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Chirality-induced bacterial rheotaxis in bulk shear flows
Interaction of swimming bacteria with flows controls their ability to explore complex environments, crucial to many societal and environmental challenges and relevant for microfluidic applications as cell sorting. Combining experimental, numerical and theoretical analysis, we present a comprehensive study of the transport of motile bacteria in shear flows. Experimentally, we obtain with high accuracy and for a large range of flow rates, the spatially resolved velocity and orientation distributions. They are in excellent agreement with the simulations of a kinematic model accounting for stochastic and microhydrodynamic properties and in particular the flagella chirality. Theoretical analysis reveals the scaling laws behind the average rheotactic velocity at moderate shear rates using a chirality parameter and explains the reorientation dynamics leading to a saturation at large shear rates from the marginal stability of a fixed point. Our findings constitute a full understanding of the physical mechanisms and relevant parameters of bacteria bulk rheotaxis.
A cardiovascular disease-linked gut microbial metabolite acts via adrenergic receptors
Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and β2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.
Screen printed sensor design for thermal flow velocity measurement with intrinsic compensation of thermal fluid conductivity
For keeping control of processes in lab-on-chip applications, monitoring the flow velocity inside channels is an important task. One popular approach to this is to consider heat transfer inside the channel as a flow velocity-dependent effect, which can be measured by relatively easy means. Additionally, these thermal measurement methods can be designed and applied in a way that the sensors and actuators do not disturb the flow profile. A fabrication technique suitable to fabricate such sensors is screen printing, which combines cost-efficient and fast processing with layers thin enough to not alter the fluid flow. In this work, we report on a devised sensor that is fabricated by screen printing and embedded into a 3D printed channel. The sensor layout enables measuring the flow velocity in a microchannel while enabling the user to compensate for the change in thermal conductivity when switching to a different fluid. This design requires calibration only for one fluid since a simple empirical model is presented to extrapolate the calibration data to fluids with varying thermal properties. The sensor was tested for one calibration fluid and two fluids with differing thermal properties. The flow velocities of these fluids were measured with an error below 5%.
Increasing the relative permittivity of dielectric materials can be useful in many applications, including capacitive sensing in microfluidics. In order to be able to efficiently integrate capacitive sensors with high sensitivity into all-polymer microfluidic devices, polymeric layers with high dielectric constants are required. In this contribution, a dielectric coating made from a polymeric base with mixed in ceramic particles, which exhibits enhanced dielectric properties compared to the polymer itself, is presented. Poly(methyl methacrylate) is chosen as a polymeric base material due to its processing properties. Its relative permittivity is increased by mixing in barium titanate particles at concentrations ranging from 0 vol% to 90 vol%. The dielectric properties of each fabricated mixture are investigated and the results are compared to theoretical values derived from standard mixing rules. To demonstrate the sensitivity enhancement of sensors due to the use of the fabricated dielectric coating, a microfluidic device is presented featuring a capacitive sensor for detection of fluids with different dielectric constants. It is shown that the sensitivity of the capacitive sensor is significantly increased by using the custom dielectric mixture when compared to pure poly(methyl methacrylate).
Adenosine Receptor Agonists Exhibit Anti-platelet effects and the potential to overcome resistance to P2Y12 receptor antagonists
Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.
Myricetin, the main flavonoid in Syzygium cumini leaf, is a novel inhibitor of platelet thiol isomerases PDI and ERp5
Background: Flavonoids have been characterized as a prominent class of compounds to treat thrombotic diseases through the inhibition of thiol isomerases. Syzygium cumini is a flavonoid-rich medicinal plant that contains myricetin and gallic acid. Little is known about the potential anti-platelet properties of S. cumini and its constituent flavonoids. Objective: To evaluate the anti-platelet effects and mechanism of action of a polyphenol-rich extract (PESc) from S. cumini leaf and its most prevalent polyphenols, myricetin and gallic acid. Methods PESc, myricetin and gallic acid were incubated with platelet-rich plasma and washed platelets to assess platelet aggregation and activation. In vitro platelet adhesion and thrombus formation as well as in vivo bleeding time were performed. Finally, myricetin was incubated with recombinant thiol isomerases to assess its potential to bind and inhibit these, whilst molecular docking studies predicted possible binding sites. Results: PESc decreased platelet activation and aggregation induced by different agonists. Myricetin exerted potent anti-platelet effects, whereas gallic acid did not. Myricetin reduced the ability of platelets to spread on collagen, form thrombi in vitro without affecting haemostasis in vivo. Fluorescence quenching studies suggested myricetin binds to different thiol isomerases with similar affinity, despite inhibiting only protein disulphide isomerase (PDI) and ERp5 reductase activities (IC50~3.5 μM). Finally, molecular docking studies suggested myricetin formed non-covalent bonds with PDI and ERp5. Conclusions: PESc and its most abundant flavonoid myricetin strongly inhibit platelet function. Additionally, myricetin is a novel inhibitor of ERp5 and PDI, unveiling a new therapeutic perspective for the treatment of thrombotic disorders.
Low dose Btk inhibitors selectively block platelet activation by CLEC-2
The CD38low natural killer cell line KHYG1 transiently expressing CD16F158V in combination with daratumumab targets multiple myeloma cells with minimal effector NK cell fratricide
Multiple myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of the CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (MoAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells that mediate in vivo tumour immunosurveillance. NK cells also play an important role during MoAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their FcγRIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring variant of CD16 harbouring a single-point polymorphism (F158V). However, the contribution of NK cells to the efficacy of daratumumab remains debatable as clinical data clearly indicate the rapid depletion of CD38high peripheral blood NK cells in patients upon daratumumab administration. In contrast, CD38low peripheral blood NK cells have been shown to survive daratumumab mediated fratricide in vivo, while still retaining their potent anti-MM cytolytic effector functions ex vivo. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a “safe” mRNA electroporation-based approach on CD38low NK cells in combination with daratumumab could represent a novel therapeutic option for treatment of MM. In the present study, we investigate a NK cell line (KHYG-1), derived from a patient with aggressive NK cell leukemia, as a platform for generating CD38low NK cells expressing CD16F158V which can be administered as an “off-the-shelf” therapy to target both CD38high and CD38low tumour clones in patients receiving daratumumab.
A more efficient preparation system for HLA-eliminated platelets
BACKGROUND AND OBJECTIVES: Although HLA-eliminated platelets can facilitate transfusions to patients possessing HLA antibodies, no such products are currently available commercially perhaps because the platelet collection rate is not yet economically viable. We have improved this process' efficiency by employing a hollow-fibre system at the last step of the production process after an acid and a reaction buffer have been washed out conventionally by centrifugation.
MATERIALS AND METHODS: HLA-eliminated platelets were prepared via four distinct steps: chilled on ice, treated with an acid solution, diluted and finally washed using the hollow-fibre system. The efficiency of this platelet recovery process was determined. The resulting products' platelet characteristics, including a capacity for HLA expression, were evaluated in vitro and compared in detail to their corresponding originals.
RESULTS: The average efficiency of platelet recovery was 91%. Although the expression levels of CD62P, a molecular marker for platelet activation, were approximately threefold higher on new platelets than on the original platelets, their HLA expression levels were lower. The phagocytosis assay, with monoclonal antibodies and cognate HLA antibody-containing sera, suggested that HLA-ABC molecules on the cell surface were sufficiently removed. The platelet functions, including the agonist-induced aggregability and adherence/aggregability of the collagen-coated plates under certain conditions, were conserved and not significantly different from the original ones.
CONCLUSION: We propose a novel preparation system for producing HLA-eliminated platelets without centrifugation, which ensures a highly efficient, and therefore, much more economical method of platelet recovery that also retains their key functionality.
Impaired spontaneous/endogenous fibrinolytic status as new cardiovascular risk factor?: JACC review topic of the week
Endogenous fibrinolysis is a powerful natural defense mechanism against lasting arterial thrombotic occlusion. Recent prospective studies have shown that impaired endogenous fibrinolysis (or hypofibrinolysis) can be detected in a significant number of patients with acute coronary syndrome (ACS) using global assays and is a strong marker of future cardiovascular risk. This novel risk biomarker is independent of traditional cardiovascular risk factors and unaffected by antiplatelet therapy. Most prospective prognostic data have been obtained using a global assay using native whole blood at high shear or plasma turbidimetric assays, which are described herein. Tests of endogenous fibrinolysis could be used to identify patients with ACS who, despite antiplatelet therapy, remain at high cardiovascular risk. This review discusses the impact of currently available medications and those in development that favorably modulate fibrinolytic status and may offer a potential new avenue to improve outcomes in ACS.
Glycosylation sterically inhibits platelet adhesion to von Willebrand factor without altering intrinsic conformational dynamics
BACKGROUND: A molecular basis for VWF self-inhibition has been proposed by which the N- and C-terminal flanking sequences of the globular A1 domain disulfide loop bind to and suppress the conformational dynamics of A1. These flanking sequences are rich in O-linked glycosylation (OLG) which is known to suppress platelet adhesion to VWF, presumably by steric hinderance. The inhibitory mechanism remains unresolved as to whether inhibition is due to steric exclusion by OLG's or a direct self-association interaction that stabilizes the domain. OBJECTIVES: The platelet adhesive function, thermodynamic stability, and conformational dynamics of the wild-type and type 2M G1324S A1 domain lacking glycosylation (E. coli) are compared with the wild-type glycosylated A1 domain (HEK293 cell culture) to decipher the self-inhibitory mechanism. METHODS: SPR and analytical rheology are utilized to assess GPIbα binding at equilibrium and platelet adhesion under shear flow. The conformational stability is assessed through a combination of protein unfolding thermodynamics and HXMS. RESULTS: A1 glycosylation inhibits both GPIbα binding and platelet adhesion. Glycosylation increases the hydrodynamic size of A1. Glycosylation stabilizes the thermal unfolding of A1 without changing its equilibrium stability. Glycosylation does not alter the intrinsic conformational dynamics of the A1 domain. Conclusions These studies invalidate the proposed inhibition through conformational suppression since glycosylation within these flanking sequences does not alter the native state stability or the conformational dynamics of A1. Rather, they confirm a mechanism by which glycosylation sterically hinders platelet adhesion to the A1 domain at equilibrium and under rheological shear stress.
Blockade of the kallikrein-kinin system reduces endothelial complement activation in vascular inflammation
The complement and kallikrein-kinin systems (KKS) are activated during vascular inflammation. The aim of this study was to investigate if blockade of the KKS can affect complement activation on the endothelium during inflammation. Vasculitis patient plasma had significantly more C3- and C9-positive endothelial microvesicles than controls. Perfusion of patient acute-phase plasma samples over glomerular endothelial cells induced the release of significantly more complement-positive microvesicles, in comparison to remission or control plasma. Complement activation on endothelial microvesicles was reduced by kinin B1- and B2-receptor antagonists or by C1-inhibitor (the main inhibitor of the classical pathway and the KKS). Likewise, perfusion of glomerular endothelial cells with C1-inhibitor-depleted plasma induced the release of complement-positive microvesicles, which was significantly reduced by kinin-receptor antagonists or C1-inhibitor. Mice with nephrotoxic serum-induced glomerulonephritis exhibited significantly reduced glomerular C3 deposition when treated with a B1-receptor antagonist.
Insights into determinants of spleen injury in sickle cell anemia
Spleen dysfunction is central to morbidity and mortality in children with sickle cell anemia (SCA). The initiation and determinants of spleen injury, including acute splenic sequestration (ASS) have not been established. We investigated splenic function longitudinally in a cohort of 57 infants with SCA enrolled at 3 to 6 months of age and followed up to 24 months of age and explored the respective contribution of decreased red blood cell (RBC) deformability and increased RBC adhesion on splenic injury, including ASS. Spleen function was evaluated by sequential 99mTc heated RBC spleen scintigraphy and high-throughput quantification of RBCs with Howell-Jolly bodies (HJBs). At 6 and 18 months of age, spleen filtration function was decreased in 32% and 50% of infants, respectively, whereas the median %HJB-RBCs rose significantly (from 0.3% to 0.74%). An excellent correlation was established between %HJB-RBCs and spleen scintigraphy results. RBC adhesion to laminin and endothelial cells increased with time. Adhesion to endothelial cells negatively correlated with splenic function. Irreversibly sickled cells (ISCs), used as a surrogate marker of impaired deformability, were detected at enrollment and increased significantly at 18 months. % ISCs correlated positively with %HJB-RBCs and negatively with splenic uptake, indicating a relationship between their presence in the circulation and spleen dysfunction. In the subgroup of 8 infants who subsequently experienced ASS, %ISCs at enrollment were significantly higher compared with the asymptomatic group, suggesting a major role of impaired deformability in ASS. Higher levels of %HJB-RBCs were observed after the occurrence of ASS, demonstrating its negative impact on splenic function.
The Metabolites of the Dietary Flavonoid Quercetin Possess Potent Antithrombotic Activity, and Interact with Aspirin to Enhance Antiplatelet Effects
Quercetin, a dietary flavonoid, has been reported to possess antiplatelet activity. However, its extensive metabolism following ingestion has resulted in difficulty elucidating precise mechanisms of action. In this study, we aimed to characterize the antiplatelet mechanisms of two methylated metabolites of quercetin—isorhamnetin and tamarixetin—and explore potential interactions with aspirin. Isorhamnetin and tamarixetin inhibited human platelet aggregation, and suppressed activatory processes including granule secretion, integrin αIIbβ3 function, calcium mobilization, and spleen tyrosine kinase (Syk)/linker for activation of T cells (LAT) phosphorylation downstream of glycoprotein VI with similar potency to quercetin. All three flavonoids attenuated thrombus formation in an in vitro microfluidic model, and isoquercetin, a 3-O-glucoside of quercetin, inhibited thrombosis in a murine laser injury model. Isorhamnetin, tamarixetin, and quercetin enhanced the antiplatelet effects of aspirin more-than-additively in a plate-based aggregometry assay, reducing aspirin IC50 values by an order of magnitude, with this synergy maintained in a whole blood test of platelet function. Our data provide mechanistic evidence for the antiplatelet activity of two quercetin metabolites, isorhamnetin and tamarixetin, and suggest a potential antithrombotic role for these flavonoids. In combination with their interactions with aspirin, this may represent a novel avenue of investigation for the development of new antithrombotic strategies and management of current therapies.
Platelet CD 40 ligand and bleeding during P2Y12 inhibitor treatment in acute coronary syndrome
Antiplatelet therapy through inhibition of the adenosine diphosphate (ADP)/P2Y12 pathway is commonly used in the treatment of acute coronary syndrome (ACS). Although efficient in preventing platelet activation and thrombus formation, it increases the risk of bleeding complications. In patients with ACS receiving platelet aggregation inhibitors, that is, P2Y12 blockers (n = 923), we investigated the relationship between plasma and platelet‐associated CD40L levels and bleeding events (n = 71). Treatment with P2Y12 inhibitors in patients with ACS did not affect plasma‐soluble CD40L levels, but decreased platelet CD40L surface expression (pCD40L) and platelet‐released CD40L (rCD40L) levels in response to stimulation as compared to healthy controls. In vitro inhibition of the ADP pathway in healthy control platelets reduced both pCD40L and rCD40L levels. In a multivariable analysis, the reduced pCD40L level observed in ACS patients was significantly associated with the risk of bleeding occurrence (adjusted odds ratio = 0.15; 95% confidence interval = 0.034‐0.67). P2Y12 inhibitor‐treated (ticagrelor) mice exhibited a 2.5‐fold increase in tail bleeding duration compared with controls. A significant reduction in bleeding duration was observed on CD40L+/+ but not CD40L−/− platelet infusion. In addition, CD40L blockade in P2Y12 inhibitor–treated blood samples from a healthy human reduced thrombus growth over immobilized collagen under arterial flow. In conclusion, measurement of pCD40L may offer a novel approach to assessing bleeding risk in patients with ACS who are being treated with P2Y12 inhibitors.
Research and Practice in Thrombosis and Haemostasis
Solvodynamic Printing As A High Resolution Printing Method
Printing techniques are becoming increasingly prevalent in modern manufacturing. However, its biggest drawback is the limit in printing resolution. In this paper, we present solvodynamic printing as a novel printing system which aims to improve print resolution by incorporating an additional immiscible carrier solvent into the ink delivery system. The resolution is improved due to the solvent-solvent interactions between the ink and the carrier solvent which alter the contact angle of the ink on the substrate and limit the printed feature size. We demonstrate the proof of concept of solvodynamic printing by printing silver nanoparticle inks on a polyethylene naphthalate substrate. Silver nanoparticle tracks with widths of 35.2 ± 7.0 μm were achieved using a 300 μm nozzle. This is equivalent to 11.7 ± 2.3% of the nozzle diameter. The result shows great potential in solvodynamic printing as not many modern printing techniques can achieve such nozzle to feature size ratios.
Atherogenic Lipid Stress Induces Platelet Hyperactivity Through CD36-Mediated Hyposensitivity To Prostacyclin-; The Role Of Phosphodiesterase 3A
Prostacyclin (PGI2) controls platelet activation and thrombosis through a cyclic adenosine monophosphate (cAMP) signalling cascade. However, in patients with cardiovascular diseases this protective mechanism fails for reasons that are unclear. Using both pharmacological and genetic approaches we describe a mechanism by which oxidised low density lipoproteins (oxLDL) associated with dyslipidaemia promote platelet activation through impaired PGI2 sensitivity and diminished cAMP signalling. In functional assays using human platelets, oxLDL modulated the inhibitory effects of PGI2, but not a PDE-insensitive cAMP analogue, on platelet aggregation, granule secretion and in vitro thrombosis. Examination of the mechanism revealed that oxLDL promoted the hydrolysis of cAMP through the phosphorylation and activation of phosphodiesterase 3A (PDE3A), leading to diminished cAMP signalling. PDE3A activation by oxLDL required Src family kinases, Syk and protein kinase C. The effects of oxLDL on platelet function and cAMP signalling were blocked by pharmacological inhibition of CD36, mimicked by CD36-specific oxidised phospholipids and ablated in CD36-/- murine platelets. The injection of oxLDL into wild type mice strongly promoted FeCl3 induced carotid thrombosis in vivo, which was prevented by pharmacological inhibition of PDE3A. Furthermore, blood from dyslipidaemic mice was associated with increased oxidative lipid stress, reduced platelet sensitivity to PGI2 ex vivo and diminished PKA signalling. In contrast, platelet sensitivity to a PDE-resistant cAMP analogue remained normal. Genetic deletion of CD36, protected dyslipidaemic animals from PGI2 hyposensitivity and restored PKA signalling. These data suggest that CD36 can translate atherogenic lipid stress into platelet hyperactivity through modulation of inhibitory cAMP signalling.
Dynamin 2 Is Required For GPVI Signaling And Platelet Hemostatic Function In Mice
Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemostatic function using both pharmacological and genetic approaches. Dnm2fl/f PF4-Cre (Dnm2Plt–/–) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2Plt platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα down-regulation, α-granule release, integrin αIIbβ3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2Plt–/– platelets, which were dysfunctional in response to stimulation via GPVI, and to a lesser extent to thrombin. Dnm2Plt–/– platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.
Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels
Endothelial surface and circulating glycoprotein von Willebrand factor(vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.
Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives
A renewable source of human monocytes and macrophages would be a valuable alternative to primary cells from peripheral blood (PB) in biomedical research. We developed an efficient protocol to derive monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) and performed a functional comparison with PB-derived cells. hiPSC-derived monocytes were functional after cryopreservation and exhibited gene expression profiles comparable with PB-derived monocytes. Notably, hiPSC-derived monocytes were more activated with greater adhesion to endothelial cells under physiological flow. hiPSC-derived monocytes were successfully polarized to M1 and M2 macrophage subtypes, which showed similar pan- and subtype-specific gene and surface protein expression and cytokine secretion to PB-derived macrophages. hiPSC-derived macrophages exhibited higher endocytosis and efferocytosis and similar bacterial and tumor cell phagocytosis to PB-derived macrophages. In summary, we developed a robust protocol to generate hiPSC monocytes and macrophages from independent hiPSC lines that showed aspects of functional maturity comparable with those from PB.
Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA
Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparumvirulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM.
Quantifying Ca2+ signaling and contraction in vascular pericytes and smooth muscle cells
Vascular pericytes and smooth muscle cells surround many blood vessels of the body. Their primary roles include vessel stabilization and regulation of the blood flow. The high degree of heterogeneity among these cells is dictated by (1) differences in their developmental origin and (2) their location in the vascular bed. Phenotype switching contributes to this heterogeneity especially following in vitro culture. In the absence of distinguishing molecular markers, functional assays that capture their heterogeneity in vitro are needed. Spatiotemporal changes in intracellular Ca2+ levels and contraction in response to vasoconstrictors reflect the differences between vascular pericyte and smooth muscle cell. In order to capture this heterogeneity in vitro, large ensembles of cells need to be analyzed. Here we developed an automated image processing method to measure intracellular Ca2+ and contraction in large cell groups which in combination with a computational approach for integrative analysis allowed vascular pericytes and smooth muscle cells to be distinguished without knowledge of their anatomical origin.
Biochemical and Biophysical Research Communications
Physical proximity and functional cooperation of glycoprotein 130 and glycoprotein VI in platelet membrane lipid rafts
Objective: Clinical and laboratory studies have demonstrated that platelets become hyperactive and prothrombotic in conditions of inflammation. We have previously shown that the proinflammatory cytokine interleukin (IL)-6 forms a complex with soluble IL-6 receptor α (sIL-6Rα) to prime platelets for activation by subthreshold concentrations of collagen. Upon being stimulated with collagen, the transcription factor signal transducer and activator of transcription (STAT) 3 in platelets is phosphorylated and dimerized to act as a protein scaffold to facilitate the catalytic action between the kinase Syk and the substrate phospholipase Cγ2 (PLCγ2) in collagen-induced signaling. However, it remains unknown how collagen induces phosphorylation and dimerization of STAT3.
Methods and Results: We conducted complementary in vitro experiments to show that the IL-6 receptor subunit glycoprotein 130 (GP130) was in physical proximity to the collagen receptor glycoprotein VI (GPVI in membrane lipid rafts of platelets. This proximity allows collagen to induce STAT3 activation and dimerization, and the IL-6-sIL-6Rα complex to activate the kinase Syk and the substrate PLCγ2 in the GPVI signal pathway, resulting in an enhanced platelet response to collagen. Disrupting lipid rafts or blocking GP130-Janus tyrosine kinase (JAK)-STAT3 signaling abolished the cross-activation and reduced platelet reactivity to collagen.
Conclusion: These results demonstrate cross-talk between collagen and IL-6 signal pathways. This cross-talk could potentially provide a novel mechanism for inflammation-induced platelet hyperactivity, so the IL-6-GP130-JAK-STAT3 pathway has been identified as a potential target to block this hyperactivity.
Biochemical and Biophysical Research Communications
Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis.
Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43-67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
A novel medical device coating prevents Staphylococcus aureus biofilm formation on medical device surfaces
Prevention of device related infections due to Staphylococcus aureus biofilms on devices represents a significant challenge. Such infections have recently been shown to be dependent on the coagulation pathway via activation of pro-thrombin and fibrin production. Three direct-thrombin inhibitors, argatroban, hirudin and dabigatran, were examined to determine their effect on preventing S. aureusbiofilm on plastic biochip surfaces under shear stress using an in vivo relevant model of infection. Surface functionalization of polyurethane discs via dityrosine covalent crosslinking with hirudin was performed and changes in bacterial density and microscopic appearances determined. The three direct-thrombin inhibitors prevented S. aureus biofilm formation on plasma-coated surfaces treated with these agents. Coating of polyurethane with one of these agents, hirudin, significantly inhibited biofilm formation on the modified surface. These findings reveal the exciting potential for coating biomaterial surfaces with direct thrombin inhibitors to prevent staphylococcal binding and subsequent device-related infections.
Sialyltransferase Inhibition Leads To Inhibition Of Tumor Cell Interactions With E-Selectin, VCAM1, And MADCAM1, And Improves Survival In A Human Multiple Myeloma Mouse Model
Aberrant glycosylation resulting from altered expression of sialyltransferases, such as ST3 beta-galactoside alpha2-3-sialyltransferase 6, plays an important role in disease progression in multiple myeloma. Hypersialylation can lead to increased immune evasion, drug resistance, tumor invasiveness, and disseminated disease. In this study, we explore the in vitro and in vivo effects of global sialyltransferase inhibition on myeloma cells using the pan-sialyltransferase inhibitor 3Fax-Neu5Ac delivered as a peracetylated methyl ester pro-drug. Specifically, we show in vivo that 3Fax-Neu5Ac improves survival by enhancing bortezomib sensitivity in an aggressive mouse model of multiple myeloma. However, 3Fax-Neu5Ac treatment of multiple myeloma cells in vitro did not reverse bortezomib resistance conferred by bone marrow stromal cells. Instead, 3Fax-Neu5Ac significantly reduced interactions of myeloma cells with E-selectin, MADCAM1 and VCAM1, suggesting that reduced sialylation impairs extravasation and retention of myeloma cells in the bone marrow. Finally, we showed that 3Fax-Neu5Ac alters the post-translational modification of the α4 integrin, which may explain the reduced affinity of α4β1/α4β7 integrins for their counter-receptors. We propose that inhibiting sialylation may represent a valuable strategy to restrict myeloma cells from entering the protective bone marrow microenvironment, a niche in which they are normally protected from chemotherapeutic agents such as bortezomib. Thus, our work demonstrates that targeting sialylation to increase the ratio of circulating to bone marrow-resident multiple myeloma cells represents a new avenue that could increase the efficacy of other anti-myeloma therapies and holds great promise for future clinical applications.
Fitness of Spontaneous Rifampicin-Resistant Staphylococcus aureus Isolates in a Biofilm Environment
Biofilms of S. aureus accumulate cells resistant to the antibiotic rifampicin. We show here that the accumulation of rifampicin resistant mutants (RifR) in biofilms is not equable but rather is a local event, suggesting that the growth of a few locally emerged mutants is responsible for this. Competition assays demonstrated that, compared to wild-type bacteria, the isolated RifR mutants have a growth advantage in biofilms, but not in planktonic culture. To gain insight into the mechanism of the growth advantage, we tested the involvement of the two-component systems (TCS) that sense and respond to environmental changes. We found that a deletion of SrrAB or NreBC has a drastic effect on the growth advantage of RifR mutants, suggesting the importance of oxygen/respiration responses. All six of the RifR isolates tested showed increased resistance to at least one of the common stresses found in the biofilm environment (i.e., oxidative, nitric acid, and organic acid stress). The RifR mutants also had a growth advantage in a biofilm flow model, which highlights the physiological relevance of our findings.
Extracellular Mitochondria Released From Traumatized Brains Induced Platelet Procoagulant Activity
Coagulopathy often develops soon after acute traumatic brain injury and its cause remains poorly understood. We have shown that injured brains release cellular microvesicles that disrupt the endothelial barrier and induce consumptive coagulopathy. Morphologically intact extracellular mitochondria accounted for 55.2% of these microvesicles, leading to the hypothesis that these extracellular mitochondria are metabolically active and serve as a source of oxidative stress that activates platelets and renders them procoagulant. In testing the hypothesis experimentally, we found that the extracellular mitochondria purified from brain trauma mice and those released from brains subjected to freeze-thaw injury remained metabolically active and produced reactive oxygen species. These extracellular mitochondria bound platelets through the phospholipid-CD36 interaction and induced α-granule secretion, microvesiculation, and procoagulant activity in an oxidant-dependent manner, but failed to induce aggregation. These results define an extracellular mitochondria-induced and redox-dependent intermediate phenotype of platelets that contribute to the pathogenesis of traumatic brain injury-induced coagulopathy and inflammation.
The formyl peptide fMLF primes platelet activation and augments thrombus formation
The role of formyl peptide receptor 1 (FPR1) and its ligand, fMLF, in the regulation of platelet function, hemostasis, and thrombosis is largely unknown. Fpr1-deficient mice and selective inhibitors for FPR1 were used to investigate the function of fMLF and FPR1 in platelets. N-formyl-methionyl-leucyl-phenylalanine primes platelet activation and augments thrombus formation, mainly through FPR1 in platelets. Formyl peptide receptor 1 plays a pivotal role in the regulation of platelet function.
Structural and Functional Plasticity of Collagen Fibrils
Collagen is a major component of the subendothelial matrix and participates in bleeding arrest by activating and aggregating platelets at the site of vascular injury. The most common type I collagen exists in both soluble and fibrillar forms, but structural exchangeability between the two forms is currently unknown. Using atomic force microscopy, we show that type I collagen switches between soluble and fibrillar forms in a pH-dependent and ion-independent manner. Fibrillar collagen is rope like with characteristic “D-bands.” The collagen fibrils can be disrupted with 0.1 M acetic acid and will reform when the pH is adjusted to 7.4. This structural plasticity leads to drastically different activities, with fibrillar collagen being significantly more active for platelets under static and flow conditions. More important, by probing with noncontact hopping probe ion-conductance microscopy, we find that platelets adherent to fibrillar collagen present primarily as high-density bubble shapes that have undergone rapid microvesiculation.
Amyloid Peptide β1-42 Induces Integrin αIIbβ3 Activation, Platelet Adhesion, and Thrombus Formation in a NADPH Oxidase-Dependent Manner
The progression of Alzheimer’s dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of β amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide β1-42 (Aβ1-42), Aβ1-40, and Aβ25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aβ1-42 compared to other Aβ peptides. In addition, significant platelet spreading was observed over Aβ1-42, while Aβ1-40, Aβ25-35, and the scAβ1-42 control did not seem to induce any platelet spreading, which suggested that only Aβ1-42 activates platelet signalling in our experimental conditions. Aβ1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aβpeptides did not. The molecular mechanism of Aβ1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbβ3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aβ1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aβ1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbβ3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide β1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer’s patients.
Oxidative Medicine and Cellular Longevity
Straight Channel Microfluidic Chips for the Study of Platelet Adhesion under Flow
Microfluidic devices have become an integral method of cardiovascular research as they enable the study of shear force in biological processes, such as platelet function and thrombus formation. Furthermore, microfluidic chips offer the benefits of ex vivo testing of platelet adhesion using small amounts of blood or purified platelets. Microfluidic chips compromise flow channels of varying dimensions and geometries which are connected to syringe pump. The pump draws blood or platelets suspensions through the channel(s) allowing for imaging of platelet adhesion and thrombus formation by fluorescence microscopy. The chips can be fabricated from various blood-compatible materials. The current protocol uses commercial plastic or in-house polydimethylsiloxane (PDMS) chips. Commericail biochips offer the advantage of standardization whereas in-house chips offer the advantage of decreased cost and flexibility in design. Microfluidic devices are a powerful tool to study the biorheology of platelets and other cell types with the potential of a diagnostic and monitoring tool for cardiovascular diseases.
Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia
Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.
Cleavage of anti-PF4/heparin IgG by a bacterial protease and potential benefit in heparin-induced thrombocytopenia
Heparin-induced thrombocytopenia (HIT) is due IgG antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes directly activate platelets via FcgammaRIIA receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy chain IgG, abolishing its ability to bind FcgammaR, including FcgammaRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcgammaRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor mRNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor-coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcgammaRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment for patients with severe HIT
Functional Validation of microRNA-126-3p as a Platelet Reactivity Regulator Using Human Haematopoietic Stem Cells
BACKGROUND: Platelets are an abundant source of micro-ribonucleic acids (miRNAs) that may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, have been noted as potential biomarkers of platelet reactivity and the recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain, and the functional validation of candidate miRNAs on human-derived cells is lacking.
OBJECTIVE: This article functionally validates miR-126-3p as a regulator of platelet reactivity in platelet-like structures (PLS) derived from human haematopoietic stem cells.
MATERIALS AND METHODS: CD34+-derived megakaryocytes were transfected with miR-126-3p and differentiated in PLS. PLS reactivity was assessed using perfusion in a fibrinogen-coated flow chamber. miR-126-3p's selected gene targets were validated using quantitative polymerase chain reaction, protein quantification and a reporter gene assay.
RESULTS: CD34+-derived megakaryocytes transfected with miR-126-3p generated PLS exhibiting 156% more reactivity than the control. These functional data were in line with those obtained analysing CD62P expression. Moreover, miR-126-3p transfection was associated with the down-regulation of a disintegrin and metalloproteinase-9 (ADAM9) messenger RNA (mRNA), a validated target of miR-126-3p, and of Plexin B2 (PLXNB2) mRNA and protein, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results to miR-126-3p transfection. Finally, using a reporter gene assay, we validated PLXNB2 as a direct target of miR-126-3p.
CONCLUSION: We functionally validated miR-126-3p as a regulator of platelet reactivity in PLS derived from human haematopoietic stem cells. Moreover, PLXNB2 was validated as a new gene target of miR-126-3p in human cells, suggesting that miR-126-3p mediates its effect on platelets, at least in part, through actin dynamics regulation.
Differences And Similarities In Ibrutinib And Acalabrutinib Effects On Platelet Functions
While efficient to treat B-cell malignancies, Bruton's tyrosine kinase inhibitors are consistently reported to increase the bleeding risk. Analyzing platelet aggregation response to collagen in platelet-rich-plasma allowed us to identify two groups in the healthy population characterized by low or high sensitivity to ibrutinib in vitro. Inhibition of drug efflux pumps induced a shift from ibrutinib low to high sensitive platelets. At clinically relevant dose, the second-generation inhibitor, acalabrutinib, did not affect maximal collagen-induced platelet aggregation in the ibrutinib low sensitive group and inhibited aggregation in a small fraction of the ibrutinib high sensitive group. Consistently, acalabrutinib delayed aggregation, particularly in the ibrutinib high sensitive group. In chronic lymphocytic leukemia patients, acalabrutinib inhibited maximal platelet aggregation only in the ibrutinib high sensitive group. Acalabrutinib inhibited collagen-induced tyrosine-753 phosphorylation of phospholipase Cγ2 in both groups, but, in contrast to ibrutinib, did not affect Src-family kinases. Acalabrutinib impacted thrombus growth under flow only in the ibrutinib high sensitive group and potentiated the effect of cyclooxygenase and P2Y12 receptor blockers in both groups. The better profile of acalabrutinib being mainly observed in the ibrutinib low sensitive group, replacement therapy in patients may not systematically reduce the bleeding risk.
Effects of three‐month streptozotocin‐induced diabetes in mice on blood platelet reactivity, COX‐1 expression and adhesion potential
Diabetes is associated with an increased risk of cardiovascular disease. This is partially attributed to an altered activation status of blood platelets in this disease. Previously, alterations have been shown in COX‐1 and protease activated receptor (PAR)‐3 receptor expression in platelets in two animal models of diabetes, there have not been studies which address expression of these proteins in mice with long‐term streptozotocin (STZ)‐induced diabetes. We have also addressed the effect of diabetes on platelet adhesion under flow conditions. With the use of flow cytometry, we have shown that certain markers of platelet basal activation, such as active form of αIIbβ3 and of CD40L were increased in STZ‐induced diabetic mice. Platelets from STZ‐induced diabetic mice were also more reactive when stimulated with PAR‐4 activating peptide as revealed by higher expression of active form of αIIbβ3, membrane‐bound on vWillebrand Factor and binding of exogenous fluorescein isothyanate‐labelled fibrinogen. Expression of COX‐1 and production of thromboxane A2 in platelets of STZ‐induced diabetic mice were higher than in control animals. We observed no effect of diabetes on ability of platelets to form stable adhesions with fibrinogen in flow conditions. We conclude that although certain similarities exist between patterns of activation of platelets in animal models of diabetes, the differences should also be taken into account.
Functionalized polymer microbubbles as new molecular ultrasound contrast agent to target P-selectin in thrombus
Thrombotic diseases rarely cause symptoms until advanced stage and sudden death. Thus, early detection of thrombus by a widely spread imaging modality can improve the prognosis and reduce mortality. Here, polymer microbubbles (MBs) made of degradable poly(IsoButylCyanoAcrylate) and functionalized with fucoidan (Fucoidan-MBs) were designed as a new targeted ultrasound contrast agent to image venous thrombus. The physicochemical characterizations demonstrate that the MBs with fucoidan surface exhibit a size of 2–6 μm and stability in suspension at 4 °C up to 2 months. MBs exhibit high echogenicity and could be completely burst under high destructive pulse. Flow chamber experiments on activated human platelets show a higher affinity of Fucoidan-MBs than control anionic MBs (CM-Dextran-MBs) under shear stress conditions. In vivo analysis by ultrasound and histological results demonstrate that Fucoidan-MBs are localized in rat venous thrombotic wall, whereas few CM-Dextran-MBs are present. In addition, the binding of Fucoidan-MBs in healthy vein is not observed. Collectively, Fucoidan-MBs appear as a promising functionalized carrier for ultrasound molecular imaging in thrombotic diseases.