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Background: Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. During thrombotic events, TFPI is proteolytically inactivated by neutrophil elastase while bound to neutrophil extracellular traps (NETs). Protein arginine deiminase 4 (PAD4) catalyzes the conversion of arginine to citrulline and is crucial for NET formation. Objectives: Here, we show that PAD4 inactivates full-length TFPIα by citrullination of its functional arginines. Methods: Citrullination of TFPIα and of TFPI-constructs by PAD4 was studied using western blotting and mass spectrometry. Binding of TFPIα to PAD4 was investigated using a solid-phase assay. Functional consequences were investigated by factor Xa inhibition and thrombin generation assays. Results: Nanomolar PAD4 amounts eliminated factor Xa inhibition by TFPIα. A citrullinated mutant Kunitz 2 domain did not inhibit factor Xa. Citrullination of TFPIα was found to be time- and concentration-dependent. Immunoprecipitation of citrullinated proteins from whole blood after neutrophil activation suggested the presence of TFPIα. Negatively charged phospholipids inhibited citrullination and truncated variants K1K2 and TFPI 1-161, and the isolated K2 domain were less efficiently citrullinated by PAD4. TFPIα bound to PAD4 with nanomolar affinity and involved the basic C-terminus. Thrombin generation in TFPI-deficient plasma demonstrated reduced anticoagulant activity of citrullinated TFPI. Mass spectrometry demonstrated citrullination of surface-exposed arginine residues in TFPIα after incubation with PAD4. Conclusion: Full-length TFPIα is sensitive to citrullination by PAD4, which causes loss of factor Xa inhibition. This process may play a role in the increased thrombosis risk associated with inflammation.

Liver international 42(5), 1185-1203 (2022). doi:10.1111/liv.15187, Published by Wiley-Blackwell, Oxford

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine–chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

Hepatic steatosis and chronic hepatocyte damage ultimately lead to liver fibrosis. Key pathophysiological steps are the activation and transdifferentiation of hepatic stellate cells. We assessed the interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions. We hypothesized that hepatocyte-derived extracellular vesicles (EVs) modify the phenotype of stellate cells. By high speed centrifugation, EVs were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2 under baseline conditions (C-EVs) or after induction of steatosis by linoleic and oleic acids for 24 h (FA-EVs). Migration of the human stellate cell line TWNT4 and of primary human stellate cells towards the respective EVs and sera of MAFLD patients were investigated using Boyden chambers. Phenotype alterations after incubation with EVs were determined by qRT-PCR, Western blotting and immunofluorescence staining. HepG2 cells released more EVs after treatment with fatty acids. Chemotactic migration of TWNT4 and primary hepatic stellate cells was increased, specifically towards FA-EVs. Prolonged incubation of TWNT4 cells with FA-EVs induced expression of proliferation markers and a myofibroblast-like phenotype. Though the expression of the collagen type 1 α1 gene did not change after FA-EV treatment, expression of the myofibroblast markers, e.g., α-smooth-muscle-cell actin and TIMP1, was significantly increased. We conclude that EVs from steatotic hepatocytes can influence the behavior, phenotypes and expression levels of remodeling markers of stellate cells and guides their directed migration. These findings imply EVs as operational, intercellular communicators in the pathophysiology of steatosis-associated liver fibrosis and might represent a novel diagnostic parameter and therapeutic target.

Background and aims: Platelets can release extracellular vesicles (EVs) upon stimulation with various agonists. Interestingly, platelets from patients with Glanzmann thrombasthenia have reduced EV release. These platelets lack functional alpha(IIb)beta(3) integrins, indicating that alpha(IIb)beta(3) integrin is critical in vesicle release. Integrin activation is central in platelet function and is associated with e.g. adhesion, aggregation and cytoskeletal rearrangement. However, while platelet activation pathways are widely known, the mechanisms underlying EV release remain uncharacterized. We investigated the role of integrin alpha(IIb)beta(3), phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release.Methods: EVs were isolated from activated platelets. Platelet activation status was measured by multicolour flow cytometry. A panel of pharmacologic inhibitors was used to interfere in specific signalling pathways. EV release was quantified enzymatically based on membrane PS content and nanoparticle tracking analysis. In addition, real-time visualization of EV shedding with confocal microscopy and EVs with Cryo-TEM imaging was performed.Results: Platelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of alpha(IIb)beta(3) integrin activation and subsequent closure paralleled by PS exposure. Prevention of alpha(IIb)beta(3) activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using a alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann thrombasthenia. Inhibition of actin cytoskeleton rearrangement decreased EV release, whereas inhibition of individual signalling targets upstream of cytoskeletal rearrangement showed no such effects.Conclusion: Platelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.

Atherosclerosis is one of the leading causes of mortality in developed and developing countries. The onset of atherosclerosis development is accompanied by overexpression of several inflammatory chemokines. Neutralization of these chemokines by chemokine-binding agents attenuates atherosclerosis progression. Here, we studied structural binding features of the tick protein Evasin-3 to chemokine (C-X-C motif) ligand 1 (CXCL1). We showed that Evasin-3-bound CXCL1 is unable to activate the CXCR2 receptor, but retains affinity to glycosamino-glycans. This observation was exploited to detect inflammation by visualizing a group of closely related CXC-type chemokines deposited on cell walls in human endothelial cells and murine carotid arteries by a fluorescent Evasin-3 conjugate. This work highlights the applicability of tick-derived chemokine-binding conjugates as a platform for the development of new agents for inflammation imaging.

To fulfil their orchestrating function in immune cell trafficking in homeostasis and disease, a network of 49 chemokines and 23 receptors capitalizes on features of specificity, redundancy, and functional selectivity such as biased agonism. The discovery of the chemokine interactome, i.e. heteromeric chemokine-chemokine interactions, even across CC- and CXC-class borders, has further expanded the complexity within the network. Moreover, some inflammatory mediators, which are not structurally linked to classical CC-, CXC-, CX3C-, or C-chemokines, can bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified the cytokine macrophage migration inhibitory factor (MIF) as an ACK that binds to the chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF may form heterocomplexes with classical chemokines. We tested this hypothesis, applying an unbiased chemokine protein binding array. The platelet chemokine CXCL4L1, but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12, was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, which also established high-affinity binding (KD≍100-150 nM). The binding interface was predicted by peptide array-based mapping and molecular docking. We next determined whether heterocomplex formation modulates inflammatory and atherogenic activities of MIF. MIF-elicited T-cell chemotaxis as assessed in a 3D-matrix-based live cell-imaging set-up was abrogated, when cells were co-incubated with MIF and CXCL4L1. Heterocomplexation also blocked MIF-triggered migration of Egfp+ microglia in cortical cultures in situ. Of note, CXCL4L1 blocked the binding of Alexa-MIF to a soluble ectodomain mimic of CXCR4 and co-incubation with CXCL4L1 attenuated MIF-triggered dynamic mass redistribution in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. As MIF and CXCL4L1 are abundant platelet products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM- FRET, and proximity ligation assay visualized heterocomplexes in platelet aggregates and clinical human thrombus sections. Moreover, heterocomplex formation inhibited MIF- stimulated thrombus formation under flow and skewed the morphology of adhering platelets from a large to a small lamellipodia phenotype. Together, our study establishes a novel molecular interaction, adding to the complexity of the chemokine interactome and chemokine/receptor network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be promising target structures to modulate inflammation and thrombosis.

The cause of atherothrombosis is rupture or erosion of atherosclerotic lesions, leading to an increased risk on myocardial infarction or stroke. Here, platelet activation plays a major role, leading to the release of bioactive molecules, e.g. chemokines and coagulation factors, and to platelet clot formation. Several antiplatelet therapies have been developed for secondary prevention of cardiovascular events, in which anticoagulant drugs are often combined. Besides playing a role in haemostasis, platelets are also involved in inflammation. However, it is unclear whether current antiplatelet therapies also affect platelet immune functions. In this study, the possible anti-inflammatory effects of antiplatelet medications on chemokine release were investigated using ELISA and on the chemotaxis of THP-1 cells towards platelet releasates. The cause of atherothrombosis is rupture or erosion of atherosclerotic lesions, leading to an increased risk on myocardial infarction or stroke. Here, platelet activation plays a major role, leading to the release of bioactive molecules, e.g. chemokines and coagulation factors, and to platelet clot formation. Several antiplatelet therapies have been developed for secondary prevention of cardiovascular events, in which anticoagulant drugs are often combined. Besides playing a role in haemostasis, platelets are also involved in inflammation. However, it is unclear whether current antiplatelet therapies also affect platelet immune functions. In this study, the possible anti-inflammatory effects of antiplatelet medications on chemokine release were investigated using ELISA and on the chemotaxis of THP-1 cells towards platelet releasates. We found that antiplatelet medication acetylsalicylic acid (ASA) led to reduced chemokine (C-C motif) ligand 5 (CCL5) and chemokine (C-X-C motif) ligand 4 (CXCL4) release from platelets, while leukocyte chemotaxis was not affected. Depending on the agonist, αIIbβ3- and P2Y12-inhibitors also affected CCL5 or CXCL4 release. The combination of ASA with a P2Y12 inhibitor or a phosphodiesterase inhibitor did not lead to an additive reduction on CCL5 or CXCL4 release. Interestingly, these combinations did reduce leukocyte chemotaxis. This study provides evidence that combined therapy of ASA and a P2Y12 or PDE3 inhibitor can decrease the inflammatory leukocyte recruiting potential of the releasate of activated platelets.

The healthy endothelium controls platelet activity through release of prostaglandin I2 (PGI2) and nitric oxide. The loss of this natural brake on platelet activity can cause platelets to become hyperreactive. PGI2 attenuates platelet activation by adenosine diphosphate (ADP) through stimulation of cyclic adenosine monophosphate (cAMP) production and subsequent phosphorylation changes by protein kinase A (PKA). We hypothesize that proteins/processes involved in platelet hyperactivity downstream of the cAMP-PKA pathway can serve as a “switch” in platelet activation and inhibition. We designed a network biology approach to explore the entangled platelet signaling pathways downstream of PGI2 and ADP. The STRING database was used to build a protein-protein interaction network from proteins of interest in which we integrate a quantitative platelet proteome dataset with pathway information, relative RNA expression of hematopoietic cells, the likelihood of the proteins being phosphorylated by PKA, and drug-target information from DrugBank in a biological network. We distilled 30 proteins from existing phosphoproteomics datasets (PXD000242 and PXD001189) that putatively can be “turned on” after ADP-mediated platelet activation and subsequently switched “off” after platelet inhibition with iloprost. Enrichment analysis revealed biological processes related to vesicle secretion and cytoskeletal reorganization to be overrepresented coinciding with topological clusters in the network. Our method highlights novel proteins related to vesicle transport, platelet shape change, and small GTPases as potential switch proteins in platelet activation and inhibition. Our novel approach demonstrates the benefit of data integration by combining tools and datasets and visualization to obtain a more complete picture of complex molecular mechanisms.

Objective: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets’ inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s−1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. Conclusions: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

International journal of molecular sciences 22(14), 7332 (2021). doi:10.3390/ijms22147332 special issue: "Special Issue "Molecular Research in Cardiovascular Disease" / Special Issue Editors: Prof. Dr. Maria I. Dorobantu, Prof. Dr. Maya Simionescu", Published by Molecular Diversity Preservation International, Basel

Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation. Ticks are blood-sucking parasites that secrete numerous immune-modulatory agents in their saliva to evade host immune responses. Evasin-3 is a small salivary protein that belongs to a class of chemokine-binding proteins isolated from the brown dog tick, Rhipicephalus sanguineus. Evasin-3 has been shown to have a high affinity for chemokines CXCL1 and CXCL8 and to diminish inflammation in mice. In the present study, solution NMR spectroscopy was used to investigate the structure of Evasin-3 and its CXCL8-Evasin-3 complex. Evasin-3 is found to disrupt the glycosaminoglycan-binding site of CXCL8 and inhibit the interaction of CXCL8 with CXCR2. Structural data were used to design two novel CXCL8-binding peptides. The linear tEv3 17-56 and cyclic tcEv3 16-56 dPG Evasin-3 variants were chemically synthesized by solid-phase peptide synthesis. The affinity of these newly synthesized variants to CXCL8 was measured by surface plasmon resonance biosensor analysis. The K-d values of tEv3 17-56 and tcEv3 16-56 dPG were 27 and 13 nm, respectively. Both compounds effectively inhibited CXCL8-induced migration of polymorphonuclear neutrophils. The present results suggest utility of synthetic Evasin-3 variants as scaffolds for designing and fine-tuning new chemokine-binding agents that suppress immune responses and inflammation.

With the incidence and impact of atherosclerotic cardiovascular disease and its clinical manifestations still rising, therapeutic options that target the causal mechanisms of this disorder are highly desired. Since the CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) has demonstrated that lowering inflammation can be beneficial, focusing on mechanisms underlying inflammation, for example, leukocyte recruitment, is feasible. Being key orchestrators of leukocyte trafficking, chemokines have not lost their attractiveness as therapeutic targets, despite the difficult road to drug approval thus far. Still, innovative therapeutic approaches are being developed, paving the road towards the first chemokine-based therapeutic against inflammation. In this overview, recent developments for chemokines and for the chemokine-like factor MIF (macrophage migration inhibitory factor) will be discussed.

Galectins are versatile glycan-binding proteins involved in immunomodulation. Increasing evidence suggests that galectins can control the immunoregulatory function of cytokines and chemokines through direct binding. Here, we report a new inverse mechanism by which chemokines control the immunomodulatory function of galectins. In a galectin-chemokine interaction screen we identified several specific galectin-chemokine binding pairs, including galectin-1/CXCL4. NMR analyses showed that CXCL4 binds on the surface edge of the galectin-1 ß-sheet causing changes in the galectin-1 carbohydrate binding site. Consequently, the interaction with CXCL4 altered the glycan binding affinity and specificity of galectin-1. With regard to immunomodulation, CXCL4 potentiated the apoptotic activity of galectin-1 on activated peripheral blood mononuclear cells. The potentiation of apoptosis specifically affected CD8+ T cells, while no effect was observed in CD4+ T cells. An opposite regulatory activity was found for another galectin-chemokine pair, i.e., galectin-9/CCL5. While CCL5 reduced the apoptosis induction by galectin-9 in activated PBMCs, this was only statistically significant for CD4+ T cells and not for CD8+ T cells. Collectively, the current study describes a novel immunomodulatory mechanism in which specific galectin-chemokine interactions control the glycan-binding activity and immunoregulatory function of galectins.

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

Atherosclerosis is an underlying cause of a broad array of cardiovascular diseases characterized by plaques, arterial wall thickening initiated by hyperlipidemia, pro-inflammatory signals, endothelial dysfunction and the influx of inflammatory cells. By still incompletely characterized mechanisms, these plaques can destabilize or erode, leading to thrombosis and blood vessel occlusion and becomes clinically manifest as angina pectoris, myocardial infarction (MI) or stroke. Among the several blood cell types that are involved in the development of atherosclerosis, the role of platelets during the thrombotic occlusion of ruptured or eroded plaques is well established and clinically exploited as evident by the extensive use of platelet inhibitors. However, there is increasing evidence that platelets are also involved in the earlier stages of atheroma development by exhibiting pro-inflammatory activities. The scope of this review is to describe the role of platelets in the initiation and propagation stages of atherosclerosis and beyond; in atherothrombotic complications.

Background and aimsHigh caloric dietary intake is associated with hepatic steatosis and chronic hepatocyte damage leading ultimately to liver fibrosis and cirrhosis with organ failure. Although the pathophysiologic process orchestrating liver fibrosis is not completely clarified, pivotal steps are the activation and transdifferentiation of hepatic stellate cells. In this study, we aim to assess the direct interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions and hypothesize that extracellular vesicles (EV) isolated from hepatocytes can directly manipulate the phenotype of stellate cells.MethodsBy high speed centrifugation, EV were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2, under baseline conditions (C-EV) or after induction of steatosis by linoleic and oleic acid for 24 hours (FA-EV). Migration of the stellate cell line TWNT4 towards respective EV as well as sera of NASH patients was investigated using Boyden chambers. TWNT4 phenotype alterations after incubation with EV was determined by qPCR, western blotting and immunofluorescence staining. ResultsHepG2 cells released more EV after treatment with fatty acids. Chemotactic migration of TWNT4 cells was increased specifically towards FA-EV. Prolonged incubation of TWNT4 cells with FA-EV induce expression of proliferation markers and a myofibroblast-like phenotype. Whereas the expression of the collagen type 1 1 gene did not change after FA-EV-treatment, expression of the myofibroblast markers e.g. -smooth muscle cell actin and TIMP1 were significantly increased. ConclusionWe concluded that EV from steatotic HepG2 cells can influence the behavior and phenotype of TWNT4 cells as well as the expression of remodeling markers and guides directed migration. These findings imply EV as operational, intercellular communicators in the pathophysiology of steatosis associated liver fibrosis.

Platelets can release extracellular vesicles (EVs) upon stimulation with various agonists. Interestingly, platelets from patients with Glanzmann thrombasthenia have reduced EV release. These platelets lack functional αEVs were isolated from activated platelets. Platelet activation status was measured by multicolour flow cytometry. A panel of pharmacologic inhibitors was used to interfere in specific signalling pathways. EV release was quantified enzymatically based on membrane PS content and nanoparticle tracking analysis. In addition, real-time visualization of EV shedding with confocal microscopy and EVs with Cryo-TEM imaging was performed.Platelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of αPlatelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.

Objective— The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations. Approach and Results— Flow-dependent endothelial dysfunction was induced in apolipoprotein E–deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A–targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [ P P Conclusions— Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A–targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.

Platelets play an important role in the pathogenesis of vascular remodelling after injury. Junctional adhesion molecule A (JAM-A) was recently described to regulate platelet activation. Specific deletion of JAM-A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet-derived JAM-A to neointima formation after vascular injury. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background underwent wire-induced injury of the common carotid artery. Ex vivo imaging by two-photon microscopy revealed increased platelet coverage at the site of injury in trJAM-A-deficient mice. Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM-A-deficient platelets than to control platelets. Inhibition of alpha(M)beta(2) or GPIb alpha, but not of CD62P, suppressed those differences. Up to 4 weeks after wire injury, intimal neoplasia and neointimal cellular content were analysed. Neointimal lesion area was increased in trJAM-A(-/-) apoe(-/-) mice and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM-A(+/+) apoe(-/-) littermates 2 weeks, but not 4 weeks after injury. Re-endothelialization was decreased in trJAM-A(-/-) apoe(-/-) mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks. A platelet gain of function by deletion of JAM-A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages.

With the incidence and impact of atherosclerotic cardiovascular disease and its clinical manifestations still rising, therapeutic options that target the causal mechanisms of this disorder are highly desired. Since the CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) has demonstrated that lowering inflammation can be beneficial, focusing on mechanisms underlying inflammation, for example, leukocyte recruitment, is feasible. Being key orchestrators of leukocyte trafficking, chemokines have not lost their attractiveness as therapeutic targets, despite the difficult road to drug approval thus far. Still, innovative therapeutic approaches are being developed, paving the road towards the first chemokine-based therapeutic against inflammation. In this overview, recent developments for chemokines and for the chemokine-like factor MIF (macrophage migration inhibitory factor) will be discussed.

Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

SummaryPlatelets not only serve as essential haemostatic cells, they also have important roles in immune defence and inflammation. Despite not having a nucleus, platelets contain physiologically relevant amounts of RNA, which can be spliced and translated into functional proteins. In addition, platelets have the ability to bind to numerous other cells, such as leukocytes and vascular cells. During those interactions, platelets can modulate cellular responses, resulting in e. g. inflammatory activation or apoptosis. Recent studies have demonstrated that platelets can influence the outcomes of bacterial and viral infection, as well as the extent of tissue injury after ischaemia. Platelets also carry considerable amounts of cytokines and growth factors in their secretory granules, preformed for rapid secretion. Those properties in combination with the sheer amount of platelets circulating in the blood stream make them an important force in the immune response during health and disease. In this overview, recent findings concerning those interesting properties of platelets beyond haemostasis are discussed.

Background: The Kunitz type tissue factor pathway inhibitor (TFPI) is an important regulator in hemostasis. Decreased concentrations are a risk factor for thrombosis and complete TFPI deficiency is associated with lethality. TFPI is also reported to be an important link between inflammation and thrombosis. Neutrophil extracellular traps (NETs) formed by NETosis can bind TFPI which then can be cleaved and inactivated by neutrophil elastase (NE) during thrombotic events. In neutrophils, the enzyme peptide arginine deiminase 4 (PAD4) is central in the citrullination of histones prior to the externalization of DNA during NETosis. In this study, we provide evidence that PAD4 also might regulate the activity of TFPI by posttranslational modification of its functional arginines into citrulline. Aim: To study the effect of citrullination of TFPI and various TFPI constructs on their functional activity on FXa or thrombin generation. Methods: Citrullination of TFPI by the neutrophil protein PAD4 was studied in a model system (FXa inhibition) and in plasma system (thrombin generation). Various TFPI constructs, Kunitz (K) domains K1K2, K2, and TFPI1-161 were used to study the effects of citrullination on inhibition of FXa. LC-MS was used to locate the specific sites of citrullination. Results: This study shows that PAD4 very efficiently citrullinates full lenght TFPI. Very low concentrations of PAD4 were sufficient (Ki 0.4 nM) to abolish FXa inhibition by TFPI. Citrullination is calcium-ion, time- and concentration-dependent. The truncated variants K1K2 and TFPI 1-161 and the isolated K2 domain were citrullinated less efficiently by PAD4 than TFPI, implying the presence of specific binding sites for PAD4 at the C-terminus of TFPI. The presence of phospholipids inhibited the citrullination reaction, an effect only seen for TFPI and not for all the other TFPI variants. Thus, the presence of the C-terminus in TFPI appears to be favourable for citrullination by PAD4. Thrombin generation in TFPI-deficient plasma triggered with TF or Russell's viper venom (RVV)-X showed almost complete absence of anticoagulant activity of citrullinated TFPI Conclusions: To conclude, only TFPI is very sensitive to citrullination by PAD4. Citrullinated TFPI has lost its ability to inhibit FXa. This process might play a role in the increased thrombosis risk with inflammation. Further experiments are needed to determine the physiologic or pathologic relevance of this process. Disclosures No relevant conflicts of interest to declare.

Myocardial infarction (MI) is a major cause of death in Western countries and finding new strategies for its prevention and treatment is thus of high priority. In a previous study, we have demonstrated a pathophysiologic relevance for the heterophilic interaction of CCL5 and CXCL4 in the progression of atherosclerosis. A specifically designed compound (MKEY) to block this CCL5-CXCR4 interaction is investigated as a potential therapeutic in a model of myocardial ischemia/reperfusion (I/R) damage. 8 week-old male C57BL/6 mice were intravenously treated with MKEY or scrambled control (sMKEY) from 1 day before, until up to 7 days after I/R. By using echocardiography and intraventricular pressure measurements, MKEY treatment resulted in a significant decrease in infarction size and preserved heart function as compared to sMKEY-treated animals. Moreover, MKEY treatment significantly reduced the inflammatory reaction following I/R, as revealed by specific staining for neutrophils and monocyte/macrophages. Interestingly, MKEY treatment led to a significant reduction of citrullinated histone 3 in the infarcted tissue, showing that MKEY can prevent neutrophil extracellular trap formation in vivo. Disrupting chemokine heterodimers during myocardial I/R might have clinical benefits, preserving the therapeutic benefit of blocking specific chemokines, and in addition, reducing the inflammatory side effects maintaining normal immune defence.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin–angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet–fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia–reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C–based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.