Thrombosis and Haemostasis Thrombosis and Haemostasis th de-de Sat, 19 Aug 17 07:21:33 +0200 Ahead of print: Clinical impact of major bleeding in patients with venous thromboembolism treated... Factor Xa (fXa)-inhibitors are as effective and safer than vitamin-K–antagonists (VKA) in the treatment of venous thromboembolism (VTE). We previously classified the severity of clinical presentation and course of all major bleeding events from the EINSTEIN, AMPLIFY and HOKUSAI-VTE trials separately. The current aim was to combine these findings in order to increase precision, assess a class effect and analyse presentation and course for different types of bleeding, i. e. intracranial, gastro-intestinal, and other. We classified the clinical presentation and course of all major bleeding events using pre-defined criteria. Both classifications comprised four categories; one being the mildest, and four the most severe. Odds ratios (OR) were calculated for all events classified as category three or four between fXa-inhibitors and VKA recipients. Also, ORs were computed for different types of bleeding. Major bleeding occurred in 111 fXa-inhibitor recipients and in 187 LMWH/VKA recipients. The clinical presentation was classified as category three or four in 35 % and 48 % of the major bleeds in fXa inhibitor and VKA recipients, respectively (OR 0.59, 95 % CI 0.36–0.97). For intracranial, gastro-intestinal and other bleeding a trend towards a less severe presentation was observed for patients treated with fXa inhibitors. Clinical course was classified as severe in 22 % of the fXa inhibitor and 25 % of the VKA associated bleeds (OR 0.83, 95 % CI 0.47–1.46). In conclusion, FXa inhibitor associated major bleeding events had a significantly less severe presentation and a similar course compared to VKA. This finding was consistent for different types of bleeding.... S. M. Bleker (1),  M. P. A. Brekelmans (1), E. S. Eerenberg (1), A. T. Cohen (3), S. Middeldorp (1), G. Raskob (4), H. R. Büller (1) 27831 2017-08-17 14:10:59 Ahead of print: Hypofibrinogenaemia associated with novel Aα126Val→Asp mutation in the fibrinogen... S. O. Brennan (1, 2), S. Brennan (3), A. D. Laurie (2) 27830 2017-08-17 14:06:13 Ahead of print: Coagulation factor XII regulates inflammatory responses in human lungs Increased procoagulant activity in the alveolar compartment and uncontrolled inflammation are hallmarks of the acute respiratory distress syndrome (ARDS). Here, we investigated whether the contact phase system of coagulation is activated and may regulate inflammatory responses in human lungs. Components of the contact phase system were characterized in bronchoalveolar lavage fluids (BALF) from 54 ARDS patients and 43 controls, and their impact on cytokine/chemokine expression in human precision cut lung slices (PCLS) was assessed by a PCR array. Activation of the contact system, associated with high levels of coagulation factor XIIa (Hageman factor, FXIIa), plasma kallikrein and bradykinin, occurred rapidly in ARDS lungs after the onset of the disease and virtually normalized within one week from time of diagnosis. FXII levels in BALF were higher in ARDS non-survivors than survivors and were positively correlated with tumor necrosis factor (TNF)-α concentration. FXII induced the production and release of interleukin (IL)-8, IL-1β, IL-6, leukemia inhibitory factor (LIF), CXCL5 and TNF-α in human PCLS in a kallikrein-kinin-independent manner. In conclusion, accumulation of FXII in ARDS lungs may contribute to the release of pro-inflammatory mediators and is associated with clinical outcome. FXII inhibition may thus offer a novel and promising therapeutic approach to antagonize overwhelming inflammatory responses in ARDS lungs without interfering with vital haemostasis.... R. Hess (1), L. Wujak (1), C. Hesse (2), K. Sewald (2), D. Jonigk (3), G. Warnecke (3), H. Fieguth (4), S. de Maat (5), C. Maas (5), F. Bonella (6), K. T. Preissner (1), B. Weiss (7), L. Schaefer (8), W. M. Kuebler (9, 10), P. Markart (11), M. Wygrecka (1) 27829 2017-08-17 13:58:51 Ahead of print: Paracelsus, poison, and colistin H. Herwald (1) 27820 2017-08-11 11:41:44 Ahead of print: Long-term bleeding risk prediction in ‘real world’ patients with atrial... Risk scores in patients with atrial fibrillation (AF) based on clinical factors alone generally have only modest predictive value for predicting high risk patients that sustain events. Biomarkers might be an attractive prognostic tool to improve bleeding risk prediction. The new ABC-Bleeding score performed better than HAS-BLED score in a clinical trial cohort but has not been externally validated. It was the aim of this study to compare the predictive performance of the ABC-Bleeding score compared to HAS-BLED score in an independent “real-world” anticoagulated AF patients with long-term follow-up. We enrolled 1,120 patients stable on vitamin K antagonist treatment. The HAS-BLED and ABC-Bleeding scores were quantified. Predictive values were compared by c-indexes, IDI, NRI, as well as decision curve analysis (DCA). Median HAS-BLED score was 2 (IQR 2–3) and median ABC-Bleeding was 16.5 (IQR 14.3–18.6). After 6.5 years of follow-up, 207 (2.84 %/year) patients had major bleeding events, of which 65 (0.89 %/year) had intracranial haemorrhage (ICH) and 85 (1.17 %/year) had gastrointestinal bleeding events (GIB). The c-index of HAS-BLED was significantly higher than ABC-Bleeding for major bleeding (0.583 vs 0.518; p=0.025), GIB (0.596 vs 0.519; p=0.017) and for the composite of ICH-GIB (0.593 vs 0.527; p=0.030). NRI showed a significant negative reclassification for major bleeding and for the composite of ICH-GIB with the ABC-Bleeding score compared to HAS-BLED. Using DCAs, the use of HAS-BLED score gave an approximate net benefit of 4 % over the ABC-Bleeding score. In conclusion, in the first “real-world” validation of the ABC-Bleeding score, HAS-BLED performed significantly better than the ABC-Bleeding score in predicting major bleeding, GIB and the composite of GIB and ICH.... M. A. Esteve-Pastor (1, 2), J. M. Rivera-Caravaca (1, 3), V. Roldan (3), V. Vicente (3), M. Valdés (2), F. Marín (2), G. Y. H. Lip (1, 4) 27819 2017-08-11 11:12:33 Ahead of print: Role of replacement therapy in the evaluation of thrombosis occurring in congenital... A. Girolami (1), S. Ferrari (1), E. Cosi (1), B. Girolami (1, 2) 27817 2017-08-10 15:17:19 Ahead of print: Immature platelets as a novel biomarker for adverse cardiovascular events in... 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.... A. Anetsberger (1), M. Blobner (1), B. Haller (2), S. Schmid (1), K. Umgelter (1), T. Hager (1), C. Langgartner (1), E. F. Kochs (1), K.-L. Laugwitz (3), B. Jungwirth (1), I. Bernlochner (3) 27816 2017-08-10 15:16:08 Ahead of print: Endothelial cell-specific overexpression of developmental endothelial locus-1 does... P. Subramanian (1), M. Prucnal (1), B. Gercken (1), M. Economopoulou (2), G. Hajishengallis (3), T. Chavakis (1) 27815 2017-08-10 15:15:24 Ahead of print: Open Access: Colistin dampens fibrinolysis and endothelial activation during... Colistin electrostatically interacts with lipopolysaccharides (LPS). Pre-clinical studies demonstrated beneficial effects of colistin on LPS-induced coagulation and fibrinolysis. The objective of this trial was to investigate the effects of colistin during experimental endotoxaemia. In this randomised, double-blind, placebo-controlled, crossover trial 16 healthy volunteers received a 2 ng/kg LPS bolus after infusion of 2.5 million IU colistin or placebo. Plasma levels of F1+2 prothrombin fragments, thrombin-antithrombin complexes (TAT), von Willebrand factor antigen levels (vWF), E-selectin, plasmin-antiplasmin complexes (PAP), tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) were measured. Infusion of colistin significantly reduced peak concentrations of PAP complexes by 70 %, t-PA antigen levels by 63 % and t-PA activity by 48 %, while PAI-1 levels decreased numerically by 63 %. Two hours after the LPS bolus F1+2 levels and TAT complexes were slightly reduced in the colistin period, but peak concentrations were similar in both periods. Colistin blunted the LPS induced four-fold increase in soluble E-Selectin levels by ~50 % and the two-fold increase in vWF antigen levels by ~70 %. The LPS-scavenging actions of colistin significantly reduce endothelial activation and fibrinolytic response in the human endotoxaemia model, while the activation of the coagulation system remains largely unaffected.... C. Schoergenhofer (1), P. Matzneller (1), M. Mußbacher (2), J. A. Schmid (2), P. Jilma-Stohlawetz (3), M. Zeitlinger (1), B. Jilma (1) 27814 2017-08-10 15:06:33 Ahead of print: Superoxide Dismutase 2 is dispensable for platelet function Increased intracellular reactive oxygen species (ROS) promote platelet activation. The sources of platelet-derived ROS are diverse and whether or not mitochondrial derived ROS, modulates platelet function is incompletely understood. Studies of platelets from patients with sickle cell disease, and diabetes suggest a correlation between mitochondrial ROS and platelet dysfunction. Therefore, we generated mice with a platelet specific knockout of superoxide dismutase 2 (SOD2-KO) to determine if increased mitochondrial ROS increases platelet activation. SOD2-KO platelets demonstrated decreased SOD2 activity and increased mitochondrial ROS, however total platelet ROS was unchanged. Mitochondrial function and content were maintained in non-stimulated platelets. However SOD2-KO platelets demonstrated decreased mitochondrial function following thrombin stimulation. In vitro platelet activation and spreading was normal and in vivo, deletion of SOD2 did not change tail-bleeding or arterial thrombosis indices. In pathophysiological models mediated by platelet-dependent immune mechanisms such as sepsis and autoimmune inflammatory arthritis, SOD2-KO mice were phenotypically identical to wildtype controls. These data demonstrate that increased mitochondrial ROS does not result in platelet dysfunction.... T. P. Fidler (1, 2, 3), J. W. Rowley (2), C. Araujo (2), L. H. Boudreau (4), A. Marti (3), R. Souvenir (3), K. Dale (2), E. Boilard (4), A. S. Weyrich (2), E. D. Abel (2, 3) 27795 2017-08-03 14:07:31 Ahead of print: Latency transition of plasminogen activator inhibitor type 1 is evolutionarily... Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin superfamily and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central β–sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI–1 variants undergo latency transition with a similar rate. Since the functional stability of PAI–1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system.... A. Jendroszek (1), M. S. Sønnichsen (1), A. C. Muñoz (1), K. Leyman (1), A. Christensen (1), S. V. Petersen (2), T. Wang (3), C. Bendixen (1), F. Panitz (1), P. A. Andreasen (1), J. K. Jensen (1) 27794 2017-08-03 14:03:03 Ahead of print: Plasma fibrin clot properties in the G20210A prothrombin mutation carriers following... We sought to investigate whether the G20210A prothrombin mutation modifies plasma fibrin clot properties in patients after venous thromboembolism (VTE) and how rivaroxaban treatment affects these alterations. We studied 34 prothrombin mutation heterozygous carriers and sex- and age-matched 34 non-carriers, all at least three months since the first VTE episode, before and during treatment with rivaroxaban. Clot permeability (Ks) and clot lysis time (CLT) with or without elimination of thrombin activatable fibrinolysis inhibitor (TAFI) were assessed at baseline, 2–6 hours (h) after and 20–25 h after intake of rivaroxaban (20 mg/day). At baseline, the prothrombin mutation group formed denser clots (Ks -12 %, p=0.0006) and had impaired fibrinolysis (CLT +14 %, p=0.004, and CLT-TAFI +13 %, p=0.03) compared with the no mutation group and were similar to those observed in 15 healthy unrelated prothrombin mutation carriers. The G20210A prothrombin mutation was the independent predictor for Ks and CLT before rivaroxaban intake. At 2–6 h after rivaroxaban intake, clot properties improved in both G20210A carriers and non-carriers (Ks +38 %, and +37 %, CLT -25 % and -25 %, CLT-TAFI -20 % and -24 %, respectively, all p<0.001), but those parameters were worse in the prothrombin mutation group (Ks -12.8 %, CLT +17 %, CLT-TAFI +13 %, all p<0.001). Rivaroxaban concentration correlated with fibrin clot properties. After 20–25 h since rivaroxaban intake most clot properties returned to baseline. Rivaroxaban-related differences in clot structure were confirmed by scanning electron microscopy images. In conclusion, rivaroxaban treatment, though improves fibrin clot properties, cannot abolish more prothrombotic fibrin clot phenotype observed in prothrombin mutation carriers following VTE.... A. Janion-Sadowska (1), J. Natorska (2, 3), J. Siudut (2, 3), M. Ząbczyk (2), A. Stanisz (4), A. Undas (2, 3) 27793 2017-08-03 13:57:10 Ahead of print: Comparison of clinical probability-adjusted D-dimer and age-adjusted D-dimer... Two new strategies for interpreting D-dimer results have been proposed: i) using a progressively higher D-dimer threshold with increasing age (age-adjusted strategy) and ii) using a D-dimer threshold in patients with low clinical probability that is twice the threshold used in patients with moderate clinical probability (clinical probability-adjusted strategy). Our objective was to compare the diagnostic accuracy of age-adjusted and clinical probability-adjusted D-dimer interpretation in patients with a low or moderate clinical probability of venous thromboembolism (VTE). We performed a retrospective analysis of clinical data and blood samples from two prospective studies. We compared the negative predictive value (NPV) for VTE, and the proportion of patients with a negative D-dimer result, using two D-dimer interpretation strategies: the age-adjusted strategy, which uses a progressively higher D-dimer threshold with increasing age over 50 years (age in years × 10 µg/L FEU); and the clinical probability-adjusted strategy which uses a D-dimer threshold of 1000 µg/L FEU in patients with low clinical probability and 500 µg/L FEU in patients with moderate clinical probability. A total of 1649 outpatients with low or moderate clinical probability for a first suspected deep vein thrombosis or pulmonary embolism were included. The NPV of both the clinical probability-adjusted strategy (99.7 %) and the age-adjusted strategy (99.6 %) were similar. However, the proportion of patients with a negative result was greater with the clinical probability-adjusted strategy (56.1 % vs, 50.9 %; difference 5.2 %; 95 % CI 3.5 % to 6.8 %). These findings suggest that clinical probability-adjusted D-dimer interpretation is a better way of interpreting D-dimer results compared to age-adjusted interpretation.... S. Takach Lapner (1, 2), J. A. Julian (3), L.-A. Linkins (2, 4), S. M. Bates (2, 4), C. Kearon (2, 4) 27792 2017-08-03 13:52:17 Ahead of print: Downregulation of hypoxia-inducible factor-1α contributes to impaired... Impaired megakaryocyte maturation and exaggerated platelet destruction play a pivotal role in the pathogenesis of immune thrombocytopenia (ITP). Previous studies have shown that HIF-1α promotes the homing and engraftment of haematopoietic stem cells (HSCs), thereby stimulating HSC differentiation. However, whether HIF-1α plays a role in megakaryocytic maturation and platelet destruction in ITP remains elusive. Using enzyme-linked immunosorbent assays (ELISAs), we demonstrated that there were lower HIF-1α levels in the bone marrow (BM) of ITP patients than in that of healthy donors and patients with chemotherapy-related thrombocytopenia. Subjects with lower megakaryocyte (<100/slide) and platelet (<30 × 109/L) counts exhibited significantly decreased BM HIF-1α levels, compared to those with higher megakaryocyte (≥100/slide) and platelet (≥30 × 109/L) counts. To test whether HIF-1α regulates megakaryopoiesis and platelet production, megakaryocytes derived from mouse BM cells were treated with an HIF-1α activator (IOX-2; 50 µM) or inhibitor (PX-478; 50 µM). PX-478 significantly decreased HIF-1α expression, cell size, and the populations of CD41-positive and high-ploidy cells. Importantly, to evaluate the role of HIF-1α as a potential therapeutic target in ITP, mouse BM cells were incubated with plasma from ITP patients in the presence or absence of IOX-2. IOX-2 significantly attenuated the ITP plasma-induced decrease in cell size as well as the proportions of CD41-positive and high-ploidy cells. In addition, IOX-2 increased the number of megakaryocytes from mouse BM cells treated with ITP plasma. Our findings indicate that decreased HIF-1α may contribute to impaired megakaryopoiesis in ITP, and HIF-1α may provide a potential therapy for ITP patients.... J. Qi (1, 2, 3, 4), T. You (1, 2), T. Pan (1, 2, 3, 4), Q. Wang (4), L. Zhu (2, 3, 4), Y. Han (1, 2, 3) 27791 2017-08-03 13:50:43 Ahead of print: Co-injection of mesenchymal stem cells with endothelial progenitor cells accelerates... Endothelial colony-forming cells (ECFCs) are progenitor cells committed to endothelial lineages and have robust vasculogenic properties. Mesenchymal stem cells (MSCs) have been described to support ECFC-mediated angiogenic processes in various matrices. However, MSC-ECFC interactions in hind limb ischemia (HLI) are largely unknown. Here we examined whether co-administration of ECFCs and MSCs bolsters vasculogenic activity in nude mice with HLI. In addition, as we have previously shown that endoglin is a key adhesion molecule, we evaluated its involvement in ECFC/MSC interaction. Foot perfusion increased on day 7 after ECFC injection and was even better at 14 days. Co-administration of MSCs significantly increased vessel density and foot perfusion on day 7 but the differences were no longer significant at day 14. Analysis of mouse and human CD31, and in situ hybridization of the human ALU sequence, showed enhanced capillary density in ECFC+MSC mice. When ECFCs were silenced for endoglin, coinjection with MSCs led to lower vessel density and foot perfusion at both 7 and 14 days (p<0.001). Endoglin silencing in ECFCs did not affect MSC differentiation into perivascular cells or other mesenchymal lineages. Endoglin silencing markedly inhibited ECFC adhesion to MSCs. Thus, MSCs, when combined with ECFCs, accelerate muscle recovery in a mouse model of hind limb ischemia, through an endoglin-dependent mechanism.... E. Rossi (1, 2), D. Smadja (3), C. Goyard (1, 2, 4), A. Cras (1, 2, 5), B. Dizier (1, 2), N. Bacha (1, 2), A. lokajczyk (1, 2), C. L. Guerin (6), N. Gendron (7), B. Planquette (1, 2, 4), V. Mignon (1, 8), C. Bernabéu (9), O. Sanchez (1, 2, 4), D. M. Smadja (7) 27790 2017-08-03 13:47:14 Ahead of print: Absence of transforming growth factor beta 1 in murine platelets reduces neointima... Platelet degranulation at the site of vascular injury prevents bleeding and may affect the chronic vascular wound healing response. Transforming Growth Factor (TGF)-β1 is a major component of platelet α-granules known to accumulating in thrombi. It was our aim to determine the role of TGFβ1 released from activated platelets for neointima formation following arterial injury and thrombosis. Mice with platelet-specific deletion of TGFβ1 (Plt.TGFβ-KO) underwent carotid artery injury. Immunoassays confirmed the absence of active TGFβ1 in platelet releasates and plasma of Plt.TGFβ-KO mice. Whole blood analyses revealed similar haematological parameters, and tail cut assays excluded major bleeding defects. Platelet aggregation and the acute thrombotic response to injury in vivo did not differ between Plt.TGFβ-KO and Plt.TGFβ-WT mice. Morphometric analysis revealed that absence of TGFβ1 in platelets resulted in a significant reduction of neointima formation with lower neointima area, intima-to-media ratio, and lumen stenosis. On the other hand, the media area was enlarged in mice lacking TGFβ1 in platelets and contained increased amounts of proteases involved in latent TGFβ activation, including MMP2, MMP9 and thrombin. Significantly increased numbers of proliferating cells and cells expressing the mesenchymal markers platelet-derived growth factor receptor-β or fibroblast-specific protein-1, and the macrophage antigen F4/80, were observed in the media of Plt.TGFβ-KO mice, whereas the medial smooth muscle-actin-immunopositive area and collagen content did not differ between genotypes. Our findings support an essential role for platelet-derived TGFβ1 for the vascular remodelling response to arterial injury, apparently independent from the role of platelets in thrombosis or haemostasis.... E. Schütz (1, 3), M. L. Bochenek (1, 2, 3), D. R. Riehl (2), M. Bosmann (2, 3), T. Münzel (1, 3), S. Konstantinides (2, 3), K. Schäfer (1, 3) 27742 2017-07-20 14:47:21 Ahead of print: Interdependence between osteoprotegerin and active von Willebrand factor in... The interdependence of the predictive accuracy of serum osteoprotegerin (OPG) and von Willebrand factor (vWF) levels for long-term cardiovascular outcomes has not been investigated so far. This was a prospective observational cohort study in 361 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI). Baseline levels of OPG, vWF, active vWF (act vWF) and ristocetin cofactor activity (vWF:RICO) were measured. Cardiovascular mortality was recorded over a median of five years. OPG concentrations >3.7 µg/ml emerged as the strongest predictor of cardiovascular (CV) death: 30 % of patients died during the five-year follow-up in this group, as compared to 10 % in patients with OPG <3.7 µg/ml (p<0.001). Act vWF had a significant prognostic impact on CV mortality when OPG levels were low (<3.7 µg/ml): patients with act vWF concentration >1 µg/ml died in 14 %, whereas those with act vWF values <1 µg/ml had a mortality rate of 1 % (p=0.015). We stratified patients into three groups: high OPG, low OPG/high act vWF and low OPG/low act vWF. Patients with high OPG values had a 13-fold higher risk for CV death than those with low OPG/low act vWF concentrations (adj. HR: 12.6; 95 %CI: 1.7–94.7; p=0.014), and a two-fold higher risk as compared to those patients with low OPG/high act vWF concentrations (adj. HR: 2.0; 95 %CI: 1.1–3.7; p=0.03) in the adjusted Cox regression analysis. In conclusion, elevated OPG at the time of PCI was a strong independent predictor of five-years cardiovascular mortality, whereas act vWF had a significant prognostic impact on CV mortality when OPG levels were low.... J. Siller-Matula (1, 2), I. M. Lang (1), C. Schoergenhofer (3), M. Roest (4), B. Jilma (3) 27741 2017-07-20 14:29:20 Ahead of print: Consequences of warfarin suspension after major bleeding in very elderly patients... G. Zoppellaro (1), S. Granziera (2), G. Bertozzo (2), G. Denas (1), L. Marigo (3), F. Petruzzellis (3), S. Padayattil Jose (1), K. Rossi (3), G. Nante (3), V. Pengo (1) 27740 2017-07-20 14:06:05 Ahead of print: Analysis of the substrate specificity of Factor VII activating protease (FSAP) and... Factor VII (FVII) activating protease (FSAP) is a circulating serine protease that is likely to be involved in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To date, no systematic information is available about the substrate specificity of FSAP. Applying phage display and positional scanning substrate combinatorial library (PS-SCL) approaches we have characterised the specificity of FSAP towards small peptides. Results were evaluated in the context of known protein substrates as well as molecular modelling of the peptides in the active site of FSAP. The representative FSAP-cleaved sequence obtained from the phage display method was Val-Leu-Lys-Arg-Ser (P4-P1’). The sequence X-Lys/Arg-Nle-Lys/Arg (P4-P1) was derived from the PS-SCL method. These results show a predilection for cleavage at a cluster of basic amino acids on the nonprime side. Quenched fluorescent substrate (Ala-Lys-Nle-Arg-AMC) (amino methyl coumarin) and (Ala-Leu-Lys-Arg-AMC) had a higher selectivity for FSAP compared to other proteases from the hemostasis system. These substrates could be used to measure FSAP activity in a complex biological system such as plasma. In histone-treated plasma there was a specific activation of pro-FSAP as validated by the use of an FSAP inhibitory antibody, corn trypsin inhibitor to inhibit Factor XIIa and hirudin to inhibit thrombin, which may account for some of the haemostasis-related effects of histones. These results will aid the development of further selective FSAP activity probes as well as specific inhibitors that will help to increase the understanding of the functions of FSAP in vivo.... E. Kara (1), D. Manna (1), G. Å. Løset (2), E. L. Schneider (3), C. S. Craik (3), S. Kanse (1) 27739 2017-07-19 15:38:56 Ahead of print: Haemostasis biomarkers and risk of intracerebral haemorrhage in the REasons for... Pathologic alterations in haemostasis cause bleeding disorders, but it is unknown if variation within the normal range relates to intracerebral haemorrhage (ICH) risk. It was our objective to assess the prospective associations of haemostasis biomarkers with ICH risk. The REasons for Geographic and Racial Differences in Stroke study (REGARDS) recruited 30,239 U. S. individuals aged ≥45 years. ICH was ascertained through biannual telephone contact and review of deaths followed by medical record evaluation. Haemostasis biomarkers (factor VIII (FVIII), factor IX (FIX), factor XI (FXI), fibrinogen, protein C, and D-dimer) were measured in a case cohort study consisting of ICH and a 1,104 person cohort random sample. The hazard ratio (HR) and 95 % confidence interval (CI) by biomarker were estimated using Cox models and adjusted for ICH risk factors. Individuals with a prior history of stroke, ICH or on warfarin were excluded. Over a median 5.8 years 66 ICH occurred. Fibrinogen, FVIII, FXI, and protein C were not associated with ICH risk in any analysis. Lower FIX increased risk of ICH with the bottom versus the top tertile of FIX associated with an HR of 5.68 (95 % CI 2.30, 14.05). D-dimer demonstrated a non-linear relationship with a potential threshold effect with increased risk only in the top 5th percentile (HR 3.22; 95 % CI 1.01, 10.31; pnon-linear = 0.04).In conclusion, low FIX levels within the normal range were associated with increased ICH risk. These data suggest non-pathologic alterations in haemostasis impact intracranial bleeding risk.... N. Zakai (1), N. C. Olson (2), S. E. Judd (3), D. O. Kleindorfer (4), B. M. Kissela (4), G. Howard (3), M. Cushman (2, 4) 27713 2017-07-05 19:45:56 Open Access: Prasugrel in critically ill patients While prasugrel is indicated for the treatment of myocardial infarction, its effects in the most severely affected patients requiring intensive care is unknown, so that we measured the antiplatelet effects and sparse pharmacokinetics of prasugrel in critically ill patients. Twenty-three patients admitted to medical intensive care units, who were treated with 10 mg prasugrel once daily, were included in this prospective trial. Critically ill patients responded poorly to daily prasugrel treatment: adenosine diphosphate (ADP)-induced aggregation in whole blood classified 65 % (95 % confidence intervals (CI) 43–84 %) of patients as having high on treatment platelet reactivity, platelet function under high shear rates even 74 % (95 %CI 52–90 %). There was only limited additional inhibition provided 2 hours after the next dose of prasugrel. In contrast, insufficient inhibition of the target was only seen in 26 % (95 %CI 10–48 %) of patients as measured by the vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) assay. Low effective plasma levels of prasugrel active metabolite were measured at trough [0.5 (quartiles 0.5–1.1) ng/ml at baseline], and 2 hours after intake [5.7 (3.8–9.8) ng/ml], but showed coefficients of variation of ~70 %. In sum, inhibition of platelet aggregation by prasugrel is not uniform but highly variable in critically ill patients, similar to clopidogrel in a general population. The pharmacokinetic measurements indicate that poor absorption/metabolism of prasugrel may partly contribute while inflammation induced heightened intrinsic platelet reactivity may also play a role.... C. Schoergenhofer (1), E.- L. Hobl (1), T. Staudinger (2), W. S. Speidl (3), G. Heinz (3), J. Siller-Matula (3), C. Zauner (4), B. Reiter (5), J. Kubica (6), B. Jilma (1) 27712 2017-07-05 19:39:51 Ahead of print: A randomised trial on the effect of anti-platelet therapy on the systemic... The use of acetylsalicylic acid (ASA) is associated with improved outcome in patients with sepsis, and P2Y12 inhibitors have been suggested to also have immunomodulatory effects. Therefore, we evaluated the effects of clinically relevant combinations of antiplatelet therapy on the immune response in experimental endotoxaemia in humans in vivo. Forty healthy subjects were randomised to seven days of placebo, placebo with ASA, ticagrelor and ASA, or clopidogrel and ASA treatment. Systemic inflammation was elicited at day seven by intravenous administration of Escherichia coli endotoxin. ASA treatment profoundly augmented the plasma concentration of pro-inflammatory cytokines, but did not affect anti-inflammatory cytokines. Addition of either P2Y12 antagonist to ASA did not affect any of the circulating cytokines, except for an attenuation of the ASA-induced increase in TNFα by ticagrelor. Systemic inflammation increased plasma adenosine, without differences between groups, and although P2Y12 inhibition impaired platelet reactivity, there was no correlation with cytokine responses.... D. Kiers (1, 2), W. A. van der Heijden (1), L. van Ede (1), J. Gerretsen (1), Q. de Mast (3), A. J. van der Ven (3), S. el Messaoudi (4), G. A. Rongen (3, 4), M. Gomes (5), M. Kox (1), P. Pickkers (1), N. P. Riksen (3) 27711 2017-07-05 19:35:45 Plasma carboxypeptidase U (CPU, CPB2, TAFIa) generation during in vitro clot lysis and its interplay... Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic carboxypeptidase that is able to attenuate fibrinolysis. The inactive precursor procarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodulin complex or plasmin. The aim of this study was to investigate and characterise the time course of CPU generation in healthy individuals. In plasma of 29 healthy volunteers, CPU generation was monitored during in vitro clot lysis. CPU activity was measured by means of an enzymatic assay that uses the specific substrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation curve and calculate the parameters that define it. In all individuals, CPU generation was biphasic. Marked inter-individual differences were present and a reference range was determined. The endogenous CPU generation potential is the composite effect of multiple factors. With respect to the first CPU activity peak characteristics, we found correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximum turbidity of the clot lysis profile. In conclusion, our method offers a technique to determine the endogenous CPU generation potential of an individual. The parameters obtained by the method quantitatively describe the different mechanisms that influence CPU generation during the complex interplay between coagulation and fibrinolysis, which are in line with the threshold hypothesis.... D. Leenaerts (1), J. Aernouts (2), P. Van Der Veken (3), Y. Sim (1), A. Lambeir (1), D. Hendriks (1) 27710 2017-07-05 19:34:45 Ahead of print: Open Access: Safety and efficacy of a glycoPEGylated rFVIII (turoctocog alpha pegol,... Turoctocog alfa pegol (N8-GP, Novo Nordisk, Bagsværd, Denmark), an extended half-life glycoPEGylated recombinant factor VIII (rFVIII), is being developed for prophylaxis and treatment of bleeds in haemophilia A patients. pathfinder™5 is a multinational, open-label, single-arm trial to assess safety, efficacy and pharmacokinetics of N8-GP in paediatric (<12 years), previously treated patients. Boys with severe haemophilia A (<1 % FVIII), no history of inhibitors and previously treated with FVIII products (>50 exposure days [ED] for patients aged 0–5 years [younger cohort]; >150 ED for patients aged 6–11 years [older cohort]) were included. For prophylaxis, N8-GP was dosed at 50–75 IU/kg twice weekly; bleeds were treated with 20–75 IU/kg. Half-life was estimated for the patients’ previous FVIII product and for N8-GP. Sixty-eight patients received N8-GP; none developed inhibitors and no other concerns were identified. Median annualised bleeding rate was 1.95 (1.94 and 1.97 in the younger and older cohorts, respectively). Twenty-nine patients (42.6 %; 15 younger and 14 older children, respectively) did not report any bleeding while on N8-GP prophylaxis; 39 patients (57.4 %; 19 younger and 20 older children, respectively) reported 70 bleeds (all mild/moderate). N8-GP treatment was successful for 78.6 % of bleeds in all patients, 80.0 % in younger and 77.5 % in older patients. Most bleeds (80.0 %) were treated with ≤2 injections. Half-life ratio between N8-GP and the patients’ previous FVIII product was 1.85. N8-GP was well tolerated and provided effective prophylaxis and treatment of bleeds in paediatric patients with severe haemophilia A.... S. Meunier (1), J. Alamelu (2), S. Ehrenforth (3), H. Hanabusa (4), F. Abdul Karim (5), K. Kavakli (6), M. Khodaie (3), J. Staber (7), O. Stasyshyn (8), D. L. Yee (9), L. Rageliene (10) 27709 2017-07-05 19:34:15 Whole blood ristocetin-activated platelet impedance aggregometry (Multiplate) for the rapid... Von Willebrand disease (VWD) is the most common bleeding disorder, but no bedside tests specific for Von Willebrand factor are available. The objective of this study was to evaluate the diagnostic accuracy of whole blood ristocetin-induced platelet aggregometry (WB-RIPA) in VWD. WB-RIPA was performed in VWD patients (n=100) and healthy controls (n=17) using the Multiplate® platelet impedance aggregometry platform. The diagnostic properties of the test were described as sensitivity/specificity, positive and negative predictive value, and ROC area under the curve (AUC). Patients with VWD had impaired platelet aggregation by WB-RIPA. At a cut-off of 98 U, the test sensitivity and specificity of WB-RIPA for VWD was 0.95 and 0.53. A cut-off of 60 U provided a specificity of 1.00 with reduced sensitivity of 0.76. All patients with type 3 VWD and >90 % of patients with type 2 VWD were accurately distinguished from the controls. Incorrect classifications were attributable to patients with type 1 VWD, showing partly overlapping WB-RIPA results with healthy controls. Remarkably, these patients had lower bleeding scores and higher VWF activity than other type 1 VWD patients. Overall, WB-RIPA discriminated VWD patients from healthy controls accurately with a ROC AUC of 0.94. These results show that WB-RIPA is a promising diagnostic test for VWD, especially when timely results are required. Depending on the chosen test threshold, WB-RIPA could be clinically used as a rule out test, or to suggest patients in whom further testing for VWD is warranted.... D. E. Schmidt (1, 2), M. Bruzelius (1), A. Majeed (1, 3), J. Odeberg (1), M. Holmström (1), A. Ågren (1) 27708 2017-07-05 19:33:55