Plasma levels of platelet-enriched microRNAs change during antiplatelet therapy in healthy subjects.

Platelets are the main effectors of primary hemostasis but also cause thrombosis in pathological conditions. Antiplatelet drugs are the cornerstone for the prevention of adverse cardiovascular events. Monitoring the extent of platelet inhibition is essential. Currently available platelet function tests come with constraints, limiting use in antiplatelet drug development as well as in clinical routine. With this study, we aim to investigate whether plasma miRNAs might be suitable biomarkers for monitoring antiplatelet treatment. Platelet-poor plasma was obtained from a trial including 87 healthy male volunteers that either received ticagrelor (n = 44) or clopidogrel (n = 43). Blood was collected before drug intake and after 2 h, 6 h, and 24 h. We measured a panel of 11 platelet-enriched miRNAs (thrombomiRs) by RT-qPCR and selected four biomarker candidates (i.e., miR-223-3p, miR-150-5p, miR-126-3p, miR-24-3p). To further characterize those miRNAs, we performed correlation analyses with the number of extracellular vesicles and clotting time dependent on procoagulant vesicles (PPL assay). We show that platelet-enriched miRNAs in the circulation are significantly reduced upon P2Y12-mediated platelet inhibition. This effect occurred fast, reaching its peak after 2 h. Additionally, we demonstrate that higher baseline levels of thrombomiRs are linked to a stronger reduction upon antiplatelet therapy. Finally, we show that miRNAs from our panel might be the cargo of platelet-derived and procoagulant vesicles. In conclusion, we provide evidence that thrombomiR levels change within 2 h after pharmacological platelet inhibition and circulate the body within platelet-derived and procoagulant extracellular vesicles, rendering them potential biomarker candidates for the assessment of in vivo platelet function.

Extracellular Vesicles and Citrullinated Histone H3 in Coronavirus Disease 2019 Patients.

OBJECTIVES: Pulmonary thrombus formation is a hallmark of coronavirus disease 2019 (COVID-19). A dysregulated immune response culminating in thromboinflammation has been described, but the pathomechanisms remain unclear. METHODS: We studied 41 adult COVID-19 patients with positive results on reverse-transcriptase polymerase-chain-reaction assays and 37 sex- and age-matched healthy controls. Number and surface characteristics of extracellular vesicles (EVs) and citrullinated histone H3 levels were determined in plasma upon inclusion by flow cytometry and immunoassay. RESULTS: In total, 20 patients had severe and 21 nonsevere disease. The number of EV (median [25th, 75th percentile]) was significantly higher in patients compared with controls (658.8 [353.2, 876.6] vs. 435.5 [332.5, 585.3], geometric mean ratio [95% confidence intervals]: 2.6 [1.9, 3.6]; p < 0.001). Patients exhibited significantly higher numbers of EVs derived from platelets, endothelial cells, leukocytes, or neutrophils than controls. EVs from alveolar-macrophages and alveolar-epithelial cells were detectable in plasma and were significantly higher in patients. Intercellular adhesion molecule-1-positive EV levels were higher in patients, while no difference between tissue factor-positive and angiotensin-converting enzyme-positive EV was seen between both groups. Levels of EV did not differ between patients with severe and nonsevere COVID-19. Citrullinated histone H3 levels (ng/mL, median [25th, 75th percentile]) were higher in patients than in controls (1.42 [0.6, 3.4] vs. 0.31 [0.1, 0.6], geometric mean ratio: 4.44 [2.6, 7.7]; p < 0.001), and were significantly lower in patients with nonsevere disease compared with those with severe disease. CONCLUSION: EV and citrullinated histone H3 are associated with COVID-19 and could provide information regarding pathophysiology of the disease.

MicroRNA Signatures in Plasma of Patients With Venous Thrombosis: A Preliminary Report.

BACKGROUND: Venous thromboembolism (VTE) is a frequent and potentially fatal disease, but its pathophysiology is incompletely understood. microRNAs (miR) dysregulate hemostatic proteins and influence thrombotic pathology by posttranscriptional regulation of gene expression. Consensus in defining VTE-related miR clusters and functionally relevant miR has not been reached. We aimed to generate a miR database in patients at high thrombotic risk of VTE and explored their biological functions by seeking information on their messenger RNA targets. METHODS: By high-throughput screening (Affymetrix miRNA Microarray), we identified 159 miR in venous blood of male patients who had two unprovoked VTE and in age-matched male controls. RESULTS: Of the 159 miR, 41 were significantly higher expressed in patients compared to controls. Six miR (hsa-miR-6798-3p, hsa-miR-6789-5p hsa-miR-4651, hsa-miR-6765-5p, hsa-miR-6816-5p, hsa-miR-4734) were modulated ≥ 5.0-fold higher. Higher expression levels of 4 of these miR (hsa-miR-6789-5p, hsa-miR-4651, hsa-miR-6765-5p, and hsa-miR-6816-5p; primers were unavailable for hsa-miR-6798-3p and hsa-miR-4734) were confirmed by quantitative real-time polymerase chain reaction in 10 independent patients and 10 control samples. Ingenuity Pathway Analysis identified 23 altered miR including hsa-miR-6789-5p, hsa-miR-4651, hsa-miR-6765-5p and hsa-miR-4734 as the main regulators of messenger RNAs involved in the pathology of VTE. Seven messenger RNA targets including thrombomodulin and four targets related to platelet function had a direct relationship to 4 identified miR. CONCLUSIONS: We provide evidence of distinct, independently validated miR signatures in patients with VTE and identified a subset of miR as main regulators of messenger RNA involved in disorders related to pathophysiologic processes in venous thrombosis development.

Differential Effects of Clopidogrel With or Without Aspirin on Platelet Reactivity and Coagulation Activation: A Randomized Trial in Healthy Volunteers.

Dual antiplatelet therapy (DAPT) is standard in acute coronary syndrome but confers a bleeding risk. To compare effects of clopidogrel single antiplatelet therapy (SAPT) with clopidogrel-based DAPT on hemostatic system activation we conducted a randomized clinical trial in 44 volunteers (clopidogrel (d1: 600 mg, d2-6: 150 mg) ± aspirin (100 mg)). Multiple electrode aggregometry-adenosine diphosphate (MEA-ADP) and MEA-arachidonic acid (MEA-AA) triggered aggregometry, vasodilator-stimulated phosphoprotein (VASP), beta thromboglobulin, p-selectin, thromboxane B2 , d-Dimer, prothrombin fragment 1.2 (f1.2), and a phospholipid-dependent clotting time were measured in venous blood. Changes are described by mean differences (Δmean (95% confidence interval (CI)) or geometric mean ratios (95% CI)). DAPT and SAPT comparably and significantly decreased MEA-ADP at 2 hours (-60% vs. -63%; P = 0.35, Δmean -4.9, 95% CI -15.4 to 5.5). At 24 hours (-59% vs. -47%, P = 0.04, Δmean -11.1, 95% CI -21.7 to -0.4]) and 8 days (-61% vs. -53%, P = 0.04, Δmean -11.3, 95% CI -22.0 to -0.6). Both treatments significantly reduced VASP and MEA-AA after 2 hours and 8 days. DAPT inhibited MEA-AA significantly stronger at 2 hours (-77% vs. -30%; P < 0.0001, Δmean -39.6, 95% CI -54.2 to -25.0), at 24 hours (-80% vs. -27%, P < 0.0001, Δmean -47.8, 95% CI -62.3 to -33.3), and 8 days (-79% vs. -27%, P < 0.0001, Δmean -48.9, 95% CI -62.5 to -35.4). Neither treatment significantly influenced beta thromboglobulin or p-selectin. DAPT abolished and SAPT reduced thromboxane B2 after 24 hours and 8 days. The d-Dimer was reduced by DAPT (0.94, 95% CI 0.89-1.00, P = 0.04) at 2 hours but not after 24 hours and 8 days. SAPT did not decrease d-Dimer. Neither treatment affected f1.2. DAPT and SAPT comparably affect platelet and coagulation activation in venous blood.

Low dose acetylsalicylic acid and shedding of microparticles in vivo in humans.

BACKGROUND: Microparticles (MPs) have procoagulant properties as shown in vitro and in animal models. Their role in haemostatic system activation at the site of a vascular injury in vivo in humans has not been studied. MATERIAL AND METHODS: In a prospective randomized crossover study, 13 healthy volunteers were given 100 mg acetylsalicylic acid or placebo daily for 7 days. Number and cellular origin, expression of tissue factor (TF) and phosphatidylserine on MPs, and platelet and coagulation activation markers [beta-thromboglobulin (beta-TG), prothrombin fragment f1.2 (f1.2)] were measured in venous blood and in blood from a vascular injury (shed blood) by flow cytometry and immunoassays, respectively. RESULTS: The number of MPs was significantly higher in shed blood than in venous blood. The majority of MPs were platelet derived. The expression of TF on MPs was low, but higher in shed blood than in venous blood. TF positive MPs were significantly higher in shed blood, which was due to an increase of MPs from platelets (PMPs). In shed blood, the number of TF expressing platelet-derived MPs correlated with beta-TG, but not with f1.2. Low dose acetylsalicylic acid did not affect shedding of PMPs, neither in venous blood nor in shed blood. CONCLUSIONS: The release of PMPs locally at the site of platelet plug formation in humans indicates a possible role of MPs for haemostatic system activation in vivo. Low dose acetylsalicylic acid might not be strong enough to suppress shedding of PMPs in the microcirculation.

ProCGlobal and endogenous thrombin potential during pregnancy.

OBJECTIVE: Pregnancy is a hypercoagulable state. We evaluated global markers of coagulation activation, ProCGlobal (Siemens Healthcare Diagnostics, Eschborn, Germany) and endogenous thrombin potential (ETP), in pregnant women with and without low-molecular-weight (LMW) heparin prophylaxis. STUDY DESIGN: We prospectively followed 113 healthy women and 61 women receiving LMW heparin prophylaxis throughout pregnancy. ProCGlobal and ETP were measured in venous blood during the first, second, and third trimester. RESULTS: ProCGlobal decreased significantly throughout pregnancy in all women and was lower in anticoagulated women (P < .001 for all comparisons). ETP levels remained unchanged until the third trimester and then significantly decreased in all women. ETP was higher in anticoagulated women than in healthy women at all time points. CONCLUSION: ProCGlobal levels decrease throughout pregnancy. In pregnant women at high thrombotic risk, coagulation activation reflected by low ProCGlobal and high ETP levels is substantial despite LMW heparin prophylaxis.

Differential Effects of Ticagrelor With or Without Aspirin on Platelet Reactivity and Coagulation Activation: A Randomized Trial in Healthy Volunteers.

Dual antiplatelet therapy (DAPT) is standard in acute coronary heart disease but confers a bleeding risk. To compare the effects of ticagrelor-monotherapy with ticagrelor-based DAPT on hemostatic system activation, we conducted a randomized controlled trial in 44 volunteers using a loading-dose regimen and measured platelet-aggregometry triggered by adenosine diphosphate (multiple electrode aggregometry (MEA)-ADP) and arachidonic acid (MEA-AA), the vasodilator-stimulated phosphoprotein (VASP), prothrombin fragment 1.2 (f1.2), and d-Dimer. Ticagrelor-based DAPT and ticagrelor-monotherapy significantly decreased MEA-ADP (Δmean: -51.4 (-56.9; -45.8) and -46.2 (-51.7; -40.7)) and VASP (Δmean: -70.3 (-76.2; -64.4) and -69.6 (-75.5; -63.7)) at 2 hours and over 24 hours. MEA-AA was reduced significantly by both treatments (Δmean: -72.9 (-80.6; -65.3) and -25.7 (-33.3; -18.0)) at 2 hours, and stronger by ticagrelor-based DAPT over 24 hours. Both treatments decreased f1.2 (geometric mean ratio (GMR): 0.92 (0.84; 1.01) and 0.88 (0.80; 0.96)) and d-Dimer (GMR: 0.89 (0.86; 0.92) and 0.91 (0.88; 0.94)) at 2 hours and d-Dimer over 24 hours. Ticagrelor-monotherapy and ticagrelor-based DAPT comparably affect hemostatic system activation.

Prediction of recurrent venous thromboembolism by endogenous thrombin potential and D-dimer.

BACKGROUND: Increased thrombin generation is associated with an increased risk of recurrent venous thromboembolism. We investigated the relation between endogenous thrombin potential (ETP) and risk of recurrent venous thromboembolism and evaluated whether prediction of recurrence can be improved by a combined analysis of ETP and D-dimer. METHODS: We followed 861 patients with first spontaneous venous thromboembolism and determined ETP and D-dimer after discontinuation of anticoagulation. Patients with natural inhibitor deficiency, lupus anticoagulant, or cancer were excluded. The study endpoint was symptomatic recurrent venous thromboembolism. RESULTS: One hundred thirty patients (15.1%) had recurrence. High ETP (> or = 100%) conferred a 1.6-fold increased risk of recurrence (95% CI 1.1-2.3) after adjustment for age, sex, factor V Leiden, factor II G20210A, and duration of anticoagulation. After adjustment for D-dimer, risk of recurrence remained significantly higher among patients with high ETP [hazard ratio 1.6 (95% CI 1.01-2.4)]. After adjustment for ETP, high D-dimer (> or = 0.5 mg/L) conferred a 1.8-fold (95% CI 1.1-2.8) increased risk of recurrence. Compared with patients with low ETP and low D-dimer, risk of recurrence was 2.8-fold (95% CI 1.5-5.3) higher among patients with both high ETP and high D-dimer after adjustment for potential confounders. CONCLUSIONS: ETP and D-dimer are independent predictors of recurrent venous thromboembolism. Assessing risk of recurrence can be optimized by combining these indicators of thrombin generation.

Tissue factor-positive microparticles: cellular origin and association with coagulation activation in patients with colorectal cancer.

The pathogenesis of hypercoagulability in cancer is not entirely understood. We hypothesized that in cancer patients circulating tissue factor-positive microparticles (TF (+) MPs) are increased and associated with hemostatic system activation. In 20 patients with advanced colorectal cancer and in 20 age- and sex-matched controls, number and cellular origin of TF (+) MPs were determined in plasma by flow cytometry. D-dimer was determined as an indicator of hemostatic system activation. Compared to controls, the median (interquartile range) number of TF (+) MPs was two-fold higher in cancer patients: 25.9 (15.4 - 42.0) x 10 (3) /ml plasma versus 13.1 (11.9 - 19.7) x 10 (3) /ml plasma, p = 0.007. This was mainly due to a higher amount of TF (+) MPs from platelets (13.4 [5.0 - 17.4] x 10 (3) /ml plasma vs. 5.8 [4.5 - 7.5] x 10 (3) /ml plasma, p = 0.017). TF (+) MPs correlated with D-dimer ( ? = 0.48, p = 0.002). High levels of TF (+) MPs in cancer patients and their correlation with D-dimer suggest that TF (+) MPs might be involved in hemostasis activation in cancer patients.

Prediction of recurrent venous thromboembolism by measuring ProC Global.

In patients with venous thromboembolism (VTE) a laboratory assay that globally measures the overall thrombophilic tendency is not available. We hypothesized that determination of ProC((R)) Global, a plasma assay which tests the global function of the protein C pathway, could be used to stratify patients according to their risk of recurrent VTE. We prospectively followed 774 patients with first spontaneousVTE for a mean time of 52 months. ProC Global normalized ratio (NR) was measured in plasma by use of a commercially available assay based on activated partial thromboplastin time. Ninety-eight of the 774 patients had recurrentVTE. Patients with ProC Global NR > or = 0.75 had a relative risk of recurrence of 0.59 (95% CI 0.40-0.87) as compared with those with lower ratio. After four years, cumulative probability of recurrence was 8.6% in patients with ProC Global NR > or = 0.75 and 17.4% in patients with a lower ratio (p = 0.006). Patients with a high ProC Global NR have a low risk of recurrent VTE. ProC Global NR can be used to stratify patients with a first unprovoked VTE according to their risk of recurrence.

Identification of patients at low risk for recurrent venous thromboembolism by measuring thrombin generation.

CONTEXT: Screening of patients with venous thromboembolism (VTE) for thrombophilic risk factors is common clinical practice. Because of the large number of risk factors, assessing the risk of recurrence in an individual patient is complex. A method covering multicausal thrombophilia is therefore required. OBJECTIVE: To investigate the relationship between recurrence of VTE and a simple global coagulation assay measuring thrombin generation. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study of 914 patients with first spontaneous VTE who were followed up for an average of 47 months after discontinuation of vitamin K antagonist therapy. The study was conducted at the Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria, between July 1992 and July 2005. Thrombin generation was measured by a commercially available assay system. Patients with a previous or secondary VTE; antithrombin, protein C, or protein S deficiencies; presence of lupus anticoagulant; cancer; or pregnancy were excluded. MAIN OUTCOME MEASURE: Objectively documented symptomatic recurrent VTE. RESULTS: Venous thromboembolism recurred in 100 patients (11%). Patients without recurrent VTE had lower thrombin generation than patients with recurrence (mean [SD], 349.2 [108.0] nM vs 419.5 [110.5] nM, respectively; P<.001). Compared with patients who had thrombin generation greater than 400 nM, the relative risk (RR) of recurrence was 0.42 (95% confidence interval [CI], 0.26-0.67; P<.001) in patients with values between 400 nM and 300 nM; for patients with lower values, the RR was 0.37 (95% CI, 0.21-0.66; P = .001). After 4 years, the probability of recurrence was 6.5% (95% CI, 4.0%-8.9%) among patients with thrombin generation less than 400 nM compared with 20.0% (95% CI, 14.9%-25.1%) among patients with higher values (P<.001). Patients with thrombin generation less than 400 nM, representing two thirds of patients, had a 60% lower RR of recurrence than those with greater values (RR, 0.40; 95% CI, 0.27-0.60; P<.001). CONCLUSION: Measurement of thrombin generation identifies patients at low risk for recurrent VTE.

Tissue factor pathway inhibitor and the risk of recurrent venous thromboembolism.

Tissue factor pathway inhibitor (TFPI) regulates factor X activation. Low TFPI is a risk factor for a first venous thrombosis. We evaluated whether low TFPI confers an increased risk of recurrent venous thromboembolism (VTE). TFPI-free antigen was measured in 611 patients with a first spontaneous VTE, and who were prospectively followed after withdrawal of anticoagulation. The endpoint was symptomatic recurrent VTE. The relative risk (RR) of recurrence increased from 1.0 (95% CI 0.4-2.6) in patients with TFPI levels < or = 5th percentile to 2.7 (95% CI 1.0-7.4) in patients with levels < or = 2nd percentile as compared with higher levels. At five years, the probability of recurrence was 48.6% (95th CI 19.0-78.1) among patients with TFPI < or = 2nd percentile and 16.8% (95th CI 13.8-19.8) among those with higher levels (p=0.04). Compared to patients with wild type factor V and high TFPI, the RR of recurrence was 1.1 (95% CI 0.7-1.7) in patients with factor V Leiden and high TFPI, 2.3 (95% CI 0.6-9.5) in patients with wild type factor V and low TFPI and 3.5 (95% CI 0.9-14.3) in patients with factor V Leiden and low TFPI. In a multivariate analysis,the high risk of recurrence in carriers of factor V Leiden and low TFPI slightly decreased [RR 2.8 (95% CI 0.6-9.5)]. We conclude that thrombosis patients with low levels of free TFPI are at an increased risk of recurrent VTE.

Interleukin-10 inhibits in vitro hematopoietic suppression and production of interferon-gamma and tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with aplastic anemia.

INTRODUCTION: Mononuclear cells (MNC) from patients with aplastic anemia (AA) can inhibit hematopoietic colony formation from normal bone marrow (BM) cells. Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are considered as soluble mediators of BM suppression in AA. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate the hematopoietic effects of MNC from patients with AA. METHODS: Using coculture experiments we studied the effect of recombinant human IL-10 (rhIL-10) on the in vitro hematopoietic suppression by peripheral blood (PB) MNC from AA patients. RESULTS: PBMNC (5 x 10(5)/ml) from seven patients with AA caused a 40-100% reduction of normal burst-forming unit-erythroid (BFU-E) growth and a 38-91% reduction of colony-forming unit-granulocyte/macrophage (CFU-GM) growth, respectively, in semisolid cocultures. IL-10 was highly effective in reversing growth inhibition in these cocultures. Addition of 10 ng/ml IL-10 to cocultures significantly restored growth of BFU-E in all seven cases and growth of CFU-GM in five of seven cases, respectively. The effect was dose dependent and correlated with decreased IFN-gamma and TNF-alpha production in suspension cultures. Using intracellular cytokine staining it was found that increased TNF-alpha production in AA cells was derived from both CD4+ and CD8+ cells, whereas aberrant IFN-gamma synthesis was only detected in CD8+ cells. CONCLUSION: IL-10 is effective in reversing in vitro hematopoietic suppression by PBMNC from AA patients. These results suggest therapeutic evaluation of rhIL-10 in patients with AA.

Tissue factor exposing microparticles in inflammatory bowel disease.

BACKGROUND: Circulating procoagulant microparticles (MPs) are thought to be involved in the pathogenesis of venous thromboembolism in patients with inflammatory bowel disease (IBD). However, the exposure of tissue factor, the primary initiator of coagulation activation, on microparticles (TF(+)MPs) and its association with hemostasis activation has not yet been studied in IBD patients. METHODS: In this case-control study 49 IBD patients (28 Crohn's disease, 21 ulcerative colitis) and 49 sex- and age-matched, healthy controls were included. Clinical disease activity (Crohn's Disease Activity Index and Clinical Activity Index, respectively) was assessed and IBD-related data were determined by chart review. Numbers, cellular origin and procoagulant activity of TF(+)MPs in plasma were determined using flow cytometry and a chromogenic activity assay. D-dimer and high-sensitive C-reactive protein (CRP) served as markers for coagulation activation and inflammation, respectively. The primary endpoint was the number of TF(+)MPs in IBD patients compared to controls. RESULTS: Median number (interquartile range) of TF(+)MPs was higher in IBD patients than in controls (14.0 (11.9-22.8)×10(3)/mL vs. 11.9 (11.9-19.1)×10(3)/mL plasma, P=0.029). This finding was due to generally higher plasma levels of MPs from platelets and leukocytes in IBD patients. However, the number of TF(+)MPs was neither correlated with their procoagulant activity and D-dimer nor with disease activity and CRP. CONCLUSIONS: Increased numbers of circulating TF(+)MPs represent a new facet of hemostatic abnormalities in IBD. However, the lack of association with activation of the coagulation system and disease activity questions their pathogenetic role for venous thromboembolism in this patient group.

Prediction of recurrent venous thromboembolism by clot lysis time: a prospective cohort study.

Venous thromboembolism (VTE) is a chronic disease, which tends to recur. Whether an abnormal fibrinolytic system is associated with an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic capacity (reflected by clot lysis time [CLT]) and risk of recurrent VTE. We followed 704 patients (378 women; mean age 48 yrs) with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, homozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. Study endpoint was symptomatic recurrent VTE. For measurement of CLT, a tissue factor-induced clot was lysed by adding tissue-type plasminogen activator. Time between clot formation and lysis was determined by measuring the turbidity. 135 (19%) patients had recurrent VTE. For each increase in CLT of 10 minutes, the crude relative risk (RR) of recurrence was 1.13 (95% CI 1.02-1.25; p = 0.02) and was 1.08 (95% CI 0.98-1.20; p = 0.13) after adjustment for age and sex. For women only, the adjusted RR was 1.14 (95% CI, 0.91-1.42, p = 0.22) for each increase in CLT of 10 minutes. CLT values in the 4(th) quartile of the female patient population, as compared to values in the 1(st) quartile, conferred a risk of recurrence of 3.28 (95% CI, 1.07-10.05; p = 0.04). No association between CLT and recurrence risk was found in men. Hypofibrinolysis as assessed by CLT confers a moderate increase in the risk of recurrent VTE. A weak association between CLT and risk of recurrence was found in women only.