Thrombosis and Haemostasis Thrombosis and Haemostasis th de-de Sat, 24 Jun 17 14:11:15 +0200 Ahead of print: Lys 42/43/44 and Arg 12 of thrombin-activable fibrinolysis inhibitor comprise a... The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K1/2) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K1/2 by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay at physiologic levels of TAFI. Similar trends in the antifibrinolytic activity of the TAFI variants were observed when plasma clots were formed using HUVECs as the source of TM. When thrombin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1-fold compared with wild-type TAFI, suggesting that these mutations only impacted activation kinetics in the presence of TM. Surface plasmon resonance data suggest that mutation of the four residues abrogates TM binding with or without thrombin. Therefore, Lys 42, Lys 43, Lys 44 and Arg 12 are critical for the interaction of TAFI with the thrombin-TM complex, which modulates its antifibrinolytic potential.... C. Wu (1, 2), A. R. Stafford (1, 2), J. C. Fredenburgh (1, 2), J. I. Weitz (1, 2, 3), A. Gils (4), P. J. Declerck (4), P. Y. Kim (1, 2) 27689 2017-06-22 13:31:24 Ahead of print: New paradigms in venous thromboprophylaxis of medically ill patients Acutelly-ill hospitalised medical patients are at risk of venous thromboembolism (VTE), both in-hospital and in the immediate post-discharge period, and mortality from VTE is thought to be particularly high in this patient population. However, despite previous mandates from international antithrombotic guidelines such as those of the American College of Chest Physicians (ACCP) for the “universal” use of thromboprophylaxis in hospitalised medical patients, global audits suggest that implementation of thromboprophylaxis continues to be challenging because of the perceived higher risk of bleeding and lower risk of VTE than that reported in clinical trials. Recent population-based studies also reveal that a “universal” hospital-only thromboprophylactic strategy does not reduce the community burden of VTE from this population, which may constitute nearly one quarter of the attributable risk of VTE. Lastly, four large randomised placebo-controlled trials of extended thromboprophylaxis have failed to show a definitive net clinical benefit in hospitalised medical patients. Recent large-scale efforts in deriving and validating scored VTE and bleed risk assessment models (RAMs) have been completed in the medically-ill population. In addition, an elevated D-dimer as a new biomarker to identify at-VTE risk medically ill patients has also undergone prospective evaluation. This paper will review current concepts of VTE and bleed risk in hospitalised medical patients, both in the hospital as well as the post-hospital discharge period, and will discuss new paradigms of thromboprophylaxis in this population using an individualised, patient-centered approach.... A. C. Spyropoulos (1), A. Spyropoulos (2), G. E. Raskob (3) 27688 2017-06-22 13:30:24 Ahead of print: Effectiveness and safety of rivaroxaban versus warfarin for treatment and prevention... The efficacy and safety or rivaroxaban versus enoxaparin/vitamin K antagonist for treatment and prevention recurrence of venous thromboembolism (VTE) was demonstrated in the randomised EINSTEIN trials. We assessed the effectiveness and safety of rivaroxaban versus warfarin in VTE patients managed in routine practice. Using US MarketScan claims from 1/2012–6/2015, we included adults with a primary diagnosis of deep vein thrombosis (DVT) or pulmonary embolism (PE) during a hospitalisation/emergency department visit, newly-initiated on rivaroxaban or warfarin within 30-days after the VTE and with ≥180-days of continuous medical/prescription benefits prior to the VTE (baseline). Patients with a claim for anticoagulation at baseline were excluded. Recurrent VTE, major bleeding, intracranial haemorrhage (ICH) and gastrointestinal bleeding (GIB) were assessed. Differences in baseline characteristics between cohorts were adjusted for using inverse probability of treatment weights based on propensity-scores. Patients had a maximum of 12-months period of follow-up post-VTE or until endpoint occurrence, switch/discontinuation of index anticoagulation, insurance disenrollment or end-of-follow-up. Cox regression was performed and reported as hazard ratios (HRs) with 95 % confidence intervals (CIs). In total, 13,609 rivaroxaban and 32,244 warfarin users experiencing VTE were included. Rivaroxaban was associated with an 19 % (95 %CI=10–27 %) reduction in recurrent VTE and a 21 % (95 %CI=4–35 %) reduction in major bleeding hazard versus warfarin. Rivaroxaban was also associated with significantly decreased hazards of ICH (HR=0.40) and GIB (HR=0.72). Rivaroxaban appears to reduce patients’ hazard of both recurrent VTE and major bleeding in routine practice. These results appear consistent with EINSTEIN and post-marketing registry studies.... C. I. Coleman (1), C. Coleman (2), T. J. Bunz (3), A. G. G. Turpie (4) 27687 2017-06-21 18:52:44 Ahead of print: Effects of direct oral anticoagulants on lupus anticoagulant assays in a real-life... Laboratory diagnosis of lupus anticoagulant (LA) is based on prolongation in at least one coagulation assay (diluted Russell’s viper venom time – dRVVT or activated partial thromboplastin time – aPTT), which normalises after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. It has been shown (primarily in vitro) that direct oral anticoagulants (DOACs – dabigatran [DAB], rivaroxaban [RIV] and apixaban [API]) may also influence LA testing. We tested the effects of DOACs on assays routinely used for the diagnosis of LA in patients treated with these drugs in a real-life setting. Plasma from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and not known to have LA were tested using dRVVT (LA-screen and LA-confirm, Life Diagnostics) and aPTT (PTT-LA, Diagnostica Stago and aPTT Actin FS, Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, dRVVT and aPTT ratios of <1.2 were considered negative, ratios of >1.4 positive, while if the ratios were 1.2–1.4 LA could not be ruled out. Plasma concentrations varied between 8–172 µg/l for DAB, 8–437 µg/l for RIV and 36–178 µg/l for API. LA diagnosis was negative in only eight (27 %) plasma samples from patients treated with DAB, and in five (25 %) and four samples (24 %) from patients treated with RIV and API, respectively. LA Positivity (dRVVT and aPTT ratios >1.4) was found in 5 cases (17 %) among patients treated with DAB, in 10 cases (50 %) treated with RIV and in 7 cases (41 %) treated with API. A concentration-dependent effect of DOACs on dRVVT-based parameters was observed, particularly as regards DAB. At lower concentrations, RIV and API had only minor effects on the confirmatory tests (below 100 µg/l and 70 µg/l, respectively). Our results suggest that a risk of overestimation of LA detection is present in samples from patients treated with DOACs. Therefore, LA testing should not be performed during treatment with DOACs. Prolongation in confirmatory assays may be helpful for the recognition of false positivity, especially as regards DAB.... A. Antovic (1), E. Norberg (2), M. Berndtsson (2), A. Rasmuson (2), R. E. Malmström (3), M. Skeppholm (4), J. Antovic (1) 27686 2017-06-21 18:44:22 Ahead of print: Molecular signature of coronary stent thrombosis: oxidative stress and innate... The clinical impact of in-stent thrombosis is high because it is associated with high mortality and 20 % of the patients suffer a recurrent event within the two following years. The aim of this study was to characterise the morphologic and proteomic profile of in-stent thrombi (IST) in comparison to thrombi developed on native coronary arteries (CT) to identify a differential molecular signature. The study included 45 patients with ST-elevation-myocardial-infarction (STEMI) treated by primary-percutaneous-intervention and thrombus aspiration: 21 had IST and 24 had CT. Thrombi were characterised by morphologic immunohistochemical analysis and differential proteomic profiling (2-DE+MALDI-TOF/TOF). Bioinformatic analysis revealed differences in proteins related to oxidative-stress and cell death/survival. IST showed a higher content of structural proteins (gelsolin, actin-cytoplasmic-1, tropomyosin, and myosin) together with an imbalance in redox-homeostasis related proteins (increased superoxide-dismutase and decreased peroxiredoxin-2 thrombus content), and a coordinated increase of chaperones (HSP60 and HSC70) and cellular quality control-related proteins (26S-protease-regulatory-subunit-7). These changes were reflected into a significant decrease in HSC70 systemic levels and a significant increase in advanced-oxidation-protein-products (AOPP) indicative of increased oxidative stress-mediated protein damage in IST. Our results reveal an imbalance in redox-related proteins indicative of an exacerbated oxidative-stress that leads to an accumulation of AOPP serum levels in IST. Moreover, the coordinated increase in chaperones and regulatory proteins reflects the activation of intracellular protection mechanisms to maintain protein integrity in IST. The failure to counterbalance the stress situation could trigger cellular apoptosis leading to the destabilization of the thrombus and to a worse prognosis of IST-STEMI-patients.... J. Cubedo (1), A. Blasco (2), T. Padro (1), I. Ramaiola (1), O. Juan-Babot (1), J. Goicolea (2), J. Fernández-Díaz (2), J. Oteo (2), L. Badimon (1, 3) 27685 2017-06-21 18:44:01 Ahead of print: Angiogenesis in metabolic-vascular disease Angiogenesis, literally formation of new blood vessels, is the main process through which the vascular system expands during embryonic and postnatal development. Endothelial cells, which constitute the inner lining of all blood vessels, are typically in a quiescent state in the healthy adult organism. However, in vascular and metabolic diseases, the endothelium becomes unstable and dysfunctional. The resulting tissue hypoxia may thereby induce pathological angiogenesis, which is a hallmark of disease conditions like cancer or diabetic retinopathy. However, recent evidence suggests that angiogenesis is also a major player in the context of further metabolic diseases, especially in obesity. In particular, deregulated angiogenesis is linked with adipose tissue dysfunction and insulin resistance. On the other hand, signalling pathways, such as the PI3K pathway, may regulate metabolic activities in the endothelium. Endothelial cell metabolism emerges as an important regulator of angiogenesis. This review summarises the role of angiogenesis in metabolic-vascular disease, with specific focus on the role of angiogenesis in obesity-related metabolic dysfunction and on signaling pathways, especially PI3K, linking cell metabolism to endothelial function.... G. Breier (1), T. Chavakis (2), E. Hirsch (3) 27613 2017-06-08 14:37:06 Ahead of print: New developments in anticoagulants: Past, present and future Thrombosis is a leading cause of death and disability worldwide, and anticoagulants are the mainstay of its prevention and treatment. Starting with unfractionated heparin (UFH) and vitamin K antagonists (VKAs) such as warfarin, the choices of anticoagulants have exploded in the past 20 years. With over 90 % subcutaneous bioavailability, no need for coagulation monitoring and dose adjustment, and a lower risk of heparin-induced thrombocytopenia, low-molecular-weight heparin and fondaparinux have replaced UFH for prevention and initial treatment of venous thromboembolism and for secondary prevention in cancer patients. In patients undergoing percutaneous interventions, bivalirudin is often used instead of UFH. Oral anticoagulation therapy has advanced with the introduction of the non-vitamin K antagonist oral anticoagulants (NOACs), which include dabigatran, rivaroxaban, apixaban and edoxaban. With efficacy at least equal to that of VKAs but with greater safety and convenience, the NOACs are now replacing VKAs for many indications. This paper a) highlights these advances, b) outlines how specific reversal agents for the NOACs will enhance their safety, c) reviews some of the ongoing trials with the NOACs, and d) describes the inhibitors of factor XII and XI that are under investigation as anticoagulants.... J. I. Weitz (1), J. Harenberg (2) 27612 2017-06-08 14:26:30 Ahead of print: Venous thromboembolism: Past, present and future Venous thromboembolism (VTE), the third most frequent acute cardiovascular syndrome, is associated with a considerable disease burden which continues to grow along with the longer life expectancy of the population worldwide. In the past century, parenteral heparin prophylaxis was established for hospitalised patients at elevated risk of VTE. More recently, non-vitamin K antagonist oral anticoagulants (NOACs) with a direct inhibiting effect on factor Xa or thrombin, underwent extensive testing in clinical trials and have been approved for patients undergoing hip or knee replacement. Clinical investigation is ongoing in further areas of thromboprophylaxis, including medical prophylaxis in patients and high-risk situations in the outpatient setting. The diagnostic approach to suspected VTE is now based on advanced imaging techniques and robust diagnostic algorithms which ensure high sensitivity and specificity. Nevertheless, the role of clinical, or pre-test, probability assessment remains crucial to avoid overdiagnosis and treatment errors. Advances in reperfusion strategies, along progressive establishment of the NOACs as the new standard of anticoagulation treatment, have simplified the management of VTE, improving outcomes and particularly safety. While new molecular targets for anticoagulation are being investigated in the quest to further reduce bleeding risk, adjusting the initial regimen to the patient’s risk and finding the optimal duration of anticoagulation after an index VTE event will be some of the top priorities in the years to come. Importantly, and in parallel to new drugs and technical advances in imaging, incentives such as hospital accreditation and funding based on evidence-based practice need to be implemented to increase guideline adherence.... S. Schulman (1, 2), W. Ageno (3), S. V. Konstantinides (4, 5) 27611 2017-06-08 13:56:04 Ahead of print: Therapeutic strategies for atherosclerosis and atherothrombosis: Past, present and... Even two centuries after they were first described, atherosclerosis and atherothrombosis remain among the leading causes of death worldwide. Over the last decades it has become clear that atherosclerosis is not only a lipid-driven disease but also a multifactorial process largely driven by inflammatory mediators, an insight that has instigated additional research and drug development focussing on anti-inflammatory therapies. In this review, we will provide a brief historical overview, followed by a more general synopsis of the range of currently available state-of-the-art therapies for atherosclerosis and atherothrombosis. Finally, we will highlight some of the promising therapeutic strategies that are currently under intense investigation. We believe that the next years will witness highly interesting developments and clinical trials investigating yet more novel therapies, and at the same time looking into potential combinations of all available therapies. This prospect closes in on the ultimate goal, which is to reduce the residual risk that still persists despite present therapeutic options.... C. Weber (1, 2, 3), L. Badimon (4), F. Mach (4), E. P. C. van der Vorst (2) 27610 2017-06-08 13:52:38 Ahead of print: Extracellular nucleic acids in immunity and cardiovascular responses: between alert... Severe inflammatory complications are a potential consequence in patients with predetermined conditions of infections, pulmonary diseases, or cardiovascular disorders. Notably, the amplitude of the inflammatory response towards these complications can dictate the disease progression and outcome. During the recent years, evidence from basic research as well as from clinical studies has identified self-extracellular nucleic acids as important players in the crosstalk between immunity and cardiovascular diseases. These stress- or injury-induced endogenous polymeric macromolecules not only serve as “alarmins” or “Danger-associated molecular patterns” (DAMPs), but their functional repertoire goes far beyond such activities in innate immunity. In fact, (patho-) physiological functions of self-extracellular DNA and RNA are associated and in many cases causally related to arterial and venous thrombosis, atherosclerosis, ischemia-reperfusion injury or tumour progression. Yet, the underlying molecular mechanisms are far from being completely understood. Interestingly enough, however, novel antagonistic approaches in vitro and in vivo, particularly using natural endonucleases or synthetic nucleic acid binding polymers, appear to be promising and safe therapeutic options for future studies. The aim of this review article is to provide an overview of the current state of (patho-) physiological functions of self-extracellular nucleic acids with special emphasis on their role as beneficial / alerting or adverse / damaging factors in connection with immune responses, inflammation, thrombosis, and cardiovascular diseases.... K. T. Preissner (1), H. Herwald (2) 27609 2017-06-08 13:40:39 Ahead of print: Coagulation factor and protease pathways in thrombosis and cardiovascular disease The biochemical characterization of the proteolytic pathways that constitute blood coagulation was one of the most relevant achievements in biomedical research during the second half of the 20th century. Understanding these pathways was of crucial importance for improving global health through application in haemostasis and thrombosis pathologies. Immediately after the cloning of the genes corresponding to these proteins, mutations were discovered in them that were associated with imbalances in haemostasis. Later, the importance of coagulation pathways in other pathological processes was demonstrated, such as in atherosclerosis and inflammation, both essential processes involved in vascular disease. In the present review we evaluate the concepts that have allowed us to reach the integrated vision on coagulation that we have today. The thrombo-inflammation model encompassing these aspects includes a pivotal role for the proteases of the coagulation pathway as well as the regulatory proteins thereof. These concepts illustrate the importance of the coagulation cascade in cardiovascular pathology, not only in thrombotic processes, but also in atherosclerotic processes and in the response to ischemia-reperfusion injury, making it a central mechanism in cardiovascular disease.... H. ten Cate (1, 2), T. M. Hackeng (3), P. García de Frutos (4) 27608 2017-06-08 13:36:56 Ahead of print: Antithrombotic therapy for acute coronary syndrome: Past, present and future Plaque erosions and ruptures are the histopathological hallmarks of arterial thrombus formation in the coronary arteries. The clinical condition associated with this process is usually referred to as acute coronary syndrome (ACS). Importantly, both blood platelets and the coagulation cascade are key players for initiation, amplification and perpetuation of ACS. There has been great progress in ACS treatment in recent decades, both at the technical level of (percutaneous) revascularisation and at the level of antithrombotic treatment. Numerous trials have led to significant advancements in the development of effective anticoagulant and antiplatelet drugs. The large number of randomised controlled clinical trials (RCTs) and the huge number of patients enrolled in these RCTs, with mega trials including >10,000 patients, is unique in the history of medical research and also reflects the exceptional efforts associated with these huge research activities. The crucial issue, however, with respect to optimising treatment, relates to finding the delicate balance between the reduction of thrombotic events by effective drug treatment and the induction of bleeding that is linked to the use of potent or multiple antithrombotic agents. Interestingly, there is a gap in modern days between current guideline recommendations favouring potent platelet inhibition in ACS and the utilization of the respective drugs in clinical practice. In this review, we will summarise and discuss the past, present and future antithrombotic treatment for ACS patients with a focus on the development of optimised antiplatelet treatment strategies and their utilisation in the real world.... D. Sibbing (1), D. J. Angiolillo (2), K. Huber (3) 27607 2017-06-08 13:36:39 Ahead of print: Interplay between elevated cellular fibronectin and plasma fibrin clot properties in... Type 2 diabetes is associated with faster formation of poorly lysable, denser fibrin clots and elevated cellular fibronectin (cFn), a marker of vascular injury. We investigated whether cFn affects clot properties in type 2 diabetes. In 200 consecutive patients with type 2 diabetes and 100 control subjects matched for age and sex, we determined plasma cFn along with clot formation and degradation using turbidimetric and permeability assays. Diabetic patients had elevated cFn (median, 3.99 [interquartile range, 2.87–4.81] µg/ml]), increased clot density (MaxAbsC) and prolonged lysis time (LysT) compared with those without type 2 diabetes (all p<0.01). Diabetic patients with documented cardiovascular disease (CVD, n=127, 63.5 %) had increased cFn (4.53 [3.68–4.95] µg/ml), decreased clot permeability (Ks) and increased MaxAbsC compared with those without CVD (all p<0.001). Diabetic patients with cFn in the top quartile (>4.81 µg/ml) were two times more likely to have CVD compared with those in the lowest quartile (odds ratio 1.80, 95 % confidence interval 1.41–2.46, p<0.001). No differences in cFn were observed in relation to microvascular complications. After adjustment for potential confounders, cFn accounted for 10.2 % of variance in Ks, 18.2 % of variance in clot density and 10.2 % of variance in AUC in diabetic patients. This study shows that elevated cFn is associated with unfavourably modified clot properties in type 2 diabetes, especially with concomitant CVD, which indicates novel links between vascular injury and prothrombotic alterations in diabetes. Coagulation, cellular fibronectin, type 2 diabetes, cardiovascular disease... M. Konieczyńska (1), A. H. Bryk (1, 2), K. P. Malinowski (3), K. Draga (4), A. Undas (1, 2) 27581 2017-06-01 16:18:46 Ahead of print: Is a normal computed tomography pulmonary angiography safe to rule out acute... A normal computed tomography pulmonary angiography (CTPA) remains a controversial criterion for ruling out acute pulmonary embolism (PE) in patients with a likely clinical probability. We set out to determine the risk of VTE and fatal PE after a normal CTPA in this patient category and compare these risk to those after a normal pulmonary angiogram of 1.7 % (95 %CI 1.0–2.7 %) and 0.3 % (95 %CI 0.02–0.7 %). A patient-level meta-analysis from 4 prospective diagnostic management studies that sequentially applied the Wells rule, D-dimer tests and CTPA to consecutive patients with clinically suspected acute PE. The primary outcome was the 3-month VTE incidence after a normal CTPA. A total of 6,148 patients were included with an overall PE prevalence of 24 %. The 3-month VTE incidence in all 4,421 patients in whom PE was excluded at baseline was 1.2 % (95 %CI 0.48–2.6) and the risk of fatal PE was 0.11 % (95 %CI 0.02–0.70). In patients with a likely clinical probability the 3-month incidences of VTE and fatal PE were 2.0 % (95 %CI 1.0–4.1 %) and 0.48 % (95 %CI 0.20–1.1 %) after a normal CTPA. The 3-month incidence of VTE was 6.3 % (95 %CI 3.0–12) in patients with a Wells rule >6 points. In conclusion, this study suggests that a normal CTPA may be considered as a valid diagnostic criterion to rule out PE in the majority of patients with a likely clinical probability, although the risk of VTE is higher in subgroups such as patients with a Wells rule >6 points for which a closer follow-up should be considered.... T. van der Hulle (1), N. van Es (2), P. den Exter (1), J. van Es (2), I. C. M. Mos (1), R. A. Douma (2), M. J. H. A. Kruip (3), M. M. C. Hovens (4), M. ten Wolde (5), M. Nijkeuter (6), H. ten Cate (7, 8), P. W. Kamphuisen (9), H. R. Büller (2), M. V. Huisman (1), E. Klok (1) 27580 2017-06-01 16:16:14 Ahead of print: Elevated plasma TFPI activity causes attenuated TF-dependent thrombin generation in... Early onset preeclampsia (EOP) is a pregnancy-specific proinflammatory disorder that is characterised by competing thrombotic and bleeding risks. It was the aim of this study to characterise thrombin generation, a major determinant of thrombotic and bleeding risk, in order to better understand the haemostatic balance in patients with EOP. Patients with EOP were recruited at the Rotunda Hospital, Dublin. Twenty-six cases of EOP were recruited over a 21-month period, out of 15,299 deliveries at the Rotunda. Blood samples were collected into sodium citrate plus corn trypsin inhibitor anticoagulated vacutainers, platelet-poor plasma was prepared, and calibrated automated thrombography was used to assess thrombin generation. Results were compared to age and sex-matched non-pregnant controls (n=13) and age- and gestation-matched pregnant controls (n=21). The rate and extent of thrombin generation triggered by low-dose tissue factor (TF) was significantly reduced in patients with EOP compared to pregnant controls, most significantly in cases of severe EOP. EOP patients displayed a trend towards an increased response to endogenous activated protein C and thrombomodulin relative to pregnant controls. Plasma tissue factor pathway inhibitor (TFPI) activity was increased in EOP patients. Inhibition of TFPI abolished the attenuation of thrombin generation stimulated by low-dose TF. In conclusion, patients with EOP are characterised by an attenuated coagulation response characterised by reduced thrombin generation stimulated by low-dose TF and elevated plasma TFPI activity. These changes in coagulation may modulate thrombotic risk and bleeding risk in patients with EOP.... K. Egan (1, 2), H. O’Connor (3), B. Kevane (2, 3, 4), F. Malone (3, 5), A. Lennon (4), A. Al Zadjali (2), S. Cooley (1), C. Monteith (3, 4), P. Maguire (2, 6), P. Szklanna (2, 6), S. Allen (1, 2), N. McCallion (3, 4), F. Ní Áinle (1, 2, 3, 4) 27579 2017-06-01 16:13:29 Ahead of print: Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents... The efficiency of current dual antiplatelet therapy might be further improved by its combination with a glycoprotein (GP) VI-targeting strategy without increasing bleeding. GPVI-Fc, a recombinant dimeric fusion protein binding to plaque collagen and concealing binding sites for platelet GPVI, acts as a lesion-focused antiplatelet drug, and does not increase bleeding in vivo. We investigated, whether GPVI-Fc added in vitro on top of acetylsalicylic acid (ASA), the P2Y12 antagonist ticagrelor, and the fibrinogen receptor antagonist abciximab alone or in combination would increase inhibition of platelet activation by atherosclerotic plaque. Under static conditions, GPVI-Fc inhibited plaque-induced platelet aggregation by 53 %, and increased platelet inhibition by ASA (51 %) and ticagrelor (64 %) to 66 % and 80 %, respectively. Under arterial flow, GPVI-Fc inhibited plaque-induced platelet aggregation by 57 %, and significantly increased platelet inhibition by ASA (28 %) and ticagrelor (47 %) to about 81 % each. The triple combination of GPVI-Fc, ASA and ticagrelor achieved almost complete inhibition of plaque-induced platelet aggregation (93 %). GPVI-Fc alone or in combination with ASA or ticagrelor did not increase closure time measured by the platelet function analyzer (PFA)-200. GPVI-Fc added on top of abciximab, a clinically used anti-fibrinogen receptor antibody which blocks platelet aggregation, strongly inhibited total (81 %) and stable (89 %) platelet adhesion. We conclude that GPVI-Fc added on top of single or dual anti-platelet therapy with ASA and/or a P2Y12 antagonist is likely to improve anti-atherothrombotic protection without increasing bleeding risk. In contrast, the strong inhibition of platelet adhesion by GPVI-Fc in combination with GPIIb/IIIa inhibitors could be harmful.... A. Mojica Muñoz (1), J. Jamasbi (1), K. Uhland (2), H. Degen, G. Münch, M. Ungerer, R. Brandl (2), R. Megens (1, 3), C. Weber (1, 4), R. Lorenz (1), W. Siess (1, 4) 27578 2017-06-01 16:09:14 Ahead of print: Thrombin is a selective inducer of heparanase release from platelets and... Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.... M. Tatour, M. Shapira, E. Axelman (1), S. Ghanem (1), A. Keren-Politansky (2), L. bonstein (3), B. Brenner (1), Y. Nadir (1) 27577 2017-06-01 15:58:09 Microvesicles in vascular homeostasis and diseases Microvesicles are members of the family of extracellular vesicles shed from the plasma membrane of activated or apoptotic cells. Microvesicles were initially characterised by their pro-coagulant activity and described as “microparticles”. There is mounting evidence revealing a role for microvesicles in intercellular communication, with particular relevance to hemostasis and vascular biology. Coupled with this, the potential of microvesicles as meaningful biomarkers is under intense investigation. This Position Paper will summarise the current knowledge on the mechanisms of formation and composition of microvesicles of endothelial, platelet, red blood cell and leukocyte origin. This paper will also review and discuss the different methods used for their analysis and quantification, will underline the potential biological roles of these vesicles with respect to vascular homeostasis and thrombosis and define important themes for future research. V. C. Ridger, C. M. Boulanger, A. Angelillo-Scherrer, L. Badimon, O. Blanc-Brude, M.-L. Bochaton-Piallat, E. Boilard, E. I. Buzas, A. Caporali, F. Dignat-George, P. C. Evans, R. Lacroix, E. Lutgens, D. F. J. Ketelhuth, R. Nieuwland, F. Toti, J. Tunon, C. Weber, I. E. Hoefer (1) 27576 2017-06-01 15:48:09 Ahead of print: Prostaglandin-endoperoxide synthase-2 deletion affects the natural trafficking of... Deep-vein thrombosis (DVT) is a common condition that often leads to pulmonary thromboembolism (VTE) and death. The role of prostaglandin-endoperoxide synthase (PTGS)2 in arterial thrombosis has been well established, whereas its impact in venous thrombosis remains unclear. Here, we showed that PTGS2 deletion predisposes to venous thrombosis as suggested by greater clot firmness and clot elasticity, by higher plasma levels of functional fibrinogen, factor VIII and PAI-1 activity, and proved by bigger thrombi detected after inferior vena cava ligation (IVCL) compared to WT mice. PTGS2-/- thrombi have greater fibrin content, higher number of F4/80+, TF+ and ANXA2+ cells, and lower S100A10+ cells. Remarkably, monocyte depletion reduced thrombus size in mutant mice, suggesting an important role of PTGS2-/- monocytes in this experimental setting. Interestingly, PTGS2 deletion reduced membrane ANXA2, and total S100A10, promoted assembly of ANXA2/p50NF-kB complex and its nuclear accumulation, and induced TF in peritoneal macrophages, whereas ANXA2 silencing decreased dramatically TF. Finally, Carbaprostacyclin treatment prevented venous thrombus formation induced by IVCL in mutant mice, reduced the ANXA2 binding to p50NF-kB subunit and its nuclear trafficking, and decreased TF in PTGS2-/- macrophages. PTGS2 deletion, changing the natural distribution of ANXA2 in monocytes/macrophages, increases TF expression and activity predisposing to venous thrombosis. Interestingly, Carbaprostacyclin treatment, inhibiting nuclear ANXA2 trafficking, controls monocyte TF activity and prevents DVT occurrence. Our data are of help in elucidating the mechanisms by which PTGS2 inhibition increases DVT risk, and suggest a new role for ANXA2 in venous thrombosis.... P. Amadio (1), E. Tarantino (1), L. Sandrini (1), E. Tremoli (1), S. S. Barbieri (1) 27572 2017-05-24 10:31:38 Ahead of print: Open Access: Differential integrin activity mediated by platelet collagen receptor... The platelet receptors glycoprotein (Gp)VI, integrin α2β1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca2+ ([Ca2+]i) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca2+]i measurements using confocal imaging. All three collagen receptors coupled to [Ca2+]i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca2+]i signals leading to real-time increases in integrins α2β1– and αIIbβ3-mediated platelet adhesion. αIIbβ3-dependent platelet aggregation was dependent on P2Y12 signalling. Co-engagement of α2β1 and GpIb/V/IX generated transient [Ca2+]i spikes and low amplitude [Ca2+]i responses that potentiated GpVI-dependent [Ca2+]i signalling. Therefore α2β1, GpIb/V/IX and GpVI synergize to generate [Ca2+]i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for αIIbβ3 in stable platelet adhesion and aggregation.... N. Pugh (1, 2), B. D. Maddox (1), D. Bihan (1), K. A. Taylor (2), M. P. Mahaut-Smith (3), R. W. Farndale (1) 27571 2017-05-24 10:22:11 Ahead of print: Molecular and clinical profile of VWD in a large cohort of Chinese population:... Von Willebrand disease (VWD), the most common inherited bleeding disorder, is characterised by a variable bleeding tendency, heterogeneous laboratory phenotype and race specific distribution of mutations. The present study aimed to determine the correlation of genotype and phenotype in 200 Chinese individuals from 90 unrelated families with VWD. Next generation sequencing (NGS) of the whole coding VWF, copy number analysis of VWF by CNVplex® technique as well as a comprehensive phenotypic assessment were carried out in all index patients (IPs). We identified putative mutations in all IPs except five mild type 1 (85/90, 94.4 %). In total, 98 different mutations were detected, 62 (63.3 %) of which were reported for the first time (23 missense mutations, 1 regulatory mutation, 12 splice site mutations and 26 null mutations). Mutations p.Ser1506Leu and p.Arg1374His/Cys/Ser were the most frequent mutations in 2A (33 % of cases) and 2M VWD (67 % of cases), respectively. In addition, mutation p.Arg816Trp was detected repeatedly in type 2N patients, while mutation p.Arg854Gln, extremely common in Caucasians, was not found in our cohort. Thirty-three patients had two or more putative mutations. Unlike most cases of type 1 and type 2 VWD, which were transmitted dominantly, we presented seven severe type 1, two type 2A and one type 2M with autosomal recessive inheritance. Here the phenotypic data of patients with novel mutations will certainly contribute to the better understanding of the molecular genetics of VWF-related phenotypes.... Q. Liang (1), H. Qin (2), Q. Ding (1), X. Xie (3), R. Wu (4), H. Wang (3), Y. Hu (5), X. Wang (1) 27570 2017-05-24 10:21:02 Ahead of print: Apixaban pharmacodynamic activity in umbilical cord, paediatric, and adult plasma The objective was to characterise apixaban pharmacodynamic (PD) activity in umbilical cord (UC), paediatric, and adult plasma. Plasma was obtained from blood samples from six UC donors, 70 paediatric (neonates [birth–≤1 month], infants [>1–≤6 months], toddlers [>6 months–≤2 years], young children [>2–≤6 years], children [>6–≤12 years], adolescents [>12–≤18 years]), and six adult (19–45 years) subjects. Plasma spiked with apixaban 0 (baseline), 30, or 110 ng/ml was analysed for anti-factor Xa activity, factor X levels, prothrombin time (PT), and modified PT (mPT). Apixaban had similar concentration-related effects on anti-factor Xa activity across groups (30 ng/ml: 0.223–0.295 IU/ml; 110 ng/ml: 1.212–1.474 IU/ml). Endogenous baseline factor X levels were 43 %–68 % lower in plasma from UC and subjects ≤6 months versus adults. Factor Xa inhibition (percentage change from baseline in apparent factor X levels) was similar for both apixaban concentrations across groups, except UC, neonate, and infant groups, which showed greater inhibition vs adults for apixaban 110 ng/ml. Baseline PT and mPT were similar across groups. Apixaban had no effect on PT at the concentrations tested. Apixaban 110 ng/ml prolonged mPT similarly across groups (44.4–53.2 s to 64.5–70.0 s); no prolongation was found with apixaban 30 ng/ml. Apixaban demonstrated consistent concentration-related effects on other PD endpoints in plasma samples from all age groups, except factor Xa inhibition.... R. J. Yetman (1), Y. C. Barrett (2), Z. Wang (2), R. Adamczyk (2), J. Wang (2), E. Ramacciotti (2), C. Frost (2) 27569 2017-05-24 10:20:22 Ahead of print: Increased risks of venous thromboembolism in patients with psoriasis Systemic inflammation and hypercoagulability in psoriasis are related to cardiovascular morbidity. The aim of the study was to investigate the incidence and risk of venous thromboembolism (VTE) in patients with psoriasis in Taiwan. We identified inpatients aged ≥ 18 years with a diagnosis of psoriasis and controls at a 1: 1 ratio of frequency matched by sex, age, frequency of medical visits, length of stay, and comorbidities between 2000 and 2010 in the Taiwan National Health Insurance Research Database. Each patient was traced to the date of VTE occurrence, loss to follow-up, death, or the December 31, 2011, whichever occurred first. We analysed 8945 patients with psoriasis and 8945 controls. The patients with psoriasis exhibited a greater incidence rate of VTE (19.2 vs 9.88 per 10 000 person-years) than did the controls. After adjustment for covariates, the patients with psoriasis presented a 2.02-fold risk of VTE (adjusted hazard ratio [aHR] = 2.02, 95 % confidence interval [CI] = 1.42–2.88) compared with that in the control cohort. The aHR of VTE was significantly higher in the first year of follow-up (aHR = 3.30, 95 % CI = 1.45–7.55) than after one year (aHR = 1.68, 95 % CI = 1.13–2.49).... W.-S. Chung (1, 2, 3), C.-L. Lin (4, 5) 27568 2017-05-24 10:19:11 Ahead of print: Low discriminating power of the modified Ottawa VTE risk score in a cohort of... Treatment of patients with cancer-associated venous thromboembolism (VTE) remains a major challenge. The modified Ottawa score is a clinical prediction rule evaluating the risk of VTE recurrences during the first six months of anticoagulant treatment in patients with cancer-related VTE. We aimed to validate the Ottawa score using data from the RIETE registry. A total of 11,123 cancer patients with VTE were included in the analysis. According to modified Ottawa score, 2,343 (21 %) were categorised at low risk for VTE recurrences, 4,525 (41 %) at intermediate risk, and 4,255 (38 %) at high risk. Overall, 477 episodes of VTE recurrences were recorded during the course of anticoagulant therapy, with an incidence rate for low, intermediate, and high risk groups of 6.88 % (95 % CI 5.31–8.77), 11.8 % (95 % CI 10.1–13.6), and 21.3 % (95 % CI 18.8–24.1) patient-years, respectively. Overall mortality had an incidence rate of 21.1 % (95 % CI 18.2–24.3), 79.4 % (95 % CI: 74.9–84.1), and 134.7 % (95 % CI: 128.3–141.4) patient-years, respectively. The accuracy and discriminating power of the modified Ottawa score for VTE recurrence was modest, with low sensitivity, specificity and positive predictive value, and a C-statistics of 0.58 (95 % CI: 0.56–0.61). In our analysis, the modified Ottawa score did not accurately predict VTE recurrence among patients with cancer-associated thrombosis, thus hindering its use in clinical practice. It is time to define a new score including other clinical predictors.... A. Alatri (1), L. Mazzolai (1), C. Font (2), A. Tafur (3), R. Valle (4), P. J. Marchena (5), A. Ballaz (6), E. Tiraferri (7), L. Font (8), M. Monreal (9), the RIETE Investigators 27547 2017-05-18 10:26:03 Ahead of print: CD32 inhibition and high dose of rhFVIII suppress murine FVIII-specific recall... Development of neutralising antibodies (inhibitors) against factor VIII (FVIII) is a frequent and severe complication of replacement therapy in haemophilia A. Previous data from haemophilia A mouse model demonstrates that both CD32 inhibition and high doses of rhFVIII prevent the differentiation of FVIII-specific memory B cells (MBCs) into antibody secreting cells (ASCs). Here, cellular targets responsible for the suppression of ASC formation by means of CD32 inhibition and high dose of rhFVIII were analysed. We investigated apoptosis on FVIII-specific MBCs using a pan caspases inhibitor, and screened for defects in rhFVIII presentation by analysing T cell release of Th1- and Th2-cytokines in vitro. Although high-dose of rhFVIII suppressed ASC formation, cytokine response was not affected. Upon re-stimulation of splenocytes with a high dose of rhFVIII, prevention of apoptosis fully restored the FVIII-specific recall response. In contrast, genetic deletion or inhibition of CD32 significantly altered Th1– and Th2-response. CD32 blockade and inhibition of apoptosis resulted in a partial rescue of FVIII-specific ASCs. Normal cytokine secretion could not be restored. In conclusion, suppression of FVIII-specific recall response by CD32 and high doses of rhFVIII is mediated by distinct mechanisms. High dose of rhFVIII induces apoptosis in FVIII-specific MBCs but does not influence FVIII-specific T cell response. CD32 blockade, however, may suppress the FVIII-specific recall response by two ways: i) increasing apoptosis of FVIII-specific MBCs and ii) disturbing FVIII-specific T cell response by modulating presentation of rhFVIII to CD4+ T cells in vitro.... N. Vollack (1), J. Friese (1), S. Bergmann (1), A. Tiede (1), S. Werwitzke (1) 27525 2017-05-11 09:11:34