Aims Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA.Methods and results Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-kappa B activation.Conclusion We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation. Graphical Abstract RAGE is a novel receptor for extracellular CyPA on leucocytes and platelets. RAGE was found to interact with CyPA and to play an important role in CyPA-induced leucocyte and platelet functions. Platelet activation, coaggregate formation, and intracellular Ca2+ release by CyPA were abrogated when RAGE was absent or blocked. Similarly, CyPA-induced leucocyte migration and adhesion were found to be dependent on RAGE in vivo and in vitro. CyPA abundance was furthermore identified to regulate RAGE expression in vivo.

Background: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. Methods: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. Results: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer , the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y 12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in Dicer Pf4∆/Pf4∆ mice. Conclusions: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Background and aims Hemolysis is a known risk factor for thrombosis resulting in critical limb ischemia and microcirculatory disturbance and organ failure. Intravasal hemolysis may lead to life-threatening complications due to uncontrolled thrombo-inflammation. Until now, conventional antithrombotic therapies failed to control development and progression of these thrombotic events. Thus, the pathophysiology of these thrombotic events needs to be investigated to unravel underlying pathways and thereby identify targets for novel treatment strategies. Methods Here we used classical experimental set-ups as well as high-end flow cytometry, metabolomics and lipidomic analysis to in-depth analyze the effects of hemin on platelet physiology and morphology. Results Hemin does strongly and swiftly induce platelet activation and this process is modulated by the sGC-cGMP-cGKI signaling axis. cGMP modulation also reduced the pro-aggregatory potential of plasma derived from patients with hemolysis. Furthermore, hemin-induced platelet death evokes distinct platelet subpopulations. Typical cell death markers, such as ROS, were induced by hemin-stimulation and the platelet lipidome was specifically altered by high hemin concentration. Specifically, arachidonic acid derivates, such as PGE2, TXB2 or 12-HHT, were significantly increased. Balancing the cGMP levels by modulation of the sGC-cGMP-cGKI axis diminished the ferroptotic effect of hemin. Conclusion We found that cGMP modulates hemin-induced platelet activation and thrombus formation in vitro and cGMP effects hemin-mediated platelet death and changes in the platelet lipidome. Thus, it is tempting to speculate that modulating platelet cGMP levels may be a novel strategy to control thrombosis and critical limb ischemia in patients with hemolytic crisis.

INTRODUCTION:Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis.METHODS:We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin.RESULTS:We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions.CONCLUSION:In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

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 & UDelta;/Pf4 & UDelta;), 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 & UDelta;/Pf4 & UDelta; 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.

Formation of Neutrophil Extracellular Traps (NETosis), accompanied by the release of extracellular decondensed chromatin and pro-inflammatory as well as pro-thrombotic factors, is a pivotal element in the development and progression of thrombo-occlusive diseases. While the process of NETosis is based on complex intracellular signalling mechanisms, it impacts a wide variety of cells including platelets, leukocytes and endothelial cells. Consequently, although initially mainly associated with venous thromboembolism, NETs also affect and mediate atherothrombosis and its acute complications in the coronary, cerebral and peripheral arterial vasculature. In this context, besides deep vein thrombosis and pulmonary embolism, NETs in atherosclerosis and especially its acute complications such as myocardial infarction and ischemic stroke gained a lot of attention in the cardiovascular research field in the last decade. Thus, since the effect of NETosis on platelets and thrombosis in general is extensively discussed in other review articles, this review focusses on the translational and clinical relevance of NETosis research in cardiovascular thrombo-occlusive diseases. Consequently, after a brief summary of the neutrophil physiology and the cellular and molecular mechanisms underlying NETosis are presented, the role of NETosis in atherosclerotic and venous thrombo-occlusive diseases in chronic and acute settings are discussed. Finally, potential prevention and treatment strategies of NET-associated thrombo-occlusive diseases are considered.

During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.

Neutrophils with intact multilobulated nuclei show ASC speck formation and high histone H3 citrullination in patients with severe COVID-19. In murine neutrophils, ASC speck forms transiently at the microtubule organizing center, before nuclear rounding, early in NETosis. Abstract Infection by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-COV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation, (i.e. ASC speck assembly), and timing relative to NETosis in stimulated neutrophils by real time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that approximately 2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in LPS-stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center, long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.

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.

Thrombo-occlusive diseases such as myocardial infarction, ischemic stroke and deep vein thrombosis with subsequent pulmonary embolism still represent a major health burden worldwide. Besides the cells of the vasculature or other hematopoietic cells, platelets are primarily responsible for the development and progression of an occluding thrombus. The activation and function of platelets crucially depend on free cytosolic calcium (Ca2+) as second messenger, which modulates platelet secretion, aggregation and thrombus formation. Ca2+ is elevated upon platelet activation by release of Ca2+ from intracellular stores thus triggering of the subsequent store-operated Ca2+ entry (SOCE), which is facilitated by Ca2+ release-activated channels (CRACs). In general, CRACs are assembled by the pore-forming unit Orai in the plasma membrane and the Ca2+-sensing stromal interaction molecule (STIM) in the endoplasmic reticulum after the depletion of internal Ca2+ stores. In the last few years, there is a growing body of the literature demonstrating the importance of STIM and Orai-mediated mechanism in thrombo-occlusive disorders. Thus, this review provides an overview of the recent understanding of STIM and Orai signaling in platelet function and its implication in the development and progression of ischemic thrombo-occlusive disorders. Moreover, potential pharmacological implications of STIM and Orai signaling in platelets are anticipated and discussed in the end.

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.

Rationale: Platelet activation after contact to subendothelial collagen leads to acute arterial thrombosis. ANXA7 (Annexin A7) is a phospholipid-binding protein participating in the regulation of intracellular Ca2+ and exocytosis. Objective: The present study aimed to determine the role of ANXA7 in platelet Ca2+ signaling and lipid metabolism during platelet activation in arterial thrombosis using the ANXA7 inhibitor 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) and gene-targeted mice lacking Anxa7 (Anxa7(-/-)). Methods and Results: ANXA7 is strongly expressed in platelets. Functionally, luminescence aggregometry revealed significantly abrogated aggregation and secretion of ABO-treated or Anxa7(-/-) platelets when compared with untreated or Anxa7(+/+) platelets after activation with collagen or the GPVI (glycoprotein VI)-specific agonist collagen-related peptide. Furthermore, while both thrombus formation on collagen-coated surfaces under high arterial shear rates in ABO-treated or Anxa7-deficient whole blood, and thrombotic vascular occlusion after FeCl3-induced injury in vivo in Anxa7(-/-) bone marrow chimeric mice were significantly diminished, no prolongation of bleeding time was observed in ABO-treated or Anxa7(-/-) mice. Fura-2-AM spectrofluorimetry unraveled a blunted [Ca2+](i) increase in ABO-treated or Anxa7(-/-) platelets after GPVI stimulation. Due to an abolished phospholipase C gamma 2 phosphorylation, Anxa7(-/-) platelets displayed abrogated intracellular Ca2+ mobilization following collagen-related peptide-dependent platelet activation. Quantitative lipidomics analysis further revealed that ANXA7 critically affects platelet oxylipin metabolism following GPVI-dependent platelet activation. Anxa7(-/-) platelets showed a significantly reduced generation of several bioactive metabolites, particularly thromboxane A(2) and 12(S)-hydroxy-eicosatetraenoic acid. Finally, defective phospholipase C gamma 2 phosphorylation and blunted [Ca2+](i) increase in Anxa7(-/-) platelets could be rescued by exogenous addition of 12(S)-hydroxy-eicosatetraenoic acid, indicating that ANXA7 is a critical regulator of the platelet 12-lipoxygenase in GPVI-dependent platelet Ca2+ signaling during arterial thrombosis. Conclusions: The present study unravels ANXA7 as a regulator of oxylipin metabolism and Ca2+-dependent platelet activation downstream of GPVI. ANXA7 plays an important role in platelet signaling during arterial thrombosis and thus may reflect a promising target for novel antiplatelet strategies.

Upon activation, platelets release a host of soluble and vesicular signals, collectively termed the “platelet releasate” (PR). The contents of this PR play a significant role in haemostasis, inflammation, and pathologic sequelae. Despite this, proteomic studies investigating the PR in coronary artery disease have not been performed. Here, we undertook a comparative label-free quantitative (LFQ) proteomic profiling of the 1 U/ml thrombin-induced PR from 13 acute coronary syndrome vs. 14 stable angina pectoris patients using a tandem mass spectrometry approach. Data are available via ProteomeXchange with identifier PXD009356. 318 PR proteins were identified across both cohorts with 9 proteins found to be differentially released, including tetranectin (CLEC3B), protein disulfide-isomerase-A3 (PDIA3), coagulation factor V (F5), and fibronectin (FN1). Strikingly, these 9 differential proteins were all associated with the gene ontology cellular component term “extracellular vesicle” and reduced levels of EVs were detected in the corresponding plasma of ST-segment elevation myocardial infarction (STEMI) patients. Network analysis revealed 3 proteins either reduced (F5; FN1) or absent (CLEC3B) in the PR of STEMI patients that are strongly connected to both the clotting cascade and major druggable targets on platelets. This moderated proteomic signature may prove useful for non-invasive risk assessment of the progression of coronary artery disease. These data further contribute to the growing evidence-base of using the platelet releasate as a predictor of pathological state and disease severity.

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 (Pdk1(fl/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.

Neutrophils and neutrophil extracellular traps (NETs) play a pivotal role in innate immunity and inflammation, thus significantly contributing to the development and progression of autoimmune diseases and thrombo-occlusive disorders. One of the major signaling eventsduring the innate immune response and inflammation is the secretion of the pro-inflammatory cytokines IL-1ß and IL-18 after inflammasome activation. The most prominent inflammasome in the innate immune system is composed of the NLR Family Pyrin Domain-Containing 3 (NLRP3) protein. Although the NLRP3 inflammasome is highly expressed in neutrophils, nothing is known about its possible role in NET formation (NETosis). Neutrophil stimulation with nigericin, a K + ionophore and a well-known activator of the NLRP3 inflammasome, results in assembly of the NLRP3 inflammasome, observed as ASC/speck formation, and subsequent NETosis. To investigate the contribution of the NLRP3 inflammasome in NETosis, MCC950 was used to inhibit NLRP3 signaling. This resulted insignificantly decreased NET formation in both murine and human neutrophils after activation with nigericin and monosodium urate crystals (MSU) or ionomycin, a common NETosis stimulant. Since NLRP3 activation in macrophages is significantly promoted by the vimentin intermediate filament network, we investigated NET formation in vimentin null mice. Neutrophils from vimentin null mice show a significant decrease in NETosis after stimulation with nigericin, MSU, or ionomycin, as well as a reduction in the assembly of ASC/speck compared to neutrophils from wild-type mice. Taken together, our results demonstrate that the NLRP3 inflammasome activation plays an important role in the process of NETosis and may, in part, explain why many diseases aggravated by NETs are also accompanied by increased activation of NLRP3 inflammasome.

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of disorders. We hypothesized that NLRP3 inflammasome may participate in NETosis. Using confocal microscopy, we observed the formation of ASC speck in stimulated neutrophils in the absence of infection, as well as the association of ASC speck with the released NETs. Peptidylarginine deiminase (PAD) 4, a mediator of NETosis, was needed for optimal NLRP3 inflammasome assembly in neutrophils and rendered bone marrow–derived macrophages more susceptible to inflammasome formation, possibly by increasing NLRP3 and ASC protein levels. NLRP3 deficiency or specific inhibition by MCC950 resulted in significantly impaired NETosis, and time-lapse visualization revealed defects in nuclear envelope and plasma membrane breakdown. Our results reveal PAD-dependent formation of NLRP3 inflammasome in neutrophils and macrophages and implicate the NLRP3 inflammasome in NETosis.

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.

Background: Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant. Methods: We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry. Results: We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca 2+ influx. Using C3aR −/− mice or C3 −/− mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR −/− mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR −/− mice with C3aR +/+ platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation. Conclusions: Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.

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.