Platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation.

Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post-myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A-mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia-sera/immunoglobulin G-triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.

Free-breathing Arterial Spin Labeling MRI for the Detection of Pulmonary Embolism.

Background Arterial spin labeling (ASL) MRI can be used to assess organ perfusion but has yet to be implemented for perfusion evaluation of the lung. Purpose To evaluate pseudo-continuous ASL (PCASL) MRI for the detection of acute pulmonary embolism (PE) and its potential as an alternative to CT pulmonary angiography (CTPA). Materials and Methods Between November 2020 and November 2021, 97 patients (median age, 61 years; 48 women) with suspected PE were enrolled in this prospective study. PCASL MRI was performed within a 72-hour period following CTPA under free-breathing conditions and included three orthogonal planes. The pulmonary trunk was labeled during systole, and the image was acquired during diastole of the subsequent cardiac cycle. Additionally, multisection, coronal, balanced, steady-state free-precession imaging was carried out. Two radiologists blindly assessed overall image quality, artifacts, and diagnostic confidence (five-point Likert scale, 5 = best). Patients were categorized as positive or negative for PE, and a lobe-wise assessment in PCASL MRI and CTPA was conducted. Sensitivity and specificity were calculated on a patient level with the final clinical diagnosis serving as the reference standard. Interchangeability between MRI and CTPA was also tested with use of an individual equivalence index (IEI). Results PCASL MRI was performed successfully in all patients with high scores for image quality, artifact, and diagnostic confidence (κ ≥ .74). Of the 97 patients, 38 were positive for PE. PCASL MRI depicted PE correctly in 35 of 38 patients with three false-positive and three false-negative findings, resulting in a sensitivity of 35 of 38 patients (92% [95% CI: 79, 98]) and a specificity of 56 of 59 patients (95% [95% CI: 86, 99]). Interchangeability analysis revealed an IEI of 2.6% (95% CI: 1.2, 3.8). Conclusion Free-breathing pseudo-continuous arterial spin labeling MRI depicted abnormal lung perfusion caused by acute pulmonary embolism and may be useful as a contrast material-free alternative to CT pulmonary angiography for selected patients. German Clinical Trials Register no. DRKS00023599 © RSNA, 2023.

Antibody-induced procoagulant platelets in severe COVID-19 infection.

The pathophysiology of COVID-19-associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA-dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.

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

[Figure: see text].

Recovery of systemic hyperinflammation in patients with severe SARS-CoV-2 infection.

INTRODUCTION: Patients with cardiovascular disease (CVD) and acute SARS-CoV-2 infection might show an altered immune response during COVID-19. MATERIAL AND METHODS: Twenty-three patients with CVD and SARS-CoV-2 infection were prospectively enrolled and received a cardiological assessment at study entry and during follow-up visit. Inclusion criteria of our study were age older than 18 years, presence of CVD, and acute SARS-CoV-2 infection. The median age of the patient cohort was 69 (IQR 55-79) years. 12 (52.2%) patients were men. Peripheral monocytes and chemokine/cytokine profiles were analysed. RESULTS: Numbers of classical and non-classical monocytes were significantly decreased during acute SARS-CoV-2 infection compared to 3-month recovery. While classical monocytes reached the expected level in peripheral blood after 3 months, the number of non-classical monocytes remained significantly reduced. DISCUSSION: All three monocyte subsets exhibited changes of established adhesion and activation markers. Interestingly, they also expressed higher levels of pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF) at the time of recovery, although MIF was only slightly increased during the acute phase. CONCLUSION: Changes of monocyte phenotypes and increased MIF expression after 3-month recovery from acute SARS-CoV-2 infection may indicate persistent, possibly long-lasting, pro-inflammatory monocyte function in CVD patients.

Platelet miRNAs: differential expression in coronary artery disease and associations with course of left ventricular systolic function.

BACKGROUND: MicroRNAs are paramount in post transcriptional gene regulation. We investigated platelet miRNAs in patients with CAD and examined potential associations with course of left ventricular ejection fraction (LVEF%). MATERIALS AND METHODS: In a first cohort, 62 MiRNAs were measured in platelets of 100 patients suffering from CAD. Expression profiles of individuals with chronic coronary syndrome (CCS) and MI were compared (CCS n = 67, MI n = 33). Also, associations between miRNA profiles and change in left ventricular ejection fraction (LVEF%) were investigated. In a second cohort of patients suffering from CCS (n = 10), MI (n = 11) or no CAD (n = 13), we measured miRNA expression in platelets, platelet supernatant and serum. This was carried out before and after in vitro platelet activation with CRP. RESULTS: Platelet miRNAs 103a-3p and 155-5p demonstrated higher expression in patients with CCS then in individuals with MI. Furthermore, multiple miRNAs were significantly higher expressed in matched controls compared to MI patients. 8 miRNAs showed higher expression in patients with improving LVEF% after a 1-year follow-up. In our second cohort, we found higher concentrations of 6 miRNAs in the platelet supernatant of patients with CCS, MI and no CAD after in vitro platelet activation. Most of these miRNAs showed a higher abundance in serum of MI patients as compared to CCS. CONCLUSION: Several miRNAs show higher expression in platelets of CCS compared to MI. After in vitro platelet activation, a release of multiple miRNAs out of the thrombocyte was observed. Furthermore, upregulation of serum miRNAs was found in MI patients when compared to CCS patients and individuals without CAD. Hence, platelets could present a source of upregulated circulating miRNAs in MI and additionally affect course of LVEF%.

Platelet Activation and Plasma Levels of Furin Are Associated With Prognosis of Patients With Coronary Artery Disease and COVID-19.

[Figure: see text].

COVID-19 versus seasonal influenza: myocardial injury and prognostic importance.

BACKGROUND: Acute myocardial injury is associated with poor prognosis in respiratory tract infections. We aimed to highlight the differences in prevalence of myocardial injury and its impact on prognosis in patients with COVID-19 compared to those with seasonal influenza. METHODS: This was a single-center prospective cohort study with a historical control group. 300 age-/sex-matched SARS-CoV-2 and seasonal influenza positive patients were enrolled. Myocardial injury was assessed by electrocardiogram (ECG), transthoracic echocardiography and biomarkers including high-sensitivity troponin-I. All patients were followed-up for 30 days after enrollment for all-cause mortalitiy, admission to the intensive care unit (ICU) and mechanical ventilation. RESULTS: Right ventricular distress was more common in COVID-19 whereas pathological ECG findings and impaired left ventricular function were more prevalent among influenza patients. COVID-19 patients suffered from a higher percentage of hypertension and dyslipidaemia. Contrary to COVID-19, pericardial effusion at admission was associated with poor outcome in the influenza group. Severe course of disease and respiratory failure resulted in significantly higher rates of ICU treatment and mechanical ventilation in COVID-19 patients. Although distribution of myocardial injury was similar, significantly fewer cardiac catheterizations were performed in COVID-19 patients. However, number of cardiac catheterizations was low in both groups. Finally, 30-day mortality was significantly higher in COVID-19 compared to influenza patients. CONCLUSIONS: In adults requiring hospitalization due to COVID-19 or seasonal influenza, cardiovascular risk factors and signs of myocardial distress differ significantly. Furthermore, cardiovascular comorbidities may impair prognosis in COVID-19 patients to a higher degree than in their influenza counterparts.

Feasibility of platelet marker analysis in ischemic stroke patients and their association with one-year outcome. A pilot project within a subsample of the Stroke Induced Cardiac Failure in Mice and Men (SICFAIL) cohort study.

Patients with ischemic stroke (IS) are at increased risk of mortality and recurrent cerebro- or cardiovascular events. Determining prognosis after IS remains challenging but blood-based biomarkers might provide additional prognostic information. As platelets are crucially involved in the pathophysiology of vascular diseases, platelet surface proteins (PSP) are promising candidates as prognostic markers in the hyperacute stage. In this pilot study, feasibility of PSP analysis by flow cytometry (HMGB1, CD84, CXCR4, CXCR7, CD62p with and without ADP-stimulation, CD41, CD61, CD40, GPVI) was investigated in 99 (median 66 years, 67.5% male) acute IS patients admitted to Stroke Unit within a substudy of the Stroke-Induced Cardiac FAILure in mice and men (SICFAIL) cohort study. Association between PSP expression and unfavorable one-year outcome (cerebro- or cardiovascular event, all-cause mortality and care dependency defined as Barthel Index <60) was explored. PSP measurements were feasible. Several process- (e.g. temperatures, processing times) and patient-related factors (e.g. prestroke ischemic events, surgery, blood pressure, antiplatelet therapy) were identified to be potentially associated with PSP expression. Elevated CD40 levels above study population's median were associated with unfavorable outcome. Standardized conditions during blood draw and processing within the hyperacute stroke unit setting are required and patient-related characteristics must be considered for valid measurements of PSP.Trial registration: German Clinical Trials Register (DRKS00011615).

Platelet SR-PSOX/CXCL16-CXCR6 Axis Influences Thrombotic Propensity and Prognosis in Coronary Artery Disease.

Platelets express the transmembrane chemokine SR-PSOX/CXCL16, proteolytic cleavage of which generates the sCXCL16 soluble-(s) chemokine. The sCXCL16 engages CXCR6 on platelets to synergistically propagate degranulation, aggregation and thrombotic response. Currently, we have investigated the pro-thrombotic and prognostic association of platelet CXCL16−CXCR6 axis in CAD-(n = 240; CCS n = 62; ACS n = 178) patients. Platelet surface-associated-CXCL16 and CXCR6 surface expression ascertained by flow cytometry correlated significantly with platelet activation markers (CD62P denoting degranulation and PAC-1 binding denoting α2bß3-integrin activation). Higher platelet CXCL16 surface association (1st quartile vs. 2nd−4th quartiles) corresponded to significantly elevated collagen-induced platelet aggregation assessed by whole blood impedance aggregometry. Platelet-CXCL16 and CXCR6 expression did not alter with dyslipidemia, triglyceride, total cholesterol, or LDL levels, but higher (>median) plasma HDL levels corresponded with decreased platelet-CXCL16 and CXCR6. Although platelet-CXCL16 and CXCR6 expression did not change significantly with or correlate with troponin I levels, they corresponded with higher Creatine Kinase-(CK) activity and progressively deteriorating left ventricular ejection fraction (LVEF) at admission. Elevated-(4th quartile) platelet-CXCL16 (p = 0.023) and CXCR6 (p = 0.030) measured at admission were significantly associated with a worse prognosis. However, after Cox-PH regression analysis, only platelet-CXCL16 was ascertained as an independent predictor for all-cause of mortality. Therefore, the platelet CXCL16−CXCR6 axis may influence thrombotic propensity and prognosis in CAD patients.

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.

Platelet-Derived PCSK9 Is Associated with LDL Metabolism and Modulates Atherothrombotic Mechanisms in Coronary Artery Disease.

Platelets play a significant role in atherothrombosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of LDL metabolism and interacts with platelet function. The effect of PCSK9 in platelet function is poorly understood. The authors of this article sought to characterize platelets as a major source of PCSK9 and PCSK9's role in atherothrombosis. In a large cohort of patients with coronary artery disease (CAD), platelet count, platelet reactivity, and platelet-derived PCSK9 release were analyzed. The role of platelet PCSK9 on platelet and monocyte function was investigated in vitro. Platelet count and hyper-reactivity correlated with plasma LDL in CAD. The circulating platelets express on their surface and release substantial amounts of PCSK9. Release of PCSK9 augmented platelet-dependent thrombosis, monocyte migration, and differentiation into macrophages/foam cells. Platelets and PCSK9 accumulated in tissue derived from atherosclerotic carotid arteries in areas of macrophages. PCSK9 inhibition reduced platelet activation and platelet-dependent thrombo-inflammation. The authors identified platelets as a source of PCSK9 in CAD, which may have an impact on LDL metabolism. Furthermore, platelet-derived PCSK9 contributes to atherothrombosis, and inhibition of PCSK9 attenuates thrombo-inflammation, which may contribute to the reported beneficial clinical effects.

Optimal antiplatelet and anticoagulation strategies in acute coronary syndromes.

Antithrombotic therapy has become increasingly challenging due to the thrombotic and bleeding risk of patients presenting with acute coronary syndrome (ACS) today. Contributing factors include increasing age, underlying comorbidities (e.g., renal failure, atrial fibrillation [AF]), or concomitant interventions including transcatheter valve procedures requiring individualized antithrombotic strategies. Thanks to the development of novel stent platforms with biocompatible polymers and thin strut design allowing for a more rapid endothelialization, shortening or de-escalation of antiplatelet therapies is an attractive option for reducing bleeding events. In fact, several trials have been recently published or are currently underway that address the issue of early monotherapy after short-term dual antiplatelet therapy in ACS patients. Patients with AF and ACS are at a particularly high risk for thromboembolic and bleeding events. An individualized combination approach of antiplatelet therapy plus non-vitamin K oral anticoagulants should be favored in these patients to reduce bleeding risk according to recent randomized trials and guidelines. In contrast to de-escalation strategies in ACS patients at high bleeding risk, in patients with myocardial infarction in whom the long-term risk for ischemic events prevails, prolongation of an intensified antithrombotic therapy on top of acetylsalicylic acid is recommended. This review summarizes the recent evidence and offers practical recommendations to determine patients' bleeding versus thrombo-ischemic risk in order to tailor early and late antithrombotic therapy after ACS.

Regulation of oxidized platelet lipidome: implications for coronary artery disease.

AIMS: Hyperlipidaemia enhances susceptibility to thrombosis, while platelet oxidixed LDL (oxLDL) binding in acute coronary syndrome (ACS) correlates with activation status. This study explores the platelet lipidome in symptomatic coronary artery disease (CAD) patients and the functional consequences of the chemokine CXCL12 and its receptors CXCR-4/-7 on lipid uptake in platelets. METHODS AND RESULTS: Platelet-oxLDL detected by flow cytometry was enhanced (P = 0.04) in CAD patients, moderately correlated with platelet CXCR7 surface expression (ρ = 0.39; P < 0.001), while inversely with CXCR4 (ρ = 0.35; P < 0.001). Platelet-oxLDL was elevated (P = 0.01) in ACS patients with angiographic evidence of intracoronary thrombi. Ex vivo analysis of intracoronary thrombi sections revealed oxLDL deposition in platelet-enriched areas verified by immunofluorescence confocal microscopy. LDL-oxLDL uptake enhanced reactive oxygen species, mitochondrial superoxide generation, intraplatelet LDL to oxLDL conversion, and lipid peroxidation, counteracted by SOD2-mimetic MnTMPyP. Lipidomic analysis revealed enhanced intraplatelet-oxidized phospholipids, cholesteryl esters, sphingomyelin, ceramides, di- and triacylglycerols, acylcarnitines in CAD patients compared with age-matched controls as ascertained by liquid chromatography hyphenated to high-resolution mass spectrometry. LDL-oxLDL induced degranulation, αIIbß3-integrin activation, apoptosis, thrombin generation estimated by calibrated automated thrombinoscopy, and shape change verified by live imaging using scanning ion conductance microscopy. Further, LDL-oxLDL enhanced thrombus formation ex vivo and in vivo in mice (ferric chloride-induced carotid artery injury). LDL-oxLDL enhanced platelet CXCL12 release, differentially regulated CXCR4-CXCR7 surface exposure, while CXCL12 prompted LDL-oxLDL uptake and synergistically augmented the LDL-oxLDL-induced pro-oxidative, thrombogenic impact on platelet function. CONCLUSION: An altered platelet lipidome might be associated with thrombotic disposition in CAD, a mechanism potentially regulated by CXCL12-CXCR4-CXCR7 axis.

HMGB1 Expression Level in Circulating Platelets is not Significantly Associated with Outcomes in Symptomatic Coronary Artery Disease.

BACKGROUND/AIMS: Platelets express high-mobility group box 1 (HMGB1), a damage-associated molecular pattern molecule (DAMP) that triggers thrombosis and inflammation when present in extracellular space. The role of platelet-derived HMGB1 in coronary artery disease (CAD) remains unexplored. METHODS: In a cohort study, we measured the expression of HMGB1 on the surface of circulating platelets in 183 patients with symptomatic CAD (stable CAD: n=80, acute coronary syndrome, ACS: n=103) at the time of percutaneous coronary intervention. All patients were tracked for course of left ventricular ejection fraction (LVEF), all-cause death (ACD), and myocardial infarction (MI) for 360 days after study inclusion. RESULTS: Platelet HMGB1 expression did not significantly differ between stable CAD, unstable CAD, non-ST segment elevation myocardial infarction (NSTEMI), and ST segment elevation myocardial infarction (STEMI). Moreover, platelet HMGB1 did not significantly correlate with LVEF, neither at baseline nor at 6 months follow-up of the MI subgroup, and did not exert any significant effect on outcome (composite of ACD and/or MI as well as single events ACD and MI). Cumulative event-free survival of patients was not significantly different between HMGB1 levels above the median and HMGB1 levels below or equal to the median. CONCLUSION: These findings suggest that HMGB1 expressed on the surface of circulating platelets in patients with symptomatic CAD may not serve as a prognostic biomarker for this disease state.

Impact of Intravenous P2Y12-Receptor Inhibition with Cangrelor in Patients Presenting with Acute Coronary Syndrome and Cardiogenic Shock - a Case Series.

BACKGROUND/AIMS: Patients with acute coronary syndromes (ACS) presenting with cardiogenic shock (CS) are at particular risk for death and adverse cardiac events. Impaired effects and absorption of oral P2Y12-receptor inhibitors due to decreased organ hypoperfusion or hypothermia and challenges regarding oral administration contribute to this risk. We report a single center experience regarding the use of intravenous P2Y12-receptor inhibitor cangrelor in patients with CS treated with percutaneous coronary intervention (PCI). METHODS: Twelve patients with ACS and CS undergoing PCI, not pretreated with oral P2Y12-receptor inhibitors, were treated with cangrelor. Platelet inhibition was assessed by multiple electrode aggregometry (MEA) before and after PCI, immediately and 2 hours after stopping the cangrelor infusion. RESULTS: Nine patients recovered from their cardiogenic shock, 3 patients died. Platelet reactivity decreased from 65.9 (SD 41.0) U before PCI to 15.8 (SD 10.8) U after PCI, 13.4 (SD 7.7) U at the end of infusion and 33.8 (SD 19.9) 2 hours after stopping the cangrelor infusion. There was no non-responder under cangrelor infusion (MEA < 46 U). CONCLUSIONS: Due to its favorable PK/PD profile, cangrelor overcomes problems with reduced absorption and effects of oral P2Y12-receptor inhibitors and should be considered for periprocedural treatment of patients with CS.

The NLRP3 inflammasome and bruton's tyrosine kinase in platelets co-regulate platelet activation, aggregation, and in vitro thrombus formation.

Cleavage of interleukin-1ß (IL-1ß) is a key inflammatory event in immune cells and platelets, which is mediated by nucleotide-binding domain leucine rich repeat containing protein (NLRP3)-dependent activation of caspase-1. In immune cells, NLRP3 and caspase-1 form inflammasome complexes with the adaptor proteins apoptosis-associated speck-like protein containing a CARD (ASC) and bruton's tyrosine kinase (BTK). In platelets, however, the regulatory triggers and the functional effects of the NLRP3 inflammasome are unknown. Here, we show in vitro that the platelet NLRP3 inflammasome contributes to platelet activation, aggregation, and thrombus formation. NLRP3 activity, as monitored by caspase-1 activation and cleavage and secretion of IL-1ß, was upregulated in activated platelets, which was dependent on platelet BTK. Pharmacological inhibition or genetic ablation of BTK in platelets led to decreased platelet activation, aggregation, and in vitro thrombus formation. We identify a functionally relevant link between BTK and NLRP3 in platelets, with potential implications in disease states associated with abnormal coagulation and inflammation.

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.

Platelet surface expression of SDF-1 is associated with clinical outcomes in the patients with cardiovascular disease.

Platelet surface expression levels of stromal cell derived factor 1 (SDF-1) are elevated in acute coronary syndrome and associated with LVEF% improvement after myocardial infarction (MI). Platelet SDF-1 might facilitate thrombus formation and endomyocardial expression of SDF-1 is enhanced in inflammatory cardiomyopathy and positively correlates with myocardial fibrosis. The influence of platelet SDF-1 on outcome in the patients with symptomatic coronary artery disease (CAD) is to the best of our knowledge unknown. Blood samples of 608 consecutive CAD patients were collected during the percutaneous coronary intervention and analyzed for surface expression of SDF-1 by flow cytometry. The primary combined endpoint was defined as the composite of either MI, or ischemic stroke, or all-cause death. Secondary endpoints were defined as the aforementioned single events. The patients with baseline platelet SDF-1 levels above the third quartile showed a significantly worse cumulative event-free survival when compared to the patients with lower baseline SDF-1 levels (first to third quartile) (log rank 0.009 for primary combined endpoint and log rank 0.016 for secondary endpoint all-cause death). Multivariate Cox regression analysis showed that SDF-1 levels above the third quartile were independently associated with the primary combined endpoint and the secondary endpoint all-cause death. We provide first clinical evidence that high platelet expression levels of SDF-1 influence clinical outcomes in CAD patients in a negative way.

Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes.

Platelets are circulating cellular sensors that express and release the damage-associated molecular pattern molecule (DAMP) high-mobility group box 1 (HMGB1) at sites of disrupted vascular and tissue integrity. We have recently identified platelet-derived HMGB1 as a critical mediator of thrombosis. The role of platelet-derived HMGB1 in mediating interactions with monocytes remains unknown. In transgenic mice with platelet-specific ablation of HMGB1 and neutralization studies, we show that HMGB1 derived from platelets promotes recruitment of monocytes and prevents monocytes from undergoing apoptosis. During experimental trauma and hemorrhagic shock, infiltrated monocytes in the lung and liver were significantly attenuated in mice lacking HMGB1 in platelets. Platelet-derived HMGB1 mediated monocyte migration via the receptor for advanced glycation end products (RAGE) and suppressed apoptosis via toll-like receptor 4 (TLR4)-dependent activation of MAPK/ERK (extracellular signal-regulated kinase) in monocytes. In conclusion, we identify platelet-derived HMGB1 as a critical regulator of monocyte recruitment and apoptosis, with potential implications in disease states associated with thrombosis and inflammation.