ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion.

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

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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.

Identification of key lipids critical for platelet activation by comprehensive analysis of the platelet lipidome.

Platelet integrity and function critically depend on lipid composition. However, the lipid inventory in platelets was hitherto not quantified. Here, we examined the lipidome of murine platelets using lipid-category tailored protocols on a quantitative lipidomics platform. We could show that the platelet lipidome comprises almost 400 lipid species and covers a concentration range of 7 orders of magnitude. A systematic comparison of the lipidomics network in resting and activated murine platelets, validated in human platelets, revealed that <20% of the platelet lipidome is changed upon activation, involving mainly lipids containing arachidonic acid. Sphingomyelin phosphodiesterase-1 (Smpd1) deficiency resulted in a very specific modulation of the platelet lipidome with an order of magnitude upregulation of lysosphingomyelin (SPC), and subsequent modification of platelet activation and thrombus formation. In conclusion, this first comprehensive quantitative lipidomic analysis of platelets sheds light on novel mechanisms important for platelet function, and has therefore the potential to open novel diagnostic and therapeutic opportunities.

Inhibitory mechanisms of very low-dose rivaroxaban in non-ST-elevation myocardial infarction.

Very low-dose (VLD) factor Xa (FXa) inhibition, in combination with acetylsalicylic acid (ASA) and clopidogrel, is associated with improved outcomes in patients with acute coronary syndrome (ACS) with a tolerable bleeding risk profile. To date, there are no data documenting platelet inhibition and the anticoagulatory effects of VLD FXa inhibition on top of guideline-adherent dual-antiplatelet therapy (DAPT) in patients with ACS. Patients with non-ST-elevation myocardial infarction (NSTEMI) receiving oral DAPT (ASA + clopidogrel, n = 20; or ASA + ticagrelor, n = 20) were prospectively enrolled in a nonrandomized study. Coagulation- and platelet-dependent thrombin generation (TG), measured by means of the calibrated automated thrombogram, were significantly decreased after in vitro and in vivo addition of rivaroxaban. As shown by a total thrombus-formation analysis approach, rivaroxaban treatment led to a significantly decreased coagulation-dependent (AR-chip) thrombus formation in patients treated with ASA plus P2Y12 inhibitor (clopidogrel/ticagrelor), whereas the pure platelet-dependent (PL-chip) thrombus formation was not affected at all. Adjunctive rivaroxaban therapy was not associated with significant differences in platelet aggregation assessed by light-transmission aggregometry (LTA). Nevertheless, according to fluorescence-activated cell sorter analysis, VLD rivaroxaban treatment resulted in a significantly reduced expression of platelet HMGB-1, whereas P-selectin exposure was not affected. Furthermore, an enhanced effect of rivaroxaban on total thrombus formation and TG was observed in particular in clopidogrel nonresponder patients defined as adenosine 5'-diphosphate-induced LTA ≥40%. VLD rivaroxaban reduces thrombus formation and platelet-dependent TG in patients with ACS receiving DAPT, which can be of potential ischemic benefit. This trial was registered at www.clinicaltrials.gov as #NCT01417884.

Ceramidase critically affects GPVI-dependent platelet activation and thrombus formation.

Platelet aggregation, dense granule secretion and thrombus formation are dependent on sphingolipids like ceramide and sphingosine as well as sphingosine-1 phosphate. Sphingosine/ceramide metabolism involves ceramide synthases and ceramidases. However, the role of ceramide synthase and ceramidase in the regulation of platelet function remained ill-defined. The present study determined transmission light aggregometry, employed luciferase based ATP release measurements and studied in vitro thrombus formation under high arterial shear rates in order to define the impact of pharmacological inhibition of serine palmitoyltransferase, ceramide synthase and ceramidase on platelet function. As a result, inhibition of ceramidase significantly blunted collagen related peptide (CRP) induced glyocoprotein VI (GPVI)-dependent platelet aggregation, ATP release and thrombus formation on a collagen-coated surface under shear rates of 1700-sec. Defective platelet aggregation after ceramidase inhibition could partially be overcome by exogenous sphingosine treatment reflecting a pivotal role of ceramidase-derived sphingosine in platelet function. Inhibition of serine palmitoyltransferase and ceramide synthase did not significantly modify GPVI-dependent platelet activation. In conclusion, the present study unraveled ceramidase as a crucial player in sphingosine-induced platelet activation following GPVI-dependent signaling.

Extracellular Cyclophilin A Augments Platelet-Dependent Thrombosis and Thromboinflammation.

Cyclophilin A (CyPA) is involved in the pathophysiology of several inflammatory and cardiovascular diseases. To our knowledge, there is no specific inhibitor targeting extracellular CyPA without affecting other extracellular cyclophilins or intracellular CyPA functions. In this study, we developed an antibody-based inhibitor of extracellular CyPA and analysed its effects in vitro and in vivo. To generate a specific antibody, mice and rats were immunized with a peptide containing the extracellular matrix metalloproteinase inducer binding site and various antibody clones were selected and purified. At first, antibodies were tested for their binding capacity to recombinant CyPA and their functional activity. The clone 8H7-mAb was chosen for further experiments. 8H7-mAb reduced the CyPA-induced migration of inflammatory cells in vitro and in vivo. Furthermore, 8H7-mAb revealed strong antithrombotic effects by inhibiting CyPA-dependent activation of platelets and thrombus formation in vitro and in vivo. Surprisingly, 8H7-mAb did not influence in vivo tail bleeding time or in vitro whole blood coagulation parameters. Our study provides first evidence that antibody-based inhibition of extracellular CyPA inhibits thrombosis and thromboinflammation without affecting blood homeostasis. Thus, 8H7-mAb may be a promising compound for thrombi modulation in inflammatory diseases to prevent organ dysfunction.

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

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

Doxepin inhibits GPVI-dependent platelet Ca2+ signaling and collagen-dependent thrombus formation.

Platelet adhesion, activation, and aggregation are essential for primary hemostasis, but are also critically involved in the development of acute arterial thrombotic occlusion. Stimulation of the collagen receptor glycoprotein VI (GPVI) leads to phospholipase Cγ2-dependent inositol triphosphate (IP3) production with subsequent platelet activation, due to increased intracellular Ca2+ concentration ([Ca2+]i). Although tricyclic antidepressants have been shown to potentially impair platelet activation, nothing is hitherto known about potential effects of the tricyclic antidepressant doxepin on platelet Ca2+ signaling and thrombus formation. As shown in the present study, doxepin significantly diminished the stimulatory effect of GPVI agonist collagen-related peptide (CRP) on intracellular Ca2+ release as well as subsequent extracellular Ca2+ influx. Doxepin was partially effective by impairment of CRP-dependent IP3 production. Moreover, doxepin abrogated CRP-induced platelet degranulation and integrin αIIbß3 activation and aggregation. Finally, doxepin markedly blunted in vitro platelet adhesion to collagen and thrombus formation under high arterial shear rates (1,700-s). In conclusion, doxepin is a powerful inhibitor of GPVI-dependent platelet Ca2+ signaling, platelet activation, and thrombus formation.

Alternative Splicing Substantially Diversifies the Transcriptome during Early Photomorphogenesis and Correlates with the Energy Availability in Arabidopsis.

Plants use light as source of energy and information to detect diurnal rhythms and seasonal changes. Sensing changing light conditions is critical to adjust plant metabolism and to initiate developmental transitions. Here, we analyzed transcriptome-wide alterations in gene expression and alternative splicing (AS) of etiolated seedlings undergoing photomorphogenesis upon exposure to blue, red, or white light. Our analysis revealed massive transcriptome reprogramming as reflected by differential expression of ∼20% of all genes and changes in several hundred AS events. For more than 60% of all regulated AS events, light promoted the production of a presumably protein-coding variant at the expense of an mRNA with nonsense-mediated decay-triggering features. Accordingly, AS of the putative splicing factor REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND1, previously identified as a red light signaling component, was shifted to the functional variant under light. Downstream analyses of candidate AS events pointed at a role of photoreceptor signaling only in monochromatic but not in white light. Furthermore, we demonstrated similar AS changes upon light exposure and exogenous sugar supply, with a critical involvement of kinase signaling. We propose that AS is an integration point of signaling pathways that sense and transmit information regarding the energy availability in plants.

PDK1 Determines Collagen-Dependent Platelet Ca2+ Signaling and Is Critical to Development of Ischemic Stroke In Vivo.

OBJECTIVE: Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca(2+) concentration ([Ca(2+)]i) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca(2+) signaling and ischemic stroke in vivo. APPROACH AND RESULTS: Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2-dependent inositol-1,4,5-trisphosphate production and intracellular [Ca(2+)]i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca(2+)]i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl3-induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1(-/-) mice, reflecting an important role of PDK1 in primary hemostasis. CONCLUSIONS: PDK1 is required for Ca(2+)-dependent platelet activation on stimulation of collagen receptor glycoprotein VI, arterial thrombotic occlusion, and ischemic stroke in vivo.