies, we initiated and are currently recruiting patients for the multi-centre randomized APixaban versus PhenpRocoumon in Patients With ACS and AF: APPROACH-ACS-AF study as well as for the multi-centre phase II randomized, double-blind, placebo-controlled study of revacept in Patients With Stable Coronary Artery Disease (Revacept/CAD/02) trial.

Purpose: To investigate the cerebrospinal fluid (CSF) dynamics along the entire optic nerve (ON) in patients with normal-tension glaucoma (NTG)., Methods: Retrospective analysis of computed tomographic (CT) cisternographies in Caucasian patients with NTG. Fifty-six patients (99 of 112 eyes) fulfilled the diagnostic criteria of NTG and underwent CT-cisternography. Twelve subjects without NTG (24 eyes) served as controls. Contrast-loaded cerebrospinal fluid (CLCSF) density measurements in Hounsfield units (HU) were performed at four defined regions along the ON and in the basal cistern., Results: In NTG patients, the mean density CLCSF in the bulbar segment measured 76 ± 49 HU right and 88 ± 74 HU left, in the mid-orbital segment 117 ± 92 HU right and 119 ± 73 HU left, in the intracanalicular ON portion 209 ± 88 HU right and 216 ± 101 HU left, in the intracranial ON portion 290 ± 106 HU right and 286 ± 118 HU left and in the basal cistern 517 ± 213 HU. The distribution of CLCSF along the ON showed a statistically significant reduction in the intraorbital ON segments in NTG patients compared to controls without NTG with the far largest difference within the retrobulbar segment (-150 HU right and -117 HU left; right: p < 0.001, left: p < 0.001)., Conclusion: This study demonstrates a gradual reduction in CLCSF towards the retrobulbar segment in NTG, while in controls without NTG, no reduction in CLCSF was measured within the orbital segments. Impaired CSF dynamics along the ON may contribute to the pathophysiology of NTG., (© 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: To examine the cerebrospinal fluid (CSF) dynamics along the entire optic nerve in patients with idiopathic intracranial hypertension (IIH) and papilledema by computed tomographic (CT) cisternography. Methods: Retrospective analysis of CT cisternographies in 16 patients with a history of IIH and papilledema (14 females and 2 males, mean age: 49 ± 16 years). Contrast loaded CSF (CLCSF) was measured in Hounsfield Units (HU) at three defined regions of interest (ROI) along the optic nerve (orbital optic nerve portion: bulbar and mid-orbital segment, intracranial optic nerve portion) and additionally in the basal cistern. The density measurements in ROI 1, ROI 2, and ROI 3 consist of measurements of the optic nerve complex: optic nerve sheath, CLCSF filled SAS and optic nerve tissue. As controls served a group of patients (mean age: 60 ± 19 years) without elevated intracranial pressure and without papilledema. Results: In IIH patients the mean CLCSF density in the bulbar segment measured 65 ± 53 HU on the right and 63 ± 35 HU on the left side, in the mid-orbital segment 68 ± 37 HU right and 60 ± 21 HU left. In the intracranial optic nerve portion 303 ± 137 HU right and 323 ± 169 HU left and in the basal cistern 623 ± 188 HU. Within the optic nerve the difference of CLCSF density showed a highly statistical difference ( p < 0.001) between the intracranial optic nerve portion and the mid-orbital segment. CLCSF density was statistically significantly ( p < 0.001) reduced in both intraorbital optic nerve segments in patients with IIH compared to controls. Conclusions: The current study demonstrates reduced CLCSF density within the orbital optic nerve segments in patients with IIH and papilledema compared to 12 controls without elevated intracranial pressure and without papilledema. Impaired CSF dynamics could be involved in the pathophysiology of optic nerve damage in PE in IIH.

Leukocyte-released antimicrobial peptides contribute to pathogen elimination and activation of the immune system. Their role in thrombosis is incompletely understood. Here we show that the cathelicidin LL-37 is abundant in thrombi from patients with acute myocardial infarction. Its mouse homologue, CRAMP, is present in mouse arterial thrombi following vascular injury, and derives mainly from circulating neutrophils. Absence of hematopoietic CRAMP in bone marrow chimeric mice reduces platelet recruitment and thrombus formation. Both LL-37 and CRAMP induce platelet activation in vitro by involving glycoprotein VI receptor with downstream signaling through protein tyrosine kinases Src/Syk and phospholipase C. In addition to acute thrombosis, LL-37/CRAMP-dependent platelet activation fosters platelet-neutrophil interactions in other inflammatory conditions by modulating the recruitment and extravasation of neutrophils into tissues. Absence of CRAMP abrogates acid-induced lung injury, a mouse pneumonia model that is dependent on platelet-neutrophil interactions. We suggest that LL-37/CRAMP represents an important mediator of platelet activation and thrombo-inflammation.

Aims: Medical treatment of arterial thrombosis is mainly directed against platelets and coagulation factors, and can lead to bleeding complications. Novel antithrombotic therapies targeting immune cells and neutrophil extracellular traps (NETs) are currently being investigated in animals. We addressed whether immune cell composition of arterial thrombi induced in mouse models of thrombosis resemble those of human patients with acute myocardial infarction (AMI)., Methods and Results: In a prospective cohort study of patients suffering from AMI, 81 human arterial thrombi were harvested during percutaneous coronary intervention and subjected to detailed histological analysis. In mice, arterial thrombi were induced using two distinct experimental models, ferric chloride (FeCl3) and wire injury of the carotid artery. We found that murine arterial thrombi induced by FeCl3 were highly concordant with human coronary thrombi regarding their immune cell composition, with neutrophils being the most abundant cell type, as well as the presence of NETs and coagulation factors. Pharmacological treatment of mice with the protein arginine deiminase (PAD)-inhibitor Cl-amidine abrogated NET formation, reduced arterial thrombosis and limited injury in a model of myocardial infarction., Conclusions: Neutrophils are a hallmark of arterial thrombi in patients suffering from acute myocardial infarction and in mouse models of arterial thrombosis. Inhibition of PAD could represent an interesting strategy for the treatment of arterial thrombosis to reduce neutrophil-associated tissue damage and improve functional outcome.

Hepatitis C virus (HCV) infection is a major problem worldwide. HCV is not limited to liver disease but is frequently complicated by immune-mediated extrahepatic manifestations such as glomerulonephritis or vasculitis. A fatal complication of HCV-associated vascular disease is thrombosis. Polyriboinosinic:polyribocytidylic acid (poly (I:C)), a synthetic analog of viral RNA, induces a Toll-like receptor 3 (TLR3)-dependent arteriolar thrombosis without significant thrombus formation in venules in vivo. These procoagulant effects are caused by increased endothelial synthesis of tissue factor and PAI-1 without platelet activation. In addition to human umbilical endothelial cells (HUVEC), human mesangial cells (HMC) produce procoagulatory factors, cytokines and adhesion molecules after stimulation with poly (I:C) or HCV-containing cryoprecipitates from a patient with a HCV infection as well. Activated protein C (APC) is able to prevent the induction of procoagulatory factors in HUVEC and HMC in vitro and blocks the effects of poly (I:C) and HCV-RNA on the expression of cytokines and adhesion molecules in HMC but not in HUVEC. In vivo, protein C inhibits poly (I:C)-induced arteriolar thrombosis. Thus, endothelial cells are de facto able to actively participate in immune-mediated vascular thrombosis caused by viral infections. Finally, we provide evidence for the ability of protein C to inhibit TLR3-mediated arteriolar thrombosis caused by HCV infection.

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: Platelet function has been intensively studied in the adult organism. However, little is known about the function and hemostatic capacity of platelets in the developing fetus as suitable in vivo models are lacking., Approach and Results: To examine fetal platelet function in vivo, we generated a fetal thrombosis model and investigated light/dye-induced thrombus formation by intravital microscopy throughout gestation. We observed that significantly less and unstable thrombi were formed at embryonic day (E) 13.5 compared with E17.5. Flow cytometry revealed significantly lower platelet counts in E13.5 versus E17.5 fetuses versus adult controls. In addition, fetal platelets demonstrated changed activation responses of surface adhesion molecules and reduced P-selectin content and mobilization. Interestingly, we also measured reduced levels of the integrin-activating proteins Kindlin-3, Talin-1, and Rap1 during fetal development. Consistently, fetal platelets demonstrated diminished spreading capacity compared with adults. Transfusion of adult platelets into the fetal circulation led to rapid platelet aggregate formation even in young fetuses. Yet, retrospective data analysis of a neonatal cohort demonstrated no correlation of platelet transfusion with closure of a persistent ductus arteriosus, a process reported to be platelet dependent., Conclusions: Taken together, we demonstrate an ontogenetic regulation of platelet function in vivo with physiologically low platelet numbers and hyporeactivity early during fetal development shedding new light on hemostatic function during fetal life., (© 2017 American Heart Association, Inc.)

Double-stranded DNA (dsDNA) constitutes a potent activator of innate immunity, given its ability to bind intracellular pattern recognition receptors during viral infections or sterile tissue damage. While effects of dsDNA in immune cells have been extensively studied, dsDNA signalling and its pathophysiological implications in non-immune cells, such as the vascular endothelium, remain poorly understood. The aim of this study was to characterize prothrombotic effects of dsDNA in vascular endothelial cells. Transfection of cultured human endothelial cells with the synthetic dsDNA poly(dA:dT) induced upregulation of the prothrombotic molecules tissue factor and PAI-1, resulting in accelerated blood clotting in vitro, which was partly dependent on RIG-I signalling. Prothrombotic effects were also observed upon transfection of endothelial cells with hepatitis B virus DNA-containing immunoprecipitates as well human genomic DNA. In addition, dsDNA led to surface expression of von Willebrand factor resulting in increased platelet-endothelium-interactions under flow. Eventually, intrascrotal injection of dsDNA resulted in accelerated thrombus formation upon light/dye-induced endothelial injury in mouse cremaster arterioles and venules in vivo. In conclusion, we show that viral or endogenous dsDNA induces a prothrombotic phenotype in the vascular endothelium. These findings represent a novel link between pathogen- and danger-associated patterns within innate immunity and thrombosis.

In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro . Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo , we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy., Competing Interests: The authors have declared that no conflict of interest exists.

Introduction: Platelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context., Material and Methods: Using fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation., Results: We show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates., Conclusions: This work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Although the investigation on the importance of mitochondria-derived reactive oxygen species (ROS) in endothelial function has been gaining momentum, little is known on the precise role of the individual components involved in the maintenance of a delicate ROS balance. Here we studied the impact of an ongoing dysregulated redox homeostasis by examining the effects of endothelial cell-specific deletion of murine thioredoxin reductase 2 (Txnrd2), a key enzyme of mitochondrial redox control., Approach and Results: We analyzed the impact of an inducible, endothelial cell-specific deletion of Txnrd2 on vascular remodeling in the adult mouse after femoral artery ligation. Laser Doppler analysis and histology revealed impaired angiogenesis and arteriogenesis. In addition, endothelial loss of Txnrd2 resulted in a prothrombotic, proinflammatory vascular phenotype, manifested as intravascular cellular deposits, as well as microthrombi. This phenotype was confirmed by an increased leukocyte response toward interleukin-1 in the mouse cremaster model. In vitro, we could confirm the attenuated angiogenesis measured in vivo, which was accompanied by increased ROS and an impaired mitochondrial membrane potential. Ex vivo analysis of femoral arteries revealed reduced flow-dependent vasodilation in endothelial cell Txnrd2-deficient mice. This endothelial dysfunction could be, at least partly, ascribed to inadequate nitric oxide signaling., Conclusions: We conclude that the maintenance of mitochondrial ROS via Txnrd2 in endothelial cells is necessary for an intact vascular homeostasis and remodeling and that Txnrd2 plays a vitally important role in balancing mitochondrial ROS production in the endothelium., (© 2016 American Heart Association, Inc.)

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-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 Ca(2+)-dependent KCa3.1 Gardos 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 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) 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., (© FASEB.)