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.

Platelet-localized ST6Gal1 does not impact IgG sialylation.

The IgG antibody class forms an important basis of the humoral immune response, conferring reciprocal protection from both pathogens and autoimmunity. IgG function is determined by the IgG subclass, as defined by the heavy chain, as well as the glycan composition at N297, the conserved site of N-glycosylation within the Fc domain. For example, lack of core fucose promotes increased antibody-dependent cellular cytotoxicity, whereas α2,6-linked sialylation by the enzyme ST6Gal1 helps to drive immune quiescence. Despite the immunological significance of these carbohydrates, little is known about how IgG glycan composition is regulated. We previously reported that mice with ST6Gal1-deficient B cells have unaltered IgG sialylation. Likewise, ST6Gal1 released into the plasma by hepatocytes does not significantly impact overall IgG sialylation. Since IgG and ST6Gal1 have independently been shown to exist in platelet granules, it was possible that platelet granules could serve as a B cell-extrinsic site for IgG sialylation. To address this hypothesis, we used a platelet factor 4 (Pf4)-Cre mouse to delete ST6Gal1 in megakaryocytes and platelets alone or in combination with an albumin-Cre mouse to also remove it from hepatocytes and the plasma. The resulting mouse strains were viable and had no overt pathological phenotype. We also found that despite targeted ablation of ST6Gal1, no change in IgG sialylation was apparent. Together with our prior findings, we can conclude that in mice, neither B cells, the plasma, nor platelets have a substantial role in homeostatic IgG sialylation.

The complementary roles of VAMP-2, -3, and -7 in platelet secretion and function.

Platelet secretion requires Soluble N-ethylmaleimide Sensitive Attachment Protein Receptors (SNAREs). Vesicle SNAREs/Vesicle-Associated Membrane Proteins (v-SNAREs/VAMPs) on granules and t-SNAREs in plasma membranes mediate granule release. Platelet VAMP heterogeneity has complicated the assessment of how/if each is used and affects hemostasis. To address the importance of VAMP-7 (V7), we analyzed mice with global deletions of V3 and V7 together or platelet-specific deletions of V2, V3, and global deletion of V7. We measured the kinetics of cargo release, and its effects on three injury models to define the context-specific roles of these VAMPs. Loss of V7 minimally affected dense and α granule release but did affect lysosomal release. V3-/-7-/- and V2Δ3Δ7-/- platelets showed partial defects in α and lysosomal release; dense granule secretion was unaffected. In vivo assays showed that loss of V2, V3, and V7 caused no bleeding or occlusive thrombosis. These data indicate a role for V7 in lysosome release that is partially compensated by V3. V7 and V3, together, contribute to α granule release, however none of these deletions affected hemostasis/thrombosis. Our results confirm the dominance of V8. When it is present, deletion of V2, V3, or V7 alone or in combination minimally affects platelet secretion and hemostasis.

What did we know? V8 is the primary VAMP isoform for platelet granule secretion, but V2 and V3 play compensatory roles.V3 is important for platelet endocytosis.V7 plays a minimal role in secretion and does not affect hemostasis.What did we discover? The loss of both V3 and V7 increases α and lysosomal secretion defects.Platelet-specific deletion of V2 and V3 with global V7-deletion causes defective α and lysosomal release.Secretion deficiencies in V3^−/−7^−/− and V2^Δ3^Δ7^−/− have no effect on hemostasis or thrombosis.What is the impact? We show that endosomal v-SNAREs (V3 and V7) play minor roles in secretion.V3^−/−7^−/− and platelet-specific V2^Δ3^Δ7^−/− mice are viable and will be valuable in in vivo studies of membrane trafficking.

α-Synuclein is the major platelet isoform but is dispensable for activation, secretion, and thrombosis.

Platelets play many roles in the vasculature ensuring proper hemostasis and maintaining integrity. These roles are facilitated, in part, by cargo molecules released from platelet granules via Soluble NSF Attachment Protein Receptor (SNARE) mediated membrane fusion, which is controlled by several protein-protein interactions. Chaperones have been characterized for t-SNAREs (i.e. Munc18b for Syntaxin-11), but none have been clearly identified for v-SNAREs. α-Synuclein has been proposed as a v-SNARE chaperone which may affect SNARE-complex assembly, fusion pore opening, and thus secretion. Despite its abundance and that it is the only isoform present, α-synuclein's role in platelet secretion is uncharacterized. In this study, immunofluorescence showed that α-synuclein was present on punctate structures that co-stained with markers for α-granules and lysosomes and in a cytoplasmic pool. We analyzed the phenotype of α-synuclein-/- mice and their platelets. Platelets from knockout mice had a mild, agonist-dependent secretion defect but aggregation and spreading in vitro were unaffected. Consistently, thrombosis/hemostasis were unaffected in the tail-bleeding, FeCl3 carotid injury and jugular vein puncture models. None of the platelet secretory machinery examined, e.g. the v-SNAREs, were affected by α-synuclein's loss. The results indicate that, despite its abundance, α-synuclein has only a limited role in platelet function and thrombosis.

What did we know? The N-terminus of α-Synuclein affects SNARE-complex assembly, fusion pore opening, and granule docking.Microvascular bleeding is seen in Parkinson Disease patients where α-synuclein has a pathological role.What did we discover? α-Synuclein colocalizes with P-selectin (α-granules) and LAMP-1 (lysosomes) in platelets.The loss of α-synuclein has only a mild, agonist-dependent effect on platelet secretion.The loss of α-synuclein had no effect on thrombosis/hemostasis in 3 injury models.What is the impact? Despite its abundance, α-synuclein is not required for platelet secretion.α-Synuclein is not required for hemostasis or thrombosis.

Platelet glycogenolysis is important for energy production and function.

Although the presence of glycogen in platelets was established in the 1960s, its importance to specific functions (i.e., activation, secretion, aggregation, and clot contraction) remains unclear. Patients with glycogen storage disease often present with increased bleeding and glycogen phosphorylase (GP) inhibitors, when used as treatments for diabetes, induce bleeding in preclinical studies suggesting some role for this form of glucose in hemostasis. In the present work, we examined how glycogen mobilization affects platelet function using GP inhibitors (CP316819 and CP91149) and a battery of ex vivo assays. Blocking GP activity increased glycogen levels in resting and thrombin-activated platelets and inhibited platelet secretion and clot contraction, with minimal effects on aggregation. Seahorse energy flux analysis and metabolite supplementation experiments suggested that glycogen is an important metabolic fuel whose role is affected by platelet activation and the availability of external glucose and other metabolic fuels. Our data shed light on the bleeding diathesis in glycogen storage disease patients and offer insights into the potential effects of hyperglycemia on platelets.

What did we know? Activated platelets transition from a low-energy-requiring, resting state to a high-energy-demanding state.Platelet glycogen is degraded upon activation.Glycogen storage disorders and glycogen phosphorylase inhibitors are associated with bleeding.What did we discover? Glycogen turnover occurs in resting platelets and its degradation is important for platelet functions.Glycogen phosphorylase inhibitors block secretion and clot contraction of which the latter can be reversed with alternative metabolic fuels.Glucose derived from glycogen may be routed through TCA/OxPhos versus aerobic glycolysis.What is the impact? Glycogen breakdown contributes to the high energy requirements of platelet function.Our work offers insights into potential energy sources in activated platelets.

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.

COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation.

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

VAMP3 and VAMP8 Regulate the Development and Functionality of Parasitophorous Vacuoles Housing Leishmania amazonensis.

To colonize mammalian phagocytic cells, the parasite Leishmania remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are poorly understood. In this study, we investigated the contribution of host cell soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins to the expansion and functionality of communal vacuoles as well as the replication of the parasite. The differential patterns of recruitment of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor to communal vacuoles harboring Leishmania amazonensis and to individual vacuoles housing L. major led us to further investigate the roles of VAMP3 and VAMP8 in the interaction of Leishmania with its host cell. We show that whereas VAMP8 contributes to the optimal expansion of communal vacuoles, VAMP3 negatively regulates L. amazonensis replication, vacuole size, as well as antigen cross-presentation. In contrast, neither protein has an impact on the fate of L. major. Collectively, our data support a role for both VAMP3 and VAMP8 in the development and functionality of L. amazonensis-harboring communal parasitophorous vacuoles.

Calcium Ion Chelation Preserves Platelet Function During Cold Storage.

OBJECTIVE: Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin ß3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss. CONCLUSIONS: Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

Platelet-HIV: interactions and their implications.

While it is clear that platelets interact with viruses, the ramifications and mechanisms of those interactions are still being defined for each type of viral infection. HIV/AIDS+ represents a potentially unique example of how viremia affects platelets since the increasing efficacy of antiretroviral therapeutics (ART) has made it a chronic disease that increases the risk of cardiovascular disease. In this opinion article, we discuss some of the open questions about how platelets interact with HIV. What happens to a virion once it binds a platelet? What is the nature of virus-induced platelet activation? Are platelets a normal part of the immune response to viremia that has been co-opted to increase the spread of HIV? The answers to these and similar questions will help define how platelet-directed therapeutics might be used in treating HIV/AIDS+ patients.

Does GEC1 Enhance Expression and Forward Trafficking of the Kappa Opioid Receptor (KOR) via Its Ability to Interact with NSF Directly?

We reported previously that GEC1 (glandular epithelial cell 1), a member of microtubule-associated proteins (MAPs), interacted directly with the C-tail of KOR (KCT) and tubulin and enhanced cell surface expression of KOR in CHO cells by facilitating its trafficking along the export pathway. Two GEC1 analogs (GABARAP and GATE16) were also shown to increase KOR expression. In addition, to understand the underlying mechanism, we demonstrated that N-ethylmaleimide-sensitive factor (NSF), an essential component for membrane fusion, co-immunoprecipitated with GEC1 from brain extracts. In this study, using pull-down techniques, we have found that (1) GEC1 interacts with NSF directly and prefers the ADP-bound NSF to the ATP-bound NSF; (2) D1 and/or D2 domain(s) of NSF interact with GEC1, but the N domain of NSF does not; (3) NSF does not interact with KCT directly, but forms a protein complex with KCT via GEC1; (4) NSF and/or α-SNAP do not affect KCT-GEC1 interaction. Thus, GEC1 (vs the α-SNAP/SNAREs complex) binds to NSF in distinctive ways in terms of the ADP- or ATP-bound form and domains of NSF involved. In conclusion, GEC1 may, via its direct interactions with KOR, NSF, and tubulin, enhance trafficking and fusion of KOR-containing vesicles selectively along the export pathway, which leads to increase in surface expression of KOR. GABARAP and GATE16 may enhance KOR expression in a similar way.

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.

Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis.

The canalicular system (CS) has been defined as: 1) an inward, invaginated membrane connector that supports entry into and exit from the platelet; 2) a static structure stable during platelet isolation; and 3) the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3. Qualitative and quantitative analysis of whole platelet reconstructions, obtained from immediately fixed or washed platelets fixed post-washing, indicated that CS, even in the presence of activation inhibitors, reorganized during platelet isolation to generate a more interconnected network. Further, CS redistribution into the PM at different times, post-activation, appeared to account for only about half the PM expansion seen in thrombin-activated platelets, in vitro, suggesting that CS reorganization is not sufficient to serve as a dominant membrane reservoir for activated platelets. In sum, our analysis highlights the need to revisit past assumptions about the platelet CS to better understand how this membrane system contributes to platelet function.

Structural analysis of resting mouse platelets by 3D-EM reveals an unexpected variation in α-granule shape.

Mice and mouse platelets are major experimental models for hemostasis and thrombosis; however, important physiological data from this model has received little to no quantitative, 3D ultrastructural analysis. We used state-of-the-art, serial block imaging scanning electron microscopy (SBF-SEM, nominal Z-step size was 35 nm) to image resting platelets from C57BL/6 mice. α-Granules were identified morphologically and rendered in 3D space. The quantitative analysis revealed that mouse α-granules typically had a variable, elongated, rod shape, different from the round/ovoid shape of human α-granules. This variation in length was confirmed qualitatively by higher-resolution, focused ion beam (FIB) SEM at a nominal 5 nm Z-step size. The unexpected α-granule shape raises novel questions regarding α-granule biogenesis and dynamics. Does the variation arise at the level of the megakaryocyte and α-granule biogenesis or from differences in α-granule dynamics and organelle fusion/fission events within circulating platelets? Further quantitative analysis revealed that the two major organelles in circulating platelets, α-granules and mitochondria, displayed a stronger linear relationship between organelle number/volume and platelet size, i.e., a scaling in number and volume to platelet size, than found in human platelets suggestive of a tighter mechanistic regulation of their inclusion during platelet biogenesis. In conclusion, the overall spatial arrangement of organelles within mouse platelets was similar to that of resting human platelets, with mouse α-granules clustered closely together with little space for interdigitation of other organelles.

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis.

Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent lipid rafts when platelets are activated. Using metabolic labeling and hydroxylamine (HA)/HCl treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on cysteine 275, 279, 280, 282, 283, and 285, and SNAP-23 was modified on cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [3H]palmitate into the t-SNAREs increased although the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [3H]palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release, as well as P-selectin exposure in platelets treated with the acyltransferase inhibitor cerulenin or the thioesterase inhibitor palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose- and time-dependent manner whereas palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin, suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.

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.

In vivo modeling of docosahexaenoic acid and eicosapentaenoic acid-mediated inhibition of both platelet function and accumulation in arterial thrombi.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are associated with a variety of cellular alterations that mitigate cardiovascular disease. However, pinpointing the positive therapeutic effects is challenging due to inconsistent clinical trial results and overly simplistic in vitro studies. Here we aimed to develop realistic models of n-3 PUFA effects on platelet function so that preclinical results can better align with and predict clinical outcomes. Human platelets incubated with the n-3 PUFAs docosahexaenoic acid and eicosapentaenoic acid were stimulated with agonist combinations mirroring distinct regions of a growing thrombus. Platelet responses were then monitored in a number of ex-vivo functional assays. Furthermore, intravital microscopy was used to monitor arterial thrombosis and fibrin deposition in mice fed an n-3 PUFA-enriched diet. We found that n-3 PUFA treatment had minimal effects on many basic ex-vivo measures of platelet function using agonist combinations. However, n-3 PUFA treatment delayed platelet-derived thrombin generation in both humans and mice. This impaired thrombin production paralleled a reduced platelet accumulation within thrombi formed in either small arterioles or larger arteries of mice fed an n-3 PUFA-enriched diet, without impacting P-selectin exposure. Despite an apparent lack of robust effects in many ex-vivo assays of platelet function, increased exposure to n-3 PUFAs reduces platelet-mediated thrombin generation and attenuates elements of thrombus formation. These data support the cardioprotective value of-3 PUFAs and strongly suggest that they modify elements of platelet function in vivo.

B-cell-independent sialylation of IgG.

IgG carrying terminal α2,6-linked sialic acids added to conserved N-glycans within the Fc domain by the sialyltransferase ST6Gal1 accounts for the anti-inflammatory effects of large-dose i.v. Ig (IVIg) in autoimmunity. Here, B-cell-specific ablation of ST6Gal1 in mice revealed that IgG sialylation can occur in the extracellular environment of the bloodstream independently of the B-cell secretory pathway. We also discovered that secreted ST6Gal1 is produced by cells lining central veins in the liver and that IgG sialylation is powered by serum-localized nucleotide sugar donor CMP-sialic acid that is at least partially derived from degranulating platelets. Thus, antibody-secreting cells do not exclusively control the sialylation-dependent anti-inflammatory function of IgG. Rather, IgG sialylation can be regulated by the liver and platelets through the corresponding release of enzyme and sugar donor into the cardiovascular circulation.

Arf6 controls platelet spreading and clot retraction via integrin αIIbß3 trafficking.

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbß3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbß3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbß3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function.