Pharmacodynamic effects of EV-077 in patients with diabetes mellitus and coronary artery disease on aspirin or clopidogrel monotherapy: results of an in vitro pilot investigation.

Patients with diabetes mellitus (DM) have increased propensity to generate thromboxane A2 (TXA2) and other eicosanoids which can contribute to their heightened platelet reactivity. EV-077 is a potent thromboxane receptor antagonist and thromboxane synthase inhibitor and thus represents an attractive therapy in patients with DM. However, the effects of EV-077 on pharmacodynamic (PD) profiles in patients with DM and coronary artery disease (CAD) while on antiplatelet therapy is poorly explored and represented the aim of this in vitro pilot investigation. Patients with DM and stable CAD (n = 10) on low-dose aspirin (81 mg/day) were enrolled and then switched to clopidogrel (75 mg/day) monotherapy for 7-10 days. PD assessments were conducted while on aspirin and on clopidogrel using light transmittance aggregometry following stimuli with U-46619 [TXA2 stable analogue (7 µM)], arachidonic acid [AA (1 mM)], collagen (3 µg/mL) and adenosine diphosphate [ADP (5 µM and 20 µM)] with and without in vitro EV-077. EV-077 completely inhibited U-46619-stimulated platelet aggregation (p = 0.005 for both aspirin and clopidogrel) and also showed a significant reduction of collagen-induced aggregation (aspirin p = 0.008; clopidogrel p = 0.005). EV-077 significantly reduced AA-induced platelet aggregation in clopidogrel (p = 0.009), but not aspirin (p = 0.667) treated patients. Ultimately, EV-077 significantly reduced ADP-mediated platelet aggregation in both aspirin (ADP 5 µM p = 0.012; ADP 20 µM p = 0.032) and clopidogrel (ADP 5 µM p = 0.007; ADP 20 µM p = 0.008) treated patients. In conclusion, in DM patients with CAD on aspirin or clopidogrel monotherapy, in vitro EV-077 exerts potent platelet inhibitory effects on multiple platelet signaling pathways. These data support that EV-077 has only additive platelet inhibiting effects on top of standard antiplatelet therapies. These findings warrant further investigation in ex vivo settings.

The impact of factor Xa inhibition on axial dependent arterial thrombus formation triggered by a tissue factor rich surface.

This study was designed to assess the effect of Factor Xa antagonists on thrombus formation at various axial positions on a tissue factor rich surface under arterial blood flow conditions. Non-anticoagulated, flowing human blood, drawn directly from an antecubital vein, was perfused over a tissue factor coated cover slip in a parallel-plate perfusion chamber. Thrombus surface coverage, thrombus mean height and fibrin surface coverage were measured at six different axial positions by confocal microscopy. Both thrombus surface coverage and mean height decreased along the cover slip axis whereas the fibrin surface coverage increased. Pre-chamber treatment of blood with the direct Factor Xa inhibitors Razaxaban and 813893 resulted in significantly reduced thrombus and fibrin formation at all axial positions investigated (P < 0.05). Thrombus and fibrin deposition in a laminar flow chamber changed with axial position with surface coverage measurements being more reproducible than thrombus mean height. Data were more reproducible towards the centre of the flow chamber than at the extremities. Razaxaban and 813893 inhibited thrombus and fibrin formation at the highest concentrations tested. No difference in drug effect was apparent at different axial positions. In conclusion, axial position influences the degree of thrombus and fibrin deposition with measurements being less reproducible at the extremities of the flow chamber. This technique may prove useful for analysing anti-thrombotic drug effects before progression to clinical trials.

Global thrombosis test for assessing thrombotic status and efficacy of antithrombotic diet and other conditions.

Because of the high mortality from myocardial infarction and stroke, there is a great demand for finding novel methods of diagnosis, prevention and treatment of these diseases. Most of the current tests measure important determinants of thrombosis such as platelet function, coagulation and fibrinolysis in isolation; therefore, a global test measuring the actual thrombotic status would be more useful in clinical conditions. We obtained considerable experience by using the global thrombosis test, which determines the actual thrombotic status by taking into account the measured platelet reactivity, coagulation and fibrinolytic activities. In animal experiments, we found significant correlation between the ex vivo global thrombosis test measurements and the in vivo thrombotic status. The published evidence for the benefit of an antithrombotic diet with regular physical exercise is also described.

Prevention of thrombotic disorders by antithrombotic diet and exercise: evidence by using global thrombosis tests.

Prevention of thrombotic disorders has priority over treatment. There are only two pathologically relevant tests which are suitable for measuring the overall thrombotic status both in experimental conditions and in humans. The Global Thrombosis Test (GTT) and the Global Parallel-Plate Thrombosis Test can detect the pathologically relevant global thrombotic status. These tests have been successfully used for monitoring the effect of antithrombotic drugs and for developing novel antithrombotic agents. By using GTT, varieties of fruits, vegetables, and regular physical exercise have been tested for the effect on global thrombotic status. This review discusses the published evidence for the benefit of diet of selected fruit and vegetable varieties and doing regular physical exercise on improving thrombotic status. Future clinical trials monitored by GTT or Global Parallel-Plate Thrombosis Test could decide on the effectiveness of an experimentally proven antithrombotic diet with regular physical exercise in the prevention of thrombotic diseases.

Institutional profile: the national Swedish academic drug discovery & development platform at SciLifeLab.

The Science for Life Laboratory Drug Discovery and Development Platform (SciLifeLab DDD) was established in Stockholm and Uppsala, Sweden, in 2014. It is one of ten platforms of the Swedish national SciLifeLab which support projects run by Swedish academic researchers with large-scale technologies for molecular biosciences with a focus on health and environment. SciLifeLab was created by the coordinated effort of four universities in Stockholm and Uppsala: Stockholm University, Karolinska Institutet, KTH Royal Institute of Technology and Uppsala University, and has recently expanded to other Swedish university locations. The primary goal of the SciLifeLab DDD is to support selected academic discovery and development research projects with tools and resources to discover novel lead therapeutics, either molecules or human antibodies. Intellectual property developed with the help of SciLifeLab DDD is wholly owned by the academic research group. The bulk of SciLifeLab DDD's research and service activities are funded from the Swedish state, with only consumables paid by the academic research group through individual grants.

The impact of blood shear rate on arterial thrombus formation.

The shear rate and corresponding shear stress have impacts on arterial thrombus formation. In particular, the effects of increasing concentration of platelets at the vessel wall and activation of platelets at this site increase the growth and stability of the thrombi which may result in a fatal narrowing of the arterial lumen. The efficacy of many antithrombotic agents is shear dependent as well. It is apparent that there is a need for a point-of-care device to rapidly monitor the risk for arterial thrombosis and to optimize antithrombotic therapy in vitro. The present review focuses on the essential role of shear rate on arterial thrombus formation in native human blood drawn directly from an antecubital vein.

A cyclic pentapeptide derived from the second EGF-like domain of Factor VII is an inhibitor of tissue factor dependent coagulation and thrombus formation.

We have previously reported the finding of a cyclic dodecapeptide representing loop I of the second EGF-like domain of FVII, which inhibited TF-dependent FX activation (Orning et al. 1997). The biological activity was localized to the tripeptide motif, Glu-Gln-Tyr. We have now synthesized a cyclic analog of this motif, Cys-Glu-Gln-Tyr-Cys (PN7051), evaluated its anticoagulant and antithrombotic properties and performed a detailed structural characterization of the peptide. PN7051 is a dose-dependent inhibitor of TF-dependent FX activation and coagulation of plasma with IC50 values of 10+/-2 microM and 1.3+/-0.2 mM, respectively. It shows inhibitory efficacy on acute thrombus formation in an ex vivo model of human thrombosis using native blood. Fibrin deposition, platelet-fibrin adhesion, platelet-thrombus formation, and thrombin-antithrombin complex formation were all inhibited by PN7051 at IC50 values between 0.3 and 0.7 mM. The cyclic peptide is a non-competitive inhibitor of FX activation with no significant active-site effects on FXa or FVIIa, indicating it affects FVII/TF/FX complex formation and function. Studies on the structure activity relationship revealed that Gln3-Tyr4, but not Glu2 were of importance for inhibition. In line with biological results, NMR measurements of PN7051 suggested that the Gln and Tyr residues configure a structural feature that contributes to the anticoagulant activity. Modeling of the Glu99Gln100Tyr101 motif in FVII and comparison with the solution structure of PN705 I suggest that the cyclic pentapeptide exerts its antithrombotic effect by interfering with the docking of Tyr101 into a hydrophobic pocket in the catalytic domain thereby disrupting an essential interaction between the second EGF-like and the catalytic domains of FVII.

Strenuous but not moderate exercise increases the thrombotic tendency in healthy sedentary male volunteers.

We have investigated the effect of moderate and strenuous exercise on experimental arterial thrombus formation in men. Thrombogenesis was measured in 15 sedentary healthy male volunteers at rest or immediately after two standardized exercise tests performed for 30 min on a bicycle ergometer. The exercises were performed at a constant load corresponding to either 50 or 70% maximal oxygen uptake. Thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native nonanticoagulated blood for 3 min. The shear rate at the collagen surface was 2,600 s(-1). Platelet and fibrin deposition was quantified by immunoenzymatic methods. The results show that moderate exercise did not affect arterial thrombus formation. In contrast, platelet thrombus formation on collagen was increased on the average by 20% after 30 min at 70% maximal oxygen uptake (P = 0.03). Fibrin deposition on collagen remained unchanged with exercise, regardless of its intensity. Thus, with the use of a clinically relevant human experimental model of thrombosis, the present study suggests that exercise of heavy intensity may increase the risk for arterial thrombogenesis in sedentary young healthy male volunteers.

Effect of radiologic contrast media on cell volume regulatory mechanisms in human red blood cells.

RATIONALE AND OBJECTIVES: The authors performed this study to evaluate cell volume regulation in human red blood cells (RBCs) after incubation in solutions of three contrast media: iohexol (830 mOsm), ioxaglate (520 mOsm), and iodixanol (300 mOsm). MATERIALS AND METHODS: Whole blood sampled from six healthy subjects was exposed to Ringer solutions containing 25% or 5% vol/vol iohexol (final osmolality, 440 or 340 mOsm, respectively), ioxaglate (final osmolality, 395 or 335 mOsm, respectively), iodixanol (final osmolality, 330 or 315 mOsm, respectively), or NaCl (control solutions with the same osmolality as that of the contrast media). In some experiments, control RBCs were subjected to a hyposmotic solution (100 mOsm). RBC volumes were obtained with a Coulter counter. RESULTS: The RBCs showed normal regulatory cell shrinkage after hyposmotically induced swelling. All 25% vol/vol contrast material solutions and their control solutions induced RBC shrinkage (range, 6% +/- 1 [standard error] to 22% +/- 3). The same was true for cells exposed to 5% vol/vol contrast material (range, 4% +/- 1 to 7% +/- 1). The shrinkage phase was followed by cell swelling (10% +/- 2 to 20% +/- 2 for 25% contrast material and their control solutions and 8% +/- 1 to 15% +/- 2 for 5% contrast material and their control solutions). No contrast material-exposed RBCs increased their volumes to the level reached with their control solutions. CONCLUSION: RBCs exposed to hyperosmotic iohexol, ioxaglate, or iodixanol solutions shrank and then swelled. The degree of shrinkage and subsequent swelling could not be explained simply with the osmolality of the test solutions. Physicochemical properties of the contrast media must be involved, putatively affecting electrolyte fluxes over the RBC membrane. Possible targets of these effects are the K+/Cl- symporter, K+ channels, and the Na+/K+/Cl- symporter.

EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin.

INTRODUCTION: This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. MATERIALS AND METHODS: Fifty-two type-2 diabetics with CAD on chronic aspirin (100 mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100 nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). RESULTS: Arachidonic acid (1 mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63 ± 4 AU), which was significantly reduced by in vitro exposure to EV-077 (38 ± 3 AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). CONCLUSIONS: Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077.

Effect of pharmaceutical interventions targeting thromboxane receptors and thromboxane synthase in cardiovascular and renal diseases.

The present review focuses on the roles of thromboxane A2 (TxA2) in arterial thrombosis, atherogenesis, vascular stent-related ischemic events and renal proteinuria. Particular emphasis is laid on therapeutic interventions targeting the TxA2 (TP) receptors and TxA2 synthase (TS), including dual TP-receptor antagonists and TS inhibitors. Their significant inhibitory efficacies on arterial thrombogenesis, atherogenesis, restenosis after stent placement, vasoconstriction and proteinuria indicate novel and improved treatments for cardiovascular and selected renal diseases. New therapeutic interventions of the TxA2 pathway may also be beneficial for patients with poor biological antiplatelet drug response, for example, to aspirin and/or clopidogrel. These new TP/TS agents offer novel improved treatments to efficiently and simultaneously interfere with thrombogenesis and atherogenesis, and to enlarge the existing panel of platelet inhibitors for efficient prophylaxis and treatment of arterial thrombosis and renal proteinuria.

Blood flow devices in medical research and clinical testing in humans: are we approaching personalized medicine?

This review focuses on studies of blood flow devices employed in man to unravel the mechanisms of bleeding and thrombotic disorders, and on the characterization of novel experimental antithrombotic entities and drug candidates in biopharmaceutical research and development. Clinical studies with drug candidates and new therapeutic strategies have also been performed, and the predictability of these experimental approaches to clinical situations is excellent. Based on the solid validation of these flow devices, miniature flow devices employing nonanticoagulated blood drawn directly from an antecubital vein should be developed for diagnostic purposes. It is anticipated that such a diagnostic flow device could develop into a personalized medicine approach.

Thrombus formation on apex of arterial stenoses: the need for a fluid high shear stenosis diagnostic device.

This review is focused upon the studies of thrombus formation in human non-anticoagulated blood on an apex of an eccentric stenosis positioned in the blood flow channel of a parallel-plate perfusion chamber. Thrombus formation in blood from healthy individuals and patients with various bleeding disorders, as well as the effects of a diet supplement and pharmacological interventions, are discussed in view of thrombus-forming mechanisms under these complex blood-flow conditions. Hallmarks of this significantly enhanced thrombus formation are the apparent dependence on thrombin generation, shear-induced platelet activation, induction of platelet procoagulant activity and pronounced platelet microparticle formation that parallel the growth of these fibrin-rich thrombi. The development of miniature models of these blood-flow devices for diagnostic purposes is suggested for the assessment and monitoring of the efficacy of antithrombotic regimens in blood from patients with atherosclerotic disease in parallel with assessments of platelet microparticle formation, shear-induced platelet activation and platelet procoagulant activity.

Validation of the human tissue factor/FVIIa complex as an antithrombotic target and the discovery of a synthetic peptide.

This review focuses on the validation of the principal initiator of human coagulation, the tissue factor (TF)/coagulation factor (F)VIIa complex, as an antithrombotic target, as well as on the discovery of a cyclic pentapeptide (PN7051), which dose-dependently inhibits TF/FVIIa-induced coagulation and thrombus formation. Target validation and studies of antithrombotic efficacy were performed with a human thrombosis model employing non-anticoagulated blood from severe homozygous FVII-deficient patients and healthy individuals at blood-flow conditions mimicking those in healthy and diseased vessels. Additional validation included an anti-TF monoclonal antibody, recombinant TF pathway inhibitor, recombinant inactivated-active site FVIIa and all-trans retinoic acid. Structural and biological characterization of PN7051 and other peptides from the same FVII domain indicate that PN7051 interferes with an essential interaction between the epidermal growth factor domain-2-like and the catalytic domains of FVIIa. A peptidomimetics approach is suggested to further improve the antithrombotic potency of PN7051.