Thrombosis and Haemostasis Thrombosis and Haemostasis th de-de Sun, 30 Apr 17 09:10:06 +0200 Ahead of print: F7 gene variants modulate protein levels in a large cohort of patients with factor... Congenital factor VII (FVII) deficiency is a rare bleeding disorder caused by mutations in F7 gene with autosomal recessive inheritance. A clinical heterogeneity with poor correlation with FVII:C levels has been described. It was the objective of this study to identify genetic defects and to evaluate their relationships with phenotype in a large cohort of patients with FVII:C<50 %. One hundred twenty-three probands were genotyped for F7 mutations and three polymorphic variants and classified according to recently published clinical scores. Forty out of 123 patients (33 %) were symptomatic (43 bleedings). A severe bleeding tendency was observed only in patients with FVII:C<0.10 %. Epistaxis (11 %) and menorrhagia (32 % of females in fertile age) were the most frequent bleedings. Molecular analysis detected 48 mutations, 20 not reported in the F7 international databases. Most mutations (62 %) were missense, large deletions were 6.2 %. Compound heterozygotes/homozygotes for mutations presented lower FVII:C levels compared to the other classes (Chi2=43.709, p<0,001). The polymorphisms distribution was significantly different among the three F7 genotypic groups (Chi2=72.289, p<0,001). The presence of truncating mutations was associated with lowest FVII:C levels (Chi2=21.351, p=0.002). This study confirms the clinical and molecular variability of the disease and the type of symptoms. It shows a good correlation between the type of F7 mutation and/or polymorphisms and FVII:C levels, without a direct link between FVII:C and bleeding tendency. The results suggest that large deletions are underestimated and that they represent a common mechanism of F7 gene inactivation which should always be investigated in the diagnostic testing for FVII deficiency.... G. Quintavalle (1), F. Riccardi (1), G. F. Rivolta (1), D. Martorana (2), C. Di Perna (1), A. Percesepe (2), A. Tagliaferri (1), on behalf of the Ad-Hoc Study Group 27460 2017-04-27 09:08:14 Ahead of print: Khorana score and histotype predicts incidence of early venous thromboembolism in... Current data suggests that the risk of venous thromboembolism (VTE) in patients with non-Hodgkin lymphoma (NHL) is comparable to that observed in patients with solid tumours, although more robust confirmatory analyses are required. With that in mind, we investigated the occurrence of VTE in a pooled analysis of 12 “Fondazione Italiana Linfomi” (FIL) prospective clinical studies. Specifically, we wished to assess the cumulative incidence of VTE in NHL patients, evaluate the predictive value of the Khorana Score (KS), and identify other potential risk factors for VTEs. Data for VTE occurrence were retrieved from study databases and pharmacovigilance reports. Our analysis includes 1717 patients from 12 prospective phase II and III trials, including newly diagnosed NHL. We observed 53 VTEs (any grade) in 46 patients, with 20 severe VTEs in 17 patients. The cumulative incidences for „all-grade“ or grade ≥3 VTEs were 2.9 % (95 % CI: 2.1–3.8) and 1.1 % (95 % CI: 0.6–1.6), respectively. KS categories were positively associated with the risk of VTE of any grade, and with severe events (i. e. grade ≥3; Gray’s test p-values = 0.048 and 0.012, respectively). Among NHL patients, those with diffuse large B-cell lymphoma (DLBCL) showed a greater risk of (any grade) VTE (HR: 3.42, 95 % CI: 1.32–8.84, p-value = 0.011). Our study indicates that 1) VTE is a relevant complication for NHL patients, 2) KS is predictive of VTE events and 3) DLBCL histotype is an independent risk factor for VTE incidence, for which preventative interventions could be considered.... R. M. Santi (1), M. Ceccarelli (2), E. Bernocco (1), C. Monagheddu (2), A. Evangelista (2), F. Valeri (1), F. Monaco (1), U. Vitolo (3), S. Cortelazzo (4), M. G. Cabras (5), M. Spina (6), L. Baldini (7), C. Boccomini (3), A. Chiappella (3), A. Bari (8), S. Luminari (8), C. Visco (9), M. Calabrese (10), G. Limberti (1), A. Levis (10), L. Contino (1), G. Ciccone (2), M. Ladetto (1) 27459 2017-04-27 09:07:01 Ahead of print: Endogenous developmental endothelial locus-1 limits ischaemia-related angiogenesis... We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of β2-integrin–dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1–deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1–proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularization in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted β2-integrin–dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the β2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1–dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.... A. Klotzsche – von Ameln (1), S. Cremer (2), J. Hoffmann (2), P. Schuster (3), S. Khedr (4), I. Korovina (1), M. Troullinaki (1), A. Neuwirth (1), D. Sprott (1), A. Chatzigeorgiou (1), M. Economopoulou (5, 6), A. Orlandi (3), A. Hain (3), A. M. Zeiher (2, 7), A. Deussen (4), G. Hajishengallis (8), S. Dimmeler (1, 7), T. Chavakis (1, 6), E. Chavakis (2, 3, 7) 27458 2017-04-27 09:06:00 Ahead of print: NOACs for treatment of venous thromboembolism in clinical practice Randomised controlled trials have provided important information on the efficacy and safety of the non-vitamin K antagonist oral anticoagulants (NOACs) for treatment of venous thromboembolism (VTE), leading to registration and increasing use in clinical practice. Many questions remain to be answered, and observational studies are often more suitable for answering ”real-world” questions than randomised controlled trials. Patient satisfaction, quality of life, and adherence and persistence in clinical practice with the drug regimen can only be assessed with an open-label design. Evaluation of risk for long-term sequelae of the disease requires much longer follow-up than is possible in registration trials. Treatment patterns and utilisation of health care resources can be assessed from observations in the clinical practice setting. We will review published as well as currently active observational studies with NOACs in VTE, with or without a comparator anticoagulant. These studies are based on cohorts of different sizes, registries, or administrative health care databases. We will also discuss some limitations in analysis and interpretation of observational studies.... S. Schulman (1), D. Singer (2), W. Ageno (3), I. B. Casella (4), M. Desch (5), S. Z. Goldhaber (6) 27454 2017-04-20 09:44:59 Ahead of print: Microparticles during long-term follow-up after acute myocardial infarction Microparticles (MPs) are formed from platelets (PMPs), endothelial cells (EMPs) and monocytes (MMPs), and in acute myocardial infarction (MI), there is an increase of MPs in the culprit artery. In this study MPs were evaluated in whole blood in 105 patients with MI at five time-points during a two-year follow-up (FU). Patients with non-ST-elevated MI had higher concentrations of CD41+MPs compared to ST-elevated MI patients (p=0.024). The concentrations of PMPs in whole blood increased during the time period (p<0.001), but no significant change over time was found for EMPs and MMPs. CD62P+MP counts were higher in MI patients with diabetes (p=0.020), and patients with hypertension had increased levels of CD14+MPs (p=0.004). The amount of CD62P+TF+MPs increased significantly during FU (p<0.001). Patients with atherosclerosis in three arterial beds, i. e. coronary, carotid and peripheral arteries, had lower concentrations of CD62P+TF+MPs (p=0.035) and CD144+TF+MPs (p=0.004) compared to patients with atherosclerosis in one or two arterial beds. Higher concentrations of CD62P+MPs early after MI were associated with an increased risk of cardiovascular events during FU, hazard ratio 3.32 (95 %CI1.20–9.31). Only small variations in PMP, EMP and MMP concentrations were found during long-term FU after MI and their levels seem to reflect the underlying cardiovascular disease rather than the acute MI. PMPs expressing P-selectin might be a promising biomarker for predicting future cardiovascular events, but further studies are needed to confirm these results.... C. Christersson (1), Å. Thulin (2), A. Siegbahn (2) 27453 2017-04-20 09:44:11 Ahead of print: Dose requirements for idarucizumab reversal of dabigatran in a lethal porcine trauma... Idarucizumab is licensed for emergency reversal of dabigatran. A single 5 g dose is usually sufficient, but higher doses may sometimes be required and optimum dosing has not been defined. It was the aim of this study to investigate the effect of idarucizumab, given once or as a split dose, after double trauma in pigs anticoagulated with dabigatran. Dabigatran etexilate (30 mg/kg bid) was given to 18 male pigs orally for 3 days. On day 4, animals were randomised 1:1:1 to receive idarucizumab 60+0, 60+60 or 120+0 mg/kg. Doses were administered 15 and 75 minutes after initial liver trauma. At 60 minutes, a second liver injury was undertaken. Animals were monitored for 5 hours after initial trauma or until death. Blood loss during the first hour was 990 ± 109 ml, 988 ± 84 ml and 964 ± 75 ml in the 60+0, 60+60 and 120+0 groups, respectively. In the 120+0 and 60+60 groups, total blood loss was 1659 ± 346 and 1426 ± 106 ml, respectively, and survival at 5 hours was 100 %. However, in the 60+0 group, total blood loss was 3561 ± 770 ml and survival was 50 %. Analysis of dabigatran plasma concentrations showed that equimolar concentrations of idarucizumab are necessary to bind all dabigatran and achieve sufficient thrombin generation. At sufficient doses, idarucizumab rapidly reduced blood loss and improved survival in this lethal porcine model of double trauma with dabigatran anticoagulation. In clinical practice, should bleeding continue after initial treatment with the approved 5 g dose of idarucizumab, a second dose may potentially be effective to control bleeding caused by redistribution of unbound dabigatran.... M. Honickel (1), H. M. Spronk (2), R. Rossaint (1), C. Stoppe (1), J. van Ryn (3), H. ten Cate (2), O. Grottke (1) 27452 2017-04-20 09:43:18 Ahead of print: Plasma microRNAs characterising patients with immune thrombocytopenic purpura Altered microRNA (miRNA) expression has been reported in patients with immune thrombocytopenic purpura (ITP). However, the detailed expression profiling of cell-free circulating miRNAs in ITP patients has not been fully investigated. In this study, we aimed to examine plasma miRNAs in ITP patients and evaluate their diagnostic values. Plasma samples from 74 ITP patients and 58 healthy controls were obtained and allocated into discovery, validation, and therapy-response sets. Initial screen with a miRNA microarray assay identified 23 miRNAs with different levels between ITP patients and healthy controls (>1.5-fold changes; p<0.01). Subsequent quantitative real-time PCR confirmed eight up-regulated miRNAs (miR-320c, miR-642b-3p, miR-1275, miR-3141, miR-4270, miR-4499, miR-4739 and miR-6126) and three down-regulated miRNAs (miR-144–3p, miR-1281 and miR-3162–3p) in ITP patients. The levels of these circulating miRNAs varied, depending on ITP subtypes, i.e. newly-diagnosed, persistent and chronic ITP, and between treatment responders and non-responders. In receiver operator characteristic analysis, 10 miRNAs had positive diagnostic values (p<0.05) when tested individually. The diagnostic value improved when the miRNAs were analysed as a panel or together with the analysis of anti-platelet autoantibodies. Plasma miR-3162–3p levels were also found to positively correlate with platelet counts in ITP patients (r=0.338, p=0.01). Our results indicate that plasma miRNA profiles are altered in ITP patients and that the differentially expressed miRNAs may be used as biomarkers to improve the diagnosis of ITP.... B. Zuo (1), J. Zhai (1), L. You (2), Y. Zhao (1), J. Yang (1), Z. Weng (1), l. Dai (1), Q. Wu (1), C. Ruan (1), Y. He (1) 27451 2017-04-20 09:42:19 Ahead of print: Gly74Ser mutation in protein C causes thrombosis due to a defect in protein... Protein C is a vitamin K–dependent serine protease zymogen in plasma which upon activation by thrombin in complex with thrombomodulin (TM) down-regulates the clotting cascade by a feedback loop inhibition mechanism. Activated protein C (APC) exerts its anticoagulant function through protein S-dependent degradation of factors Va and VIIIa. We recently identified a venous thrombosis patient whose plasma level of protein C antigen is normal, but its anticoagulant activity is only 34 % of the normal level. Genetic analysis revealed that the proband and her younger brother carry a novel heterozygous mutation c.346G>A, p.Gly74Ser (G74S) in PROC. Thrombin generation assay indicated that the TM-dependent anticoagulant activity of the proband’s plasma has been significantly impaired. We expressed protein C-G74S in mammalian cells and characterised its properties in established coagulation assays. We demonstrate that the protein C variant can be normally activated by the thrombin-TM complex and the resulting APC mutant also exhibits normal amidolytic and proteolytic activities toward both FVa and FVIIIa. However, it was discovered the protein S-dependent catalytic activity of APC variant toward both procoagulant cofactors has been significantly impaired. Protein S concentration-dependence of FVa degradation revealed that the capacity of APC variant to interact with the cofactor has been markedly impaired. The same results were obtained for inactivation of FVa-Leiden suggesting that the protein S-dependent activity of APC variant toward cleavage of Arg-306 site has been adversely affected. These results provide insight into the mechanism through which G74S substitution in APC causes thrombosis in the proband carrying this mutation.... C. Chen (1), L. Yang (2), B. O. Villoutreix (3), X. Wang (4), Q. Ding (4), A. R. Rezaie (2) 27435 2017-04-13 08:23:09 Ahead of print: Predictors of oral cavity bleeding and clinical outcome after dental procedures in... Patients on vitamin K antagonists (VKA) often undergo invasive dental procedures. International guidelines consider all dental procedures as low-risk procedures, while bleeding risk may differ between standard low-risk (e. g. extraction 1–3 elements) and extensive high-risk (e. g. extraction of >3 elements) procedures. Therefore current guidelines may need refinement. In this cohort study, we identified predictors of oral cavity bleeding (OCB) and evaluated clinical outcome after low-risk and high-risk dental procedures in patients on VKA. Perioperative management strategy, procedure risk, and 30-day outcomes were assessed for each procedure. We identified 1845 patients undergoing 2004 low-risk and 325 high-risk procedures between 2013 and 2015. OCB occurred after 67/2004 (3.3 %) low-risk and 21/325 (6.5 %) high-risk procedures (p=0.006). In low-risk procedures, VKA continuation with tranexamic acid mouthwash was associated with a lower OCB risk compared to continuation without mouthwash [OR=0.41, 95 %CI 0.23–0.73] or interruption with bridging [OR=0.49, 95 %CI 0.24–1.00], and a similar risk as interruption without bridging [OR=1.44, 95 %CI 0.62–3.64]. In high-risk procedures, VKA continuation was associated with an increased OCB risk compared to interruption [OR=3.08, 95 %CI 1.05–9.04]. Multivariate analyses revealed bridging, antiplatelet therapy, and a supratherapeutic or unobjectified INR before the procedure as strongest predictors of OCB. Non-oral cavity bleeding (NOCB) and thromboembolic event (TE) rates were 2.1 % and 0.2 %. Bridging therapy was associated with a two-fold increased risk of NOCB [OR=1.93, 95 %CI 1.03–3.60], but not with lower TE rates. In conclusion, predictors of OCB were mostly related to perioperative management and differed between low-risk and high-risk procedures. Perioperative management should be differentiated accordingly.... J. S. Biedermann (1, 2), W. M. H. Rademacher (3), H. C. A. M. Hazendonk (1), D. E. van Diermen (3), F. W. G. Leebeek (1), F. Rozema (3), M. J. H. A. Kruip (1, 2) 27434 2017-04-13 08:22:07 Ahead of print: Open Access: Factor VIIIa-mimetic cofactor activity of a bispecific antibody to... Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab–antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (KD = 1.58, 1.52, 1.85, and 0.978 µM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens’ epidermal growth factor (EGF)-like domains. We then performed KD-based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX–emicizumab–FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab’s cofactor activity. The simulation also predicted that at 10.0–100 µg/ml of emicizumab–levels shown in a previous study to be clinically effective–the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The KD-based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab’s cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.... T. Kitazawa (1), K. Esaki (2), T. Tachibana (1), S. Ishii (2), T. Soeda (2), A. Muto (2), Y. Kawabe (2), T. Igawa (2), H. Tsunoda (1), K. Nogami (3), M. Shima (3), K. Hattori (1) 27433 2017-04-13 08:21:13 Ahead of print: A long-acting PAI-1 inhibitor reduce thrombus formation Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in the fibrinolytic system. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we tried to fuse PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which combined fused HSA and a H37R mutation in PAItrap, gave a six-fold improvement IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis mediated by PAI-1.... S. Peng (1, 2), G. Xue (1, 3), L. Gong (1), C. Fang (4), J. Chen (5), C. Yuan (1), Z. Chen (1), L. Yao (5), B. Furie (4), M. Huang (1, 6) 27432 2017-04-13 08:20:20 Ahead of print: Open Access: The management of acute venous thromboembolism in clinical practice Venous thromboembolism (VTE) is a significant cause of morbidity and mortality in Europe. Data from real-world registries are necessary, as clinical trials do not represent the full spectrum of VTE patients seen in clinical practice. We aimed to document the epidemiology, management and outcomes of VTE using data from a large, observational database. PREFER in VTE was an international, non-interventional disease registry conducted between January 2013 and July 2015 in primary and secondary care across seven European countries. Consecutive patients with acute VTE were documented and followed up over 12 months. PREFER in VTE included 3,455 patients with a mean age of 60.8 ± 17.0 years. Overall, 53.0 % were male. The majority of patients were assessed in the hospital setting as inpatients or outpatients (78.5 %). The diagnosis was deep-vein thrombosis (DVT) in 59.5 % and pulmonary embolism (PE) in 40.5 %. The most common comorbidities were the various types of cardiovascular disease (excluding hypertension; 45.5 %), hypertension (42.3 %) and dyslipidaemia (21.1 %). Following the index VTE, a large proportion of patients received initial therapy with heparin (73.2 %), almost half received a vitamin K antagonist (48.7 %) and nearly a quarter received a DOAC (24.5 %). Almost a quarter of all presentations were for recurrent VTE, with >80 % of previous episodes having occurred more than 12 months prior to baseline. In conclusion, PREFER in VTE has provided contemporary insights into VTE patients and their real-world management, including their baseline characteristics, risk factors, disease history, symptoms and signs, initial therapy and outcomes.... A. T. Cohen (1), A. K. Gitt (2), R. Bauersachs (3, 4), E.-M. Fronk (5), P. Laeis (5), P. Mismetti (6), M. Monreal (7), S. N. Willich (8), P. Bramlage (9), G. Agnelli (10), on behalf of the PREFER in VTE Scientific Steering Committee and the PREFER in VTE Investigators 27431 2017-04-13 08:17:59 Ahead of print: Open Access: Glucagon-like peptide 1-related peptides increase nitric oxide effects... Glucagon-like peptide 1 (GLP-1) is object of intensive investigation for not only its metabolic effects but also the protective vascular actions. Since platelets exert a primary role in the pathogenesis of atherosclerosis, inflammation and vascular complications, we investigated whether GLP-1 directly influences platelet reactivity. For this purpose, in platelets from 72 healthy volunteers we evaluated GLP-1 receptor (GLP-1R) expression and the effects of a 15-minute incubation with the native form GLP-1(7–36), the N-terminally truncated form GLP-1(9–36) and the GLP-1 analogue Liraglutide (100 nmol/l) on: i) aggregation induced by collagen or arachidonic acid (AA); ii) platelet function under shear stress; iii) cGMP and cAMP synthesis and cGMP-dependent protein kinase (PKG)-induced Vasodilator-Stimulated-Phosphoprotein (VASP) phosphorylation; iv) activation of the signalling molecules Phosphatidylinositol 3-Kinase (PI3-K)/Akt and Mitogen Activated Protein Kinase (MAPK)/ERK-1/2; and v) oxidative stress. Experiments were repeated in the presence of the nitric oxide donor Na–nitroprusside. We found that platelets constitutively express GLP-1R and that, independently of GLP-1R, GLP-1(7–36), GLP-1(9–36) and Liraglutide exert platelet inhibitory effects as shown by: a) increased NO-antiaggregating effects, b) increased the activation of the cGMP/PKG/VASP pathway, c) reduced the activation of PI3-K/Akt and MAPK/ERK-2 pathways, d) reduced the AA-induced oxidative stress. When the experiments were repeated in the presence of the antagonist of GLP-1R Exendin(9–39), the platelet inhibitory effects were maintained, thus indicating a mechanism independent of GLP-1R. In conclusion, GLP-1(7–36), its degradation product GLP-1(9–36) and Liraglutide exert similar inhibitory effects on platelet activation, suggesting a potential protective effect on the cardiovascular system.... C. Barale (1, 2), S. Buracco (2), F. Cavalot (1, 2), C. Frascaroli (1, 2), A. Guerrasio (1, 2), I. Russo (1, 2) 27430 2017-04-13 08:17:00 Ahead of print: On-clopidogrel platelet reactivity as predictor for long-term clinical outcome in... It is unknown whether the known association of high on-treatment platelet reactivity (HTPR) with worse clinical outcome in patients on clopidogrel following coronary stent implantation persists after planned discontinuation of clopidogrel. This study investigated the association of HTPR with major ischaemic events after planned discontinuation of clopidogrel. Consecutive patients undergoing elective coronary stent implantation after loading with clopidogrel 600 mg were followed for up to seven years (n=765). Platelet reactivity was tested on day 1 after coronary intervention. Clopidogrel was continued for six months after implantation of drug-eluting stents and for one month if only bare-metal stents were used. The combined primary endpoint was death of any cause or non-fatal myocardial infarction (MACE). HTPR was found in 217 of 765 patients (28 %). During a median follow-up of 5.7 years, the primary endpoint occurred in 145 subjects after planned discontinuation of clopidogrel. Patients with HTPR showed a higher incidence of MACE after discontinuation of clopidogrel. There was a significant interaction of HTPR and time following discontinuation of clopidogrel beyond one year (p for interaction 0.08). Landmark analyses confirmed that the association of HTPR and MACE was only significant within the first year (HR: 2.93, 95 %-CI 1.13–7.60, p=0.03), but not beyond the first year following discontinuation of clopidogrel (HR: 1.19, 95 %-CI 0.82–1.72, p=0.37). In conclusion, patients with HTPR persist to be at high risk for death or myocardial infarction even following planned discontinuation of clopidogrel. However, this association was only significant for the first year following discontinuation of clopidogrel.... T. Bömicke (1), C. M. Valina (1), C. Stratz (1), M. Amann (1), F.-J. Neumann (1), W. Hochholzer (1) 27393 2017-04-06 08:55:48 Ahead of print: von Willebrand factor deficiency leads to impaired blood flow recovery after... Neovascularisation, i. e. arteriogenesis and angiogenesis, is an inflammatory process. Therefore attraction and extravasation of leukocytes is essential for effective blood flow recovery after ischaemia. Previous studies have shown that von Willebrand factor (VWF) is a negative regulator of angiogenesis. However, it has also been shown that VWF facilitates leukocyte attraction and extravasation. We aimed to investigate the role of VWF in arteriogenesis and angiogenesis during post-ischaemic neovascularisation. Wild-type (WT) and VWF deficient (VWF-/-) C57BL/6 mice were subjected to hindlimb ischaemia via double ligation of the left femoral artery, and blood flow recovery was followed over time, using Laser Doppler Perfusion Imaging. Blood flow recovery was impaired in VWF-/- mice. After 10 days, VWF-/- mice showed a 43 ± 5 % recovery versus 68 ± 5 % in WT. Immunohistochemistry revealed that both arteriogenesis in the adductor muscles and angiogenesis in the gastrocnemius muscles were reduced in VWF-/- mice. Furthermore, leukocyte infiltration in the affected adductor muscles was reduced in VWF-/- mice. Residual paw perfusion directly after artery ligation was also reduced in VWF-/- mice, indicating a decrease in pre-existing collateral arteriole density. When we quantified collateral arterioles, we observed a 31 % decrease in the average number of collateral arterioles in the pia mater compared to WT mice (57 ± 3 in WT vs 40 ± 4 pial collaterals in VWF-/-). We conclude that VWF facilitates blood flow recovery in mice. VWF deficiency hampers both arteriogenesis and angiogenesis in a hindlimb ischaemia model. This is associated with impaired leukocytes recruitment and decreased pre-existing collateral density in the absence of VWF.... M. R. de Vries (1, 2), E. A. B. Peters (1, 2), P. H. A. Quax (1, 2), A. Y. Nossent (1, 2) 27392 2017-04-06 08:54:39 Ahead of print: Fibrin clot characteristics in acute ischaemic stroke patients treated with... Fibrin clot properties in acute ischaemic stroke (AIS) are unfavourably altered, including faster formation of denser and poorly lysable fibre networks. We investigated clot properties in AIS patients treated with recombinant tissue plasminogen activator (rtPA) and their impact on clinical outcome. In 74 consecutive AIS patients eligible for rtPA treatment, we assessed ex vivo plasma fibrin clot formation, permeability (Ks), and rtPA-induced lysis, along with peak thrombin generation, fibrinolysis proteins and inhibitors at three time points – on admission, after 24 hours and 3 months since stroke. Clinical outcome was assessed using the NIHSS and mRS scores. Compared with the pretreatment values, fibrin networks assessed 24 hours since thrombolysis were formed more slowly (+20.5 % lag phase on turbidimetry), were less compact (+36.9 % Ks), composed of thinner fibres (-10.6 % lower maximum absorbancy [ΔAb]), which were lysed more rapidly (-20.8 % clot lysis time [CLT] and +7.1 % the rate of rtPA-induced D-dimer release from clots [D-Drate]). Thrombin generation and fibrinolysis proteins remained elevated. Lower ΔAb (<0.86 at 405 nm), shorter CLT (<105 min), and higher D-Drate (>0.072 mg/l/min) assessed at baseline predicted good outcome (mRS 0–2) at 3 months after adjustment for age and fibrinogen. Logistic regression adjusted for potential confounders showed that good outcome at 3 months was predicted by pretreatment D-Drate, while pretreatment CLT predicted excellent outcome (mRS of 0–1). In conclusion, formation of denser fibrin clots displaying impaired lysability and pattern of their changes induced by thrombolysis may affect clinical outcome in AIS patients.... J. P. Bembenek (1), M. Niewada (1), J. Siudut (2, 3), K. Plens (4), A. Członkowska (1), A. Undas (2, 3) 27391 2017-04-06 08:53:42 Ahead of print: Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial... The system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.... L. B. Mann Dosier (1), V. J. Premkumar (2), H. Zhu (2), I. Akosman (2), M. F. Wempe (3), T. J. McMahon (2, 4) 27390 2017-04-06 08:52:05 Ahead of print: Characteristics of dyspnoea and associated clinical outcomes in the CHAMPION... Dyspnoea may be induced by some reversibly-binding P2Y12 inhibitors, including cangrelor and ticagrelor. Dyspnoea was not associated with any compromise to the efficacy of ticagrelor in the PLATO study. The CHAMPION PHOENIX study (NCT01156571) compared initial treatment with cangrelor versus initial treatment with clopidogrel in patients undergoing PCI. We investigated the incidence, characteristics, and associated clinical outcomes in patients with dyspnoea in CHAMPION PHOENIX. Adverse events (AEs) of dyspnoea to 48 hours were recorded in patients randomised to cangrelor or clopidogrel in CHAMPION PHOENIX. The composite primary endpoint of death, myocardial infarction, ischaemia-driven revascularisation (IDR), or stent thrombosis (ST) as well its individual components were assessed in patients who did or did not report dyspnoea. A total of 68 (1.2 %) cangrelor-treated patients and 18 (0.3 %) clopidogrel-treated patients reported dyspnoea (p W. A. Parker (1), D. L. Bhatt (2), J. Prats (3), J. R. S. Day (4), P. G. Steg (5, 6), G. W. Stone (7), C. W. Hamm (8), K. W. Mahaffey (9), M. J. Price (10), C. M. Gibson (11), H. D. White (12), R. F. Storey (1), on behalf of the CHAMPION PHOENIX Investigators 27389 2017-04-06 08:49:07 Ahead of print: Mechanism of plasmin generation by S100A10 Plasminogen (Pg) is cleaved to form plasmin by the action of specific plasminogen activators such as the tissue plasminogen activator (tPA). Although the interaction of tPA and Pg with the surface of the fibrin clot has been well characterised, their interaction with cell surface Pg receptors is poorly understood. S100A10 is a cell surface Pg receptor that plays a key role in cellular plasmin generation. In the present report, we have utilised domain-switched/deleted variants of tPA, truncated plasminogen variants and S100A10 site-directed mutant proteins to define the regions responsible for S100A10-dependent plasmin generation. In contrast to the established role of the finger domain of tPA in fibrin-stimulated plasmin generation, we show that the kringle-2 domain of tPA plays a key role in S100A10-dependent plasmin generation. The kringle-1 domain of plasminogen, indispensable for fibrin-binding, is also critical for S100A10-dependent plasmin generation. S100A10 retains activity after substitution or deletion of the carboxyl-terminal lysine suggesting that internal lysine residues contribute to its plasmin generating activity. These studies define a new paradigm for plasminogen activation by the plasminogen receptor, S100A10.... V. A. Miller (1), P. A. Madureira (2), A. A. Kamaludin (3), J. Komar (3), V. Sharma (4), G. Sahni (4), C. Thelwell (5), C. Longstaff (5), D. M. Waisman (1, 3) 27388 2017-04-06 08:45:51 Ahead of print: Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT... The combined action of collagen and thrombin induces the formation of COAT platelets, which are characterised by a coat of procoagulant and adhesive molecules on their surface. Although recent work has started to highlight their clinical relevance, the exact mechanisms regulating the formation of procoagulant COAT platelets remain unclear. Therefore, we employed flow cytometry in order to visualise in real time surface and intracellular events following simultaneous platelet activation with convulxin and thrombin. After a rapid initial response pattern characterised by the homogenous activation of the fibrinogen receptor glycoprotein IIb/IIIa in all platelets, starting with a delay of about 2 minutes an increasing fraction transforms to procoagulant COAT platelets. Their surface is characterised by progressive loss of PAC-1 binding, expression of negative phospholipids and retention of α-granule von Willebrand factor. Intracellular events in procoagulant COAT platelets are a marked increase of free calcium into the low micromolar range, concomitantly with early depolarisation of the mitochondrial membrane and activation of caspase-3, while non-COAT platelets keep the intracellular free calcium in the nanomolar range and maintain an intact mitochondrial membrane. We show for the first time that the flow-cytometrically distinct fractions of COAT and non-COAT platelets differentially phosphorylate two signalling proteins, PKCα and p38MAPK, which may be involved in the regulation of the different calcium fluxes observed in COAT versus non-COAT platelets. This study demonstrates the utility of concomitant cellular and signalling evaluation using flow cytometry in order to further dissect the mechanisms underlying the dichotomous platelet response observed after collagen/thrombin stimulation.... L. Alberio (1, 2), C. Ravanat (1), B. Hechler (1), P. H. Mangin (1), F. Lanza (1), C. Gachet (1) 27387 2017-04-06 08:44:32 Ahead of print: Open Access: Impact of fibrinogen carbamylation on fibrin clot formation and... Carbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.... V. Binder (1), B. Bergum (1), S. Jaisson (2), P. Gillery (2), C. Scavenius (3), E. Spriet (4), A. K. Nyhaug (4), H. M. Roberts (5), I. L. C. Chapple (5), A. Hellvard (6), N. Delaleu (1, 7), P. Mydel (1, 8) 27386 2017-04-06 08:36:41 Erratum to Parasrampuria et al. "Pharmacokinetics, safety, and tolerability of edoxaban in end-stage... 27379 2017-04-03 09:40:47 Ahead of print: Tailoring treatment of haemophilia B: accounting for the distribution and clearance... The prophylactic administration of factor IX (FIX) is considered the most effective treatment for haemophilia B. The inter-individual variability and complexity of the pharmacokinetics (PK) of FIX, and the rarity of the disease have hampered identification of an optimal treatment regimens. The recent introduction of extended half-life recombinant FIX molecules (EHL-rFIX), has prompted a thorough reassessment of the clinical efficacy, PK and pharmacodynamics of plasma-derived and recombinant FIX. First, using longer sampling times and multi-compartmental PK models has led to more precise (and favourable) PK for FIX than was appreciated in the past. Second, investigating the distribution of FIX in the body beyond the vascular space (which is implied by its complex kinetics) has opened a new research field on the role for extravascular FIX. Third, measuring plasma levels of EHL-rFIX has shown that different aPTT reagents have different accuracy in measuring different FIX molecules. How will this new knowledge reflect on clinical practice? Clinical decision making in haemophilia B requires some caution and expertise. First, comparisons between different FIX molecules must be assessed taking into consideration the comparability of the populations studied and the PK models used. Second, individual PK estimates must rely on multi-compartmental models, and would benefit from adopting a population PK approach. Optimal sampling times need to be adapted to the prolonged half-life of the new EHL FIX products. Finally, costs considerations may apply, which is beyond the scope of this manuscript but might be deeply connected with the PK considerations discussed in this communication.... A. Iorio (1), K. Fischer (2), V. Blanchette (3), S. Rangarajan (4), G. Young (5), M. Morfini (6), for the Pharmacokinetic (PK) Expert Working Group of the International Prophylaxis Study Group (the IPSG) 27373 2017-03-30 09:41:29 Ahead of print: Mean platelet volume is associated with lower risk of overall and non-vascular... Larger mean platelet volume (MPV) has been associated with adverse health outcomes in high-risk populations or patients with cardiovascular disease (CVD). We tested the association of MPV with mortality in a prospective cohort study including 17,402 subjects randomly recruited from an adult general population within the Moli-sani study (2005–2010). Two distinct subgroups (with or without CVD at baseline) were subsequently analysed. Hazard ratios (HR) were calculated using multivariable Cox-proportional hazard models. Over a median follow up of eight years (137,547 person-years), 925 all-cause deaths occurred (330 vascular, 351 cancer and 244 other deaths). In a multivariable model, the highest MPV quintile (mean MPV=10.0 fL), as compared to the lowest one, was associated with reduced risk of overall mortality (HR=0.79; 95 % confidence interval 0.64–0.98), cancer death (HR=0.70; 0.49–1.00) and death from other non- vascular/non cancer causes (HR=0.55; 0.36–0.84) but not with vascular mortality. The inverse association with overall death appeared even stronger in the subgroup without CVD at baseline (HR=0.64; 0.50–0.81). In contrast, within 920 subjects reporting a previous CVD event, larger MPV was associated with higher risk of total mortality (HR=1.69; 1.05–2.72; p for interaction=0.048) and with a trend of risk for other cause-specific deaths. In conclusion, larger MPV is associated with lower risk of overall and non-vascular death in subjects apparently free from CVD, but appears to be a predictive marker of death in patients with CVD history. The latter is a likely effect modifier of the association between MPV and death.... M. Bonaccio (1), A. Di Castelnuovo (1), S. Costanzo (1), A. de Curtis (1), M. Persichillo (1), C. Cerletti (1), M. B. Donati (1), G. de Gaetano (1), L. Iacoviello (1, 2), on behalf of the MOLI-SANI study Investigators 27372 2017-03-30 09:36:03 Ahead of print: High on treatment platelet reactivity as a risk factor for adverse left ventricular... G. M. Podda (1), M. Cattaneo (1) 27371 2017-03-30 09:33:04