Platelet mass cytometry reveals dysregulation of prothrombotic pathways in essential thrombocythemia.

Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients.

Essential thrombocythemia (ET) is a rare disease characterized by an increased number of platelets in the blood. As a complication, many of these patients develop a blood clot, which can be life-threatening. So far, the reason behind the higher risk of blood clots is unclear. In this study, we analyzed platelet surface markers that play a critical role in platelet function and platelet activation using a modern technology called mass cytometry. For this purpose, blood samples from 6 patients with ET and 6 healthy control individuals were analyzed. We found significant differences between ET platelets and healthy platelets. ET platelets had higher expression levels of p-Selectin (CD62P), a key marker of platelet activation, and of the collagen receptor GPVI, which is important for clot formation. These results may be driven by a specific platelet subcluster overrepresented in ET. Other surface markers, such as the fibrinogen receptor GPIIb/IIIa CD41, CD61, and the von Willebrand factor receptor CD42b, were lower expressed in ET platelets. When ET platelets were treated with the clotting factor thrombin (thrombin receptor-activating peptide, TRAP), we found a differential response in platelet activation compared to healthy platelets. In conclusion, our results show an increased activation and clotting potential of ET platelets. The platelet surface protein GPVI may be a potential drug target to prevent abnormal blood clotting in ET patients.

Plasma Soluble Glycoprotein VI, Platelet Function, Bleeding, and Ischemic Events in Patients Undergoing Elective Percutaneous Coronary Intervention.

BACKGROUND AND AIMS: Glycoprotein VI (GPVI) is the major platelet-specific collagen receptor. GPVI shedding with generation of soluble GPVI (sGPVI) is an endogenous feedback mechanism preventing platelet overstimulation. sGPVI has not been investigated in patients with chronic coronary syndrome (CCS) undergoing percutaneous coronary intervention (PCI), especially regarding its potential value as a predictor of ischemic and bleeding risk. METHODS: Baseline plasma sGPVI levels were available in 318 patients with CCS undergoing PCI. Platelet function was assessed by measuring both adenosine diphosphate (ADP) and collagen-induced platelet aggregation. Co-primary endpoints were a composite of death or myocardial injury at 48 hours after PCI, and Bleeding Academic Research Consortium (BARC) type 1 to 5 bleeding at 30 days. RESULTS: There was no significant correlation between sGPVI and platelet function at baseline or at 48 hours after PCI and loading with antiplatelet drugs. Baseline plasma sGPVI levels were not associated with the ischemic risk: the incidence of the ischemic endpoint was 25.0% in the lower, 22.9% in the middle, and 26.7% in the upper sGPVI tertile (p = 0.82). There was a significant nonlinear relationship between sGPVI and the risk of bleeding: the incidence of the bleeding endpoint was 11.8% in the lower, 12.6% in the middle, and 26.4% in the upper sGPVI tertile (p = 0.006). CONCLUSION: In patients with CCS undergoing PCI, plasma levels of sGPVI did not correlate with ADP- or collagen-induced platelet aggregation. Patients with higher baseline levels of sGPVI may carry an increased risk of bleeding at 30 days after PCI but no excess risk of ischemic events.

Association of immature platelets with perioperative complications in neurosurgery.

Immature platelets are newly formed platelets with an increased prothrombotic potential. This study evaluates whether immature platelets are associated with relevant complications in neurosurgical patients. Data were obtained in the frame of a prospectively conducted observational study exploring the association between immature platelets and major cardiovascular events after surgery. Immature platelet fraction (IPF) and H-IPF (highly fluorescent immature platelet fraction) were measured preoperatively and postoperatively at the neurosurgical ward (24-72 hours after surgery). Therapy-relevant complications after surgery were stratified using the Clavien-Dindo Grade (CDG >2) as primary outcome. Data were analyzed in 391 neurosurgical patients. While preoperatively there were no differences in IPF or H-IPF, patients with higher therapy-complication grades had higher values post-op compared to patients with lower grade complications (≤2 CDG). Cut-off values identified by receiver operating characteristic curve analysis revealed that there were significantly more patients with H-IPF ≥0.95% in the group with serious complications (CDG >2) [odds ratio OR (95% confidence interval CI) = 2.06 (1.09-3.9), p = .025], whereas this association was not present for the IPF cutoff value. In a multivariate model, H-IPF≥0.95% was independently associated with serious complications after surgery [OR (95% CI) = 1.97 (1.03-3.78), p = .041]. These findings suggest that H-IPF is associated with surgical complications and may improve risk stratification of neurosurgical patients (clinicaltrials.gov: NCT02097602, registration date: 27/03/2014).

What is the context?Immature platelets are newly formed platelets with a higher thrombotic potential and play an important role in atherothrombotic events.Higher levels of immature platelets were observed in patients with acute coronary syndrome or stroke.Lately, the focus in immature platelet research shifted from observation to outcomes. Immature platelets were identified as independent predictors of major cardiovascular events in cardiologic patients with coronary artery disease. Besides, an association between immature platelets and major cardiovascular events was described in surgical patients after non-cardiac surgery.What is new?This study builds on these findings and extends the focus to perioperative complications after neurosurgery.The data were obtained prospectively in the frame of an observational clinical trial exploring the association of immature platelets and major cardiovascular events in general. Data measured in the neurosurgical cohort of that study (391 neurosurgical patients) were analyzed in the present work.Within the limitations of our study, our analyses suggest that the postoperative IPF (immature platelet fraction) and H-IPF (highly fluorescent immature platelet fraction) values, which were measured at the neurosurgical ward after surgery are both associated with higher therapy-relevant complication grades (>2 according to Clavien-Dindo Grade), whereas preoperatively obtained values were not.What is the impact?This is the first study showing a relationship between immature platelets and therapy-relevant perioperative complications in neurosurgical patients. It could be a pilot trial for varied scientific questions including risk stratification of neurosurgical patients.

JCAD promotes arterial thrombosis through PI3K/Akt modulation: a translational study.

AIMS: Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. METHODS AND RESULTS: JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. CONCLUSIONS: JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.

Platelet Surface Protein Expression and Reactivity upon TRAP Stimulation after BNT162b2 Vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet-leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.

Ticagrelor or Prasugrel in Patients With Acute Coronary Syndrome in Relation to Estimated Glomerular Filtration Rate.

OBJECTIVES: The aim of this study was to assess the safety and efficacy of ticagrelor versus prasugrel for patients with acute coronary syndrome (ACS) according to their estimated glomerular filtration rates (eGFRs). BACKGROUND: The outcomes of ticagrelor versus prasugrel in patients with ACS according to eGFR have not been defined. METHODS: Patients (n = 4,012) were categorized into 3 groups: low eGFR (<60 mL/min/1.73 m2), intermediate eGFR (≥60 and <90 mL/min/1.73 m2), and high eGFR (≥90 mL/min/1.73 m2). The primary endpoint was a composite of all-cause death, myocardial infarction, and stroke; the secondary safety endpoint was Bleeding Academic Research Consortium types 3 to 5 bleeding, both at 1 year. RESULTS: Patients with low eGFRs had a higher risk for the primary endpoint compared with patients with intermediate eGFRs (adjusted HR: 1.89; 95% CI: 1.46-2.46]) and those with high eGFRs (adjusted HR: 2.33; 95% CI: 1.57-3.46). A risk excess for low eGFR was also observed for bleeding (adjusted HR: 1.55 [95% CI: 1.12-2.13] vs intermediate eGFR; adjusted HR: 1.59 [95% CI: 1.01-2.50] vs high eGFR). However, eGFR did not affect the relative efficacy and safety of ticagrelor versus prasugrel. In patients with low eGFR, the primary endpoint occurred in 20.5% with ticagrelor and in 14.7% with prasugrel (HR: 1.47; 95% CI: 1.04-2.08; P = 0.029); there was no significant difference in bleeding. CONCLUSIONS: These results show that among patients with ACS, reduction of eGFR is associated with increased risk for ischemic and bleeding events but has no significant impact on the relative efficacy and safety of ticagrelor versus prasugrel. (Prospective, Randomized Trial of Ticagrelor Versus Prasugrel in Patients With Acute Coronary Syndrome [ISAR-REACT 5]; NCT01944800).

Efficacy and safety of ticagrelor versus prasugrel in smokers and nonsmokers with acute coronary syndromes.

BACKGROUND: The efficacy and safety of ticagrelor versus prasugrel according to smoking status in patients with acute coronary syndromes (ACS) are not known. We assessed the efficacy and safety of ticagrelor versus prasugrel according to smoking status in patients with ACS undergoing invasive management. METHODS: This pre-specified analysis of the ISAR-REACT 5 trial included 1349 smokers and 2652 nonsmokers randomized to receive ticagrelor or prasugrel. The primary endpoint was the incidence of death, myocardial infarction, or stroke; the secondary endpoint was the incidence of Bleeding Academic Research Consortium (BARC) type 3 to 5 bleeding (both endpoints assessed at 12 months). RESULTS: There was no significant treatment arm-by-smoking status interaction regarding the efficacy outcome. The primary endpoint occurred in 47 patients (7.0%) in the ticagrelor group and 41 patients (6.2%) in the prasugrel group in smokers (hazard ratio [HR] = 1.15; 95% confidence interval [CI] 0.76-1.75; P = 0.510) and in 133 patients (10.2%) in the ticagrelor group and 94 patients (7.2%) in the prasugrel group in nonsmokers (HR = 1.44 [1.10-1.87]; P = 0.007; P for interaction = 0.378). The secondary endpoint occurred in 27 patients (4.6%) in the ticagrelor group and 33 patients (5.6%) in the prasugrel group in smokers (HR = 0.81 [0.49-1.35]; P = 0.412) and in 66 patients (6.0%) in the ticagrelor group and 46 patients (4.4%) in the prasugrel group in nonsmokers (HR = 1.38 [0.94-2.01]; P = 0.097). CONCLUSIONS: In patients with ACS undergoing an invasive management strategy, the smoking status did not significantly interact with the relative treatment effect of ticagrelor vs. prasugrel. CLINICAL TRIAL REGISTRATION: NCT01944800.

Efficacy and Safety of Revacept, a Novel Lesion-Directed Competitive Antagonist to Platelet Glycoprotein VI, in Patients Undergoing Elective Percutaneous Coronary Intervention for Stable Ischemic Heart Disease: The Randomized, Double-blind, Placebo-Controlled ISAR-PLASTER Phase 2 Trial.

Importance: The assessment of new antithrombotic agents with a favorable safety profile is clinically relevant. Objective: To test the efficacy and safety of revacept, a novel, lesion-directed antithrombotic drug, acting as a competitive antagonist to platelet glycoprotein VI. Design, Setting, and Participants: A phase 2 randomized clinical trial; patients were enrolled from 9 centers in Germany from November 20, 2017, to February 27, 2020; follow-up ended on March 27, 2020. The study included patients with stable ischemic heart disease (SIHD) undergoing elective percutaneous coronary intervention (PCI). Interventions: Single intravenous infusion of revacept, 160 mg, revacept, 80 mg, or placebo prior to the start of PCI on top of standard antithrombotic therapy. Main Outcomes and Measures: The primary end point was the composite of death or myocardial injury, defined as an increase in high-sensitivity cardiac troponin to at least 5 times the upper limit of normal within 48 hours from randomization. The safety end point was bleeding type 2 to 5 according to the Bleeding Academic Research Consortium criteria at 30 days. Results: Of 334 participants (median age, 67.4 years; interquartile range, 60-75.1 years; 253 men [75.7%]; and 330 White participants [98.8%]), 120 were allocated to receive the 160-mg dose of revacept, 121 were allocated to receive the 80-mg dose, and 93 received placebo. The primary end point showed no significant differences between the revacept and placebo groups: 24.4%, 25.0%, and 23.3% in the revacept, 160 mg, revacept, 80 mg, and placebo groups, respectively (P = .98). The high dose of revacept was associated with a small but significant reduction of high-concentration collagen-induced platelet aggregation, with a median 26.5 AU × min (interquartile range, 0.5-62.2 AU × min) in the revacept, 160 mg, group; 43.5 AU × min (interquartile range, 22.8-99.5 AU × min) in the revacept, 80 mg, group; and 41.0 AU × min (interquartile range, 31.2-101.0 AU × min) in the placebo group (P = .02), while adenosine 5'-diphosphate-induced aggregation was not affected. Revacept did not increase Bleeding Academic Research Consortium type 2 or higher bleeding at 30 days compared with placebo: 5.0%, 5.9%, and 8.6% in the revacept, 160 mg, revacept, 80 mg, and placebo groups, respectively (P = .36). Conclusions and Relevance: Revacept did not reduce myocardial injury in patients with stable ischemic heart disease undergoing percutaneous coronary intervention. There were few bleeding events and no significant differences between treatment arms. Trial Registration: ClinicalTrials.gov Identifier: NCT03312855.

Ticagrelor or Prasugrel in Patients With Non-ST-Segment Elevation Acute Coronary Syndromes.

BACKGROUND: Current guidelines recommend intensified platelet inhibition by prasugrel or ticagrelor in patients with unstable angina (UA) or non-ST-segment elevation (NSTE) myocardial infarction (MI). OBJECTIVES: This study sought to investigate the benefits and risks of ticagrelor as compared with prasugrel in patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS) and planned invasive management. METHODS: This post hoc analysis combines the pre-specified subgroups of UA and NSTEMI of the randomized ISAR-REACT 5 trial. It included 1,179 patients assigned to ticagrelor and 1,186 assigned to prasugrel. Ticagrelor was started immediately after randomization and prasugrel after coronary angiography. The primary endpoint was a composite of death, MI, or stroke during 1-year follow-up, and the safety endpoint was Bleeding Academic Research Consortium class 3-5. RESULTS: The primary endpoint was reached in 101 (8.7%) patients in the ticagrelor and in 73 (6.3%) patients in the prasugrel group (hazard ratio [HR]: 1.41; 95% confidence interval [CI]: 1.04 to 1.90). The HR for all-cause death was 1.43 (95% CI: 0.93 to 2.21) and that for MI 1.43 (95% CI: 0.94 to 2.19). The safety endpoint occurred in 49 (5.2%) patients in the ticagrelor and in 41 (4.7%) patients in the prasugrel group (HR: 1.09; 95% CI: 0.72 to 1.65). Landmark analysis revealed persistence of the efficacy advantage with prasugrel after the first month. CONCLUSIONS: In patients with NSTE-ACS, we found that prasugrel was superior to ticagrelor in reducing the combined 1-year risk of death, MI, and stroke without increasing the risk of bleeding. Due to the post hoc nature of the analysis, these findings need confirmation by further studies. (Prospective, Randomized Trial of Ticagrelor Versus Prasugrel in Patients With Acute Coronary Syndrome; NCT01944800).

Ticagrelor or Prasugrel in Patients With ST-Segment-Elevation Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention.

BACKGROUND: Data on the comparative efficacy and safety of ticagrelor versus prasugrel in patients with ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention are limited. We assessed the efficacy and safety of ticagrelor versus prasugrel in a head-to-head comparison in patients with ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention. METHODS: In this prespecified subgroup analysis, we included 1653 patients with ST-segment-elevation myocardial infarction randomized to receive ticagrelor or prasugrel in the setting of the ISAR REACT-5 trial (Intracoronary Stenting and Antithrombotic Regimen: Rapid Early Action for Coronary Treatment 5). The primary end point was the incidence of death, myocardial infarction, or stroke at 1 year after randomization. The secondary end point was the incidence of bleeding defined as BARC (Bleeding Academic Research Consortium) type 3 to 5 bleeding at 1 year after randomization. RESULTS: The primary end point occurred in 83 patients (10.1%) in the ticagrelor group and in 64 patients (7.9%) in the prasugrel group (hazard ratio, 1.31 [95% CI, 0.95-1.82]; P=0.10). One-year incidence of all-cause death (4.9% versus 4.7%; P=0.83), stroke (1.3% versus 1.0%; P=0.46), and definite stent thrombosis (1.8% versus 1.0%; P=0.15) did not differ significantly in patients assigned to ticagrelor or prasugrel. One-year incidence of myocardial infarction (5.3% versus 2.8%; hazard ratio, 1.95 [95% CI, 1.18-3.23]; P=0.010) was higher with ticagrelor than with prasugrel. BARC type 3 to 5 bleeding occurred in 46 patients (6.1%) in the ticagrelor group and in 39 patients (5.1%) in the prasugrel group (hazard ratio, 1.22 [95% CI, 0.80-1.87]; P=0.36). CONCLUSIONS: In patients with ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention, there was no significant difference in the primary end point between prasugrel and ticagrelor. Ticagrelor was associated with a significant increase in the risk for recurrent myocardial infarction. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01944800.

Ticagrelor or Prasugrel in Patients with Acute Coronary Syndromes.

BACKGROUND: The relative merits of ticagrelor as compared with prasugrel in patients with acute coronary syndromes for whom invasive evaluation is planned are uncertain. METHODS: In this multicenter, randomized, open-label trial, we randomly assigned patients who presented with acute coronary syndromes and for whom invasive evaluation was planned to receive either ticagrelor or prasugrel. The primary end point was the composite of death, myocardial infarction, or stroke at 1 year. A major secondary end point (the safety end point) was bleeding. RESULTS: A total of 4018 patients underwent randomization. A primary end-point event occurred in 184 of 2012 patients (9.3%) in the ticagrelor group and in 137 of 2006 patients (6.9%) in the prasugrel group (hazard ratio, 1.36; 95% confidence interval [CI], 1.09 to 1.70; P = 0.006). The respective incidences of the individual components of the primary end point in the ticagrelor group and the prasugrel group were as follows: death, 4.5% and 3.7%; myocardial infarction, 4.8% and 3.0%; and stroke, 1.1% and 1.0%. Definite or probable stent thrombosis occurred in 1.3% of patients assigned to ticagrelor and 1.0% of patients assigned to prasugrel, and definite stent thrombosis occurred in 1.1% and 0.6%, respectively. Major bleeding (as defined by the Bleeding Academic Research Consortium scale) was observed in 5.4% of patients in the ticagrelor group and in 4.8% of patients in the prasugrel group (hazard ratio, 1.12; 95% CI, 0.83 to 1.51; P = 0.46). CONCLUSIONS: Among patients who presented with acute coronary syndromes with or without ST-segment elevation, the incidence of death, myocardial infarction, or stroke was significantly lower among those who received prasugrel than among those who received ticagrelor, and the incidence of major bleeding was not significantly different between the two groups. (Funded by the German Center for Cardiovascular Research and Deutsches Herzzentrum München; ISAR-REACT 5 ClinicalTrials.gov number, NCT01944800.).

Transcriptome Analysis of Reticulated Platelets Reveals a Prothrombotic Profile.

Reticulated platelets (RPs) are larger, hyperreactive platelets that contain significantly more ribonucleic acid (RNA) compared with mature platelets (MPs). High levels of RPs in peripheral blood are predictors of an insufficient response to dual antiplatelet therapy in cardiovascular patients and of adverse cardiovascular events. However, the mechanisms underlying these correlations remain widely unknown and the biology of RPs has not been investigated yet. Here, we compared for the first time the transcriptomic profiles of RPs and MPs isolated from peripheral blood of healthy donors. Total RNA sequencing revealed 1,744 differentially expressed genes (670 downregulated, 1,074 upregulated) in RPs compared with MPs. In particular, transcripts for the collagen receptor GP6, thromboxane receptor A2 (TBXA2R), thrombin receptor PAR4 (F2RL3), and adenosine triphosphate receptors P2RX1, ORAI2, and STIM1 (both involved in calcium signaling) were significantly upregulated in RPs, whereas several RNA regulators as the ribonuclease PARN, the RISC-component TNRC6A, and the splicing factor LUC7L3 were downregulated in RPs. Gene ontology analysis revealed an enrichment of relevant biological categories in RPs including platelet activation and blood coagulation. Gene Set Enrichment Analysis showed an overrepresentation of several platelet activation pathways like thrombin, thromboxane, and glycoprotein IIb/IIIa signaling in RPs. Small-RNA sequencing reported 9 micro-RNAs significantly downregulated in RPs with targets involved in platelet reactivity. Our data show for the first time an enrichment of several prothrombotic transcripts in RPs providing a first biological explanation for their hyperreactive phenotype.

Btk Inhibitors as First Oral Atherothrombosis-Selective Antiplatelet Drugs?

Bruton's tyrosine kinase (Btk) is essential for B cell differentiation and proliferation, but also platelets express Btk. Patients with X-linked agammaglobulinemia due to hereditary Btk deficiency do not show bleeding, but a mild bleeding tendency is observed in high dose therapy of B-cell malignancies with ibrutinib and novel second-generation irreversible Btk inhibitors (acalabrutinib and ONO/GS-4059). This review discusses recent studies that may explain this apparent paradox and gives mechanistic insights that suggest a unique potential of low dose irreversible Btk inhibitors as atherothrombosis-focused antiplatelet drugs.

Immature platelets as a novel biomarker for adverse cardiovascular events in patients after non-cardiac surgery.

This study evaluates whether immature platelets (IPF) determined in the post anesthesia care unit (PACU) can predict major adverse cardiovascular events (MACE) or other thromboembolic events after intermediate and high-risk surgery. IPF are increased in patients with acute coronary syndrome and recently gained interest as novel biomarker for risk stratification. In this prospective observational trial 732 patients undergoing intermediate or high-risk non-cardiac surgery were enrolled (NCT02097602). IPF was measured preoperatively and postoperatively in the PACU. Primary outcome was a composite endpoint defined as MACE, deep vein thrombosis or pulmonary embolism during hospital stay (modMACE). A cut off for IPF identifying a threshold between a low and high risk for modMACE was calculated by log-rank optimization. A multivariate Cox regression was calculated in a forward stepwise manner to assess the relation between this IPF cut off and modMACE as well as other established risk factors (inclusion if p<0.05). Preoperatively, there were no differences in IPF between patients with and without modMACE (3.1 % [2.2 % - 4.7 %](median [interquartile range]) vs. 2.8 % [1.9 % - 4.3 %]. Patients with modMACE (28 of 730 patients; 3.8 %) had higher IPF values in the PACU compared to patients without modMACE (3.6 % [2.6-6 %] vs. 2.9 % [2-4.4 %]; p=0.011). The optimal cut off of IPF > 5.4 % was associated with an increased risk for modMACE after adjustment for covariates (hazard ratio: 2.528; 95 % confidence interval: 1.156 to 5.528, p=0.02). In conclusion, IPF is an independent predictor of modMACE after surgery and might improve risk stratification of surgical patients.

Ticagrelor versus prasugrel in patients with high on-clopidogrel treatment platelet reactivity after PCI: The ISAR-ADAPT-PF study.

Patients with high on-treatment platelet reactivity (HTPR) on clopidogrel are at high risk for adverse cardiovascular events after percutaneous coronary intervention (PCI). The aim of the ISAR-ADAPT-PF study was to assess the antiplatelet efficacy of ticagrelor versus prasugrel in patients with HTPR on clopidogrel. In a prospective and randomized clinical study, 70 patients with HTPR on clopidogrel loading dose (LD) within 24 h post PCI were assigned to receive either ticagrelor [180 mg LD followed by 90 mg maintenance dose (MD) twice daily] or prasugrel (60 mg LD followed by 10 mg MD once daily). The adenosine diphosphate-induced platelet aggregation assessed on the Multiplate analyzer on day 2 after randomization (primary end point) was as follows: the mean difference between the two treatment groups was 6 aggregation units (AU) × min with an upper 95% confidence interval (CI) of 41 AU × min, which was greater than the predefined noninferiority margin of 18 AU × min (P for noninferiority = 0.29). However, no significant differences in absolute platelet reactivity levels between ticagrelor- versus prasugrel-treated patients at that time point were observed (138 ± 100 AU × min vs. 132 ± 64 AU × min, P for superiority = 0.77). In conclusion, neither drug was statistically more effective for inhibition of platelet aggregation in patients with HTPR on clopidogrel post PCI, although the study could not formally demonstrate the assumed noninferiority of ticagrelor versus prasugrel.

High on-treatment platelet reactivity and outcomes after percutaneous endovascular procedures in patients with peripheral artery disease.

BACKGROUND: High on-treatment platelet reactivity (HPR) predicts adverse cardiovascular events in patients with coronary artery disease. The impact of HPR in patients with peripheral artery disease (PAD) after peripheral endovascular procedures is unclear. PATIENTS AND METHODS: A total of 385 patients with PAD and successful percutaneous endovascular procedure were included. Patients received aspirin as a long-term treatment in addition to the P2Y12 receptor antagonist clopidogrel, as recommended after such a procedure for at least 1 month. Platelet function was assessed on a Multiplate analyzer. The primary endpoint was target lesion revascularization (TLR) at one year. Restenosis (≥ 75 %) in duplex sonography, mortality at one year and identification of independent predictors of TLR were secondary endpoints. RESULTS: TLR rates were similar in HPR and no-HPR patients (14.3 % vs. 12.7 %, hazard rate (HR) 0.94, 95 % CI 0.48 - 1.84, P = 0.86). Restenosis (≥ 75 %) in duplex sonography did not differ between the two study groups (15.6 % vs. 16.9 %, HR 1.16, 95% CI 0.62 - 2.12, P = 0.64). Independent predictors of TLR were intervention of restenotic lesions, total vessel occlusions and critical limb ischemia, but not HPR (adjusted HR 1.07, 95% CI 0.55 - 2.10, P = 0.84). No difference in mortality at one year was observed (1.3 % vs. 1.6 %, HR 1.28, 95 % CI 0.15 - 11.0, P = 0.82). CONCLUSIONS: In patients with PAD, HPR did not have a significant impact on outcomes within the first year after percutaneous endovascular intervention.

Combination of factor Xa inhibition and antiplatelet therapy after stenting in patients with iliofemoral post-thrombotic venous obstruction.

OBJECTIVES: Studies addressing optimal postprocedural pharmacological management after endovascular stenting of iliofemoral post-thrombotic venous obstruction are lacking. We report our early clinical experience with a combination of rivaroxaban and clopidogrel in patients after iliofemoral post-thrombotic venous obstruction stenting. METHODS: Demographic, procedural, and follow-up data of nine patients (seven women; mean age of 32 ± 11 years) undergoing 10 procedures for iliofemoral post-thrombotic venous obstruction performed between August 2012 and January 2014 were retrospectively reviewed. After endovascular intervention, all patients were administered 20 mg rivaroxaban once daily (s.i.d.) and 75 mg clopidogrel s.i.d. or every second day depending on the individual drug responsiveness for at least six months. The adenosine diphosphate-induced platelet aggregation (platelet aggregation, in aggregation units × min) was assessed on a Multiplate analyzer. Patency was verified venographically at procedure end and was evaluated with duplex ultrasound in regular follow-ups. RESULTS: Iliofemoral venous flow was successfully re-established by percutaneous endovascular angioplasty and stent implantation in nine left-sided and one bilateral iliofemoral post-thrombotic venous obstruction. Under dual treatment strategy of rivaroxaban and clopidogrel with platelet aggregation control (median (range): 285 aggregation units × min (192; 402)), none of the patients experienced restenosis or stent thrombosis, respectively. After a median follow-up of 14 months (range: 6-26 months), the primary patency rate was 100% and no in-stent restenosis, stent occlusion or relevant minor or major bleeding occurred. CONCLUSION: Combined factor Xa inhibition and tailored antiplatelet therapy after stenting of iliofemoral post-thrombotic venous obstruction were safe and performed favorably in terms of vessel patency.

Impact of immature platelets on platelet response to ticagrelor and prasugrel in patients with acute coronary syndrome.

AIMS: The influence of reticulated platelets (RPs) on platelet inhibition by ticagrelor when compared with prasugrel is unknown. We aimed to determine the influence of RPs on adenosine diphosphate- (ADP-) induced platelet aggregation in patients with acute coronary syndrome who were randomly assigned to receive either ticagrelor or prasugrel for P2Y12 receptor inhibition. METHODS AND RESULTS: One hundred and twenty-four patients were prospectively enrolled. The immature platelet fraction (IPF) was measured by an automated haematoanalyzer and platelet aggregation was assessed by multiple electrode aggregometry. In a subgroup of patients (n = 28) ADP-induced P-selectin expression was measured in dependence of the time point of study drug intake in RPs that were discriminated from mature platelets by thiazole-orange (TO) staining. The primary endpoint was the correlation of platelet aggregation and IPF in ticagrelor- vs. prasugrel-treated patients. Platelet aggregation significantly correlated with IPF in patients who received prasugrel (r = 0.41, P < 0.001). In contrast, no correlation between IPF and platelet aggregation was observed in ticagrelor-treated patients (r = 0.08, P = 0.51, P for difference of correlation coefficients P = 0.05). Within the TO-positive RP fraction, P-selectin expression was significantly higher in prasugrel when compared with ticagrelor-treated individuals (prasugrel 18.6 ± 16.0% vs. ticagrelor 11.5 ± 6.0%, P = 0.047). A time-dependent P-selectin expression was observed in prasugrel but not in ticagrelor-treated patients (prasugrel early 11.9 ± 9.4% vs. late 26.3 ± 19.0%, P = 0.031, ticagrelor early 9.6 ± 4.9% vs. late 13.5 ± 6.6%, P = 0.127). CONCLUSION: Reticulated platelets show a greater impact on platelet reactivity in response to prasugrel when compared with ticagrelor.

The impact of therapeutic hypothermia on on-treatment platelet reactivity and clinical outcome in cardiogenic shock patients undergoing primary PCI for acute myocardial infarction: Results from the ISAR-SHOCK registry.

INTRODUCTION: Mild therapeutic hypothermia (TH) is standard of care after cardiac arrest of any cause. However, its impact on on-treatment platelet reactivity and clinical outcome in patients with acute myocardial infarction (AMI) complicated by cardiogenic shock and undergoing PCI with P2Y12 receptor inhibitor treatment is less clear. METHODS AND RESULTS: For the ISAR-SHOCK registry, 145 patients with AMI, cardiogenic shock and primary PCI in two centers (Deutsches Herzzentrum München and Klinikum rechts der Isar, Technical University Munich) between January 2009-May 2012 were analysed. Of these, 64 (44%) patients received TH treatment. The median [IQR] ADP-induced platelet aggregation following thienopyridine loading dose administration (clopidogrel in 95 and prasugrel in 50 patients) did not differ between the two groups (419 [283-684] for TH vs. 355 [207-710] AU x min for non-TH patients, P=0.22). After 30days follow-up, no significant differences were observed between both groups for mortality (42 vs. 44 %, HR: 0.93, 95% CI [0.56-1.53], p=0.77), MI (6 vs. 6%, HR: 0.99 95% CI [0.27-3.7], p=0.99) and TIMI minor bleedings (17 vs. 17%, HR 0.99 95% CI [0.45-2.18], p=0.98). TIMI major bleedings were numerically higher in the TH vs. non-TH cohort (25 % vs. 12 %, HR: 2.1 95% CI [0.95-4.63], p=0.07). Three definite stent thrombosis (ST) were observed in this registry and all STs occurred in the TH group of patients (p=0.09). CONCLUSION: Results of this registry suggest that TH does not negatively impact on platelet reactivity in shock patients receiving either clopidogrel or prasugrel. The numerically higher rate of major bleedings and the clustering of STs in the TH cohort warrant further investigation.