Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets

Essentials

• Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF).
• Salivary EVs expose CD24, a ligand of P‐selectin.
• CD24 and coagulant TF co‐localize on salivary EVs.
• TF⁺/CD24⁺ salivary EVs bind to activated platelets and trigger coagulation.

Summary

Background

Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF‐exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell‐derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P‐selectin glycoprotein ligand‐1 (PSGL‐1) and P‐selectin.

Objectives

We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P‐selectin.

Methods

We investigated the presence of two ligands of P‐selectin on salivary EVs, PSGL‐1 and CD24.

Results

Salivary EVs expose CD24 but PSGL‐1 was not detected. Immune depletion of CD24‐exposing EVs completely abolished the TF‐dependent coagulant activity of cell‐free saliva, showing that coagulant TF and CD24 co‐localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24.

Conclusions

A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P‐selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action.     

Role of P-selectin, β2-integrins, and Src tyrosine kinases in mouse neutrophil–platelet adhesion

Summary. Background: The initial interaction of human polymorphonuclear leukocytes (PMN) with activated human platelets is mediated by P‐selectin and its leukocyte ligand PSGL‐1; subsequently the interaction is strengthened by activation of αMβ2 via protein tyrosine phosphorylation mediated by Src kinases and binding of activated αMβ2 to its platelet counterreceptor(s). Objectives: Because mouse models are being used to define the role of PMN–platelet interactions in thrombosis and the response to vascular injury, we investigated the molecular determinants responsible for the interaction of murine PMNs with activated murine platelets. Methods: Mouse platelets were labeled with the green fluorescent dye BCECF and then activated with thrombin and fixed with 1% paraformaldehyde. Mouse PMNs were labeled with the red fluorescent dye hydroethidine and then stirred with the fixed platelets. After stopping the reaction with paraformaldehyde, formation of mixed cell conjugates was analyzed by flow cytometry. Results: In time course experiments, 90 ± 1.9% of PMNs formed mixed conjugates with platelets after 2 min and the mean (± SEM) number of platelets per positive PMN was 8.4 ± 1.5. A monoclonal antibody to P‐selectin reduced the percentage of PMNs with attached platelets to 16 ± 2.4% (P = 0.001), and only 8 ± 5% of PMNs interacted with platelets from P‐selectin?/? mice. In contrast, monoclonal antibodies to PSGL‐1, β₂‐integrin, and αIIbβ3 had much less or no effect on the production of mixed cell aggregates. To better identify a secondary contribution of β₂‐integrins, P‐selectin interactions were disrupted by briefly adding 5 mm EGTA to already‐formed mixed cell aggregates. Brief EGTA treatment alone reduced the percentage of PMNs with attached platelets to 70 ± 3.5% (P = 0.004 vs. no treatment), but did not modify the number of platelets per positive PMN (9.5 ± 1.7). The combination of brief EGTA treatment and a monoclonal antibody to β₂‐integrin lowered the percentage of PMN with attached platelets to 50 ± 7% and reduced the number of platelets attached per positive PMN to 3.6 ± 0.7 (P = 0.03 vs. brief EGTA treatment only). Brief EGTA treatment did not modify the effect of the other antibodies. When the incubation was stopped with EGTA the Src inhibitors PP1 and PP2 reduced PMN–platelet adhesion, while the inactive analog PP3 was ineffective. Conclusions: These results confirm that P‐selectin plays a prominent role in mediating the initial interactions between mouse PMN and platelets, and provide support for additional contributions from β₂‐integrins and Src family kinases.     

Differential contributions of monocyte‐ and platelet‐derived microparticles towards thrombin generation and fibrin formation and stability

Summary. Background: Microparticles (MPs) are sub‐micron vesicles shed by activated or apoptotic cells, including platelets and monocytes. Increased circulating MPs are associated with thrombosis; however, their role in thrombogenesis is poorly understood. Objective: To determine how MPs promote thrombin generation and modulate fibrin density and stability. Methods: Platelets and monocytes were isolated from healthy donors. Platelets were stimulated with calcium ionophore, thrombin receptor agonist peptide (TRAP) or TRAP/convulxin. Monocytes and human monocytic THP‐1 cells were stimulated with lipopolysaccharide (LPS). MPs were isolated, washed by high‐speed centrifugation and assessed using the following: transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA), flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density and stability. Results: MPs from monocytes (M‐MPs) and platelets (PMPs) had similar shapes and diameters (100–300 nm). M‐MPs had TF activity (16.7 ± 2.4 pm TF per 10⁶ MP), supported prothrombinase activity and triggered shorter thrombin generation lag times than buffer controls (5.4 ± 0.5 vs. 84.2 ± 4.8 min, respectively). Compared with controls, M‐MPs supported faster fibrin formation (0.24 ± 0.24 vs. 76.7 ± 15.1 mOD min^?1, respectively), 38% higher fibrin network density and higher clot stability (3.8‐fold higher turbidity in the presence of tissue plasminogen activator). In contrast, PMPs did not have TF activity and supported 2.8‐fold lower prothrombinase activity than M‐MPs. PMPs supported contact‐dependent thrombin generation, but did not independently increase fibrin network density or stability. Interestingly, PMPs increased rates of thrombin generation and fibrin formation (1.7‐ and 1.3‐fold, respectively) when mixed with THP‐1‐derived MPs. Conclusion: MPs from platelets and monocytes differentially modulate clot formation, structure and stability, suggesting unique contributions to thrombosis.     

Decorin mimic promotes endothelial cell health in endothelial monolayers and endothelial–smooth muscle co‐cultures

Non‐specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti‐restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC–SMC co‐cultures, we investigated the ability of DS–SILY₂₀, a decorin mimic, to promote EC health. DS–SILY₂₀ increased EC proliferation and migration by 1.5‐ and 2‐fold, respectively, which corresponded to increased phosphorylation of ERK‐1/2. Interestingly, IL‐6 secretion and the production of both E‐selectin and P‐selectin were reduced in the presence of 10 μm DS–SILY₂₀, even in the presence of the potent pro‐inflammatory cytokine platelet‐derived growth factor (PDGF). In hyperplastic and quiescent EC–SMC co‐cultures, DS–SILY₂₀ treatment reduced the secretion of IFNγ, IL‐1β, IL‐6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E‐selectin and P‐selectin expression was further reduced following DS–SILY₂₀ treatment in both co‐culture models. These results indicate that DS–SILY₂₀ promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.     

P-selectin polymorphisms' influences on P-selectin serum concentrations and on their familial correlation: the STANISLAS family study

Summary. Background: P‐selectin is an adhesion molecule known to be involved in the pathogenesis of several diseases through its major role in the initial phase of leukocytes recruitment during inflammation. However, genetic characterization of soluble P‐selectin remains unclear. Objectives: In the STANISLAS cohort, we study the familial correlations of P‐selectin levels and investigate the association of six P‐selectin polymorphisms (C‐2123G, A‐1969G, S290N, N562D, V599L and T715P) and cardiovascular risk factors with P‐selectin concentrations. Patients/Methods: Full phenotypic and genotypic information was available for 136 healthy families composed of both natural parents and at least one child (boys, n = 125; and girls, n = 139) aged more than 4 years. Results: While no correlation was observed between spouses, family correlations of P‐selectin concentrations were highly significant for sibling (0.50 ± 0.12, P < 10^?3) and child‐parent pairs (0.42 ± 0.04, P < 10^?3). P‐selectin haplotypes explained about 25% of the variability of P‐selectin concentrations, this effect being mainly due to the additive effects of two polymorphisms, V599L and T715P. After adjusting for the effect of the P‐selectin polymorphisms, the sibling and child‐parent correlations decreased to (0.39 ± 0.08, P < 10^?4) and (0.32 ± 0.06, P < 10^?4), respectively. Conclusions: In the present study, we showed that two P‐selectin polymorphisms, V599L and T715P, explained about 25% of the variability of P‐selectin concentrations and accounted for about 40% of their family resemblance. These results would suggest a genetic influence on P‐selectin concentrations beyond the contribution of the P‐selectin gene.     

A new macromolecular paramagnetic MR contrast agent binds to activated human platelets

A new functionalized macromolecular magnetic resonance (MR) contrast agent has been developed from a carboxymethyldextran‐Gd(DOTA) devoid of biospecificity. The functionalized contrast agent was synthesized in order to mimic PSGL‐1, the main ligand of P‐selectin, a glycoprotein mainly expressed on the surface of activated platelets. The starting compound, CM1, was first carboxymethylated by monochloroacetic acid leading to a series of 10 derivatives varying in their carboxymethyl content. CM8 derivative, with a degree of substitution in carboxymethyl of 0.84, was chosen for subsequent fluorolabeling and sulfation to give CM8FS. CM8FS has an average number molecular weight of 27 000 ± 500 g/mol, a hydrodynamic radius of 5.7 ± 0.2 nm and a high relaxivity (r₁ = 11.2/mM (Gd)/s at 60 MHz). Flow cytometry experiments on whole human blood or on isolated platelets evidenced in vitro a preferential binding of CM8FS on TRAP‐activated human platelets. Interestingly, CM8FS did not bind to other blood cells or to resting platelets. Pellets of TRAP‐activated human platelets have also been imaged in tubes with a 1.5 T MR imager. A MR signal was observed for activated platelets incubated with CM8FS. Altogether, these in vitro results evidenced the recognition of activated human platelets by a fluorescent paramagnetic contrast agent grafted with carboxyl and sulfate groups. This biomimetic approach associated with the versatile macromolecular platform appears promising for the development of new contrast agents for molecular imaging of activated platelets in cardiovascular diseases such as atherosclerosis and aneurysms. Copyright © 2007 John Wiley & Sons, Ltd.     

Splice variants of tissue factor promote monocyte‐endothelial interactions by triggering the expression of cell adhesion molecules via integrin‐mediated signaling

Summary. Background: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. Objective: To examine the role of integrin signaling in TF‐dependent recruitment of monocytes by endothelial cells. Methods: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ‐TF) and recombinant asTF were assessed. Results: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid‐rich plaques. Tumor lesions, as well as stromal CD68⁺ monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ‐TF, and this was completely blocked by anti‐β1 integrin antibody. asTF‐ and LZ‐TF‐treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF‐elicited adhesion was more pronounced than that elicited by LZ‐TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E‐selectin, ICAM‐1 and VCAM‐1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ～3‐fold under MCP‐1 gradient. Conclusions: TF splice variants ligate β1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non‐proteolytic, integrin‐mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.     

Inflammatory markers in relation to insulin resistance and the metabolic syndrome

Background Inflammation has repeatedly been demonstrated to be associated with the metabolic syndrome (MetS) and insulin resistance, but the relative importance of different aspects of the inflammatory process is largely unexplored. Design We measured circulating interleukins (IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10); other cytokines (tumour necrosis factor‐α, interferon gamma and monocyte chemotactic protein‐1), cell adhesion molecules [vascular cell adhesion molecule‐1 (VCAM‐1), intercellular adhesion molecule‐1, E‐selectin, P‐selectin, l ‐selectin], and systemic inflammation markers [C‐reactive protein (CRP) and leukocyte count] in 943 70 year old participants (50% women) of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study. We related these biomarkers to MetS and the homeostasis model assessment insulin resistance index (HOMA‐IR). Results In a multivariable model including all inflammatory markers conjointly together with sex, log VCAM‐1 [odds ratio (OR), 1·45; 95% confidence interval (CI), 1·22–1·72 per 1 SD increase; P < 0·0001], log E‐selectin (OR, 1·33; 95% CI, 1·12–1·57 per 1SD increase; P = 0·001), and log CRP (OR, 1·41; 95% CI, 1·20–1·66 per 1‐SD increase; P < 0·0001) were independently associated with MetS. These biomarkers were also independently associated with HOMA‐IR. Conclusions Among 17 inflammatory biomarkers, most of them previously not examined in relation to MetS and insulin resistance, VCAM‐1, E‐selectin and CRP demonstrated the strongest associations with MetS and insulin resistance in our community based sample of the elderly. The relative importance of these biomarkers in predicting the development of MetS, insulin resistance and cardiovascular disease needs to be further examined in a longitudinal setting.     

Enhanced monocyte expression of tissue factor by oxidative stress in patients with antiphospholipid antibodies: effect of antioxidant treatment

Summary. In a first study, we performed a cross‐sectional analysis of urinary excretion of isoprostanes, IPF_2α‐III and _VI, and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody‐positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day^?1, vitamin C at 2000 mg day^?1) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti‐β₂ glycoprotein 1 (β₂GP₁) or control IgG, either with or without vitamin E. APL‐positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL‐negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF_2α‐III (rho 0.79; P < 0.003) and IPF_2α‐VI (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti‐β₂GP₁ antibodies dose‐dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL‐positive patients, oxidative stress contributes to activate the clotting system via over‐expression of monocyte TF. We suggest that anti‐β₂GP₁ antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.     

Elevated plasma levels of P‐selectin glycoprotein ligand‐1‐positive microvesicles in patients with unprovoked venous thromboembolism

Essentials

• PSGL‐1⁺ microvesicles (MVs) may be important in venous thromboembolism (VTE).
• We measured plasma levels and parental origin of PSGL‐1⁺ MVs in patients with unprovoked VTE.
• VTE patients had higher plasma levels of PSGL‐1⁺ MVs than healthy controls.
• The PSGL‐1⁺ MVs originated mainly from monocytes and endothelial cells.

Summary

Background

Microvesicles (MVs) express antigens from their parental cells and have a highly procoagulant surface. Animal studies suggest that P‐selectin glycoprotein ligand‐1‐positive (PSGL‐1⁺) MVs play a role in the pathogenesis of venous thromboembolism (VTE).

Objective

The aim of this study was to determine plasma levels, the cellular origin and the morphological characteristics of PSGL‐1⁺ MVs in patients with unprovoked VTE.

Methods

We conducted a population‐based case–control study in 20 patients with a history of unprovoked VTE and 20 age‐ and sex‐matched healthy controls recruited from the general population. Plasma levels, the cellular origin and the morphological characteristics of PSGL‐1⁺ MVs were evaluated using flow cytometry, electron microscopy and confocal microscopy.

Results

Plasma levels of PSGL‐1⁺ MVs were associated with increased risk of VTE. The odds ratio per one standard deviation increase in PSGL‐1⁺ MVs was 3.11 (95% confidence interval [CI], 1.41–6.88) after adjustment for age and sex, and 2.88 (95% CI, 1.29–6.41) after further adjustment for body mass index. The PSGL‐1⁺ MVs originated mainly from monocytes and endothelial cells determined by double staining with markers of parental cells using flow cytometry and transmission electron microscopy. Scanning electron microscopy of PSGL‐1‐labeled plasma‐derived MVs displayed dominantly spherical vesicles that varied between 50 and 300 nm in diameter.

Conclusions

Increased plasma levels of PSGL‐1⁺ MVs are associated with the risk of unprovoked VTE. Large population‐based prospective studies are required to validate our findings.

     

Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo

Summary. Background: Endothelial activation and dysfunction are associated with several diseases. However, hardly any specific markers are available. Microparticles (MP) from endothelial cells (EC; EMP) were reported in patient groups and healthy individuals. The antibodies used to detect EMP, however, were mainly directed against antigens without EC specificity. Objectives: We evaluated the antigens on EC and EMP to establish proper markers for EMP detection. Methods: EMP were isolated from supernatants of resting and interleukin (IL)‐1α activated human umbilical vein EC (HUVEC; n = 3; 0–72 h), stained with annexin V and monoclonal antibodies, and analyzed by flow cytometry. Human platelet‐MP (PMP), the main MP population in plasma, were prepared in vitro. EMP and PMP were studied in plasma from systemic lupus erythematosus (SLE) patients (n = 11) and healthy individuals (n = 10). Results: Platelet–endothelial cell adhesion molecule‐1 (PECAM‐1), α_ν and β₃ were constitutively exposed on HUVEC, but (almost) absent on EMP (<15% positive for α_ν and β₃), or only exposed on a subpopulation (PECAM‐1; 30–60%). Activated HUVEC (>80%) and (subpopulations of) EMP exposed E‐selectin and tissue factor. PMP strongly exposed PECAM‐1, β₃, and glycoprotein (GP)Ib (CD42b), but not α_ν or E‐selectin. GPIb and P‐selectin (CD62P) were absent on EMP. Plasma samples contained 0.5% MP staining for E‐selectin and/or α_ν. Plasma from one SLE patient contained E‐selectin exposing MP (21%), but little α_ν‐positive MP. Conclusions: EC release EMP in vitro. The antigenic phenotype of EMP released from resting and IL‐1α‐stimulated EC differs among each other as well as from resting and stimulated EC, respectively. E‐selectin exposed on IL‐1α‐stimulated EC is a valid marker for EMP detection ex vivo to establish endothelial cell activation.     

Serum brain‐derived neurotrophic factor and platelet activation evaluated by soluble P‐selectin and soluble CD‐40‐ligand in patients with acute myocardial infarction

Little is known about the role of neurotrophins (NT) under adult vascular homeostasis in normal and pathological conditions. The NT family, including nerve growth factor and brain‐derived neurotrophic factor (BDNF) are expressed in atherosclerotic vessels. Previous studies demonstrated that plasma BDNF levels were increased in the coronary circulation in patients with unstable angina. However, the role of BDNF during the onset and evolution of unstable angina remains to be elucidated. The objective of this study was to evaluate the relationship between BDNF, functional parameters and biological markers associated with inflammatory processes and platelet activation. BDNF serum levels were assessed in patients with acute myocardial infarction (MI) (n = 20) or stable angina pectoris (SAP) (n = 20) who underwent coronary angiography. Serum levels of IL‐6, MCP1, sVCAM, soluble CD‐40‐ligand (sCD40L) and soluble P‐selectin (sP‐selectin) were measured simultaneously by flux cytometry. Median BDNF levels were higher in the MI than in the SAP group (1730 vs. 877 pg/mL, respectively; P = 0.025). In MI patients, we observed a significant correlation between BDNF and sP‐selectin (r = 0.58, P = 0.023), although we found a non‐significant trend between BDNF and sCD40L (r = +0.35, P = 0.144). By contrast, no such correlation was observed in SAP patients (r = ?0.22, P = 0.425). No difference was observed between the two groups regarding baseline demographics, risk factors, biological data and angiographic findings. The study suggests that BDNF serum levels in MI patients could be related to platelet activation and the inflammatory response. Further studies are needed to investigate the role of NT in the setting of acute MI.     

Glycogen synthase kinase‐3 negatively regulates tissue factor expression in monocytes interacting with activated platelets

Summary. Background: At the site of vascular injury, monocytes (MN) interacting with activated platelets (PLT) synthesize tissue factor (TF) and promote thrombus formation. Intracellular signals necessary for the expression of TF in MN, in the context of a developing thrombus, remain unknown. Objective: The study was designed to investigate the role of the glycogen synthase kinase 3 (GSK3, a serine‐threonine kinase) downstream insulin receptor pathway, in PLT‐induced TF expression in MN. Methods: To this purpose we used a well‐characterized in vitro model of human MN‐PLT interactions that allows detailed analysis of TF activity, TF protein and gene expression.Results: The results demonstrated that, in MN interacting with activated PLT: (i) TF activity, antigen and mRNA were low until 8–10 h and dramatically increased thereafter, up to 24 h; (ii) according to the kinetics of TF expression in MN, GSK3β undergoes phosphorylation on serine 9, a process associated with down‐regulation of enzyme activity; (iii) pharmacological blockade of GSK3 further increased TF expression and was accompanied by increased accumulation of NF‐kB, in the nucleus; (iv) blockade of phosphoinositide‐3 kinase (PI(3)K) by wortmannin inhibited PLT‐induced TF expression; and (v) according to the established role of the GSK3 downstream insulin receptor, insulin increased PLT‐induced TF expression in a PI(3)K‐dependent manner. Conclusion: GSK3 acts as a molecular brake on the signaling pathway, leading to TF expression in MN interacting with activated PLT. PI(3)K, through Akt‐dependent phosphorylation of GSK3, relieves this brake and allows TF gene expression. This study identifies a novel molecular link between thrombotic risk and metabolic disorders.     

Ascidian dermatan sulfates attenuate metastasis,inflammation and thrombosis by inhibition of P‐selectin

See also Zacharski LR. Controlling cancer growth from within the blood coagulation mechanism. This issue, pp 1804–6. Summary. Background: Cancer‐associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti‐inflammatory activities that are linked to inhibition of P‐selectin and/or L‐selectin. P‐selectin‐mediated platelet–tumor cell and tumor cell–endothelium interactions facilitate the initial steps of metastasis. Objectives and Methods: The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P‐selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. Results: Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2‐GalNAc)_n, but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P‐selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC‐38 colon carcinoma and B16‐BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl₃‐induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P‐selectin and thereby the binding of activated platelets during thrombus formation. Conclusions: Collectively, these findings provide evidence that specific inhibition of P‐selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.     

Immature myeloid dendritic cells capture and remove activated platelets from preformed aggregates

Summary. Background: Immature dendritic cells (DCs) patrol the circulation, where they can uptake antigens. It has been reported that mature monocyte‐derived DCs have the ability to interact with an activated platelet monolayer under high shear conditions (1500 s^?1). Objectives: In this study, we investigated whether platelets can recruit immature myeloid DCs (CD1c⁺) directly isolated from blood (BDCs) and if so, which receptors are involved. Results: Using flow cytometry and electron microscopy, we showed that BDCs interact with activated but not resting platelets in suspension. Interaction was also observed after perfusing BDCs under low flow conditions (150 s^?1) through collagen‐coated microcapillaries in which platelets had adhered and formed aggregates. No such interaction could be detected at higher shear rates. Whereas initial transient attachment required the exposure of P‐selectin on activated platelets and PSGL‐1 on BDCs, subsequent stationary adhesion was dependent on α_IIbβ₃ and α_Mβ₂ integrins on platelets and BDCs, respectively. Moreover, during their transient interaction, BDCs preferentially removed platelets located at the outer margin of the thrombus in a P‐selectin‐ and integrin‐dependent manner. Conclusion: This study provides evidence for an interaction between activated platelets and immature myeloid DCs only under low shear conditions. This could be of importance for BDC maturation and antigen uptake during normal hemostasis or in the context of atherothrombosis at sites of reduced blood flow.     

Short-term myocardial ischemia induces cardiac modified C-reactive protein expression and proinflammatory gene (cyclo-oxygenase-2, monocyte chemoattractant protein-1, and tissue factor) upregulation in peripheral blood mononuclear cells

Summary. Background: Prompt coronary thrombus resolution, reducing time of ischemia, improves cardiac recovery. The factors triggered by ischemia that contribute to the clinical outcome are not fully known. We hypothesize that unabated inflammation due to cardiac ischemia may be a contributing factor. Aims: As a proof‐of‐concept, we evaluated the effect of short‐term myocardial ischemia on the local and systemic inflammatory response. Methods: Pigs underwent either 90‐min mid‐left anterior descending (LAD) coronary artery balloon occlusion (infarct size 25% ± 1% left ventricle; 29% heart function deterioration) or a sham‐operation procedure. Peri‐infarcted and non‐ischemic cardiac tissue was obtained for histopathologic, molecular and immunohistochemical analysis of inflammatory markers [interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), modified C‐reactive protein (mCRP), and human alveolar macrophage‐56 (HAM‐56)]. Blood (femoral vein) was withdrawn prior to myocardial infarction (MI) induction (t = 0) and at 30 and 90 min to evaluate: (i) systemic cytokine levels (IL‐6, TNF‐α, CRP); (ii) proinflammatory gene and protein expression in peripheral blood mononuclear cells (PBMCs) of tissue factor (TF), cyclo‐oxygenase‐2 (Cox‐2), monocyte chemoattractant protein‐1 (MCP‐1), and CRP; and (iii) platelet activation (assessed by perfusion studies and RhoA activation). Results: Short‐term ischemia triggered cardiac IL‐6 and TNF‐α expression, recruitment of inflammatory cells, and mCRP expression in infiltrated macrophages (P < 0.05 vs. t = 0 and sham). PBMC mRNA and protein expression of MCP‐1, Cox‐2 and TF was significantly increased by ischemia, whereas no differences were detected in CRP. Ischemia increased cardiac troponin‐I, IL‐6 and TNF‐α systemic levels, and was associated with higher platelet deposition and RhoA activation (P < 0.001 vs. t = 0 and sham). Conclusion: Short‐term myocardial ischemia, even without atherosclerosis, induces an inflammatory phenotype by inducing local recruitment of macrophages and systemic activation of mononuclear cells, and renders platelets more susceptible to activation.     

Platelet microparticles and soluble P selectin in peripheral artery disease: Relationship to extent of disease and platelet activation markers

Background. There is increased platelet activation in many cardiovascular diseases. This observation may explain the presence of increased levels of platelet microparticles (PMP) in these diseases. However, whether or not levels of PMPs inter‐relate with other markers of platelet activation, such as soluble P‐selectin, or with disease severity, is unknown. We therefore hypothesized raised PMP levels in stable peripheral artery disease (PAD) intermittent claudication (IC), with an additional increase in severe PAD critical limb ischaemia (CLI). Furthermore, we tested the hypothesis that PMP levels are correlated with other markers of platelet activation, such as soluble P‐selectin, membrane bound P‐selectin (CD62P) and CD63.

Methods. Patients with PAD were recruited from the vascular outpatient and inpatient facilities at a teaching hospital. Age‐ and sex‐matched controls were also recruited from healthy volunteers. Venous blood was obtained from 23 patients with severe disease (CLI), 36 with moderate disease (IC), and from 30 healthy controls. The percentage of platelets positive for CD62P and CD63, as well as the numbers of PMPs were defined by flow cytometry. Plasma soluble P selectin was measured by enzyme‐linked immunosorbent assay (ELISA).

Results. PMPs were increased relative to healthy controls in patients with IC, with a further increase in CLI (P<0.001). Soluble P selectin and CD62+ve platelets were raised in both patient groups, but there was no difference amongst the two patient groups. CD63+ve cells were raised only in CLI compared to healthy controls. In multivariate analysis, only PMP and soluble P selectin independently predicted disease severity, and the two markers correlated modestly (r?=?0.345, P<0.001).

Conclusion. Increased PMP and soluble P selectin are both related to the severity of symptomatic PAD. However, it is uncertain if this relationship is a cause or effect of atherosclerosis. This finding may have clinical implications as PMPs have the potential to influence the progression of atheroma as well as promote thrombosis.     

Decreased platelet inhibition by P2Y12 receptor blockers in anaemia

Background

Anaemic patients undergoing angioplasty and stenting are at an increased risk of ischaemic events, which may be caused by an inadequate response to antiplatelet therapy with adenosine diphosphate (ADP) P2Y₁₂ inhibitors. In the current study, we investigated the associations between anaemia and on‐treatment platelet reactivity in clopidogrel‐treated (group 1, n = 306) and prasugrel‐/ticagrelor‐treated (group 2, n = 109) patients undergoing elective and acute angioplasty with stent implantation, respectively.

Materials and methods

Monocyte‐platelet aggregate (MPA) formation was determined by flow cytometry in both groups. On‐treatment residual platelet reactivity in response to ADP was assessed by light transmission aggregometry (LTA) in both groups, and by the VerifyNow P2Y₁₂ assay and the Impact‐R in group 1. P‐selectin expression was measured by flow cytometry in group 2.

Results

In both groups, anaemia was associated with significantly higher MPA formation in response to ADP (both P ≤ .02). Moreover, by LTA maximal aggregation in response to ADP was significantly higher in patients with anaemia in both groups (both P < .05), and anaemic patients in group 1 had a significantly higher on‐treatment platelet reactivity by the VerifyNow P2Y₁₂ assay and the Impact‐R than those without anaemia (both P < .001). In group 2, significantly higher platelet surface expression of P‐selectin was seen in anaemia after stimulation with ADP (P = .02).

Conclusion

Anaemia is associated with decreased platelet inhibition by ADP P2Y₁₂ receptor antagonists after elective and acute percutaneous interventions with stent implantation. However, due to inconsistencies between different platelet function tests additional data are needed to clarify the role of anaemia for platelet inhibition.     

慢性牙周炎龈沟液IL-10、IL-23、MCP-1与牙周指数的相关性分析

目的分析慢性牙周炎患者龈沟液白介素-10(IL-10)、白介素-23(IL-23)、单核细胞趋化蛋白-1(MCP-1)水平及其与牙周指数的相关性。方法选取2017年5月至2020年4月本院口腔诊疗中心患者134例,依据口腔情况分为对照组(n=30)、牙龈炎组(n=48)、慢性牙周炎组(n=56),记录菌斑指数(PLI)、出血指数(BI)、探诊深度(PD)、附着丧失(AL)。依据病变程度将慢性牙周炎组分为轻度病变亚组(n=30)、中重度病变亚组(n=26),收集龈沟液后采用酶联免疫吸附试验测定各组IL-10、IL-23、MCP-1水平,分析其与牙周指数相关性。结果牙龈炎组、慢性牙周炎组PLI、BI、PD、AL均高于对照组,慢性牙周炎组PLI、BI、PD、AL高于牙龈炎组,差异均具有统计学意义(P<0.05)。牙龈炎组、慢性牙周炎组龈沟液IL-10低于对照组,而IL-23、MCP-1水平高于对照组,慢性牙周炎组龈沟液IL-10低于牙龈炎组,而IL-23、MCP-1水平高于牙龈炎组,差异均具有统计学意义(P<0.05)。慢性牙周炎患者中,轻度病变亚组患者龈沟液IL-10高于中重度病变亚组患者,而PLI、BI、PD、AL、IL-23、MCP-1水平低于中重度病变亚组患者,差异均具有统计学意义(P<0.05)。慢性牙周炎患者龈沟液IL-10水平与各项牙周指数均呈负相关(P<0.05),IL-23、MCP-1水平均与各项牙周指数呈正相关(P<0.05)。结论慢性牙周炎患者龈沟液中IL-10呈低表达,IL-23、MCP-1呈高表达,且三者均与牙周指数有明显相关性。