Cyclophilin A Is Not Acetylated at Lysine-82 and Lysine-125 in Resting and Stimulated Platelets.

Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed-neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets.

Pivotal role of PDK1 in megakaryocyte cytoskeletal dynamics and polarization during platelet biogenesis.

During thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1-/-) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1-/- BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1-/- BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis.

Mitochondrial Dynamics in Tachycardiomyopathy.

BACKGROUND/AIMS: Tachycardiomyopathy (TCM) is a largely reversible form of non-ischemic heart failure. The underlying mechanism are, however, still today poorly understood. Recent data indicate distinct changes in mitochondrial distribution in these patients, compared to other non-ischemic cardiomyopathies.This study investigated underlying mechanisms in mitochondrial dynamics in endomyocardial biopsy samples (EMB) from patients with TCM and compared them to patients with dilated cardiomyopathy (DCM), which show similar clinical features. METHODS: Focused mRNA analyses were performed on routinely obtained paraffinfixed EMB specimen from patients fulfilling TCM diagnosis criteria, as well as patients with DCM to elucidate regulatory changes in mitochondrial fusion, fission and mitophagy. RESULTS: In patients with TCM we were able to identify mRNA of Mitofusin 1 and 2, two effector proteins regulating mitochondrial fusion, to be strongly upregulated compared to patients with DCM. Conclusively, we did not find differences in the mRNA expression of mitochondrial fission regulators including DRP1, Fis1, MFF, MiD49, and MiD51. Furthermore, we did not find significant changes in PINK1 expression, an important mediator for mitochondrial autophagy. CONCLUSION: The mRNA upregulation of Mitofusin 1 and 2 provides first insight into the complex changes of mitochondrial dynamics in cardiomyocytes of patients with reversible heart failure due to TCM.

Cryoballoon ablation for persistent atrial fibrillation in patients without left atrial fibrosis.

INTRODUCTION: The role of cryoballoon (CB) pulmonary vein isolation (PVI) for patients with persistent atrial fibrillation (AF) is controversial, since long-term success can be poor. We performed left atrial voltage mapping before CB PVI and determined AF-free survival depending on the extent of low-voltage areas (LVAs). METHODS AND RESULTS: We consecutively enrolled 60 patients with persistent AF (average age, 60.6 ± 12.9 years; CHA2 DS 2 VASc score, 2.3 ± 1.6; and left atrial size 46.0 ± 5.2 mm) who were planned for CB PVI. Before ablation, we performed left atrial voltage mapping (Abbott EnSite Precision or Velocity). LVAs were defined if local bipolar signal amplitudes were less than 0.5 mV during sinus rhythm. Thirty-seven patients did not show significant LVAs (<10%), while 12 patients had LVAs between 10% and 30% and 11 patients showed substantial LVAs greater than 30% of the left atrial area. CB PVI could be successfully performed in all patients. A 7-day holter monitoring was obtained 3, 6, and 12 months after ablation. After a 12-month follow-up time, 83.8% of patients without LVAs (<10%) were free of atrial fibrillation, while 50.0% of patients with 10% to 30% LVAs and 9.1% of patients with LVAs more than 30% had stable sinus rhythm. The degree of atrial fibrosis correlated with the risk of AF recurrence. CONCLUSION: In patients with persistent AF undergoing CB PVI, the extent of left atrial LVAs predicts an AF-free survival. CB PVI seems to be a highly effective treatment for patients with persistent AF without atrial fibrosis.

TGFß Is Specifically Upregulated on Circulating CD14++ CD16+ and CD14+ CD16++ Monocytes in Patients with Atrial Fibrillation and Severe Atrial Fibrosis.

BACKGROUND/AIMS: Fibrotic remodeling of the atria plays a key role in the pathogenesis of atrial fibrillation (AF). As little is known about the contribution of circulating monocytes in atrial remodeling and the pathophysiology of AF, we investigated profibrotic factors in different subsets of circulating monocytes obtained from patients with atrial fibrillation undergoing catheter ablation. METHODS: A 3D high density voltage mapping was performed in sinus rhythm to evaluate the extent of low-voltage areas (LVAs) in the atria of 71 patients with persistent AF. Low-voltage was defined as signals of < 0.5mV during sinus rhythm. Prior to ablation, blood was drawn and monocytes were analyzed by FACS. Based on the expression of CD14 and CD16, three subgroups including CD14++ CD16- ('classical'), CD14++ CD16+ ('intermediate'), and CD14+ CD16++ ('non-classical') were analyzed for the expression of TGFb, CD147, and MMP-9, representing pivotal profibrotic pathways in myocardial remodeling. RESULTS: Expression of TGFb was increased in CD14+ monocytes of patients with extensive LVAs compared to patients with a low extend of LVAs. While CD14++ CD16- monocytes showed no difference, CD14++ CD16+ and CD14+ CD16++ monocytes showed a strong increase of TGFb abundance. Although CD147 and MMP-9 are strongly associated with myocardial fibrosis, we found no difference in expression between the two groups in any monocyte subsets. CONCLUSION: TGFb is specifically upregulated on CD14++ CD16+ and CD14+ CD16++ monocytes in patients with extensive LVAs undergoing catheter ablation.

Extracellular Matrix Metalloproteinase Inducer EMMPRIN (CD147) in Cardiovascular Disease.

The receptor EMMPRIN is involved in the development and progression of cardiovascular diseases and in the pathogenesis of myocardial infarction. There are several binding partners of EMMPRIN mediating the effects of EMMPRIN in cardiovascular diseases. EMMPRIN interaction with most binding partners leads to disease progression by mediating cytokine or chemokine release, the activation of platelets and monocytes, as well as the formation of monocyte-platelet aggregates (MPAs). EMMPRIN is also involved in atherosclerosis by mediating the infiltration of pro-inflammatory cells. There is also evidence that EMMPRIN controls energy metabolism of cells and that EMMPRIN binding partners modulate intracellular glycosylation and trafficking of EMMPRIN towards the cell membrane. In this review, we systematically discuss these multifaceted roles of EMMPRIN and its interaction partners, such as Cyclophilins, in cardiovascular disease.

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.

Pivotal role of serum- and glucocorticoid-inducible kinase 1 in vascular inflammation and atherogenesis.

OBJECTIVE: Atherosclerosis, an inflammatory disease of arterial vessel walls, requires migration and matrix metalloproteinase (MMP)-9-dependent invasion of monocytes/macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor nuclear factor-κB, which is regulated by inhibitor κB (IκB) and thus IκB kinase. Regulators of nuclear factor-κB include serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored involvement of SGK1 in vascular inflammation and atherogenesis. APPROACH AND RESULTS: Gene-targeted apolipoprotein E (ApoE)-deficient mice without (apoe(-/-)sgk1(+/+)) or with (apoe(-/-)sgk1(-/-)) additional SGK1 knockout received 16-week cholesterol-rich diet. According to immunohistochemistry atherosclerotic lesions in aorta and carotid artery, vascular CD45(+) leukocyte infiltration, Mac-3(+) macrophage infiltration, vascular smooth muscle cell content, MMP-2, and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe(-/-)sgk1(-/-)mice than in apoe(-/-)sgk1(+/+)mice. As determined by Boyden chamber, thioglycollate-induced peritonitis and air pouch model, migration of SGK1-deficient CD11b(+)F4/80(+) macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1(-/-) than in sgk1(+/+)macrophages and in control plasmid-transfected or inactive (K127N)SGK1-transfected than in constitutively active (S422D)SGK1-transfected THP-1 cells. Confocal microscopy revealed reduced macrophage number and macrophage MMP-9 content in plaques of apoe(-/-)sgk1(-/-) mice. In THP-1 cells, MMP-inhibitor GM6001 (25 µmol/L) abrogated (S422D)SGK1-induced MMP-9 production and invasion. According to reverse transcription polymerase chain reaction, MMP-9 transcript levels were significantly reduced in sgk1(-/-)macrophages and strongly upregulated in (S422D)SGK1-transfected THP-1 cells compared with control plasmid-transfected or (K127N)SGK1-transfected THP-1 cells. According to immunoblotting and confocal microscopy, phosphorylation of IκB kinase and inhibitor κB and nuclear translocation of p50 were significantly lower in sgk1(-/-)macrophages than in sgk1(+/+)macrophages and significantly higher in (S422D)SGK1-transfected THP-1 cells than in control plasmid-transfected or (K127N)SGK1-transfected THP-1 cells. Treatment of (S422D)SGK1-transfected THP-1 cells with IκB kinase-inhibitor BMS-345541 (10 µmol/L) abolished (S422D)SGK1-induced increase of MMP-9 transcription and gelatinase activity. CONCLUSIONS: SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of monocyte/macrophage migration and MMP-9 transcription via regulation of nuclear factor-κB.

Platelet-bound cyclophilin A in patients with stable coronary artery disease and acute myocardial infarction.

OBJECTIVE: Recently, we reported that extracellular cyclophilin A (CyPA) is an important agonist for platelets. Whereas soluble CyPA-levels have been associated with cardiovascular risk factors, cell-bound CyPA has not been investigated yet. In this study, we analyzed for the first time platelet-bound CyPA in patients with symptomatic coronary artery disease (CAD). METHODS AND RESULTS: blood was obtained from 388 consecutive patients: 204 with stable CAD and 184 with acute coronary syndrome (76 with unstable angina, 78 with non ST-elevation myocardial infarction (NSTEMI), and 30 with STEMI). In vitro stimulation of platelets with classical agonists revealed an enhanced expression of CyPA on the platelet surface. In patients with stable CAD, platelet-bound CyPA correlated excellently with platelet activity measured by P-selectin exposure in flow cytometry. The analysis of classical risk factors for atherosclerosis revealed that patients with hypertension and hypercholesterolemia had significantly enhanced platelet-bound CyPA, whereas diabetes and smoking were not associated with enhanced CyPA-binding to the platelet surface. In multivariate analysis, hypercholesterolemia was the only significant predictor of enhanced platelet-bound CyPA. Interestingly, in patients with acute myocardial infarction (AMI) platelet-bound CyPA was significantly decreased compared with patients with stable CAD. CONCLUSIONS: Enhanced platelet-bound CyPA is associated with hypertension and hypercholesterolemia in stable CAD patients. In patients with AMI platelet-bound CyPA is significantly decreased.

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.

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.

Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation.

OBJECTIVE: Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation. APPROACH AND RESULTS: Collagen-related peptide-induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1(-/-)) when compared with platelets from wild-type mice (Smpd1(+/+)). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1(-/-) platelets than in Smpd1(+/+) platelets. In contrast, platelet integrin αIIbß3 activation and aggregation, as well as activation-dependent Ca(2+) flux, were not significantly different between Smpd1(-/-) and Smpd1(+/+) platelets. In vitro thrombus formation at shear rates of 1700 s(-1) and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1(-/-) mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1(+/+) platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 µmol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1(-/-) platelets were again overcome by exogenous bacterial sphingomyelinase. CONCLUSIONS: Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.

Anticoagulation after catheter ablation of atrial fibrillation guided by implantable cardiac monitors.

BACKGROUND: Discontinuation of oral anticoagulation (OAC) after catheter ablation of atrial fibrillation (AF) is not recommended in patients with elevated CHADS2 scores. However, a low incidence of thromboembolic events is reported when OAC is stopped in these patients. We introduce an algorithm for discontinuation of OAC after ablation based on the AF burden documented by implantable cardiac monitors (ICM). METHODS: Sixty-five patients with CHADS2 scores 1-3 free from AF 3 months after ablation (AF ablation [n = 49] or ablation of possible AF triggers [n = 16]) were included. One day after implantation of the ICM, OAC was stopped. Patients performed a daily interrogation of the ICM which was programmed to alarm the patient if daily AF burden exceeded 1 hour. Study end point was the first recurrence of a daily AF burden ≥1 hour or a thromboembolic event, which both triggered reinitiation of OAC. RESULTS: During a follow-up time of 32 ± 12 months (126 patient-years), 41 of the 65 patients (63%) had an AF burden <1 h/day and were able to stay off OAC. Twenty-one patients (32%) had to reinitiate OAC due to an AF burden ≥1 hour and three patients due to other reasons. No stroke, transitory ischemic attack, or other thromboembolic event was observed during follow-up. CONCLUSIONS: Rhythm monitoring by ICM in patients who have stopped OAC after catheter ablation of AF or ablation of possible AF triggers seems to be a safe and promising method to monitor for AF recurrence. Within 1.3 years after ablation, about two-thirds of patients were able to stay off OAC.

Sphingosine kinase 1 (Sphk1) negatively regulates platelet activation and thrombus formation.

Sphingosine 1-phosphate (S1P) is a powerful regulator of platelet formation. Enzymes generating S1P include sphingosine kinase 1. The present study thus explored the role of sphingosine kinase 1 in platelet formation and function. Activation-dependent platelet integrin αIIbß3 activation and secretion of platelets lacking functional sphingosine kinase 1 (sphk1(-/-)) and of wild-type platelets (sphk1(+/+)) were determined utilizing flow cytometry and chronolume luciferin assay. Cytosolic Ca(2+) activity ([Ca(2+)]i) and aggregation were measured using fura-2 fluorescence and aggregometry, respectively. In vitro platelet adhesion and thrombus formation were evaluated using a flow chamber with shear rates of 1,700 s(-1). Activation-dependent increase of [Ca(2+)]i, degranulation (release of alpha and dense granules), integrin αIIbß3 activation, and aggregation were all significantly increased in sphk1(-/-) platelets compared with sphk1(+/+) platelets. Moreover, while platelet adhesion and thrombus formation under arterial shear rates were significantly augmented in Sphk1-deficient platelets, bleeding time and blood count were unaffected in sphk1(-/-) mice. In conclusion, sphingosine kinase 1 is a powerful negative regulator of platelet function counteracting degranulation, aggregation, and thrombus formation.

Gremlin-1 is an inhibitor of macrophage migration inhibitory factor and attenuates atherosclerotic plaque growth in ApoE-/- Mice.

Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE(-/-) mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (KD = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE(-/-) mice with a dimeric recombinant fusion protein, mGremlin1-Fc, but not with equimolar control Fc or inactivated mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.

Intracellular cyclophilin A is an important Ca(2+) 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.

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 α(IIb)ß(3) activation, and shape change. CXCL16 increased Akt phosphorylation (Thr(308)/Ser(473)), 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 α(IIb)ß(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 P(2)Y(1) (MRS2179, 100 µmol/L) and especially P(2)Y(12) (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.

Focal Pulsed Field Ablation for Atrial Arrhythmias: Efficacy and Safety under Deep Sedation.

Focal pulsed field ablation (PFA) is a novel technique for treating cardiac arrhythmias. It has demonstrated positive results in initial studies and has a good safety profile. In recent studies, PFA was often utilized for first-time pulmonary vein isolation (PVI) and was performed under general anesthesia. In our study, we assessed the feasibility, safety, acute procedural efficacy, and efficiency of focal PFA under deep sedation in patients, 80% of whom had undergone at least one left atrial ablation previously. We treated 30 patients (71 ± 7, 46% male) using the CENTAURI system for various atrial arrhythmias, including atrial fibrillation, typical and atypical atrial flutter, and focal atrial tachycardia. The average procedure and fluoroscopy times were 122 ± 43 min and 9 ± 7 min, respectively. A total of 83.33% of patients received additional line ablations beyond PVI, specifically targeting the posterior box and anterior mitral line. All ablations were successfully performed in deep sedation with only one major and one minor complication observed. The major complication was a vasospasm of the right coronary artery during ablation of the cavotricuspid isthmus, which was treated successfully with intracoronary nitroglycerin. All patients could be discharged in sinus rhythm. Moreover, adenosine appears effective in identifying dormant conduction in some patients after focal PFA. In conclusion, focal PFA is an effective approach for complex left atrial ablations under deep sedation, offering both high efficacy and efficiency with a reliable safety profile. Studies on long-term outcomes are needed.