Platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation.

Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post-myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A-mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia-sera/immunoglobulin G-triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.

Platelet SR-PSOX/CXCL16-CXCR6 Axis Influences Thrombotic Propensity and Prognosis in Coronary Artery Disease.

Platelets express the transmembrane chemokine SR-PSOX/CXCL16, proteolytic cleavage of which generates the sCXCL16 soluble-(s) chemokine. The sCXCL16 engages CXCR6 on platelets to synergistically propagate degranulation, aggregation and thrombotic response. Currently, we have investigated the pro-thrombotic and prognostic association of platelet CXCL16−CXCR6 axis in CAD-(n = 240; CCS n = 62; ACS n = 178) patients. Platelet surface-associated-CXCL16 and CXCR6 surface expression ascertained by flow cytometry correlated significantly with platelet activation markers (CD62P denoting degranulation and PAC-1 binding denoting α2bß3-integrin activation). Higher platelet CXCL16 surface association (1st quartile vs. 2nd−4th quartiles) corresponded to significantly elevated collagen-induced platelet aggregation assessed by whole blood impedance aggregometry. Platelet-CXCL16 and CXCR6 expression did not alter with dyslipidemia, triglyceride, total cholesterol, or LDL levels, but higher (>median) plasma HDL levels corresponded with decreased platelet-CXCL16 and CXCR6. Although platelet-CXCL16 and CXCR6 expression did not change significantly with or correlate with troponin I levels, they corresponded with higher Creatine Kinase-(CK) activity and progressively deteriorating left ventricular ejection fraction (LVEF) at admission. Elevated-(4th quartile) platelet-CXCL16 (p = 0.023) and CXCR6 (p = 0.030) measured at admission were significantly associated with a worse prognosis. However, after Cox-PH regression analysis, only platelet-CXCL16 was ascertained as an independent predictor for all-cause of mortality. Therefore, the platelet CXCL16−CXCR6 axis may influence thrombotic propensity and prognosis in CAD patients.

Targeted Profiling of Short-, Medium-, and Long-Chain Fatty Acyl-Coenzyme As in Biological Samples by Phosphate Methylation Coupled to Liquid Chromatography-Tandem Mass Spectrometry.

Fatty acyl-coenzyme As (acyl-CoAs) are of central importance in lipid metabolism pathways. Short-chain acyl-CoAs are usually part of metabolomics, and medium- to (very) long-chain acyl-CoAs are focus of lipidomics studies. However, owing to the specific complex and amphiphilic nature contributed by fatty acyl chains and hydrophilic CoA moiety, lipidomic analysis of acyl-CoAs is still challenging, especially in terms of sample preparation and chromatographic coverage. In this work, we propose a derivatization strategy of acyl-CoAs based on phosphate methylation. After derivatization, full coverage (from free CoA to C25:0-CoA) and good peak shape in liquid chromatography were achieved. At the same time, analyte loss due to the high affinity of phosphate groups to glass and metallic surfaces was resolved, which is beneficial for routine analysis in large-scale lipidomics studies. A sample preparation method based on mixed-mode SPE was developed to optimize extraction recoveries and allow optimal integration of the derivatization process in the analytical workflow. LC-MS/MS was performed with targeted data acquisition by SRM transitions, which were constructed based on similar fragmentation rules observed for all methylated acyl-CoAs. To achieve accurate quantification, uniformly 13C-labeled metabolite extract from yeast cells was taken as internal standards. Odd-chain and stable isotope-labeled acyl-CoAs were used as surrogate calibrants in the same matrix. LOQs were between 16.9 nM (short-chain acyl-CoAs) and 4.2 nM (very-long-chain acyl-CoAs). This method was validated in cultured cells and was applied in HeLa cells and human platelets of coronary artery disease patients. It revealed distinct acyl-CoA profiles in HeLa cells and platelets. The results showed that this method can effectively detect acyl-CoAs in biological samples. Considering their central importance in many de novo lipid biosynthesis and remodeling processes, this targeted method offers a valid foundation for future lipidomics analysis of acyl-CoA profiles in biological samples, particularly those concerning metabolic syndrome.

Impact of Amyloid-ß on Platelet Mitochondrial Function and Platelet-Mediated Amyloid Aggregation in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by an accumulation of amyloid ß (Aß) peptides in the brain and mitochondrial dysfunction. Platelet activation is enhanced in AD and platelets contribute to AD pathology by their ability to facilitate soluble Aß to form Aß aggregates. Thus, anti-platelet therapy reduces the formation of cerebral amyloid angiopathy in AD transgenic mice. Platelet mitochondrial dysfunction plays a regulatory role in thrombotic response, but its significance in AD is unknown and explored herein. METHODS: The effects of Aß-mediated mitochondrial dysfunction in platelets were investigated in vitro. RESULTS: Aß40 stimulation of human platelets led to elevated reactive oxygen species (ROS) and superoxide production, while reduced mitochondrial membrane potential and oxygen consumption rate. Enhanced mitochondrial dysfunction triggered platelet-mediated Aß40 aggregate formation through GPVI-mediated ROS production, leading to enhanced integrin αIIbß3 activation during synergistic stimulation from ADP and Aß40. Aß40 aggregate formation of human and murine (APP23) platelets were comparable to controls and could be reduced by the antioxidant vitamin C. CONCLUSIONS: Mitochondrial dysfunction contributes to platelet-mediated Aß aggregate formation and might be a promising target to limit platelet activation exaggerated pathological manifestations in AD.

Molecular Drivers of Platelet Activation: Unraveling Novel Targets for Anti-Thrombotic and Anti-Thrombo-Inflammatory Therapy.

Cardiovascular diseases (CVDs) are the leading cause of death globally-partly a consequence of increased population size and ageing-and are major contributors to reduced quality of life. Platelets play a major role in hemostasis and thrombosis. While platelet activation and aggregation are essential for hemostasis at sites of vascular injury, uncontrolled platelet activation leads to pathological thrombus formation and provokes thrombosis leading to myocardial infarction or stroke. Platelet activation and thrombus formation is a multistage process with different signaling pathways involved to trigger platelet shape change, integrin activation, stable platelet adhesion, aggregation, and degranulation. Apart from thrombotic events, thrombo-inflammation contributes to organ damage and dysfunction in CVDs and is mediated by platelets and inflammatory cells. Therefore, in the past, many efforts have been made to investigate specific signaling pathways in platelets to identify innovative and promising approaches for novel antithrombotic and anti-thrombo-inflammatory strategies that do not interfere with hemostasis. In this review, we focus on some of the most recent data reported on different platelet receptors, including GPIb-vWF interactions, GPVI activation, platelet chemokine receptors, regulation of integrin signaling, and channel homeostasis of NMDAR and PANX1.

CK2ß regulates thrombopoiesis and Ca2+-triggered platelet activation in arterial thrombosis.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

Mononuclear and Dinuclear Ruthenium Complexes of cis- and trans-Thioindigo: Geometrical and Electronic Structure Analyses.

The compounds [Ru(acac)2(L)] (1) and [Ru2(acac)4(µ-L)] (2) with acac- = acetylacetonato and L = thioindigo were characterized crystallographically with a cis-configurated L and O,O'-coordinated metal in 1 and with trans-configurated L and two S,O-coordinated bridged ruthenium centers for 2. The electronic structures of 1 and 2 were confirmed by spectroscopy and density functional theory calculations, suggesting considerable metal-to-ligand electron transfer resulting in the formation of the thioindigo radical anion and oxidized metal(s). UV-Vis-Near-IR and IR (spectro)electrochemistry was used to investigate charged forms 1 n ( n = 1+, 1-) and 2 n ( n = 1+, 1-, 2-), which reveal electron transfer activity of both the metal and the thioindigo ligand as well as sizable orbital mixing. An intense near-IR absorption is observed for 2- at 2180 nm. The remarkable ligand properties of thioindigo are being discussed in connection to related coordination compounds of indigo derivatives such as dehydroindigo and corresponding ("Nindigo") diimines.

Regulation of oxidized platelet lipidome: implications for coronary artery disease.

AIMS: Hyperlipidaemia enhances susceptibility to thrombosis, while platelet oxidixed LDL (oxLDL) binding in acute coronary syndrome (ACS) correlates with activation status. This study explores the platelet lipidome in symptomatic coronary artery disease (CAD) patients and the functional consequences of the chemokine CXCL12 and its receptors CXCR-4/-7 on lipid uptake in platelets. METHODS AND RESULTS: Platelet-oxLDL detected by flow cytometry was enhanced (P = 0.04) in CAD patients, moderately correlated with platelet CXCR7 surface expression (ρ = 0.39; P < 0.001), while inversely with CXCR4 (ρ = 0.35; P < 0.001). Platelet-oxLDL was elevated (P = 0.01) in ACS patients with angiographic evidence of intracoronary thrombi. Ex vivo analysis of intracoronary thrombi sections revealed oxLDL deposition in platelet-enriched areas verified by immunofluorescence confocal microscopy. LDL-oxLDL uptake enhanced reactive oxygen species, mitochondrial superoxide generation, intraplatelet LDL to oxLDL conversion, and lipid peroxidation, counteracted by SOD2-mimetic MnTMPyP. Lipidomic analysis revealed enhanced intraplatelet-oxidized phospholipids, cholesteryl esters, sphingomyelin, ceramides, di- and triacylglycerols, acylcarnitines in CAD patients compared with age-matched controls as ascertained by liquid chromatography hyphenated to high-resolution mass spectrometry. LDL-oxLDL induced degranulation, αIIbß3-integrin activation, apoptosis, thrombin generation estimated by calibrated automated thrombinoscopy, and shape change verified by live imaging using scanning ion conductance microscopy. Further, LDL-oxLDL enhanced thrombus formation ex vivo and in vivo in mice (ferric chloride-induced carotid artery injury). LDL-oxLDL enhanced platelet CXCL12 release, differentially regulated CXCR4-CXCR7 surface exposure, while CXCL12 prompted LDL-oxLDL uptake and synergistically augmented the LDL-oxLDL-induced pro-oxidative, thrombogenic impact on platelet function. CONCLUSION: An altered platelet lipidome might be associated with thrombotic disposition in CAD, a mechanism potentially regulated by CXCL12-CXCR4-CXCR7 axis.

Platelets as a Novel Source of Pro-Inflammatory Chemokine CXCL14.

OBJECTIVE: Platelets are a major source of chemokines. Here, we demonstrate for the first time that platelets express significant amounts of CXCL14 and disclose powerful effects of platelet-derived CXCL14 on monocyte and endothelial migration. METHODS: The expression of CXCL14 in platelets and in the supernatant of activated platelets was analysed by immunoblotting, ELISA, and flow cytometry. The effect of platelet-derived CXCL14 on monocyte migration was evaluated using a modified Boyden chamber. The effect of CXCL14 on monocyte phagocytosis was tested by using fluorochrome-labelled E.coli particles. The effect of platelet-derived CXCL14 on endothelial migration was explored by the use of an endothelial scratch assay. RESULTS: Hitherto unrecognized expression of CXCL14 in human and murine platelets was uncovered by immunoblotting. Activation with platelet agonists such as adenosine-di-phosphate (ADP), collagen-related peptide (CRP), or thrombin-receptor activating peptide (TRAP), increased CXCL14 surface expression (flow cytometry) and release into the supernatant (immunoblotting, ELISA). Since CXCL14 is known to be chemotactic for CD14+ monocytes, we investigated the chemotactic potential of platelet-derived CXCL14 on human monocytes. Activated platelet supernatant induced monocyte migration, which was counteracted upon neutralization of platelet-derived CXCL14 as compared to IgG control. Blocking of the chemokine receptor CXCR4, but not CXCR7, reduced the number of migratory monocytes towards recombinant CXCL14, suggesting the involvement of CXCR4 in the CXCL14-directed monocyte chemotaxis. Recombinant CXCL14 enhanced the phagocytic uptake of E.coli particles by monocytes. In scratch assays with cultured endothelial cells (HUVECs), platelet-derived CXCL14 counteracted the pro-angiogenic effects of VEGF, supporting its previously recognized angiostatic potential. CONCLUSIONS: Platelets are a relevant source of CXCL14. Platelet-derived CXCL14 at the site of vascular lesions might play an important role in vascular repair/regeneration.

Clinical significance of receptor shedding-platelet GPVI as an emerging diagnostic and therapeutic tool.

Platelet membrane bedecked with a wide array of receptors offers a platform to regulate platelet responsiveness, thrombotic propensity, inflammatory disposition, and immune reactivity under diverse pathophysiological conditions. Ectopic proteolytic cleavage of such receptors irreversibly inactivates receptor-mediated intracellular signaling governing cellular functions, further releases soluble fragments into circulation which might modulate functions of target cells. Glycoprotein VI-(GPVI) is a membrane glycoprotein expressed in platelets and megakaryocytes. Platelet GPVI surface expression is enhanced following acute ischemic events like myocardial infarction and cerebral stroke, serves as an imminent diagnostic tool independent of markers of tissue necrosis, and is associated with poor prognosis. Platelets undergo GPVI shedding and thereby contribute to soluble plasma levels of sGPVI, with distinct diagnostic and prognostic attributes. This review summarizes the functional significance and mechanistic basis whereby GPVI surface availability is up- or downregulated on platelets and the impact of GPVI in diagnostic, prognostic, and therapeutic strategies in diseases where platelets play a regulatory role. Further, we also highlight how novel non-invasive platelet-based diagnostic and therapeutic strategies have evolved utilizing GPVI for lesion-directed antithrombotic therapy or to counteract atherosclerotic disposition to ameliorate care of patients particularly in the context of cardio-cerebro-vascular medicine.

Platelet surface expression of SDF-1 is associated with clinical outcomes in the patients with cardiovascular disease.

Platelet surface expression levels of stromal cell derived factor 1 (SDF-1) are elevated in acute coronary syndrome and associated with LVEF% improvement after myocardial infarction (MI). Platelet SDF-1 might facilitate thrombus formation and endomyocardial expression of SDF-1 is enhanced in inflammatory cardiomyopathy and positively correlates with myocardial fibrosis. The influence of platelet SDF-1 on outcome in the patients with symptomatic coronary artery disease (CAD) is to the best of our knowledge unknown. Blood samples of 608 consecutive CAD patients were collected during the percutaneous coronary intervention and analyzed for surface expression of SDF-1 by flow cytometry. The primary combined endpoint was defined as the composite of either MI, or ischemic stroke, or all-cause death. Secondary endpoints were defined as the aforementioned single events. The patients with baseline platelet SDF-1 levels above the third quartile showed a significantly worse cumulative event-free survival when compared to the patients with lower baseline SDF-1 levels (first to third quartile) (log rank 0.009 for primary combined endpoint and log rank 0.016 for secondary endpoint all-cause death). Multivariate Cox regression analysis showed that SDF-1 levels above the third quartile were independently associated with the primary combined endpoint and the secondary endpoint all-cause death. We provide first clinical evidence that high platelet expression levels of SDF-1 influence clinical outcomes in CAD patients in a negative way.

Influence of γ-Secretase Inhibitor 24-Diamino-5-Phenylthiazole DAPT on Platelet Activation.

BACKGROUND/AIMS: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca(2+)-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α(IIb)ß3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by α(IIb)ß3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. CONCLUSIONS: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation.

Inflammatory Contribution of Platelets Revisited: New Players in the Arena of Inflammation.

Platelet-derived mediators, either in an autocrine or paracrine mode of action, regulate systemic and vascular inflammation as well as contribute to innate immune defense and also to regenerative mechanisms. This review reevaluates the impact of inflammatory mediators such as CXC-chemokine-Ligands, their receptors in modulating platelet functions and platelet survival, thereby influencing inflammatory or regenerative processes. We further explore the contribution of cyclophilin A and C-reactive protein in regulating thrombotic and hemostatic attributes of platelets. Moreover, we emphasize on the role of platelets as active components bridging the innate and adaptive immune responses, toll-like receptors on platelets, platelet interactions with the complement system, and platelet-derived thrombocidins exhibiting direct antimicrobial properties. As highlighted in this review, the multifaceted aspects of platelets and platelet-derived factors encourage further investigations in this intriguing and expansive but largely uncharted area of research in platelet biology.

Macrophage migration inhibitory factor limits activation-induced apoptosis of platelets via CXCR7-dependent Akt signaling.

RATIONALE: Macrophage migration inhibitory factor (MIF) is released on platelet activation. Circulating MIF could potentially regulate platelets and thereby platelet-mediated inflammatory and regenerative mechanisms. However, the effect of MIF on platelets is unknown. OBJECTIVE: The present study evaluated MIF in regulating platelet survival and thrombotic potential. METHODS AND RESULTS: MIF interacted with CXCR4-CXCR7 on platelets, defining CXCR7 as a hitherto unrecognized receptor for MIF on platelets. MIF internalized CXCR4, but unlike CXCL12 (SDF-1α), it did not phosphorylate Erk1/2 after CXCR4 ligation because of the lack of CD74 and failed in subsequent CXCR7 externalization. MIF did not alter the activation status of platelets. However, MIF rescued platelets from activation and BH3 mimetic ABT-737-induced apoptosis in vitro via CXCR7 and enhanced circulating platelet survival when administered in vivo. The antiapoptotic effect of MIF was absent in Cxcr7(-/-) murine embryonic cells but pronounced in CXCR7-transfected Madin-Darby canine kidney cells. This prosurvival effect was attributed to the MIF-CXCR7-initiated PI3K-Akt pathway. MIF induced CXCR7-Akt-dependent phosphorylation of BCL-2 antagonist of cell death (BAD) both in vitro and in vivo. Consequentially, MIF failed to rescue Akt(-/-) platelets from thrombin-induced apoptosis when challenged ex vivo, also in prolonging platelet survival and in inducing BAD phosphorylation among Akt(-/-) mice in vivo. MIF reduced thrombus formation under arterial flow conditions in vitro and retarded thrombotic occlusion after FeCl3-induced arterial injury in vivo, an effect mediated through CXCR7. CONCLUSION: MIF interaction with CXCR7 modulates platelet survival and thrombotic potential both in vitro and in vivo and thus could regulate thrombosis and inflammation.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

Noninnocence of Indigo: Dehydroindigo Anions as Bridging Electron-Donor Ligands in Diruthenium Complexes.

Complexes of singly or doubly deprotonated indigo (H2Ind) with one or two [Ru(pap)2](2+) fragments (pap = 2-phenylazopyridine) have been characterized experimentally [molecular structure, voltammetry, electron paramagnetic resonance (EPR), and UV-vis-near-IR spectroelectrochemistry] and by time-dependent density functional theory calculations. The compound [Ru(pap)2(HInd(-))]ClO4 ([1]ClO4) was found to contain an intramolecular NH---O hydrogen bond, whereas [{Ru(pap)2}2(µ-Ind(2-))](ClO4)2 ([2](ClO4)2), isolated as the meso diastereoisomer with near-IR absorptions at 1162 and 991 nm, contains two metals bridged at 6.354 Å distance by the bischelating indigo dianion. The spectroelectrochemical study of multiple reversible reduction and oxidation processes of 2(n) (n = 4+, 3+, 2+, 1+, 0, 1-, 2-, 3-, 4-) reveals the stepwise addition of electrons to the terminal π-accepting pap ligands, whereas the oxidations occur predominantly at the anionic indigo ligand, producing an EPR-identified indigo radical intermediate and revealing the suitability of deprotonated indigo as a σ- and π-donating bischelating bridge.

Activated platelets interfere with recruitment of mesenchymal stem cells to apoptotic cardiac cells via high mobility group box 1/Toll-like receptor 4-mediated down-regulation of hepatocyte growth factor receptor MET.

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.

Role of chemokine receptors CXCR4 and CXCR7 for platelet function.

Platelet-derived SDF-1α (stromal cell derived factor-α) mediates inflammation, immune defence and repair mechanisms at site of tissue injury. This review summarizes the relative expression of CXC chemokine receptor 4 (CXCR4) and CXCR7 in platelets, their dynamic trafficking in presence of ligands like chemokine C-X-C-motif ligand 11 (CXCL11), CXCL12 and MIF (macrophage migration inhibitory factor); how these receptors differentially mediate the functional and survival response to the chemokines CXCL11, CXCL12 and MIF. We further elaborate and emphasize the prognostic significance of platelet surface expression of CXCR4-CXCR7 in the context of coronary artery disease (CAD). SDF-1α/CXCL12, CXCL11, MIF effects mediated through CXCR4 and CXCR7 may play a regulatory role at the site of vascular and tissue inflammation, immune defence and repair where activated platelets reach as forerunners and function as critical players.

SDF-1α induces differential trafficking of CXCR4-CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival.

Platelet-derived SDF-1α (CXCL12) mediates inflammatory and regenerative mechanisms. The present study characterizes the effect of SDF-1α ligation in platelets. SDF-1α (0-100 µM) dose and time dependently caused internalization of its receptor CXCR4 (28.9 ± 1.6 vs. 16.1 ± 1.9 in SDF-1α-treated platelets), coupled to the surface externalization of CXCR7 (65.5 ± 8 vs. 162.8 ± 27.6 following SDF-1α treatment), both in vitro and in vivo. This was inhibited in the presence of AMD3100 (100 µM), CXCR4 blocking and vesicular transport inhibitors (brefeldin A, 10 µM; rapamycin, 100 nM). SDF-1α/CXCR-4-mediated CXCR7 translocation was significantly reduced by inhibitors of ERK1/2-(U0126-10 µM) and cyclophilinA (CyPA)-(NIM811-10 µM) by 28 and 46%, respectively. Further, SDF-1α-induced downstream phosphorylation of Erk1/2 led to CyPA-dependent ubiquitination of CXCR7, which is essential for its surface translocation. CyPA-PPIase-activity inhibitor NIM-811, Erk1/2, and E1-ligase inhibitor-(PYR-41-25 µM) significantly abolished SDF-1α-driven CXCR7 ubiquitination and subsequent surface translocation. SDF-1α induced CXCR7 ubiquitination, and its surface exposure was observed in wild-type murine platelets, but not in CyPA-deficient platelets. SDF-1α/CXCR4-CyPA-dependent CXCR7 translocation and its subsequent ligation attenuated activation-induced apoptosis both in vitro and when administered in vivo. This antiapoptotic effect of SDF-1α was abrogated by blocking CXCR7, also significantly affected in Cypa(-/-) platelets. Thus, we decipher a novel mechanism, whereby SDF-1α regulates relative receptor availability in circulating platelets and exerts its prosurvival benefits.-Chatterjee, M., Seizer, P., Borst, O., Schönberger, T., Mack, A., Geisler, T., Langer, H. F., May, A. E., Vogel, S., Lang, F., Gawaz, M. SDF-1α induces differential trafficking of CXCR4-CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival.