Impact of Surface Ligand on the Biocompatibility of InP/ZnS Quantum Dots with Platelets.

InP/ZnS quantum dots (QDs) have received a large focus in recent years as a safer alternative to heavy metal-based QDs. Given their intrinsic fluorescent imaging capabilities, these QDs can be potentially relevant for in vivo platelet imaging. The InP/ZnS QDs are synthesized and their biocompatibility investigated through the use of different phase transfer agents. Analysis of platelet function indicates that platelet-QD interaction can occur at all concentrations and for all QD permutations tested. However, as the QD concentration increases, platelet aggregation is induced by QDs alone independent of natural platelet agonists. This study helps to define a range of concentrations and coatings (thioglycolic acid and penicillamine) that are biocompatible with platelet function. With this information, the platelet-QD interaction can be identified using multiple methods. Fluorescent lifetime imaging microscopy (FLIM) and confocal studies have shown QDs localize on the surface of the platelet toward the center while showing evidence of energy transfer within the QD population. It is believed that these findings are an important stepping point for the development of fluorescent probes for platelet imaging.

Practical Considerations of Dissolved Oxygen Levels for Platelet Function under Hypoxia.

Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 108/mL versus 2 × 107/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.

CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane.

Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane.

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets.

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by human platelets, understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually, identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

Quantum Dot Imaging Agents: Haematopoietic Cell Interactions and Biocompatibility.

Quantum dots (QDs) are semi-conducting nanoparticles that have been developed for a range of biological and non-biological functions. They can be tuned to multiple different emission wavelengths and can have significant benefits over other fluorescent systems. Many studies have utilised QDs with a cadmium-based core; however, these QDs have since been shown to have poor biological compatibility. Therefore, other QDs, such as indium phosphide QDs, have been developed. These QDs retain excellent fluorescent intensity and tunability but are thought to have elevated biological compatibility. Herein we discuss the applicability of a range of QDs to the cardiovascular system. Key disease states such as myocardial infarction and stroke are associated with cardiovascular disease (CVD), and there is an opportunity to improve clinical imaging to aide clinical outcomes for these disease states. QDs offer potential clinical benefits given their ability to perform multiple functions, such as carry an imaging agent, a therapy, and a targeting motif. Two key cell types associated with CVD are platelets and immune cells. Both cell types play key roles in establishing an inflammatory environment within CVD, and as such aid the formation of pathological thrombi. However, it is unclear at present how and with which cell types QDs interact, and if they potentially drive unwanted changes or activation of these cell types. Therefore, although QDs show great promise for boosting imaging capability, further work needs to be completed to fully understand their biological compatibility.

Microplastics in human blood: Polymer types, concentrations and characterisation using µFTIR.

Microplastics (MPs) are an everyday part of life, and are now ubiquitous in the environment. Crucially, MPs have not just been found within the environment, but also within human bodies, including the blood. We aimed to provide novel information on the range of MP polymer types present, as well as their size and shape characteristics, in human whole blood from 20 healthy volunteers. Twenty-four polymer types were identified from 18 out of 20 (90 %) donors and quantified in blood, with the majority observed for the first time. Using an LOQ approach, five polymer types met the threshold with a lower mean ± SD of 2466 ± 4174 MP/L. The concentrations of plastics analysed in blood samples ranged from 1.84 - 4.65 µg/mL. Polyethylene (32 %), ethylene propylene diene (14 %), and ethylene-vinyl-acetate/alcohol (12 %) fragments were the most abundant. MP particles that were identified within the blood samples had a mean particle length of 127.99 ± 293.26 µm (7-3000 µm), and a mean particle width of 57.88 ± 88.89 µm (5-800 µm). The MPs were predominantly categorised as fragments (88 %) and were white/clear (79 %). A variety of plastic additive chemicals were identified including endocrine disrupting-classed phthalates. The procedural blank samples comprised 7 polymer types, that were distinct from those identified in blood, mainly resin (25 %), polyethylene terephthalate (17 %), and polystyrene (17 %) with a mean ± SD of 4.80 ± 5.59 MP/L. This study adds to the growing evidence that MPs are taken up into the human body and are transported via the bloodstream. The shape and sizes of the particles raise important questions with respect to their presence and associated hazards in terms of potential detrimental impacts such as vascular inflammation, build up within major organs, and changes to either immune cell response, or haemostasis and thrombosis.

The Arp2/3 activator WASH regulates α5ß1-integrin-mediated invasive migration.

The actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for α5ß1 integrin recycling. WASH regulates the trafficking of endosomal α5ß1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.

Should aspirin be replaced with ADP blockers and anti-GPVI to manage thrombosis?

Platelets have a pivotal role in maintaining cardiovascular homeostasis. They are kept docile by endothelial-derived mediators. Aberration in haemostatic balance predisposes an individual to an elevated risk of a prothrombotic environment. Anti-platelet therapy has been a key component to reduce this risk. However, understanding how these medications affect the balance between the activation and inhibition of platelets is critical. There is no evidence that a key anti-platelet therapy - aspirin, may not be the most efficacious medicine of choice, as it can compromise both platelet inhibition and activation pathways. In this review, the rationale of aspirin as an anti-thrombotic drug has been critically discussed. This review looks at how recently published trials are raising key questions about the efficacy and safety of aspirin in countering cardiovascular diseases. There is an increasing portfolio of evidence that identifies that although aspirin is a very cheap and accessible drug, it may be used in a manner that is not always beneficial to a patient, and a more nuanced and targeted use of aspirin may increase its clinical benefit and maximize patient response. The questions about the use of aspirin raise the potential for changes in its clinical use for dual anti-platelet therapy. This highlights the need to ensure that treatment is targeted in the most effective manner and that other anti-platelet therapies may well be more efficacious and beneficial for CVD patients in their standard and personalized approaches.

Platelet zinc status regulates prostaglandin-induced signaling, altering thrombus formation.

BACKGROUND: Approximately 17.3% of the global population exhibits an element of zinc (Zn2+) deficiency. One symptom of Zn2+ deficiency is increased bleeding through impaired hemostasis. Platelets are crucial to hemostasis and are inhibited by endothelial-derived prostacyclin (prostaglandin I2 [PGI2]), which signals via adenylyl cyclase (AC) and cyclic adenosine monophosphate signaling. In other cell types, Zn2+ modulates cyclic adenosine monophosphate concentrations by changing AC and/or phosphodiesterase activity. OBJECTIVES: To investigate if Zn2+ can modulate platelet PGI2 signaling. METHODS: Platelet aggregation, spreading, and western blotting assays with Zn2+ chelators and cyclic nucleotide elevating agents were performed in washed platelets and platelet-rich plasma conditions. In vitro thrombus formation with various Zn2+ chelators and PGI2 was assessed in whole blood. RESULTS: Incubation of whole blood or washed platelets with Zn2+ chelators caused either embolization of preformed thrombi or reversal of platelet spreading, respectively. To understand this effect, we analyzed resting platelets and identified that incubation with Zn2+ chelators elevated pVASPser157, a marker of PGI2 signaling. In agreement that Zn2+ affects PGI2 signaling, addition of the AC inhibitor SQ22536 blocked Zn2+ chelation-induced platelet spreading reversal, while addition of Zn2+ blocked PGI2-mediated platelet reversal. Moreover, Zn2+ specifically blocked forskolin-mediated AC reversal of platelet spreading. Finally, PGI2 inhibition of platelet aggregation and in vitro thrombus formation was potentiated in the presence of low doses of Zn2+ chelators, increasing its effectiveness in inducing platelet inhibition. CONCLUSION: Zn2+ chelation potentiates platelet PGI2 signaling, elevating PGI2's ability to prevent effective platelet activation, aggregation, and thrombus formation.

NIR-quantum dots in biomedical imaging and their future.

Fluorescence imaging has gathered interest over the recent years for its real-time response and high sensitivity. Developing probes for this modality has proven to be a challenge. Quantum dots (QDs) are colloidal nanoparticles that possess unique optical and electronic properties due to quantum confinement effects, whose excellent optical properties make them ideal for fluorescence imaging of biological systems. By selectively controlling the synthetic methodologies it is possible to obtain QDs that emit in the first (650-950 nm) and second (1000-1400 nm) near infra-red (NIR) windows, allowing for superior imaging properties. Despite the excellent optical properties and biocompatibility shown by some NIR QDs, there are still some challenges to overcome to enable there use in clinical applications. In this review, we discuss the latest advances in the application of NIR QDs in preclinical settings, together with the synthetic approaches and material developments that make NIR QDs promising for future biomedical applications.

Vav family proteins are required for optimal regulation of PLCgamma2 by integrin alphaIIbbeta3.

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.

Methods to Study the Roles of Rho GTPases in Platelet Function.

Platelets are a critical cell for prevention of bleeding. Part of the response to the formation of the thrombus is the activation of the actin cytoskeleton, with an inability to effectively activate the cytoskeleton linked to thrombus formation defects and instability. The control of this process is linked to activation of the Rho GTPases, Cdc42, Rac1, and RhoA, although additional small GTPases such as Rif and Rap have been shown to play roles in platelet function.Here we describe the methodology to accurately understand how Rho GTPases are activated in platelets. Due to the technical limitations of working with platelets, such as their lack of ability to be transfected, the majority of work has been carried out either using inhibitors of Rho GTPases or within knockout mouse models. Studies can be conducted both in suspension samples and in spread platelets. In suspension the platelets will undergo a shape change response, but will not be able to spread. In spread platelets it is possible to examine the effects of the matrix environment, such as concentration, type, and stiffness on Rho GTPase function within platelet activation and platelet spreading.

Studies on the actin-binding protein HS1 in platelets.

BACKGROUND: The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1-/- mice. RESULTS: The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1-/- mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay. CONCLUSION: This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.

Podosomes in adhesion, migration, mechanosensing and matrix remodeling.

Cells use various actin-based motile structures to allow them to move across and through matrix of varying density and composition. Podosomes are actin cytoskeletal structures that form in motile cells and that mediate adhesion to substrate, migration, and other specialized functions such as transmigration through cell and matrix barriers. The podosome is a unique and interesting entity, which appears in the light microscope as an individual punctum, but is linked to other podosomes like a node on a network of the underlying cytoskeleton. Here, we discuss the signals that control podosome assembly and dynamics in different cell types and the actin organising proteins that regulate both the inner actin core and integrin-rich surrounding ring structures. We review the structure and composition of podosomes and also their functions in various cell types of both myeloid and endothelial lineage. We also discuss the emerging idea that podosomes can sense matrix stiffness and enable cells to respond to their environment.

Actin on trafficking: could actin guide directed receptor transport?

Here, we present emerging ideas surrounding the interplay between the actin cytoskeleton and receptor transport and activation. The bulk of actin dynamics in cells is thought to contribute to architecture and mobility. Actin also contributes to trafficking, acting as a molecular scaffold, providing force to deform membranes, facilitating vesicle abscission or propelling a vesicle through the cytoplasm ( 1) (,) ( 2) and recent studies highlight important connections between the directed trafficking of receptors and the impact on cell migration and actin dynamics. Additionally, a number of newly described actin nucleation promoting factors, such as the vesicle associated protein WASH, reveal unexpected roles of actin in membrane traffic and suggest that the cell dedicates a significant proportion of its regulation of actin dynamics to controlling trafficking.

Lineage tracing of Pf4-Cre marks hematopoietic stem cells and their progeny.

The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15-60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage.

Tissue inducible Lifeact expression allows visualization of actin dynamics in vivo and ex vivo.

We describe here the development and characterization of a conditionally inducible mouse model expressing Lifeact-GFP, a peptide that reports the dynamics of filamentous actin. We have used this model to study platelets, megakaryocytes and melanoblasts and we provide evidence that Lifeact-GFP is a useful reporter in these cell types ex vivo. In the case of platelets and megakaryocytes, these cells are not transfectable by traditional methods, so conditional activation of Lifeact allows the study of actin dynamics in these cells live. We studied melanoblasts in native skin explants from embryos, allowing the visualization of live actin dynamics during cytokinesis and migration. Our study revealed that melanoblasts lacking the small GTPase Rac1 show a delay in the formation of new pseudopodia following cytokinesis that accounts for the previously reported cytokinesis delay in these cells. Thus, through use of this mouse model, we were able to gain insights into the actin dynamics of cells that could only previously be studied using fixed specimens or following isolation from their native tissue environment.

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.

WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

Analysis of intact phosphoinositides in biological samples.

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.