Patrick Münzer

The Inflammatory Chemokine CXC Motif Ligand 16 Triggers Platelet Activation and Adhesion Via CXC Motif Receptor 6–Dependent Phosphatidylinositide 3-Kinase/Akt Signaling

Rationale: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. Objective: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. Methods and Results: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin &agr;IIb&bgr;3 activation, and shape change. CXCL16 increased Akt phosphorylation (Thr308/Ser473), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin &agr;IIb&bgr;3 activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000−s) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P2Y1 (MRS2179, 100 µmol/L) and especially P2Y12 (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6–dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.Rationale: The recently discovered chemokine CXCL16 is highly expressed in atherosclerotic lesions and a potential pathogenic mediator in coronary artery disease. Objective: To test the role of CXCL16 on platelet activation and vascular adhesion as well as the underlying mechanism and signaling pathway. Methods and Results: RT-PCR, western blotting, confocal microscopy and flow cytometry revealed that CXCL16-specific receptor CXCR6 is highly expressed on platelets. According to flow cytometry and confocal microscopy stimulation of platelets with CXCL16 induced platelet degranulation, integrin α IIb β 3 activation and shape change. CXCL16 increased Akt phosphorylation (Thr 308 /Ser 473 ), an effect abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (100nM) and LY294002 (25 µM). The PI3K inhibitors and Akt inhibitor SH-6 (20 µM) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α IIb β 3 activation and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXCR6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro following high arterial shear stress (2000 -s ) and to injured vascular wall in vivo following carotis ligation. CXCL16-induced stimulation of platelet adhesion was again prevented by PI3K and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P 2 Y 1 (MRS2179, 100µM) and especially P 2 Y 12 (Cangrelor, 10µM) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotis ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXCR6-dependent PI3K/Akt signaling and paracrine activation suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes

Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)β3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

The Inflammatory Chemokine CXCL16 Triggers Platelet Activation and Adhesion via CXCR6-Dependent PI3K/Akt Signaling

Rationale: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. Objective: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. Methods and Results: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin &agr;IIb&bgr;3 activation, and shape change. CXCL16 increased Akt phosphorylation (Thr308/Ser473), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin &agr;IIb&bgr;3 activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000−s) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P2Y1 (MRS2179, 100 µmol/L) and especially P2Y12 (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6–dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.Rationale: The recently discovered chemokine CXCL16 is highly expressed in atherosclerotic lesions and a potential pathogenic mediator in coronary artery disease. Objective: To test the role of CXCL16 on platelet activation and vascular adhesion as well as the underlying mechanism and signaling pathway. Methods and Results: RT-PCR, western blotting, confocal microscopy and flow cytometry revealed that CXCL16-specific receptor CXCR6 is highly expressed on platelets. According to flow cytometry and confocal microscopy stimulation of platelets with CXCL16 induced platelet degranulation, integrin α IIb β 3 activation and shape change. CXCL16 increased Akt phosphorylation (Thr 308 /Ser 473 ), an effect abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (100nM) and LY294002 (25 µM). The PI3K inhibitors and Akt inhibitor SH-6 (20 µM) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α IIb β 3 activation and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXCR6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro following high arterial shear stress (2000 -s ) and to injured vascular wall in vivo following carotis ligation. CXCL16-induced stimulation of platelet adhesion was again prevented by PI3K and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P 2 Y 1 (MRS2179, 100µM) and especially P 2 Y 12 (Cangrelor, 10µM) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotis ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXCR6-dependent PI3K/Akt signaling and paracrine activation suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

Platelet-derived HMGB1 is a critical mediator of thrombosis

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1)

Ca2+ signaling includes store‐operated Ca2+ entry (SOCE) following depletion of endoplas‐mic reticulum (ER) Ca2+ stores. On store depletion, the ER Ca2+ sensor STIM1 activates Orai1, the pore‐forming unit of Ca2+‐release‐activated Ca2+ (CRAC) channels. Here, we show that Orai1 is regulated by serum‐ and glucocorticoid‐inducible kinase 1 (SGK1), a growth factor‐regulated kinase. Membrane Orai1 protein abundance, /CRAC, and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active SGK1 (by+81, +378, and+136%, respectively) but not by inactive K12 NSGK1. Coexpression of the ubiquitin ligase Nedd4‐2, an established negatively regulated SGK1 target, down‐regulated SOCE (by –48%) and /CRAc (by –60%), an effect reversed by expression of SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1‐knockout (sgk1) mice (by –37% and –52%, respectively) than in mast cells from wild‐type (sgk1+/+) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase‐inhibitor thapsi‐gargin (2 μΜ) stimulated migration, an effect significantly higher (by +306%) in SGKl‐expressing than in SGKl‐expressing HEK293 cells, and also significantly higher (by +108%) in sgk1+/+ than in sgk1−/− mast cells. SGK1 is thus a novel key player in the regulation of SOCE.—Eylenstein, A., Geh‐ring, E.‐M., Heise, N., Shumilina, E., Schmidt, S., Szteyn, K., Münzer, P., Nurbaeva, M. K., Eichenmüller, M., Tyan, L., Regel, I., Föller, M., Kuhl, D., Soboloff, J., Penner, R., Lang, R. Stimulation of Ca2+‐channel Orai1/STIM1 by serum‐ and glucocorticoid‐inducible kinase 1 (SGK1). FASEB J. 25, 2012‐2021 (2011). www.fasebj.org

Blood Platelets in the Progression of Alzheimer's Disease

Alzheimer’s disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.

Intracellular cyclophilin A is an important Ca2+ regulator in platelets and critically involved in arterial thrombus formation

Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca(2+) activity ([Ca(2+)](i)). Activation-dependent depletion of intracellular Ca(2+) stores triggers Ca(2+) entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca(2+) sensor that regulates store-operated Ca(2+) entry through activation of the pore-forming subunit Orai1, the major store-operated Ca(2+) entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca(2+) modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca(2+) mobilization from intracellular stores and Ca(2+) influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca(2+) sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca(2+) regulator in platelets, a critical mechanism for arterial thrombosis.

Regulation of STIM1/Orai1-dependent Ca2+ signalling in platelets

Platelet secretion and aggregation as well as thrombus formation of blood platelets critically depend on increase of cytosolic Ca2+ concentration ([Ca2+]i) mainly resulting from intracellular Ca2+ release followed by store operated Ca2+ entry (SOCE) through Ca2+ release activated channels (CRAC). SOCE is in part accomplished by the pore forming unit Orai and its regulator stromal interaction molecule (STIM). Orai1 and STIM1 transcription is stimulated by NF-κB (nuclear factor kappa B). Serum- and glucocorticoid-inducible kinase 1 (SGK1) up-regulates NF-κB-activity in megakaryocytes and thus Orai1-expression and SOCE in platelets. SGK1 is thus a powerful regulator of platelet Ca2+-signalling and thrombus formation and presumably participates in the regulation of platelet activation by a variety of hormones as well as clinical conditions (e.g. type 2 diabetes or metabolic syndrome) associated with platelet hyperaggregability and increased risk of thromboocclusive events. SOCE in platelets is further regulated by scaffolding protein Homer and chaperone protein cyclophilin A (CyPA). Additional potential regulators of Orai1/STIM1 and thus SOCE in platelets include AMP activated kinase (AMPK), protein kinase A (PKA), reactive oxygen species, lipid rafts, pH and mitochondrial Ca2+ buffering. Future studies are required defining the significance of those mechanisms for platelet Orai1 abundance and function, for SOCE into platelets and for platelet function in cardiovascular diseases.

Expression and functional significance of the Ca(2+)-activated Cl(-) channel ANO6 in dendritic cells.

Background/Aims: Migration of dendritic cells (DCs), antigen presenting cells that link innate and adaptive immunity, is critical for initiation of immune responses. DC migration is controlled by the activity of different ion channels, which mediate Ca2+ flux or set the membrane potential. Moreover, cell migration requires local volume changes at the leading and rear end of travelling cells, which might be mediated by the fluxes of osmotically active solutes, including Cl-. The present study explored the functional expression, regulation and role of Cl- channels in mouse bone marrow-derived DCs. Methods/Results: In whole-cell patch clamp experiments we detected outwardly rectifying Cl- currents which were activated by elevation of cytosolic Ca2+, triggered either by ionomycin in the presence of extracellular Ca2+ or mobilization of Ca2+ by IP3 Most importantly, Ca2+-activated Cl- channels (CaCCs) were activated by CCL21 (75 ng/ml), an agonist of the chemokine receptor CCR7. The currents showed sensitivity to Cl- channel blockers such as tannic acid (10 µM), digallic acid (100 µM) and more specific CaCC blockers niflumic acid (300 µM) and AO1 (20 µM). According to RT-PCR and Western blot data, Anoctamin 6 (ANO6) is expressed in DCs. Knock-down of ANO6 with siRNA led to inhibition of CaCC currents in DCs. Moreover, chemokine-induced migration of both immature and LPS-matured DCs was reduced upon ANO6 knock-down. Conclusion: Our data identify ANO6 as a Ca2+-activated Cl- channel in mouse DCs, show its activation upon chemokine receptor ligation and establish an important role of ANO6 in chemokine-induced DC migration.

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 Ca2+-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.