ANXA7 Regulates Platelet Lipid Metabolism and Ca2+ Release in Arterial Thrombosis.

[Figure: see text].

Acid Sphingomyelinase (ASM) is a Negative Regulator of Regulatory T Cell (Treg) Development.

BACKGROUND/AIMS: Regulatory T cell (Treg) is required for the maintenance of tolerance to various tissue antigens and to protect the host from autoimmune disorders. However, Treg may, indirectly, support cancer progression and bacterial infections. Therefore, a balance of Treg function is pivotal for adequate immune responses. Acid sphingomyelinase (ASM) is a rate limiting enzyme involved in the production of ceramide by breaking down sphingomyelin. Previous studies in T-cells have suggested that ASM is involved in CD28 signalling, T lymphocyte granule secretion, degranulation, and vesicle shedding similar to the formation of phosphatidylserine-exposing microparticles from glial cells. However, whether ASM affects the development of Treg has not yet been described. METHODS: Splenocytes, isolated Naive T lymphocytes and cultured T cells were characterized for various immune T cell markers by flow cytometery. Cell proliferation was measured by Carboxyfluorescein succinimidyl ester (CFSE) dye, cell cycle analysis by Propidium Iodide (PI), mRNA transcripts by q-RT PCR and protein expression by Western Blotting respectively. RESULTS: ASM deficient mice have higher number of Treg compared with littermate control mice. In vitro induction of ASM deficient T cells in the presence of TGF-ß and IL-2 lead to a significantly higher number of Foxp3+ induced Treg (iTreg) compared with control T-cells. Further, ASM deficient iTreg has less AKT (serine 473) phosphorylation and Rictor levels compared with control iTreg. Ceramide C6 led to significant reduction of iTreg in both ASM deficient and WT mice. The reduction in iTreg leads to induction of IL-1ß, IL-6 and IL-17 but not IFN-γ mRNA levels. CONCLUSION: ASM is a negative regulator of natural and iTreg.

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.

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.

1,25(OH)2 vitamin D3-dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho-deficient mice.

Platelets are activated by increased cytosolic Ca(2+) concentration ([Ca(2+)]i) following store-operated calcium entry (SOCE) accomplished by calcium-release-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca(2+) transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca(2+) signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl-mice were analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist-induced [Ca(2+)]i increase, activation-dependent degranulation, integrin αIIbß3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) and 1,25(OH)2D3-treated WT megakaryocytes. Nuclear NF-κB subunit p50/p65 abundance was significantly reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca(2+) signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-κB activity and STIM1/Orai1 expression in megakaryocytes.

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.

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.

Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors.

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 µM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

Chorein sensitivity of cytoskeletal organization and degranulation of platelets.

Chorea-acanthocytosis (ChAc), a lethal disease caused by defective chorein, is characterized by neurodegeneration and erythrocyte acanthocytosis. The functional significance of chorein in other cell types remained ill-defined. The present study revealed chorein expression in blood platelets. As compared to platelets from healthy volunteers, platelets from patients with ChAc displayed a 47% increased globular/filamentous actin ratio, indicating actin depolymerization. Moreover, phosphoinositide-3-kinase subunit p85 phosphorylation, p21 protein-activated kinase (PAK1) phosphorylation, as well as vesicle-associated membrane protein 8 (VAMP8) expression were significantly reduced in platelets from patients with ChAc (by 17, 22, and 39%, respectively) and in megakaryocytic (MEG-01) cells following chorein silencing (by 16, 54, and 11%, respectively). Activation-induced platelet secretion from dense granules (ATP release) and α granules (P-selectin exposure) were significantly less (by 55% after stimulation with 1 µg/ml CRP and by 33% after stimulation with 5 µM TRAP, respectively) in ChAc platelets than in control platelets. Furthermore, platelet aggregation following stimulation with different platelet agonists was significantly impaired. These observations reveal a completely novel function of chorein, i.e., regulation of secretion and aggregation of blood platelets.

Skepinone-L, a novel potent and highly selective inhibitor of p38 MAP kinase, effectively impairs platelet activation and thrombus formation.

BACKGROUND/AIMS: Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A2 via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), on platelet activation and thrombus formation. METHODS: Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B2 synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca(2+) concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber. RESULTS: Skepinone-L (1 µM) virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 µg/ml), thrombin (5 mU/ml) or thromboxane A2 analogue U-46619 (1 µM). Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A2 analogue U-46619. Skepinone-L did not impair platelet Ca(2+) signaling but prevented agonist-induced thromboxane A2 synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A2 (cPLA2). Skepinone-L further markedly blunted thrombus formation under low (500-s) and high (1700-s) arterial shear rates. CONCLUSIONS: The present study discloses a powerful inhibiting effect of p38 MAPK-blocker Skepinone-L on platelet activation and thrombus formation.

Sgk1-dependent stimulation of cardiac Na+/H+ exchanger Nhe1 by dexamethasone.

BACKGROUND/AIMS: The serum- and glucocorticoid-inducible kinase Sgk1 contributes to cardiac remodeling and development of heart failure, which is paralelled by Sgk1-dependent stimulation of the cardiac Na(+)/H(+) exchanger Nhe1. Glucocorticoids are powerful stimulators of Sgk1 expression and influence cardiac remodeling. The present study thus explored whether the glucocorticoid receptor agonist dexamethasone influenced cardiac Sgk1 expression, as well as activity, expression and phosphorylation at Ser(703) of the cardiac Na(+)/H(+) exchanger Nhe1. METHODS: Experiments were performed in HL-1 cardiomyocytes and gene targeted mice lacking functional Sgk1 (sgk1(-/-)) and respective wild type mice (sgk1(+/+)). Gene expression was determined by quantitative RT-PCR and Nhe1 phosphorylation was determined utilizing a specific antibody against a 14-3-3 binding motif at P-Ser(703), which represents a putative phosphorylation site recognition motif for Sgk1 and is involved in Nhe1 activation. Cytosolic pH (pHi) was determined utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Nhe activity by the Na(+)-dependent realkalinization after an ammonium pulse. RESULTS: Treatment of HL-1 cardiomyocytes with dexamethasone was followed by a significant increase in Sgk1 mRNA expression, parallelled by increased Na(+)/H(+) exchanger activity. Furthermore, dexamethasone significantly increased Nhe1 and Spp1 mRNA expression. The effects of dexamethasone were blunted by cotreatment of HL-1 cardiomyocytes with the Sgk1 inhibitor EMD638683. Cotreatment with Nhe1 inhibitor cariporide similarly prevented dexamethasone-stimulated Spp1 mRNA expression. In sgk1(+/+) mice, dexamethasone significantly increased cardiac Sgk1 mRNA levels. In sgk1(+/+) mice, but not in sgk1(-/-) mice, dexamethasone significantly increased cardiac Nhe1 mRNA expression and Nhe1 phosphorylation at Ser(703). Furthermore, cardiac Spp1, Ctgf, Nppa and Nppb mRNA levels were significantly increased in dexamethasone treated sgk1(+/+) mice, effects significantly blunted in sgk1(-/-) mice. CONCLUSIONS: Sgk1 is critically involved in the phosphorylation and activation of the cardiac Na(+)/H(+) exchanger Nhe1.

Thrombin-sensitive expression of the store operated Ca(2+) channel Orai1 in platelets.

Thrombin activates pore forming channel protein Orai1 resulting in store operated Ca(2+) entry (SOCE) with subsequent Ca(2+)-dependent release of platelet granules, activation of integrin αIIbß3, adhesion, aggregation and thrombus formation. Platelets lack nuclei and are thus unable to modify protein abundance by transcriptional regulation. Nevertheless, they still contain pre-mRNA and mRNA and are thus able to express protein by stimulation of rapid translation. Platelet translation is sensitive to phosphoinositide-3-kinase (PI3K) and actin polymerization. The present study explored whether platelet activation via thrombin modifies Orai1 protein abundance. According to RT-PCR platelets contain pre-mRNA and mRNA encoding Orai1. Activation with thrombin (0.1 U/ml) results in a significant decline of pre-mRNA, which is, according to Western blotting and confocal microscopy, paralleled by a marked and statistically significant increase of Orai1 protein abundance. The increase of Orai1 protein abundance is insensitive to inhibition of transcription with actinomycin (4 µg/ml), but is significantly blunted by inhibition of translation with puromycin (100 nM) and by inhibition of PI3K with wortmannin (100 nM) or LY294002 (25 µM). In conclusion, activation of platelets stimulates the translational expression of Orai1, thus augmenting platelet Ca(2+) signaling.

PKB/SGK-resistant GSK-3 signaling following unilateral ureteral obstruction.

BACKGROUND/AIMS: Renal tissue fibrosis contributes to the development of end-stage renal disease. Causes for renal tissue fibrosis include obstructive nephropathy. The development of renal fibrosis following unilateral ureteral obstruction (UUO) is blunted in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase SGK1. Similar to Akt isoforms, SGK1 phosphorylates and thus inactivates glycogen synthase kinase GSK-3. The present study explored whether PKB/SGK-dependent phoshorylation of GSK-3α/ß impacts on pro-fibrotic signaling following UUO. METHODS: UUO was induced in mice carrying a PKB/SGK-resistant GSK-3α/ß (gsk-3(KI)) and corresponding wild-type mice (gsk-3(WT)). Three days after the obstructive injury, expression of fibrosis markers in kidney tissues was analyzed by quantitative RT-PCR and western blotting. RESULTS: GSK-3α and GSK-3ß phosphorylation was absent in both, the non-obstructed and the obstructed kidney tissues from gsk-3(KI) mice but was increased by UUO in kidney tissues from gsk-3(WT) mice. Expression of α-smooth muscle actin, type I collagen and type III collagen in the non-obstructed kidney tissues was not significantly different between gsk-3(KI) mice and gsk-3(WT) mice but was significantly less increased in the obstructed kidney tissues from gsk-3(KI) mice than from gsk-3(WT) mice. After UUO treatment, renal ß-catenin protein abundance and renal expression of the ß-catenin sensitive genes: c-Myc, Dkk1, Twist and Lef1 were again significantly less increased in kidney tissues from gsk-3(KI) mice than from gsk-3(WT) mice. CONCLUSIONS: PKB/SGK-dependent phosphorylation of glycogen synthase kinase GSK-3 contributes to the pro-fibrotic signaling leading to renal tissue fibrosis in obstructive nephropathy.

Niemann-Pick C1 protein regulates platelet membrane-associated calcium ion signaling in thrombo-occlusive diseases in mice.

BACKGROUND: Pathophysiologic platelet activation leads to thrombo-occlusive diseases such as myocardial infarction or ischemic stroke. Niemann-Pick C1 protein (NPC1) is involved in the regulation of lysosomal lipid trafficking and calcium ion (Ca2+) signaling, and its genetic mutation causes a lysosomal storage disorder. Lipids and Ca2+ are key players in the complex orchestration of platelet activation. OBJECTIVES: The present study aimed to determine the impact of NPC1 on Ca2+ mobilization during platelet activation in thrombo-occlusive diseases. METHODS: Using MK/platelet-specific knockout mice of Npc1 (Npc1Pf4∆/Pf4∆), ex vivo and in vitro approaches as well as in vivo models of thrombosis, we investigated the effect of Npc1 on platelet function and thrombus formation. RESULTS: We showed that Npc1Pf4∆/Pf4∆ platelets display increased sphingosine levels and a locally impaired membrane-associated and SERCA3-dependent Ca2+ mobilisation compared to platelets from wildtype littermates (Npc1lox/lox). Further, we observed decreased platelet. CONCLUSION: Our findings highlight that NPC1 regulates membrane-associated and SERCA3-dependent Ca2+ mobilization during platelet activation and that MK/platelet-specific ablation of Npc1 protects against experimental models of arterial thrombosis and myocardial or cerebral ischemia/reperfusion injury.

Translational regulation of the serum- and glucocorticoid-inducible kinase-1 (SGK1) in platelets.

Activation of platelets by thrombin opens pore forming channel protein Orai1 with subsequent store operated Ca(2+) entry (SOCE) and Ca(2+) dependent platelet granule release, integrin α(IIb)ß(3) activation, adhesion, aggregation and thrombus formation. Orai1 and thus SOCE as well as platelet activation are up-regulated by the serum- and glucocorticoid-inducible kinase-1 (SGK1), which transcriptionally regulates Orai1 expression in megakaryocytes and thus determines Orai1 protein abundance in mature, circulating platelets. As platelets are devoid of nuclei, they are unable to modify protein abundance by regulation of transcription. However, they contain mRNA and thus could express novel protein by stimulation of protein translation. Translation is sensitive to actin polymerization and phosphoinositide-3-kinase (PI3K). Translational regulation of SGK1 expression has never been described before. The present study thus explored whether thrombin regulates SGK1 expression in platelets. As a result, according to RT-PCR mRNA encoding SGK1 is present in circulating platelets and significantly decreased by activation of platelets with thrombin (1 U/ml). The protein abundance of SGK1 is significantly enhanced by thrombin treatment, an effect significantly decreased by inhibition of translation with puromycin (100 nM) but not by inhibition of transcription with actinomycin (4 µg/ml). The increase of SGK1 protein abundance is blunted by inhibition of PI3K with wortmannin (100 nM) or LY294002 (25 µM), and by disruption of the cytoskeleton with cytochalasin B (1 µM). In conclusion, activation of platelets with thrombin stimulates the translation of SGK1.

Enhanced FGF23 serum concentrations and phosphaturia in gene targeted mice expressing WNK-resistant SPAK.

BACKGROUND: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na+, K+, 2Cl- cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. METHODS: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak(tg/tg)) and in mice expressing wild type SPAK (spak(wt/wt)). RESULTS: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spak(tg/tg) mice than in spak(wt/wt) mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spak(tg/tg) mice compared to spak(wt/wt) mice. Treatment of spak(wt/wt) mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. CONCLUSIONS: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca2+ and phosphate excretion.

ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions.

BACKGROUND: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks. METHODS AND RESULTS: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo. CONCLUSIONS: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

CD44 sensitivity of platelet activation, membrane scrambling and adhesion under high arterial shear rates.

CD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca(2+)-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44(-/-)) were compared to platelets from corresponding wild-type mice (cd44(+/+)). In resting platelets, P-selectin abundance, α(IIb)ß3 integrin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44(-/-) and cd44(+/+) platelets. Platelet degranulation and α(IIb)ß3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca(2+)-store depletion with thapsigargin (1 µM), effects more pronounced in cd44(-/-) than in cd44(+/+) platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44(-/-) than in cd44(+/+) platelets. Thrombin further decreased forward scatter in cd44(-/-) and cd44(+/+) platelets, an effect which tended to be again more pronounced in cd44(-/-) than in cd44(+/+) platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s(-1)) were significantly augmented in cd44(-/-) mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and pro-thrombotic potential of platelets.

Endomyocardial expression of SDF-1 predicts mortality in patients with suspected myocarditis.

BACKGROUND: Risk stratification in patients with suspected myocarditis is pivotal for optimizing therapy. Stromal cell-derived factor 1 (SDF-1) is an inflammatory chemokine expressed in the inflamed and failing myocardium. Therefore, we aimed to investigate whether endomyocardial expression of SDF-1 identifies high-risk patients with suspected myocarditis. METHODS AND RESULTS: We prospectively enrolled 174 patients with non-ischemic HF who underwent endomyocardial biopsy for suspected myocarditis. Biopsies were analyzed using established histopathological and immunohistological criteria together with SDF-1 staining. SDF-1 was significantly enhanced in patients with inflammatory cardiomyopathy (65.4 % positive biopsies) as compared to patients with non-inflammatory cardiomyopathy (19.1 %, p < 0.001). SDF-1 expression levels correlated significantly with the degree of myocardial fibrosis (correlation coefficient r = 0.196; p = 0.010) since patients with severe myocardial fibrosis displayed high myocardial SDF-1 expression. During a mean follow-up of 27.5 months, 20 patients (11.5 %) died. The 4-year mortality rate was 26.0 % among the 92 SDF-1-positive patients vs. 9.5 % among the 82 SDF-1-negative patients (p = 0.001). On multivariable analysis which considered clinical (NYHA functional class, left ventricular ejection fraction), laboratory (brain natriuretic peptide, troponin I) and biopsy staining, SDF-1 was the strongest independent predictor of mortality (hazard ratio 6.1; 95 % confidence interval 1.4-27.5; p = 0.018). Subgroup analysis revealed SDF-1 as a predictor of mortality in both patients with inflammatory and non-inflammatory cardiomyopathy. CONCLUSIONS: Endomyocardial expression of SDF-1 is enhanced in inflammatory cardiomyopathy, positively correlates with myocardial fibrosis and identifies high-risk patients with suspected myocarditis.

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.