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Introduction: A hypercoagulable condition was described in patients with coronavirus disease 2019 (COVID-19) and proposed as a possible pathogenic mechanism contributing to disease progression and lethality., Aim: We evaluated if in-hospital administration of heparin improved survival in a large cohort of Italian COVID-19 patients., Methods: In a retrospective observational study, 2,574 unselected patients hospitalized in 30 clinical centers in Italy from February 19, 2020 to June 5, 2020 with laboratory-confirmed severe acute respiratory syndrome coronavirus-2 infection were analyzed. The primary endpoint in a time-to event analysis was in-hospital death, comparing patients who received heparin (low-molecular-weight heparin [LMWH] or unfractionated heparin [UFH]) with patients who did not. We used multivariable Cox proportional-hazards regression models with inverse probability for treatment weighting by propensity scores., Results: Out of 2,574 COVID-19 patients, 70.1% received heparin. LMWH was largely the most used formulation (99.5%). Death rates for patients receiving heparin or not were 7.4 and 14.0 per 1,000 person-days, respectively. After adjustment for propensity scores, we found a 40% lower risk of death in patients receiving heparin (hazard ratio = 0.60; 95% confidence interval: 0.49-0.74; E-value = 2.04). This association was particularly evident in patients with a higher severity of disease or strong coagulation activation., Conclusion: In-hospital heparin treatment was associated with a lower mortality, particularly in severely ill COVID-19 patients and in those with strong coagulation activation. The results from randomized clinical trials are eagerly awaited to provide clear-cut recommendations., Competing Interests: None declared., (Thieme. All rights reserved.)

Current evidence supports the use of low-dose aspirin for secondary cardiovascular prevention. By contrast, the benefit-to-risk ratio of aspirin use in primary prevention is debated: three contemporary randomized control trials have been conflicting, and meta-analyses have concluded for an unclear clinical benefit, based on the consideration that the reduction in thromboembolic events is counterbalanced by increased bleeding. The primary prevention setting is, however, a heterogeneous mix of subjects at highly variable cardiovascular risk. One possible explanation for the uncertainty of data interpretation is the progressive reduction in risk of major adverse cardiovascular events (MACEs) in primary prevention that has accompanied global education programs, leading patients to smoke less, exercise more, and increasingly take lipid-lowering therapies. Based on a meta-regression of the benefits and harm of aspirin therapy in primary prevention as a function of the 10-year risk of MACE, we favor a nuanced approach still, however, based on the evaluation of cardiovascular risk, acknowledging differences between patients and emphasizing an individualized assessment of both benefits and harm. After optimal control of cardiovascular risk factors, and when patients are less than 70 years of age, clinicians should assess the risk of MACE and base decision on such stratification, considering the risk of bleeding and patient preferences. Clinicians would then advise the use of aspirin in primary prevention patients at the highest risk of MACE who do not have a prohibitive risk of bleeding, and in the majority of cases after initiation of properly titrated statin therapy., Competing Interests: R.D.C. declares having received fees and honoraria from Bayer related to the topic. A.A. and P.M.R. have no conflict of interest to disclose., (Georg Thieme Verlag KG Stuttgart · New York.)

Studies evaluating the relationship between platelet indices and cardiovascular (CV) outcomes yielded conflicting results. We assessed the incidence of adverse events according to baseline quintiles of platelet indices in the prospective cohort of the Malmö Diet and Cancer Study. A total of 30,314 individuals (age 57 ± 8 years) were followed for a median of 16 years (468,490 person-years). Outcome measures included all-cause death, CV death, myocardial infarction (MI), and ischemic stroke. The fifth quintile of platelet count (> 274.6 × 10 9 /L) was associated with higher incidence of all-cause death (hazard ratio [HR] 1.20, 95% confidence interval [CI] 1.09-1.32, p  < 0.001), CV death (HR 1.19, 95% CI 1.00-1.42; p  = 0.044), MI (HR 1.32, 95% CI 1.12-1.54; p  = 0.001), and ischemic stroke (HR 1.27, 95% CI 1.08-1.50, p  = 0.004) compared with the first quintile (≤ 185 × 10 9 /L), and also associated with a lower survival, regardless of previous history of MI ( p for interaction = 0.58) or stroke ( p for interaction = 0.42). In the highest quintile, history of stroke had a higher risk of CV death (HR 3.18, 95% CI 1.54-6.54) compared with no previous stroke (HR 1.12, 95% CI 0.96-1.31). The risk of MI and stroke was greatest in the fifth quintile, regardless of previous MI or previous stroke, respectively. The risk of all adverse events was similar across different quintiles of mean platelet volume. In conclusion, elevated platelet count is associated with higher mortality and risk of CV events, regardless of previous MI and stroke. Platelet count may thus be a useful marker for further stratification of CV risk, and especially of death., Competing Interests: G.P.: Speaker/consultant/advisory board for Amgen, Sanofi, Bayer, Boehringer-Ingelheim, BMS-Pfizer, Daiichi Sankyo, Astra Zeneca, Sigma-Tau, Malesci, PIAM, and MSD, outside the submitted work. G.R.: Speaker/consultant/advisory board for Boehringer-Ingelheim, Daiichi-Sankyo, and Bayer. V.H.: Educational funding from Boston Scientific and shareholder in AstraZeneca and Swedish Orphan Biovitrum, outside the submitted work. R.S.: Consultant to Medtronic Inc., member of speakers' bureau of Abbott Labs and stock holder in Edwards Lifescience, Boston Scientific Inc., and AstraZeneca PLC. R.D.C.: Fees, honoraria, and research funding from Sanofi-Aventis, Boehringer-Ingelheim, Bayer, BMS/Pfizer, Daiichi-Sankyo, Novartis, MSD, and Portola, outside the submitted work. G.R.: Personal fees from Bayer, Boehringer-Ingelheim, Daiichi Sankyo, outside the submitted work. A.F.: Speaker/consultant/advisory board for Medtronic and honoraria and royalties from Cardiome and Thermofisher, outside the submitted work. R.S.: Other from Boston Scientific, Astra Zeneca PLC, Edwards Lifescience, personal fees from Medtronic, Abbott laboratories, outside the submitted work. All other authors report no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Non-vitamin K antagonist oral anticoagulants (NOACs) include dabigatran, which inhibits thrombin, and apixaban, betrixaban, edoxaban and rivaroxaban, which inhibit factor Xa. In large clinical trials comparing the NOACs with the vitamin K antagonist (VKA) warfarin, dabigatran, apixaban, rivaroxaban and edoxaban were at least as effective for stroke prevention in atrial fibrillation and for treatment of venous thromboembolism, but were associated with less intracranial bleeding. In addition, the NOACs are more convenient to administer than VKAs because they can be given in fixed doses without routine coagulation monitoring. Consequently, the NOACs are now replacing VKAs for these indications, and their use is increasing. Although, as a class, the NOACs have a favourable benefit-risk profile compared with VKAs, choosing among them is complicated because they have not been compared in head-to-head trials. Therefore, selection depends on the results of the individual trials, renal function, the potential for drug-drug interactions and preference for once- or twice-daily dosing. In addition, several 'special situations' were not adequately studied in the dedicated clinical trials. For these situations, knowledge of the unique pharmacological features of the various NOACs and judicious cross-trial comparison can help inform prescription choices. The purpose of this position article is therefore to help clinicians choose the right anticoagulant for the right patient at the right dose by reviewing a variety of special situations not widely studied in clinical trials., Competing Interests: The authors declare the following potential conflicts of interest: Raffaele De Caterina declares fees, honoraria and research funding from Sanofi-Aventis, Boehringer Ingelheim, Bayer, BMS/Pfizer, Daiichi-Sankyo, Novartis, MSD and Portola. Walter Ageno declares honoraria from Boehringer Ingelheim, Bayer, Bristol-Myers Squibb/Pfizer, Daiichi Sankyo, Aspen, Stago, CSL Behring and Portola, and research funding from Bayer. Giancarlo Agnelli declares honoraria from Bayer, BMS/Pfizer, Daiichi Sankyo and Sanofi. Noel C. Chan declares honoraria from Bayer and Boehringer Ingelheim. Hans-Christoph Diener declares honoraria for participation in clinical trials, contribution to advisory boards or oral presentations from: Abbott, Achelios, Allergan, AstraZeneca, Bayer Vital, BMS, Boehringer Ingelheim, CoAxia, Corimmun, Covidien, Daiichi-Sankyo, D-Pharm, Fresenius, GlaxoSmithKline, Janssen-Cilag, Johnson & Johnson, Knoll, Lilly, MSD, Medtronic, MindFrame, Neurobiological Technologies, Novartis, Novo-Nordisk, Paion, Parke-Davis, Pfizer, Portola, Sanofi-Aventis, Schering-Plough, Servier, Solvay, St. Jude, Syngis, Talecris, Thrombogenics, WebMD Global, Wyeth and Yamanouchi. Financial support for research projects was provided by AstraZeneca, GSK, Boehringer Ingelheim, Lundbeck, Novartis, Janssen-Cilag, Sanofi-Aventis, Syngis and Talecris. The Department of Neurology at the University Duisburg-Essen received research grants from the German Research Council (DFG), German Ministry of Education and Research (BMBF), European Union, NIH, Bertelsmann Foundation and Heinz-Nixdorf Foundation. Hans-Christoph Diener has no ownership interest and does not own stocks of any pharmaceutical company. Within the past year he served as editor of Aktuelle Neurologie, Arzneimitteltherapie, Kopfschmerznews, Stroke News, as co-editor of Cephalalgia, and is on the editorial board of Lancet Neurology, Stroke, European Neurology and Cerebrovascular Disorders. He chairs the Treatment Guidelines Committee of the German Society of Neurology and contributed to the EHRA and ESC guidelines for the treatment of AF. Elaine Hylek declares honoraria from Bayer, Boehringer Ingelheim, Bristol Myers Squibb/Pfizer, DOASENSE, Janssen, Medtronic and Portola, and research funding from Janssen. Gary E. Raskob declares consultant fees and/or honoraria from Bayer, BMS, Boehringer-Ingelheim, Daiichi-Sankyo, Eli-Lilly, Janssen, Johnson & Johnson, Novartis, Pfizer and Portola. Deborah M. Siegal declares honoraria for presentations from Bayer, Portola, BMS-Pfizer and Servier. Freek W.A. Verheugt declares having received educational and research grants from Bayer Healthcare, as well as honoraria for consultancies/presentations from Bayer Healthcare, BMS/Pfizer, Daiichi-Sankyo and Boehringer Ingelheim. Gregory Y. H. Lip is a consultant for Bayer/Janssen, BMS/Pfizer, Medtronic, Boehringer Ingelheim, Novartis, Verseon and Daiichi-Sankyo; and speaker for Bayer, BMS/Pfizer, Medtronic, Boehringer Ingelheim and Daiichi-Sankyo. No fees are directly received personally. Jeffrey I. Weitz declares consultancy fees and honoraria from Boehringer Ingelheim, Bayer, Janssen, BMS/Pfizer, Daiichi-Sankyo, Novartis, Portola, IONIS and Servier., (Georg Thieme Verlag KG Stuttgart · New York.)

Management strategies for patients with atrial fibrillation (AF) in association with valvular heart disease (VHD) have been less informed by randomized trials, which have largely focused on ‘non-valvular AF’ patients. Thromboembolic risk also varies according to valve lesion and may also be associated with CHA2DS2-VASc score risk factor components, rather than only the valve disease being causal. Given the need to provide expert recommendations for professionals participating in the care of patients presenting with AF and associated VHD, a task force was convened by the European Heart Rhythm Association (EHRA) and European Society of Cardiology (ESC) Working Group (WG) on Thrombosis, with representation from the ESC WG on Valvular Heart Disease, Heart Rhythm Society (HRS), Asia Pacific Heart Rhythm Society (APHRS), South African Heart (SA Heart) Association and Sociedad Latinoamericana de Estimulación Cardíaca y Electrofisiología (SOLEACE) with the remit to comprehensively review the published evidence, and to produce a consensus document on the management of patients with AF and associated VHD, with up-to-date consensus statements for clinical practice for different forms of VHD, based on the principles of evidence-based medicine. This is an executive summary of a consensus document which proposes that the term ‘valvular AF’ is outdated and given that any definition ultimately relates to the evaluated practical use of oral anticoagulation (OAC) type, we propose a functional EHRA (Evaluated Heartvalves, Rheumatic or Artificial) categorization in relation to the type of OAC use in patients with AF, as follows: (1) EHRA (Evaluated Heartvalves, Rheumatic or Artificial) type 1 VHD, which refers to AF patients with ‘VHD needing therapy with a vitamin K antagonist (VKA)’ and (2) EHRA (Evaluated Heartvalves, Rheumatic or Artificial) type 2 VHD, which refers to AF patients with ‘VHD needing therapy with a VKA or a non-VKA oral anticoagulant also taking into consideration CHA2DS2-VASc score risk factor components., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Concepts and our approaches to stroke prevention in atrial fibrillation (AF) have changed markedly over the last decade. There has been an evolution over the approach to stroke and bleeding risk assessment, as well as new treatment options. An increasing awareness of AF has led to calls to improve the detection of and population screening for AF. Stroke and bleeding risk assessment continues to evolve, and the ongoing debate on balance between simplicity and practicality, against precision medicine will continue. In this review article, we provide an overview of past, present and the (likely) future concepts and approaches to stroke prevention in AF. We propose three simple steps (the Birmingham '3-step') that offers a practical management pathway to help streamline and simplify decision-making for stroke prevention in patients with AF.

The hypothesis of atherosclerosis as an inflammatory process has been a leitmotiv in cardiology for the past 20 years, and has now led to the launch of clinical trials aimed at testing whether drugs that primarily target inflammation can reduce cardiovascular events. Inflammation indeed drives all phases of atherosclerosis, from inception, through progression, and ultimately acute thrombotic complications (plaque rupture and probably plaque erosion). Since plaque rupture and erosion cause most acute coronary syndromes, appropriately tuned anti-inflammatory treatments should limit myocardial infarction and cardiovascular death. Beyond interrupting inflammation-related plaque disruption, such treatments might, however, also ameliorate the propensity to thrombosis once the trigger (plaque rupture or erosion) has occurred. Several lines of evidence support this view: experimental data document the role of inflammation in platelet activation, tissue factor-mediated coagulation, hyperfibrinogenaemia, impaired activity of natural anticoagulants (including those expressed by endothelial cells), and reduced fibrinolytic activity. Supporting evidence also derives from the involvement of inflammation in venous thrombosis, a process that commonly occurs in the absence of traditional risk factors for atherosclerosis but is associated with several inflammatory diseases including obesity. Ongoing trials, in addition to evaluating effects on primary outcomes, will afford the opportunity to probe the possibility that anti-inflammatory interventions that yield salutary changes in biomarkers of the thrombotic/fibrinolytic balance also translate into reduction of clinical events.

Until recently, vitamin K antagonists (VKAs) were the only available oral anticoagulants evaluated for long-term treatment of patients with coronary heart disease (CHD), particularly after an acute coronary syndrome (ACS). Despite efficacy in this setting, VKAs are rarely used because they are cumbersome to administer. Instead, the more readily manageable antiplatelet agents are the mainstay of prevention in ACS patients. This situation has the potential to change with the introduction of non-VKA oral anticoagulants (NOACs), which are easier to administer than VKAs because they can be given in fixed doses without routine coagulation monitoring. The NOACs include dabigatran, which inhibits thrombin, and apixaban, rivaroxaban and edoxaban, which inhibit factor Xa. Apixaban and rivaroxaban were evaluated in phase III trials for prevention of recurrent ischaemia in ACS patients, most of whom were also receiving dual antiplatelet therapy with aspirin and clopidogrel. Although at the doses tested rivaroxaban was effective and apixaban was not, both agents increased major bleeding. The role for the NOACs in ACS management, although promising, is therefore complicated, because it is uncertain how they compare with newer antiplatelet agents, such as prasugrel, ticagrelor or vorapaxar, and because their safety in combination with these other drugs is unknown. Ongoing studies are also now evaluating the use of NOACs in non-valvular atrial fibrillation patients, where their role is established, with coexistent ACS or coronary stenting. Focusing on CHD, we review the results of clinical trials with the NOACs and provide a perspective on their future incorporation into clinical practice.

Bivalirudin is a valuable anticoagulant option in patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention. Advantages over heparin as a parenteral anticoagulant include more predictable pharmacokinetics and pharmacodynamics, shorter half-life, no need for cofactors, some degree of antiplatelet effect, and the ability to inhibit clot-bound thrombin. Clinical evidence supporting the use of bivalirudin over heparin in current ACS guidelines, however, derives mostly from early randomised trials that may no longer reflect current management patterns, now including the use of oral antiplatelet agents more potent than clopidogrel (i.e. prasugrel or ticagrelor) and a broader implementation of strategies to reduce bleeding (i.e. radial access for percutaneous coronary intervention, and use of glycoprotein IIb/IIIa inhibitors only in bailout situations). Defining the fine balance between bivalirudin efficacy and safety over heparins in the context of other antithrombotic treatments remains a challenge in clinical practice, particularly in a fast-evolving scenario, such as ACS, where numerous new trials have been presented in very recent times. Here we provide an up-to-date overview of the evidence on the use of bivalirudin in ACS, with focus on new data, open issues, and future directions.

Due to improved implementation of guidelines, new scoring approaches to improve risk categorisation, and introduction of novel oral anticoagulants, medical management of patients with atrial fibrillation (AF) is continuously improving. The PREFER in AF registry enrolled 7,243 consecutive patients with ECG-confirmed AF in seven European countries in 2012-2013 (mean age: 71.5 ± 10.7 years; 60.1% males; mean CHA2DS2-VASc score: 3.4). While patient characteristics were generally homogeneous across countries, anticoagulation management showed important differences: the proportion of patients taking vitamin K antagonists (VKAs) varied between 86.0% (in France) and 71.4% (in Italy). Warfarin was used predominantly in the UK and Italy (74.9% and 62.0%, respectively), phenprocoumon in Germany (74.1%), acenocoumarol in Spain (67.3%), and fluindione in France (61.8 %). The major sites for international normalised ratio (INR) measurements were biology laboratories in France, anticoagulation clinics in Italy, Spain, and the UK, and physicians' offices or self-measurement in Germany. Temporary VKA discontinuation and bridging with other anticoagulants was frequent (at least once in the previous 12 months for 22.9% of the patients, on average; ranging from 29.7% in Germany to 14.9% in the UK). Time in therapeutic range (TTR), defined as at least two of the last three available INR values between 2.0-3.0 prior to enrolment, ranged from 70.3% in Spain to 81.4% in Germany. TTR was constantly overestimated by physicians. While the type and half-lives of VKA as well as the mode of INR surveillance differed, overall quality of anticoagulation management by TTR was relatively homogenous in AF patients across countries.

Oral anticoagulants are a mainstay of cardiovascular therapy, and for over 60 years vitamin K antagonists (VKAs) were the only available agents for long-term use. VKAs interfere with the cyclic inter-conversion of vitamin K and its 2,3 epoxide, thus inhibiting γ-carboxylation of glutamate residues at the amino-termini of vitamin K-dependent proteins, including the coagulation factors (F) II (prothrombin), VII, IX and X, as well as of the anticoagulant proteins C, S and Z. The overall effect of such interference is a dose-dependent anticoagulant effect, which has been therapeutically exploited in heart disease since the early 1950s. In this position paper, we review the mechanisms of action, pharmacological properties and side effects of VKAs, which are used in the management of cardiovascular diseases, including coronary heart disease (where their use is limited), stroke prevention in atrial fibrillation, heart valves and/or chronic heart failure. Using an evidence-based approach, we describe the results of completed clinical trials, highlight areas of uncertainty, and recommend therapeutic options for specific disorders. Although VKAs are being increasingly replaced in most patients with non-valvular atrial fibrillation by the new oral anticoagulants, which target either thrombin or FXa, the VKAs remain the agents of choice for patients with atrial fibrillation in the setting of rheumatic valvular disease and for those with mechanical heart valves.

Anticoagulants are a mainstay of cardiovascular therapy, and parenteral anticoagulants have widespread use in cardiology, especially in acute situations. Parenteral anticoagulants include unfractionated heparin, low-molecular-weight heparins, the synthetic pentasaccharides fondaparinux, idraparinux and idrabiotaparinux, and parenteral direct thrombin inhibitors. The several shortcomings of unfractionated heparin and of low-molecular-weight heparins have prompted the development of the other newer agents. Here we review the mechanisms of action, pharmacological properties and side effects of parenteral anticoagulants used in the management of coronary heart disease treated with or without percutaneous coronary interventions, cardioversion for atrial fibrillation, and prosthetic heart valves and valve repair. Using an evidence-based approach, we describe the results of completed clinical trials, highlight ongoing research with currently available agents, and recommend therapeutic options for specific heart diseases.

Contrary to previous models based on plasma, coagulation processes are currently believed to be mostly cell surface-based, including three overlapping phases: initiation, when tissue factor-expressing cells and microparticles are exposed to plasma; amplification, whereby small amounts of thrombin induce platelet activation and aggregation, and promote activation of factors (F)V, FVIII and FXI on platelet surfaces; and propagation, in which the Xase (tenase) and prothrombinase complexes are formed, producing a burst of thrombin and the cleavage of fibrinogen to fibrin. Thrombin exerts a number of additional biological actions, including platelet activation, amplification and self-inhibition of coagulation, clot stabilisation and anti-fibrinolysis, in processes occurring in the proximity of vessel injury, tightly regulated by a series of inhibitory mechanisms. "Classical" anticoagulants, including heparin and vitamin K antagonists, typically target multiple coagulation steps. A number of new anticoagulants, already developed or under development, target specific steps in the process, inhibiting a single coagulation factor or mimicking natural coagulation inhibitors.

Matrix metalloproteinase (MMP)-9 plays an important role in stroke by accelerating matrix degradation, disrupting the blood-brain barrier and increasing infarct size. Dipyridamole is an antiplatelet agent with recognised benefits in ischaemic stroke prevention. In addition to its antiplatelet properties, recent studies have reported that dipyridamole also features anti-inflammatory and anti-oxidant properties. We therefore investigated whether dipyridamole can ameliorate the proinflammatory profile of human monocytes, a source of MMP-9 in stroke, in terms of regulation of MMP-9 activity and expression, and explored underlying mechanisms. Human peripheral blood mononuclear cells (PBMC) and U937 cells were treated with increasing concentrations of dipyridamole (up to 10 µg/ml) for 60 minutes before stimulation with tumour necrosis factor (TNF)-α or phorbol myristate acetate (PMA). Exposure of PBMC and U937 to dipyridamole reduced TNF-α- and PMA-induced MMP-9 activity and protein release as well as MMP-9 mRNA, without significantly affecting the release of TIMP-1. This inhibitory effect was independent of dipyridamole-induced cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) increase. Correspondingly, dipyridamole also significantly inhibited TNF-α-induced nuclear factor (NF)-κB activation and nuclear translocation of the p65 NF-κB subunit through a mechanism involving the inhibition of IkBα degradation and p38 MAPK activation. In conclusion, dipyridamole, at therapeutically achievable concentrations, reduces the expression and release of MMP-9 through a mechanism involving p38 MAPK and NF-κB inhibition. These results indicate that dipyridamole exerts anti-inflammatory properties in human monocytes that may favourably contribute to its actions in the secondary prevention of stroke, independent of its antiplatelet properties.

The liver possesses impressive regenerative capacities. Grafts of embryonic liver explants and liver explant-conditioned media have been shown to enhance the mitotic activity of hepatocytes. Hepatocyte growth factor (HGF), also named scatter factor (SF), has been identified as a primary candidate in promoting and regulating liver regeneration. Although initially thought to be a liver-specific mitogen, HGF was later reported to have mitogenic, motogenic, morphogenic, and anti-apoptotic activities in various cell types. By promoting angiogenesis and inhibiting apoptosis, endogenous HGF may play an important role in cardioprotection as well as in the regeneration of endothelial cells and cardiomyocytes after myocardial infarction. Since serum concentration of HGF increases in the early phase of myocardial infarction and in heart failure, HGF may also play a key role as a prognostic and diagnostic biomarker of cardiovascular disease. Here we discuss the role of HGF as a biomarker and mediator in cardioprotection and cardiovascular regeneration.

Platelet glycoprotein IIb/IIIa inhibitors (GPI) are antithrombotic agents preventing the binding of fibrinogen to GP IIb/IIIa receptors. Thus, GPI interfere with interplatelet bridging mediated by fibrinogen. Currently, three generic GPI with different antithrombotic properties are available for intravenous administration: abciximab, eptifibatide, and tirofiban. The development of oral GPI was abandoned, whereas intravenous GPI were introduced in various clinical settings during the 1990s, yielding substantial benefit in the treatment of acute coronary syndromes, particularly during percutaneous coronary interventions. Results of the many randomised trials evidenced the efficacy of this drug class, though these trials were conducted prior to the emergence of modern oral antiplatelet therapy with efficient P2Y(12) inhibitors. Subsequent trials failed to consolidate the strongly favourable impression of GPI, and indications for their use have been more restricted in recent years. Nonetheless, GPI may still be beneficial during coronary interventions among high-risk patients including acute ST-elevation and non-ST-elevation myocardial infarctions, particularly in the absence of adequate pretreatment with oral antiplatelet drugs or when direct thrombin inhibitors are not utilised. Intracoronary GPI administration has been suggested as adjunctive therapy during primary percutaneous coronary intervention, and the results of larger ongoing trials are expected to elucidate its clinical potential. The present review outlines the key milestones of GPI development and provides an up-to-date overview of the clinical applicability of these drugs in the era of refined coronary stenting, potent antithrombotic drugs, and novel thrombin inhibiting agents.

Microvascular dysfunction occurs in insulin resistance and/or hyperinsulinaemia. Enhanced uptake of free fatty acids (FFA) and oxidised low-density lipoproteins (oxLDL) may lead to oxidative stress and microvascular dysfunction interacting with CD36, a PPARα/γ-regulated scavenger receptor and long-chain FFA transporter. We investigated CD36 expression and CD36-mediated oxLDL uptake before and after insulin treatment in human dermal microvascular endothelial cells (HMVECs), ± different types of fatty acids (FA), including palmitic, oleic, linoleic, arachidonic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. Insulin (10(-8) and 10(-7) M) time-dependently increased DiI-oxLDL uptake and CD36 surface expression (by 30 ± 13%, p<0.05 vs. untreated control after 24 hours incubation), as assessed by ELISA and flow cytometry, an effect that was potentiated by the PI3-kinase inhibitor wortmannin and reverted by the ERK1/2 inhibitor PD98059 and the PPARα/γ antagonist GW9662. A ≥ 24 hour exposure to 50 μM DHA or EPA, but not other FA, blunted both the constitutive (by 23 ± 3% and 29 ± 2%, respectively, p<0.05 for both) and insulin-induced CD36 expressions (by 45 ± 27 % and 12 ± 3 %, respectively, p<0.05 for both), along with insulin-induced uptake of DiI-oxLDL and the downregulation of phosphorylated endothelial nitric oxide synthase (P-eNOS). At gel shift assays, DHA reverted insulin-induced basal and oxLDL-stimulated transactivation of PPRE and DNA binding of PPARα/γ and NF-κB. In conclusion, omega-3 fatty acids blunt the increased CD36 expression and activity promoted by high concentrations of insulin. Such mechanisms may be the basis for the use of omega-3 fatty acids in diabetic microvasculopathy.

Hyperinsulinemia predicts future cardiovascular events, but may also contribute to atherosclerosis. We therefore studied the consequences of prolonged insulin treatment of human umbilical vein endothelial cells (HUVEC) on the phosphatidylinositol-3'-kinase(PI3K)/Akt/nitric oxide(NO)-dependent insulin signaling, together with the expression of the pro-atherogenic molecule vascular cell adhesion molecule (VCAM)-1. HUVEC were incubated with insulin (10(-11) to 10(-7) M) in short- (30 min) and long-term (24 h to 3 days) incubations. In short-term incubations, insulin did not affect constitutive Akt and eNOS at any concentration, but significantly increased their active phosphorylated forms, and NO production. In long-term incubations, however, such insulin effects on the phosphorylated forms, as well as NO production, were attenuated, promoting an effect of insulin also at concentrations otherwise ineffective. Such effects were accompanied by a boosting of insulin effect on VCAM-1 surface expression. In contrast, under similar conditions, insulin did not exert any significant effect on the surface expression of ICAM-1 and E-selectin. Therefore, prolonged exposure of HUVEC to high insulin levels induces a downregulation of the PI3K/Akt/eNOS axis. Such impairment of insulin signalling in states of prolonged hyperinsulinemia pontially contributes to detrimental effects on atherogenesis in insulin resistance states, such as the metabolic syndrome and type 2 diabetes.

Patients with critical limb ischemia (CLI) have low levels of endothelial progenitor cells (EPC). Iloprost has been demonstrated to stimulate vascular endothelial growth factor (VEGF) and promote angiogenesis. We investigated the effects of iloprost on EPC levels in vivo in CLI patients. Twenty-three patients with stage III and IV CLI were treated with iloprost for four weeks, improving clinical and instrumental parameters. Mononuclear cells isolated from peripheral blood were cultured to obtain "early" EPC, evaluated counting adherent cells with double positivity for acetylated low-density lipoprotein uptake and Ulex Europaeus lectin at flow cytometry. These cells also co-expressed the monocyte markers CD14 and CD45. Iloprost increased EPC number in the whole patient population: pre-treatment median: 13,812/ml; range: 1,263-83,648/ml; post-treatment median: 23,739/ml; range: 3,385-99,251/ml; p = 0.035, irrespective of age, sex, disease stage or atherosclerosis risk factors. In conclusion, iloprost increases EPC number in peripheral blood in vivo. Such an effect may have therapeutic relevance.

NAD(P)H oxidase is a prominent source of reactive oxygen species in the vasculature. Vascular NAD(P)H oxidase is comprised of several subunits, one of which, p22phox, is encoded by a gene exhibiting several allelic variants. Here the C(242)T nucleotide transition has been found to alter superoxide anion production and associated with an altered risk of coronary artery disease (CAD). We assessed the role of this variant in two case-control studies, and performed a meta-analysis of previously reported investigations relating it to vascular risk. Population I was comprised of 492 subjects with type 2 diabetes, with or without macrovascular disease, matched for age, sex, and duration of diabetes. Population II was comprised of 158 subjects with or without either CAD or cerebro-vascular disease, and matched for age, sex, smoking status, weight category and the presence of hypertension, dyslipidemia, and diabetes. Our findings were meta-analyzed together with additional studies retrieved from the literature. The C(242)T polymorphism distribution did not differ between cases and controls in populations I and II both at univariate and multivariate analyses, and this was confirmed in a meta-analysis with 11 previously published populations. The meta-analysis, however, suggested a protective role of the T allele on CAD as an end point in Asian populations. In conclusion, these data suggest a significant heterogeneity for a modulating role of the T allele in the C(242)T polymorphism of p22-phox for the occurrence of CAD across ethnicities, with the absence of a significant effect in Caucasians.

Tissue factor (TF) is expressed on the endothelium in response to inflammatory mediators, giving endothelial cells a pro-thrombotic phenotype. Since fish-derived n-3 fatty acids (FA) have been associated with reduced incidence of myocardial infarction, we investigated the endothelial effects of the most abundant n-3 FA, docosahexaenoate (DHA), on TF expression. Human umbilical vein endothelial cells were pre-incubated with DHA (or stearate and arachidonate as controls) for 48-72 hours, and then stimulated with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Pre-incubation of endothelial cells with DHA (but not stearate or arachidonate) concentration-dependently reduced surface protein exposure, independent of TF mRNA or total protein expression regulation. Conversely, DHA treatment in conjunction with activating stimuli, induced the release of endothelial TF-exposing microparticles from endothelial cells, quantitatively accounting for the decreased TF cell surface exposure. In conclusion, DHA treatment, with a time-course consistent with its incorporation in membrane phospholipids, increases the release of TF-exposing microparticles from endothelial cells, accounting for decreased endothelial cell TF surface exposure, thus potentially modifying the overall endothelial control of microparticle-related effects.

We previously showed that the exposure of vascular endothelium to oleate results in reduced endothelial activation. We now investigate possible mechanisms for this effect in relation to generation of reactive oxygen species (ROS). We stimulated several types of endothelial cells with cytokines or lipopolysaccharide, with or without preincubation with 10-100 mumol/L oleate. Oleate preincubation reduced VCAM-1 expression in all cell types, as well as macrophage-colony stimulating factor release. We simultaneously measured the concentration of intracellular glutathione (GSH), the activity of GSH-related antioxidant enzymes and the production of intracellular ROS. Stimulation of endothelial cells caused a decrease of GSH and an increase in intracellular ROS. The addition of oleate before stimulation, prevented the depletion of GSH and partially prevented stimuli-induced increase of intracellular ROS. This occurred without any change in the activity of GSH-related antioxidant enzymes, superoxide dismutase and catalase. Furthermore, in a cell-free superoxide anion-generating system, oleate quenched the generation of ROS. These results indicate that oleate may exert direct vascular atheroprotective effects by inhibiting endothelial activation through a quenching of stimuli-induced increase in ROS.

Because of the association of oral contraceptives (OC) and cigarette smoking with an increased thrombotic risk, we evaluated thromboxane (TX) and prostacyclin urinary (u) metabolites, as in vivo indices of platelet-vessel wall interactions, in women assigned to third generation OC. Twenty-eight women (15 smokers) underwent a 6-month trial of 30 microg ethinylestradiol plus 0.150 mg desogestrel. Cotinine plasma levels were elevated only in persons classified as smokers and serum TXB2 determination confirmed the absence of cyclooxygenase inhibition throughout the study. u-TXB2 and 11-dehydro-TXB2 were higher in smokers than in non-smokers. OC decreased u-11-dehydro-TXB2 both in smokers (from (pg/micromol creatinine) 35.1+/-6.9 to 15.8+/-2.8; P<0.025) and non-smokers (from 31.7+/-9.8 to 20.6+/-4.8, P = N.S.). u-6-keto-prostaglandin(PG)F1alpha excretion, also higher in smokers compared to non-smokers, was also reduced after OC in smokers (from (pg/micromol creatinine) 24.3+/-5.2 to 14.8+/-2.3; P<0.05). Smokers also had a trend to higher u-2,3-dinor-6-keto-PGF1alpha, marginally reduced by OC. Thus, the OC regimen used here improves - if anything - platelet vessel wall interactions as assessed by prostanoid production in vivo. The prothrombotic tendency associated with the use of OC in smokers does not appear to be mediated by changes in platelet-vessel wall interactions.

Aims: The recent publication of two large trials of secondary prevention of coronary artery disease with oral anticoagulants (WARIS and ASPECT) has caused a revival of the interest for this antithrombotic therapy in a clinical setting where the use of aspirin is common medical practice. Despite this, the preferential use of aspirin has been supported by an American cost-effectiveness analysis (JAMA 1995; 273: 965)., Methods and Results: Using the same parameters used in that analysis and incidence of events from the Antiplatelet Trialists Collaboration and the ASPECT study, we re-evaluated the economic odds in favor of aspirin or oral anticoagulants in the Italian Health System, which differs significantly in cost allocation from the United States system and is, conversely, similar to other European settings. Recalculated costs associated with each therapy were 2,150 ECU/ patient/year for oral anticoagulants and 2,187 ECU/patient/year for aspirin. In our analysis, the higher costs of oral anticoagulants versus aspirin due to a moderate excess of bleeding (about 10 ECU/ patient/year) and the monitoring of therapy (168 ECU/ patient/year) are more than offset by an alleged savings for recurrent ischemic syndromes and interventional procedures (249 ECU/ patient/year)., Conclusions: Preference of aspirin vs. oral anticoagulants in a pharmaco-economical perspective is highly dependent on the geographical situation whereupon calculations are based. On a pure cost-effectiveness basis, and in the absence of data of direct comparisons between aspirin alone versus I.N.R.-adjusted oral anticoagulants, the latter are not more expensive than aspirin in Italy and, by cost comparisons, in other European countries in the setting of post-myocardial infarction.

Indobufen ((+/-)-2-[p-(1-oxo-2-insoindolinyl)-phenyl]-butyric acid, indo) is a drug inhibiting platelet function by a reversible block of the arachidonic acid metabolism at the level of cyclooxygenase. Since tolerability profile of such drugs is mostly linked to extra-platelet cyclooxygenase inhibition, we prospectively evaluated the extent of platelet and extra-platelet cyclooxygenase inhibition by in vivo administration of indo in comparison with ASA. We assessed the effects of the two drugs on the ex vivo generation of TXB2 and 6-keto-PGF1 alpha in whole blood, as indices of the production of TXA2 and PGI2 (prostacyclin), respectively, either after spontaneous clotting at 37 degrees C for 1 h (Study 1) or after the addition of 2 micrograms/ml collagen (Study 2). Generation of 6-keto-PGF1 alpha in whole blood is a mixed index of platelet and extra-platelet cyclooxygenase activity, deriving from both platelet and white blood cell arachidonic acid metabolization. Fifteen patients with ischemic heart disease and baseline serum TXB2 levels > 300 ng/ml were allocated to receiving one single administration of either indobufen 200 mg (n = 6) or aspirin 500 mg (n = 9). Whole blood prostanoid generation was assessed at 0, 1, 2, 4, 6, 8, 12 and 24 h after drug administration (Study I). Ten healthy male volunteers were allocated to a double-blind, randomized crossover comparison of indo 200 mg b.i.d. vs. ASA 300 mg/d for 7 days (Study 2). Prostanoid generation and whole blood platelet aggregation were performed before and at the end of each study period (Day 0 and Day 7). At each time-point after single dose administration (Study 1), indobufen caused less % inhibition of whole blood 6-keto-PGF1 alpha than of TXB2. At 2 h, TXB2 was reduced to a similar extent after ASA (98 +/- 4%) and indo (97 +/- 6%) (p = N.S.), while inhibition of 6-keto-PGF1 alpha was clearly different ( > 98% after ASA, 81 +/- 2.5% after indo, p < 0.01). After one week of ASA or indo (Study 2) the maximum extent of whole blood platelet aggregation was similarly inhibited (from 17.2 +/- 1.4 ohms to 3.6 +/- 1.3 ohms with ASA; from 18.3 +/- 1.0 ohms to 1.6 +/- 0.7 ohms with indo (p ASA vs. indo = N.S.). Despite equal inhibition of whole blood TX production after collagen (from 49.0 +/- 4.3 ng/ml to 1.1 +/- 0.6 ng/ml with ASA, from 49.8 +/- 1.3 ng/ml to 1.4 +/- 0.6 ng/ml with indo), again, however, 6-keto-PGF1 alpha production was less affected by indo than by ASA (from 409 +/- 30 pg/ml to 37 +/- 13 pg/ml with ASA, inhibition = 91%; from 396 +/- 35 to 318 +/- 40 with indo, inhibition = 20%). These differential effects of indo and ASA might lead to a better platelet selectivity, tolerability and benefit/risk profile of indo vs. ASA, which are worthy of further assessment.

Indobufen is an antiplatelet drug able to inhibit thromboxane production and cyclooxygenase-dependent platelet aggregation by a reversible inhibition of cyclooxygenase. Indobufen exists in two enantiomeric forms, of which only d-indobufen is active in vitro in inhibiting cyclooxygenase. In order to verify that also inhibition of platelet function is totally accounted for by d-indobufen, ten patients with proven coronary artery disease (8 male, 2 female, age, mean +/- S.D., 58.7 +/- 7.5 years) were given, in random sequence, both 100 mg d-indobufen and 200 mg dl-indobufen as single administrations in a double-blind crossover design study with a washout period between treatments of 72 h. In all patients thromboxane (TX) B2 generation after spontaneous clotting (at 0, 1, 2, 4, 6, 8, 12, 24 h), drug plasma levels (at the same times), platelet aggregation in response to ADP, adrenaline, arachidonic acid, collagen, PAF, and bleeding time (at 0, 2, 12 h) were evaluated after each treatment. Both treatments determined peak inhibition of TXB2 production at 2 h from administration, with no statistical difference between the two treatments (97 +/- 3% for both treatments). At 12 h inhibition was 87 +/- 6% for d-indobufen and 88 +/- 6% for dl-indobufen (p = NS). Inhibition of TXB2 production correlated significantly with plasma levels of the drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Ticlopidine (T) and aspirin (ASA) are two antiplatelet drugs both capable of prolonging bleeding time (BT), with a different mechanism of action. A synergism in BT prolongation has been reported and is currently considered an argument for not recommending their combination. However, a profound suppression of platelet function might be a desirable counterpart of a marked prolongation of BT, with a possible use in selected clinical situations. We therefore studied ex vivo platelet function (aggregation by ADP 0.5-1-2.5 microM; adrenaline 0.75-2.5 microM; collagen 1.5-150 micrograms/ml; arachidonic acid 1 mM; PAF 1 microM; adrenaline 0.17 microM + ADP 0.62 microM; serum thromboxane [( TX]B2 generation) and BT (Mielke) in 6 patients with stable coronary artery disease receiving such combination. Patients underwent sequential laboratory evaluations at baseline, after 7 days of T 250 mg b.i.d., before and after the intravenous administration of ASA 500 mg, respectively, and, finally, after a minimum of 7 days of sole ASA oral administration (50 mg/day). The experimental design, therefore, allowed a comparison of T and ASA effects (2nd and 4th evaluation), and an assessment of the combination effect (3rd evaluation). Platelet aggregation in response to all doses of ADP was depressed more by T than by ASA. Conversely, responses to adrenaline, and arachidonate were affected more by ASA than by T. For all other agents, differences were not significant. T + ASA combination was more effective (p less than 0.05) than either treatment alone in depressing responses to high-dose collagen (% over control, mean +/- SEM: T: 95 +/- 3; ASA: 96 +/- 5; T + ASA: 89 +/- 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Isosorbide dinitrate inhibits platelet function in vivo at concentrations about 10 times lower than in vitro (10(-7)-10(-6) vs. 10(-6)-10(-5) M). We investigated two possible reasons for this difference. Isosorbide dinitrate and its in vivo longer-lived metabolites, isosorbide-2- and isosorbide-5-mononitrate were incubated for 5 min with human platelet-rich plasma or washed platelets; irreversible aggregation was induced with threshold doses of ADP, adrenaline, collagen, arachidonic acid and thrombin, and thromboxane (TX) B2 production was measured by radioimmunoassay. Moreover, the concentration of exogenous prostacyclin required to inhibit platelet aggregation by 50% (IC50) after preincubation with isosorbide dinitrate or vehicle was determined. At 10(-7) M, only isosorbide-2-mononitrate inhibited aggregation (-12%, p less than 0.05) and TX production (-36%, p less than 0.01) by ADP. At 10(-6) M isosorbide-2-mononitrate inhibited aggregation by adrenaline more than the dinitrate (-41% vs. -25%, p less than 0.05). In addition, at supra-threshold doses of all the aggregating agents, isosorbide dinitrate decreased IC50 of prostacyclin from 2.7 +/- 1.2 to 0.36 +/- 0.2 nM. Generation of a platelet-active metabolite and synergism with prostacyclin are new properties of isosorbide dinitrate that may account for antiplatelet effects in vivo.

To learn whether glucocorticoids inhibit prostaglandin (PG) production in vascular endothelial cells, we investigated the effects of glucocorticoids on PG synthesis by cultured human umbilical vein endothelial cells (EC). Pretreatment of EC with dexamethasone (DX, 10(-9) to 5 X 10(-5) M) caused a dose-dependent inhibition of PGI2 production when PG synthesis from endogenous arachidonate was stimulated by human thrombin (0.25-2 U/ml) or ionophore A 23187 (1-5 microM). The inhibition was detectable at 10(-7) M DX and maximal at 10(-5) M (4.0 +/- 0.7 vs. control: 7.7 +/- 1.9 ng/ml, mean +/- S.D., P less than 0.01). The production of PGE2 and the release of radiolabelled arachidonate (AA) from prelabelled cells were similarly inhibited. Prolonged incubation of EC with glucocorticoids was required to inhibit PG production or arachidonate release: ranging from 8% inhibition at 5 h to 44% at 38 h. In contrast, prostaglandin formation from exogenous AA was not altered by DX treatment. When thrombin or ionophore-stimulated EC were restimulated with exogenous AA (25 microM), DX-treated cells released more PGI2 than control cells (5.7 +/- 0.5 vs. 4.1 +/- 0.6 ng/ml, P less than 0.01). Both the decrease in PGI2 production after thrombin/ionophore and the increase after re-stimulation with AA were blunted in the presence of the protein synthesis inhibitor cycloheximide (0.1-0.2 micrograms/ml). Thus, incubation of EC with glucocorticoids inhibits PG production at the step of phospholipase activation. The time requirement for these steroid effects and their blunting by cycloheximide are consistent with the induction of regulatory proteins, possibly lipocortins, in endothelial cells.