TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation.

Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly understood. Here, we show that MHC-I selectively accumulate within phagosomes carrying microbial components, which engage Toll-like receptor (TLR) signaling. Although cross-presentation requires Sec22b-mediated phagosomal recruitment of the peptide loading complex from the ER-Golgi intermediate compartment (ERGIC), this step is independent of TLR signaling and does not deliver MHC-I. Instead, MHC-I are recruited from an endosomal recycling compartment (ERC), which is marked by Rab11a, VAMP3/cellubrevin, and VAMP8/endobrevin and holds large reserves of MHC-I. While Rab11a activity stocks ERC stores with MHC-I, MyD88-dependent TLR signals drive IκB-kinase (IKK)2-mediated phosphorylation of phagosome-associated SNAP23. Phospho-SNAP23 stabilizes SNARE complexes orchestrating ERC-phagosome fusion, enrichment of phagosomes with ERC-derived MHC-I, and subsequent cross-presentation during infection.

Platelets Endocytose Viral Particles and Are Activated via TLR (Toll-Like Receptor) Signaling.

OBJECTIVE: Thrombocytopenia is associated with many viral infections suggesting virions interact with and affect platelets. Consistently, viral particles are seen inside platelets, and platelet activation markers are detected in viremic patients. In this article, we sought mechanistic insights into these virion/platelet interactions by examining how platelets endocytose, traffic, and are activated by a model virion. Approach and Results: Using fluorescently tagged HIV-1 pseudovirions, 3-dimensional structured illumination microscopy, and transgenic mouse models, we probed the interactions between platelets and virions. Mouse platelets used known endocytic machinery, that is, dynamin, VAMP (vesicle-associated membrane protein)-3, and Arf6 (ADP-ribosylation factor 6), to take up and traffic HIV-1 pseudovirions. Endocytosed HIV-1 pseudovirions trafficked through early (Rab4+) and late endosomes (Rab7+), and then to an LC3+ (microtubule-associated protein 1A/1B-light chain 3) compartment. Incubation with virions induced IRAK4 (interleukin 1 receptor-associated kinase 4), Akt (protein kinase B), and IKK (IκB kinase) activation, granule secretion, and platelet-leukocyte aggregate formation. This activation required TLRs (Toll-like receptors) and MyD88 (myeloid differentiation primary response protein 88) but was less extensive and slower than activation with thrombin. In vivo, HIV-1 pseudovirions injection led to virion uptake and platelet activation, as measured by IKK activation, platelet-leukocyte aggregate formation, and mild thrombocytopenia. All were decreased in VAMP-3-/- and, megakaryocyte/platelet-specific, Arf6-/- mice. Similar platelet activation profiles (increased platelet-leukocyte aggregates, plasma platelet factor 4, and phospho-IκBα) were detected in newly diagnosed and antiretroviral therapy-controlled HIV-1+ patients. CONCLUSIONS: Collectively, our data provide mechanistic insights into the cell biology of how platelets endocytose and process virions. We propose a mechanism by which platelets sample the circulation and respond to potential pathogens that they take up.

Cellubrevin/vesicle-associated membrane protein-3-mediated endocytosis and trafficking regulate platelet functions.

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbß3, αvß3), and purinergic receptors (P2Y1, P2Y12), and thus normal platelet function. However, the molecular machinery required and possible trafficking routes are still ill-defined. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing, soluble N-ethylmaleimide factor attachment protein receptor (v-SNARE) called cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) in platelet function. Although not required for normal platelet exocytosis or hemostasis, VAMP-3-/- mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Other granule markers were unaffected. Direct experiments, both in vitro and in vivo, showed that loss of VAMP-3 led to a robust defect in uptake/storage of Fg in platelets and cultured megakaryocytes. Uptake of the fluid-phase marker, dextran, was only modestly affected. Time-dependent uptake and endocytic trafficking of Fg and dextran were followed using 3-dimensional-structured illumination microscopy. Dextran uptake was rapid compared with Fg, but both cargoes progressed through Rab4+, Rab11+, and von Willebrand factor (VWF)+ compartments in wild-type platelets in a time-dependent manner. In VAMP-3-/- platelets, the 2 cargoes showed limited colocalization with Rab4, Rab11, or VWF. Loss of VAMP-3 also affected some acute platelet functions, causing enhanced spreading on Fg and fibronectin and faster clot retraction compared with wild-type. In addition, the rate of Janus kinase 2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3-/- platelets. Collectively, our studies show that platelets are capable of a range of endocytosis steps, with VAMP-3 being pivotal in these processes.

The ins and outs of endocytic trafficking in platelet functions.

PURPOSE OF REVIEW: Although platelet endocytosis has been recognized in granule cargo loading and the trafficking of several platelet surface receptors, its acute physiological relevance is poorly understood as is its mechanism. The present review discusses the current understanding of platelet endocytosis and its implications for platelet function. RECENT FINDINGS: Recent studies are beginning to identify and define the proteins that mediate platelet endocytosis. These studies have shown that platelets contain different endosomal compartments and may use multiple endocytic routes to take in circulating molecules and surface proteins. The studies have also shown that platelet endocytosis is involved in several aspects of platelet function such as signaling, spreading, and granule cargo loading. SUMMARY: Mechanistic studies of platelet endocytosis have shown it to be not only involved in granule cargo loading but also in various other platelet functions important for hemostasis and beyond.

Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis.

Autophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling cascades and requires proteases, acidic compartments, and membrane fusion. To assess the physiological role of platelet autophagy, we generated a mouse strain with a megakaryocyte- and platelet-specific deletion of Atg7, an enzyme required for LC3II production. Ex vivo analysis of platelets from these mice shows modest defects in aggregation and granule cargo packaging. Although these mice have normal platelet numbers and size distributions, they exhibit a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Our results demonstrate that autophagy occurs in platelets and is important for hemostasis and thrombosis.

IκB kinase phosphorylation of SNAP-23 controls platelet secretion.

Platelet secretion plays a key role in thrombosis, thus the platelet secretory machinery offers a unique target to modulate hemostasis. We report the regulation of platelet secretion via phosphorylation of SNAP-23 at Ser95. Phosphorylation of this t-soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) occurs upon activation of known elements of the platelet signaling cascades (ie, phospholipase C, [Ca(2+)]i, protein kinase C) and requires IκB kinase (IKK)-ß. Other elements of the nuclear factor κB/IκB cascade (ie, IKK-α,-ß,-γ/NEMO and CARMA/MALT1/Bcl10 complex) are present in anucleate platelets and IκB is phosphorylated upon activation, suggesting that this pathway is active in platelets and implying a nongenomic role for IKK. Inhibition of IKK-ß, either pharmacologically (with BMS-345541, BAY11-7082, or TPCA-1) or by genetic manipulation (platelet factor 4 Cre:IKK-ß(flox/flox)), blocked SNAP-23 phosphorylation, platelet secretion, and SNARE complex formation; but, had no effect on platelet morphology or other metrics of platelet activation. Consistently, SNAP-23 phosphorylation enhanced membrane fusion of SNARE-containing proteoliposomes. In vivo studies with IKK inhibitors or platelet-specific IKK-ß knockout mice showed that blocking IKK-ß activity significantly prolonged tail bleeding times, suggesting that currently available IKK inhibitors may affect hemostasis.

Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity.

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Total Plasma Protein S Is a Prothrombotic Marker in People Living With HIV.

BACKGROUND: HIV-1 infection is associated with multiple procoagulant changes and increased thrombotic risk. Possible mechanisms for this risk include heigthened expression of procoagulant tissue factor (TF) on circulating monocytes, extracellular vesicles, and viral particles and/or acquired deficiency of protein S (PS), a critical cofactor for the anticoagulant protein C (PC). PS deficiency occurs in up to 76% of people living with HIV-1 (PLWH). As increased ex vivo plasma thrombin generation is a strong predictor of mortality, we investigated whether PS and plasma TF are associated with plasma thrombin generation. METHODS: We analyzed plasma samples from 9 healthy controls, 17 PLWH on first diagnosis (naive), and 13 PLWH on antiretroviral therapy (ART). Plasma thrombin generation, total and free PS, PC, C4b-binding protein, and TF activity were measured. RESULTS: We determined that the plasma thrombin generation assay is insensitive to PS, because of a lack of PC activation, and developed a modified PS-sensitive assay. Total plasma PS was reduced in 58% of the naive and 38% of the ART-treated PLWH samples and correlated with increased thrombin generation in the modified assay. Conversely, plasma TF was not increased in our patient population, suggesting that it does not significantly contribute to ex vivo plasma thrombin generation. CONCLUSION: These data suggest that reduced total plasma PS contributes to the thrombotic risk associated with HIV-1 infection and can serve as a prothrombotic biomarker. In addition, our refined thrombin generation assay offers a more sensitive tool to assess the functional consequences of acquired PS deficiency in PLWH.

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

In search of the 'self': Holistic rehabilitation in restoring cognition and recovering the 'self' following traumatic brain injury: A case report.

BACKGROUND: Following mild-moderate traumatic brain injury (TBI), an individual experiences a range of emotional changes. It is often difficult for the patient to reconcile with their post-injury persona, and the memory of pre-injury personhood is particularly painful. Insight into one's cognitive deficits subsequent to injury can lead to an existential crisis and a sense of loss, including loss of self. OBJECTIVE: Restoration of cognitive functions and reconciliation with loss of pre-traumatic personhood employing a holistic method of neuropsychological rehabilitation in a patient suffering from TBI. METHODS: Ms. K.S, a 25-year-old female, presented with emotional disturbances following TBI. She reported both retrograde and anterograde amnesia. A multidimensional holistic rehabilitation was planned. Treatment addressed cognitive deficits through the basic functions approach. Cognitive behavioural methods for emotional regulation like diary writing helped reduce irritability and anger outbursts. Use of social media created new modes of memory activation and interactions. Compensatory strategies were used to recover lost skills, music-based attention training helped foster an individualised approach to the sense of one's body and self. RESULTS: As a result of these differing strategies, changes were reflected in neuro-psychological tests, depression score and the patient's self-evaluation. This helped generate a coherent self-narrative. CONCLUSION: Treatment challenges in such cases are increased due to patient's actual deficits caused by neuronal/biochemical changes. Innovative and multi-pronged rehabilitation strategies which involve everyday activities provided an answer to some of these problems. This method of rehabilitation may provide an optimistic context for future research.

Differential Leukocyte and Platelet Profiles in Distinct Models of Traumatic Brain Injury.

Traumatic brain injury (TBI) affects over 3 million individuals every year in the U.S. There is growing appreciation that TBI can produce systemic modifications, which are in part propagated through blood-brain barrier (BBB) dysfunction and blood-brain cell interactions. As such, platelets and leukocytes contribute to mechanisms of thromboinflammation after TBI. While these mechanisms have been investigated in experimental models of contusion brain injury, less is known regarding acute alterations following mild closed head injury. To investigate the role of platelet dynamics and bioenergetics after TBI, we employed two distinct, well-established models of TBI in mice: the controlled cortical impact (CCI) model of contusion brain injury and the closed head injury (CHI) model of mild diffuse brain injury. Hematology parameters, platelet-neutrophil aggregation, and platelet respirometry were assessed acutely after injury. CCI resulted in an early drop in blood leukocyte counts, while CHI increased blood leukocyte counts early after injury. Platelet-neutrophil aggregation was altered acutely after CCI compared to sham. Furthermore, platelet bioenergetic coupling efficiency was transiently reduced at 6 h and increased at 24 h post-CCI. After CHI, oxidative phosphorylation in intact platelets was reduced at 6 h and increased at 24 h compared to sham. Taken together, these data demonstrate that brain trauma initiates alterations in platelet-leukocyte dynamics and platelet metabolism, which may be time- and injury-dependent, providing evidence that platelets carry a peripheral signature of brain injury. The unique trend of platelet bioenergetics after two distinct types of TBI suggests the potential for utilization in prognosis.

An unusual cyanobacterium from saline thermal waters with relatives from unexpected habitats.

Cyanobacteria that grow above seawater salinity at temperatures above 45 degrees C have rarely been studied. Cyanobacteria of this type of thermo-halophilic extremophile were isolated from siliceous crusts at 40-45 degrees C in a geothermal seawater lagoon in southwest Iceland. Iceland Clone 2e, a Leptolyngbya morphotype, was selected for further study. This culture grew only at 45-50 degrees C, in medium ranging from 28 to 94 g L(-1) TDS, It showed 3 doublings 24 h(-1) under continuous illumination. This rate at 54 degrees C was somewhat reduced, and death occurred at 58 degrees C. A comparison of the 16S rDNA sequence with all others in the NCBI database revealed 2 related Leptolyngbya isolates from a Greenland hot spring (13-16 g L(-1) TDS). Three other similar sequences were from Leptolyngbya isolates from dry, endolithic habitats in Yellowstone National Park. All 6 formed a phylogenetic clade, suggesting common ancestry. These strains shared many similarities to Iceland Clone 2e with respect to temperature and salinity ranges and optima. Two endolithic Leptolyngbya isolates, grown previously at 23 degrees C in freshwater medium, grew well at 50 degrees C but only in saline medium. This study shows that limited genotypic similarity may reveal some salient phenotypic similarities, even when the related cyanobacteria are from vastly different and remote habitats.

Augmentation of IL-6 production contributes to development of gestational diabetes mellitus: An Indian study.

AIM: Inflammatory mediators like interleukin-6 (IL-6) and acute phase protein like C-reactive protein (CRP) are supposed to contribute to development of GDM, however clinical data supporting this hypothesis is limited. This study was designed to analyze the association of IL-6 and CRP with development of GDM in Indian females. METHODS: This case control study included pregnant women diagnosed as GDM (n = 53) and those having normal glucose tolerance (n = 50). Serum levels of IL-6 and CRP were analysed and correlated with various clinical parameters. RESULTS: Serum IL-6 levels were significantly high (p < 0.05) in GDM females as compared to control females. IL-6 levels correlated with pre-pregnancy body mass index (BMI), fasting blood sugar (FBS) and postprandial sugar (PPBS). Unlike IL-6, CRP levels did not show significant differences between GDM and control females. However, positive correlation of CRP levels with BMI, FBS and PPBS was observed. CONCLUSION: High IL-6 levels in gestational diabetes may indicate a possible role for inflammation in pathophysiology of GDM.

Autophagy in Platelets.

Anucleate platelets are produced by fragmentation of megakaryocytes. Platelets circulate in the bloodstream for a finite period: upon vessel injury, they are activated to participate in hemostasis; upon senescence, unused platelets are cleared. Platelet hypofunction leads to bleeding. Conversely, pathogenic platelet activation leads to occlusive events that precipitate strokes and heart attacks. Recently, we and others have shown that autophagy occurs in platelets and is important for platelet production and normal functions including hemostasis and thrombosis. Due to the unique properties of platelets, such as their lack of nuclei and their propensity for activation, methods for studying platelet autophagy must be specifically tailored. Here, we describe useful methods for examining autophagy in both human and mouse platelets.

Alterations in platelet secretion differentially affect thrombosis and hemostasis.

We genetically manipulated the major platelet vesicle-associated membrane proteins (VAMP2, VAMP3, and VAMP8) to create mice with varying degrees of disrupted platelet secretion. As previously shown, loss of VAMP8 reduced granule secretion, and this defect was exacerbated by further deletion of VAMP2 and VAMP3. VAMP2Δ3Δ8-/- platelets also had reduced VAMP7. Loss of VAMP2 and VAMP3 (VAMP2Δ3Δ) had a minimal impact on secretion when VAMP7 and VAMP8 were present. Integrin αIIbß3 activation and aggregation were not affected, although spreading was reduced in VAMP2Δ3Δ8-/- platelets. Using these mice as tools, we asked how much secretion is needed for proper thrombosis and hemostasis in vivo. VAMP2Δ3Δ mice showed no deficiency, whereas VAMP8-/- mice had attenuated formation of occlusive thrombi upon FeCl3-induced arterial injury but no excessive bleeding upon tail transection. VAMP2Δ3Δ8-/- mice bled profusely and failed to form occlusive thrombi. Plasma-coagulation factors were normal in all of the strains, but phosphatidylserine exposure was reduced in VAMP2Δ3Δ and VAMP2Δ3Δ8-/- platelets. From our data, an ∼40% to 50% reduction in platelet secretion in vitro (dense and α granule) correlated with reduced occlusive thrombosis but no compromise in hemostasis. At a >50% reduction, thrombosis and hemostasis were defective in vivo. Our studies are the first systematic manipulation of platelet exocytic machinery to demonstrate a quantitative linkage between in vitro platelet secretion and hemostasis and thrombosis in vivo. The animals described will be invaluable tools for future investigations into how platelet secretion affects other vascular processes.

Scavenging of nickel and chromium toxicity in Aulosira fertilissima by immobilization: effect on nitrogen assimilating enzymes

The ubiquity of heavy metals in the biosphere results in the introduction of high amounts of toxic metals into the food chain from various sources. In the present study, one of the strongest nitrogen fixing cyanobacterium of the rice fields, Aulosira fertilissima, was subjected to nickel and chromium stress and the ameliorating effect of immobilization was investigated. Cell immobilization could protect the organism's growth against the toxicity of both heavy metals at LC50 as compared to lethal concentrations. The nitrate reductase activity in free cells treated with the metals was substantially inhibited but immobilized cells treated with 0.1 ppm nickel was not affected by the metal treatment. Cell immobilization also resulted in a significant protection against sub-lethal concentration of chromium but to a lesser degree than it did with sub- lethal levels of nickel. Control immobilized cells also had higher Nitrogenase activity than control free cells. Nickel and chromium addition markedly decreased the enzyme activity in free cells but immobilized cells exposed to sublethal concentrations of both metals could overcome this decrease. Glutamine synthetase showed similar response under immobilized conditions compared to free cells with both metals. The addition of algal filtrate in 3:1 ratio further increased the nitrogenase activity compared with immobilized cells treated with sublethal doses of both metals. Immobilization facilitated higher uptake of nickel as compared to chromium. The observations of the present study clearly demonstrate the protective effect of immobilization on Aulosira fertilissima against Nickel and chromium toxicity. Rice field ecosystem thus possess a bidirectional natural metal ameliorating system where Aulosira mats act as a naturally immobilized system and the decay of Aulosira along with other cyanobacteria act as natural chelators protecting the rice plants from deleterious effects of the heavy metals. Most importantly is...