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Inflammation plays a substantial role in the process of atherosclerosis. The key players are monocytes transformed to macrophages. Limited experience with anti-inflammatory drugs is described – antibodies for IL-1 (CANTOS) and colchicine (COLCOT, LoDoCo-2 trial). Cannabis extracts are known to have profound anti-inflammatory effect in diseases associated with chronic inflammation, e.g. rheumatoid arthritis. Human monocytic cell line THP-1 was differentiated into macrophages by incubation in the presence of phorbol-12-myristate-13-acetate, which leads to a macrophage-like phenotype characterized by changes in morphology and adhesion. Macrophages were polarized toward M1 pro-inflammatory phenotype responsible for production of several cytokines, esp. TNFα and IL-6.The ability of cannabinoids to decrease production of pro-inflammatory cytokines in polarized macrophages was measured by ELISA. The viability of macrophages after the treatment was verified by resazurin assay., Cannabigerol (CBG) and cannabidiol (CBD) significantly decreased the production of TNFα in dose-dependent manner. 7.5 mg/L of CBG and 1 mg/L of CBD decreased the production of TNFα by 32 and 34%, respectively. CBD, cannabidivarin (CBDV), cannabichromene (CBC), and cannabinol (CBN) significantly decreased also IL-6 production. 7.5 mg/L of CBDV and CBC decreased the production of IL-6 by 38 and 62%, respectively. 2 mg/L of CBD and CBN decreased the production of IL-6 by 33% (both).The concentration of the most potent cannabinoid (CBC) inhibiting IL-6 production by half (IC50) was determined to be 6.7 ± 0.1 mg/L, which is two-times better treatment potential than colchicine (13.4 ± 0.8 mg/L). Cannabinoids might be promising substance in the treatment of subclinical inflammation associated with atherosclerosis. The project was funded by the Grant No 22–20860S of The Czech Science Foundation.

We describe the internal structure, spatial organization and dynamic formation of coronary artery thrombi from ST-segment elevation myocardial infarction patients. Scanning electron microscopy (SEM) revealed significant differences among four groups of patients (12 hours) related to the time of ischemia. Coronary artery thrombi from patients presenting less than 2 hours after the infarction were almost entirely composed of platelets, with small amounts of fibrin and red blood cells. In contrast, thrombi from late presenters (>12 hours) consisted of mainly platelets at the distal end, where clotting was initiated, with almost no platelets at the proximal end, while the red blood cell content went from low at the initiating end to more than 90% at the proximal end. Furthermore, fibrin was present mainly on the outside of the thrombi and older thrombi contained thicker fibers. The red blood cells in late thrombi were compressed to a close-packed, tessellated array of polyhedral structures, called polyhedrocytes. Moreover, there was redistribution from the originally homogeneous composition to fibrin and platelets to the outside, with polyhedrocytes on the interior. The presence of polyhedrocytes and the redistribution of components are signs of in vivo clot contraction (or retraction). These results suggest why later thrombi are resistant to fibrinolytic agents and other treatment modalities, since the close-packed polyhedrocytes form a nearly impermeable seal. Furthermore, it is of particular clinical significance that these findings suggest specific disparate therapies that will be most effective at different stages of thrombus development.

BACKGROUND: Patients with peripheral artery disease have an increased risk of cardiovascular morbidity and mortality. Antiplatelet agents are widely used to reduce these complications.METHODS: This was a multicentre, double-blind, randomised placebo-controlled trial for which patients were recruited at 602 hospitals, clinics, or community practices from 33 countries across six continents. Eligible patients had a history of peripheral artery disease of the lower extremities (previous peripheral bypass surgery or angioplasty, limb or foot amputation, intermittent claudication with objective evidence of peripheral artery disease), of the carotid arteries (previous carotid artery revascularisation or asymptomatic carotid artery stenosis of at least 50%), or coronary artery disease with an ankle-brachial index of less than 0·90. After a 30-day run-in period, patients were randomly assigned (1:1:1) to receive oral rivaroxaban (2·5 mg twice a day) plus aspirin (100 mg once a day), rivaroxaban twice a day (5 mg with aspirin placebo once a day), or to aspirin once a day (100 mg and rivaroxaban placebo twice a day). Randomisation was computer generated. Each treatment group was double dummy, and the patient, investigators, and central study staff were masked to treatment allocation. The primary outcome was cardiovascular death, myocardial infarction or stroke; the primary peripheral artery disease outcome was major adverse limb events including major amputation. This trial is registered with ClinicalTrials.gov, number NCT01776424, and is closed to new participants.FINDINGS: Between March 12, 2013, and May 10, 2016, we enrolled 7470 patients with peripheral artery disease from 558 centres. The combination of rivaroxaban plus aspirin compared with aspirin alone reduced the composite endpoint of cardiovascular death, myocardial infarction, or stroke (126 [5%] of 2492 vs 174 [7%] of 2504; hazard ratio [HR] 0·72, 95% CI 0·57-0·90, p=0·0047), and major adverse limb events including major amputation (32 [1%] vs 60 [2%]; HR 0·54 95% CI 0·35-0·82, p=0·0037). Rivaroxaban 5 mg twice a day compared with aspirin alone did not significantly reduce the composite endpoint (149 [6%] of 2474 vs 174 [7%] of 2504; HR 0·86, 95% CI 0·69-1·08, p=0·19), but reduced major adverse limb events including major amputation (40 [2%] vs 60 [2%]; HR 0·67, 95% CI 0·45-1·00, p=0·05). The median duration of treatment was 21 months. The use of the rivaroxaban plus aspirin combination increased major bleeding compared with the aspirin alone group (77 [3%] of 2492 vs 48 [2%] of 2504; HR 1·61, 95% CI 1·12-2·31, p=0·0089), which was mainly gastrointestinal. Similarly, major bleeding occurred in 79 (3%) of 2474 patients with rivaroxaban 5 mg, and in 48 (2%) of 2504 in the aspirin alone group (HR 1·68, 95% CI 1·17-2·40; p=0·0043).INTERPRETATION: Low-dose rivaroxaban taken twice a day plus aspirin once a day reduced major adverse cardiovascular and limb events when compared with aspirin alone. Although major bleeding was increased, fatal or critical organ bleeding was not. This combination therapy represents an important advance in the management of patients with peripheral artery disease. Rivaroxaban alone did not significantly reduce major adverse cardiovascular events compared with asprin alone, but reduced major adverse limb events and increased major bleeding.FUNDING: Bayer AG.

During coagulation, the soluble fibrinogen is converted into insoluble fibrin. Fibrinogen is a multifunctional plasma protein, which is essential for hemostasis. Various oxidative posttranslational modifications influence fibrinogen structure as well as interactions between various partners in the coagulation process. The aim was to examine the effects of oxidative stress conditions on fibrin clot formation in arterial atherothrombotic disorders. We studied the changes in in vitro fibrin network formation in three groups of patients—with acute coronary syndrome (ACS), with significant carotid artery stenosis (SCAS), and with acute ischemic stroke (AIS), as well as a control group. The level of oxidative stress marker malondialdehyde measured by LC-MS/MS was higher in SCAS and AIS patients compared with controls. Turbidic methods revealed a higher final optical density and a prolonged lysis time in the clots of these patients. Electron microscopy was used to visualize changes in the in vitro-formed fibrin network. Fibers from patients with AIS were significantly thicker in comparison with control and ACS fibers. The number of fibrin fibers in patients with AIS was significantly lower in comparison with ACS and control groups. Thus, oxidative stress-mediated changes in fibrin clot formation, structure and dissolution may affect the effectiveness of thrombolytic therapy.

Background. Cardiac troponins are routinely used as markers of myocardial damage. Originally, they were only intended for use in diagnosing acute coronary syndromes; however, we now know that raised serum troponin levels are not always caused by ischemia. There are many other clinical conditions that cause damage to cardiomyocytes, leading to raised levels of troponin. However, the specificity of cardiac troponins towards the myocardium is absolute. Our work focuses on mechanical damage to the myocardium and on monitoring the factors that raise the levels of cardiospecific markers after primo-implantation of a pacemaker with an actively fixed electrode. Aims. (i) To determine whether the use of a primo-implanted pacemaker with an electrode system with active fixation will raise troponin levels over baseline. (ii) To assess whether troponin I elevation is dependent on procedure complexity. Methods. We enrolled 219 consecutive patients indicated for pacemaker primo-implantation; cardiospecific marker values (troponin I, CKMB, myoglobin) were determined before the implantation procedure and again at 6- and 18-h intervals after the procedure. We monitored duration of cardiac skiascopy, number of attempts to place the electrode (active penetration into the tissue) and intervention range (single-chamber versus dual-chamber pacing), and we assessed the clinical data. Results. The average age of the enrolled patients was 78.2 +/- 8.0 years (median age, 80 years); women constituted 45% of the group. We implanted 128 dual-chamber and 91 single-chamber devices with an average skiascopic time of 38.6 +/- 22.0 s (median, 33.5 s). Troponin I serum levels increased from an initial 0.03 +/- 0.07 mu g/L (median, 0.01) to 0.18 +/- 0.17 mu g/L (median, 0.13) and 0.09 +/- 0.18 mu g/L (median, 0.04) at 6 and 18 h, respectively. The differences were statistically significant (P < 0.001 or P < 0.001). We confirmed a correlation between troponin increase and duration of skiascopy (P < 0.001). We also demonstrated a correlation between increased troponin I and number of attempts to place a pacemaker electrode (penetration into the tissue) at 6 h (P < 0.001) post-implantation. Conclusion. We detected slightly elevated troponin I levels in patients with primo-implanted pacemakers using electrodes with active fixation. We demonstrated a direct correlation between myocardial damage (number of electrode penetrations into the myocardium) and troponin I elevation, as well as between complexity (severity) of the implantation procedure (indicated by prolonged skiascopy) and raised troponin I. The described phenomenon demonstrates the loss of the diagnostic role of troponin I early after pacemaker primo-implantation in patients with concomitant chest pain.

Atherosclerosis is a leading cause of major vascular events, myocardial infarction, and ischemic stroke. Tryptophan (TRP) catabolism was recognized as an important player in inflammation and immune response having together with oxidative stress (OS) significant effects on each phase of atherosclerosis. The aim of the study is to analyze the relationship of plasma levels of TRP metabolites, inflammation, and OS in patients with neurovascular diseases (acute ischemic stroke (AIS), significant carotid artery stenosis (SCAS)) and in healthy controls. Blood samples were collected from 43 patients (25 with SCAS, 18 with AIS) and from 25 healthy controls. The concentrations of twelve TRP metabolites, riboflavin, neopterin (NEO, marker of inflammation), and malondialdehyde (MDA, marker of OS) were measured by liquid chromatography&ndash, tandem mass spectrometry (LC-MS/MS). Concentrations of seven TRP metabolites (TRP, kynurenine (KYN), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), anthranilic acid (AA), melatonin (MEL), tryptamine (TA)), NEO, and MDA were significantly different in the studied groups. Significantly lower concentrations of TRP, KYN, 3-HAA, MEL, TA, and higher MDA concentrations were found in AIS compared to SCAS patients. MDA concentration was higher in both AIS and SCAS group (p &lt, 0.001, p = 0.004, respectively) compared to controls, NEO concentration was enhanced (p &lt, 0.003) in AIS. MDA did not directly correlate with TRP metabolites in the study groups, except for 1) a negative correlation with kynurenine acid and 2) the activity of kynurenine aminotransferase in AIS patients (r = &minus, 0.552, p = 0.018, r = &minus, 0.504, p = 0.033, respectively). In summary, TRP metabolism is clearly more deregulated in AIS compared to SCAS patients, the effect of TRP metabolites on OS should be further elucidated.

BACKGROUND: Coronary artery disease is a major cause of morbidity and mortality worldwide, and is a consequence of acute thrombotic events involving activation of platelets and coagulation proteins. Factor Xa inhibitors and aspirin each reduce thrombotic events but have not yet been tested in combination or against each other in patients with stable coronary artery disease.METHODS: In this multicentre, double-blind, randomised, placebo-controlled, outpatient trial, patients with stable coronary artery disease or peripheral artery disease were recruited at 602 hospitals, clinics, or community centres in 33 countries. This paper reports on patients with coronary artery disease. Eligible patients with coronary artery disease had to have had a myocardial infarction in the past 20 years, multi-vessel coronary artery disease, history of stable or unstable angina, previous multi-vessel percutaneous coronary intervention, or previous multi-vessel coronary artery bypass graft surgery. After a 30-day run in period, patients were randomly assigned (1:1:1) to receive rivaroxaban (2·5 mg orally twice a day) plus aspirin (100 mg once a day), rivaroxaban alone (5 mg orally twice a day), or aspirin alone (100 mg orally once a day). Randomisation was computer generated. Each treatment group was double dummy, and the patients, investigators, and central study staff were masked to treatment allocation. The primary outcome of the COMPASS trial was the occurrence of myocardial infarction, stroke, or cardiovascular death. This trial is registered with ClinicalTrials.gov, number NCT01776424, and is closed to new participants.FINDINGS: Between March 12, 2013, and May 10, 2016, 27 395 patients were enrolled to the COMPASS trial, of whom 24 824 patients had stable coronary artery disease from 558 centres. The combination of rivaroxaban plus aspirin reduced the primary outcome more than aspirin alone (347 [4%] of 8313 vs 460 [6%] of 8261; hazard ratio [HR] 0·74, 95% CI 0·65-0·86, pINTERPRETATION: In patients with stable coronary artery disease, addition of rivaroxaban to aspirin lowered major vascular events, but increased major bleeding. There was no significant increase in intracranial bleeding or other critical organ bleeding. There was also a significant net benefit in favour of rivaroxaban plus aspirin and deaths were reduced by 23%. Thus, addition of rivaroxaban to aspirin has the potential to substantially reduce morbidity and mortality from coronary artery disease worldwide.FUNDING: Bayer AG.

Background— Bioresorbable vascular scaffolds (BVS) represent promising new technology, but data on their long-term outcomes in ST-segment–elevation myocardial infarction (STEMI) setting are missing. The aim was to analyze 1-year clinical and computed tomographic angiographic outcomes after BVS implantation in STEMI. Methods and Results— PRAGUE-19 is a prospective multicenter single-arm study enrolling consecutive STEMI patients undergoing primary percutaneous coronary intervention (pPCI) with intention-to-implant BVS. A total of 343 STEMI patients were screened during 15 months enrollment period, and 70 patients (mean age 58.6±10.3 and 74% males) fulfilled entry criteria and BVS was successfully implanted in 96% of them. All patients were invited for clinical and computed tomographic angiographic control 1 year after BVS implantation. Restenosis was defined as ≥75% area stenosis within the scaffolded segment. Three events were potentially related to BVS: 1 in-stent restenosis (treated 7 months after pPCI with drug-eluting balloon), 1 stent thrombosis (treated 2 weeks after pPCI by balloon dilatation—this patient stopped all medications after pPCI), and 1 sudden death at home 9 months after pPCI. Four other patients had events definitely unrelated to BVS. Overall, 1-year mortality was 2.9%. Computed tomographic angiography after 1 year was performed in 59 patients. All BVS were widely patent, and binary restenosis rate was 2% (the only restenosis mentioned above). Mean in-scaffold minimal luminal area was 7.8±2.6 mm 2 , area stenosis was 20.1±16.3%, minimal luminal diameter was 3.0±0.6 mm, and diameter stenosis was 12.8±11.1%. Conclusions— BVS implantation in STEMI is feasible and safe and offers excellent 1-year clinical and angiographic outcomes.

Background Cardiovascular diseases are linked with oxidative stress which is the source of reactive oxidative and nitrative species, contributors of post-translational modification. Fibrinogen due to its high concentration in blood is considered as one of the most sought of targets of oxidative stress substances. Post-translational modifications of fibrinogen might influence its physiological function, thus affect hemostasis in the terms of fibrin nets forming and architecture or interaction with platelets. The aim of this study was to observe influence of in vivo fibrinogen modifications on formation of fibrin net and to identify amino acid residues prone to changes related to oxidative stress. Methods Plasma samples were collected from patients of The Military University Hospital Prague in the agreement with ethical committees of participating institutions and with informed consents from all subjects. Samples were divided into 4 groups: patients with acute coronary syndrome (A), patients with stroke (B), patients with thrombus localized in carotid vein (C) and control group (patients without coronary atherosclerosis; D). Fibrin net architecture was studied by scanning electron microscopy (Mira 3 LMH, Tescan Orsay Holding, a.s., Brno, Czech Republic). For identification of modified amino acids residue mass spectroscopy was used (Triple TOF 6600, Sciex). Molecular dynamics simulations of hydrated protein were performed in Gromacs software with Gromos force fields. Crystal structure 3GHG was used as a reference structure to which post-translational modifications were introduced manually in Yasara View. Results We found extensive both qualitative and quantitative changes in the structure of fibrinogen molecule in all groups of patients. Oxidative stress level differed among patient groups and between the control group. Different oxidative changes caused by in vivo modifications of fibrinogen affected quite distinctly the architecture of fibrin net. Modified amino acids were detected in all three fibrinogen chains. In gamma chain the localisation of modified amino acid residues correlated with the part of fibrinogen important for fibrin polymerisation. The impact of the most pronounced post-translational modifications on the secondary structure of fibrinogen was described by molecular dynamics simulations. Conclusions The results show that the degree of impairment of fibrinogen functions in the cardiovascular diseases is related to the level of oxidative stress. Characterization of oxidative fibrinogen modification and its precise meaning to the function of fibrinogen in hemostasis appears to be extremely helpful to better understanding of thrombotic/bleeding complications linked with various cardiovascular diseases. Acknowledgments This work was supported by the Ministry of Health, Czech Republic, no. 00023736, by the Academy of Sciences, Czech Republic no. P205/12/G118 and NV18-08-00149, by ERDF OPPK CZ.2.16/3.1.00/24001 and by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports). Disclosures No relevant conflicts of interest to declare.

A novel NOx control technique based on the utilization of iron-containing compounds was studied. Iron-containing additives can be injected in small amounts either into the combustion zone or, if reburning is used, into the reburning zone to reduce NOx emissions. Tests in a 300-kW Boiler Simulator Facility demonstrate that iron additives achieve 20−30% NOx reduction without reburning, whereas in conjunction with reburning, they increase NOx reduction from about 60%, typical for conventional reburning, to about 80−85%. The additives tested included powdered metallic iron, iron oxides, iron waste, Fe(CO)5, char, and ash. Up to 1000 ppm (atomic Fe basis) was added to the flue gas. Experimental variables studied include additive composition and amount, injector location, and reburning heat input. Depending on the additive, the effect is dominated by either heterogeneous or homogeneous reactions.

The advanced reburning (AR) process is an integration of basic reburning and N-agent injection. The efficiency of AR depends on many factors, including the amount of the reburning fuel, the amount and location of N-agent injection, and spray characteristics. These parameters can be optimized via computer modeling. This paper describes a model that combines a detailed chemical mechanism with a simplified representation of mixing to describe the AR-lean process, a combination of basic reburning and N-agent coinjection with overfire air (OFA). The model takes into account mixing and thermal characteristics of a 300 KW combustion facility, which was used for experimental studies in support of model development. Modeling suggested that the efficiency of AR-lean strongly depends on amounts of the reburning fuel and N-agent, flue gas temperature at the point of OFA/N-agent injection, and N-agent evaporation time. The model described the most important features of AR-lean. Application of the model for optimization of AR-lean performance in the test facility was demonstrated.

Selective Non-Catalytic Reduction (SNCR) is a well-known commercial NOx control process based on injecting a nitrogen agent into combustion products containing NO at temperatures near 1250 K. A serious limitation of the SNCR processes is that the temperature range over which nitrogen agents are effective is relatively narrow. In this work, we show that adding small amounts of sodium salts significantly improves the performance of the SNCR process. Parts per million levels of sodium compounds enhance NO removal and extend the effective SNCR temperature range in comparison with use of a nitrogen agent alone. When added in the same sodium atom amounts, the efficiencies of different sodium compounds are similar. Kinetic modeling suggests that the performance improvement can be explained as a homogeneous chain reaction ensuing after the sodium compounds are converted into NaOH. The overall result of introducing sodium compounds is conversion of H2O and inactive HO2 radicals into reactive OH radicals, with the effective stoichiometry H2O + HO2 → 3 OH, which enhances the SNCR performance of nitrogen agents by increasing the production rate of NH2 radicals.

A process has been developed for producing a pumpable slurry fuel from refuse-derived fuel (RDF). A target application for this fuel is cofiring with fossil fuels in utility boilers. Pilot scale tests were conducted to characterize the emissions of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs) and trace metals from carbonized RDF slurry combustion. Total PCDD/PCDF concentration averaged 0.38 ng/dscm at 7% O 2 for two test runs. Mercury, cadmium, and lead had average emission concentrations of 0.0066, 0.0017, and 0.035 mg/dscm at 7% O 2 , respectively. While nearly all mercury was found to be in the vapor phase, emissions were low due to low mercury concentration in the fuel itself. Cadmium and lead were evidently in the solid phase and were effectively captured by the ESP. In general, the results indicate that the carbonized RDF slurry would be expected to meet promulgated new source performance standards (NSPS) for municipal waste combustion and make an effective fuel for cofiring in utility boilers.

In this project, General Electric Energy and Environmental Research Corporation (EER) developed a novel mercury (Hg) control technology in which the sorbent for gas-phase Hg removal is produced from coal in a gasification process in-situ at a coal burning plant. The main objective of this project was to obtain technical information necessary for moving the technology from pilot-scale testing to a full-scale demonstration. A pilot-scale gasifier was used to generate sorbents from both bituminous and subbituminous coals. Once the conditions for optimizing sorbent surface area were identified, sorbents with the highest surface area were tested in a pilot-scale combustion tunnel for their effectiveness in removing Hg from coal-based flue gas. It was determined that the highest surface area sorbents generated from the gasifier process ({approx}600 m{sup 2}/g) had about 70%-85% of the reactivity of activated carbon at the same injection rate (lb/ACF), but were effective in removing 70% mercury at injection rates about 50% higher than that of commercially available activated carbon. In addition, mercury removal rates of up to 95% were demonstrated at higher sorbent injection rates. Overall, the results of the pilot-scale tests achieved the program goals, which were to achieve at least 70% Hg removal from baseline emissions levels at 25% or less of the cost of activated carbon injection.

Background Implantation of a carotid artery stent after predilation is a standard approach in the endovascular treatment of carotid artery stenoses. Stenting without predilation may be an alternative approach in a certain subset of patients. The present prospective, single-center registry was designed to evaluate the feasibility and safety of direct carotid artery stenting (DCAS) in high-risk patients. Methods and Results Symptomatic patients with stenosis >50% and asymptomatic patients with stenosis >70% were eligible for enrolment. Criteria for high-risk patients included: need or history of open heart surgery, history of myocardial infarction, multivessel coronary artery disease, left ventricular dysfunction (ejection fraction ≤40%), severe pulmonary or renal disease, significant contralateral carotid disease, previous endarterectomy, and age ≥80 years. All procedures were performed using a filter protection device. Patients underwent complete clinical examination before and after DCAS and at 30-day follow-up. A total of 83 consecutive patients (45 males, 68±9 years, 33% symptomatic) underwent 100 procedures and 103 stents were deployed successfully. The technical success rate of stenting was 100%. Predilation of carotid stenosis was necessary in 1 (1%) procedure. Carotid-artery stenoses before and after DCAS were 80±9% and 7±9%, respectively. The median fluoroscopic time for DCAS was 7 min. The overall rate of in-hospital major adverse cerebrovascular events (death, stroke, myocardial infarction) was 5% (2 minor strokes, 3 transient attacks). There was 1 (1%) minor stroke within the 30-day follow-up. Conclusion DCAS is feasible and can be performed with an acceptable risk in high-risk patients. (Circ J 2007; 71: 1468 ‐ 1472)

Background Alcohol septal ablation (ASA) decreases the left ventricular (LV) outflow gradient and relieves symptoms in patients with highly symptomatic hypertrophic obstructive cardiomyopathy (HOCM). The aim of this study was to evaluate the early course of hemodynamic, morphologic and clinical changes in younger and elderly patients. Methods and Results Forty-four consecutive patients (age, 24-81 years) underwent the ASA procedure for HOCM. Clinical and echocardiographic data were obtained at baseline and periodically up to 12 months after ASA. There was a significant correlation between septum thickness and age at baseline and in the early post procedural period (p=0.004 at baseline, p=0.0033 days postoperative, p=0.0193 weeks pos operative). The dependence of septal thickness on the duration of follow-up (p

Introduction Thrombosis is a common pathology underlying ischemic heart disease, stroke, and venous thromboembolism. D-dimer is a global indicator of blood coagulation activation and fibrinolysis and, therefore, an indirect marker of thrombotic activity. D-dimers half-life is 48 hours. D-dimer determination is the standard procedure in the treatment of thrombosis. Serine protease thrombin plays pivotal roles in thrombosis and hemostasis, blood coagulation and platelet activation. Thrombin has a short half-life and naturally occurring inhibitors, such as antithrombin, rapidly bind thrombin, which makes the direct measurements of in vivo active thrombin difficult. Thrombin binds to fibrin where it is quite efficiently protected from inhibition by heparin-antithrombin but susceptible to inactivation by different antithrombin-independent inhibitors. There are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrinogen E region, and one high affinity thrombin non-substrate binding site on fibrinogen gamma' chains inside the D region (Meh DA, Siebenlist KR, Mosesson MW, J Biol Chem. 1996, 23121-5). We have shown that thrombin bound to fibrin promotes further fibrin growth in the presence of fibrinogen and absence of free thrombin in solution (Riedel T, Brynda E, Dyr JE, Houska, M, J. Biomed. Mater. Res. Part A 2009, 437-447). The aim of this project was to look for any thrombin activity on isolated D-dimers using several commercial D-dimer kits in groups of patients with elevated D-dimers. Methods Three D-dimers kits were used (ELISA kits CEA506Hu, USCN and D-Dimer (D2D), BioAssay™; and immunoturbidimetric assay D-Dimer KAI-090, K-ASSAY). To detect thrombin activity on captured D-dimers one chromogenic (S-2238, Chromogenix) and two fluorogenic (SN-59, Haematologic Technologies, Inc.; (p-tosyl-Gly-Pro-Arg)2-R110, Thermo Fisher Scientific, Inc.) specific substrates and specific inhibitors (hirudine and PPACK) were used. Independently, bound proteins were removed from immobilized antibodies in 8 M urea and after treatment with trypsin analyzed by mass spectrometry (TripleTOF 5600, Sciex). 34 patients with high, moderate, and low levels of D-dimers and with different diagnosis were analyzed. Results Out of 18 patients with main diagnosis linked with thrombosis 61 % exhibited active thrombin on D-dimers. In these patients we found active thrombin bound to D-dimers captured by antibodies in all applied D-dimer kits. Using mass spectrometry thrombin bound to isolated D-dimers was detected. Specificity of thrombin activity related to D-dimers was determined by combination of several specific thrombin substrates and inhibitors. The activity of bound thrombin was remarkably stable over the period of more than 24 hours. It showed that precise measurement of even very low activity of thrombin was possible. In the group of 16 non-thrombotic patients with elevated D-dimers only 19 % exhibited thrombin activity. Interestingly, differences in values of thrombin activity were up to five orders of magnitude and differences in the activities related to the value of captured D-dimers were even greater. Conclusion This, to our knowledge, is the first report showing the presence of active thrombin bound to circulating D-dimers. Although the group of patients we were so far able to evaluate is too small to being statistically tested, the results are highly encouraging. The amount of bound active thrombin on fibrin degradation products reflects the way of thrombus formation and its degradation (times, durations and rates of relevant reactions and especially the rate of thrombin generation). The thrombin concentration present at the time of fibrin gelation plays an important role in formation of fibrin clot structure, including its mechanical and fibrinolytic stability. It remains to be seen in further studies with much larger cohorts of patients if the presence of active thrombin has any impact on pathophysiology of thrombosis and if its determination may be of importance for the improvement of diagnosis of thrombotic events. Supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.1.16/3.1.00/28007. Disclosures No relevant conflicts of interest to declare.

Introduction The use of thromboaspiration in primary percutaneous intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) has offered a unique opportunity to study thrombus composition, its dynamic formation, and architecture in vivo. There has been, however, several limitations, not least the fact that the technique has not yet allowed a precise transversal analysis from one side of the artery to the other, as is done in histological analysis. The dynamic process of intracoronary thrombus formation in STEMI patients is thus still not well understood. Ischemic time was hypothesized to be among the strongest independent correlates of thrombus architecture. In time the platelets are decreasing its proportion and fibrin proportion is increasing (J Silvain, J-P Collet, JW Weisel et al, J Am Coll Cardiol 2011; 57:1359). However, no real report on the internal structures of the in vivo formed thrombi has been shown so far. Therefore, we investigated both the surface and the composition of longitudinally freeze-fractured thrombi. Methods Thrombi were collected by PCI from 119 STEMI patients. Out of the patients there were "early comers " (˃12 h from symptom onset; 23 patients) and "late comers" (more than 720 min; 29 patients). The mean age of all patients was 64 years, 70% of patients were males, 51% were smokers, 50% had arterial hypertension, 20% were diabetics and 23% had chronic renal insufficiency. Scanning electron microscopy; collected thrombi obtained by PCI were thoroughly washed in saline solution and stored in 4% formaldehyde prior dehydration. To reveal the internal structures of the thrombi selected samples were longitudinally freeze fractured in liquid nitrogen and coated with platinum. Samples were examined in SEM Vega Plus TS 5135 (Tescan s.r.o., Brno, Czech Republic). Whole areas of the freeze-fractured thrombi were scanned. Results and discussion The thrombus composition of longitudinally freeze-fractured thrombi was compared between groups of "early-comers" and "late-comers. The distribution of the components in the "early comers" thrombi freeze-fracture seemed to be uniform. Platelets were far the main component (about 75 % in proportion) of the "early comers" thrombus, followed by fibrin and other compounds. The amount of red blood cells was negligible (about 2 - 8 %). We did not observe any significant differences between the thrombi in the group of early comers. Thrombi of the "late-comers" group were composed mainly of red blood cells; platelets and fibrin formed only minority of the thrombi. In contrast to the "early comers" the distribution of the main thrombus components in the "late comers" thrombi was dramatically different between individual parts of the thrombus. The number of platelets and red blood cells varied from 0% to almost 99% and vice versa. It was possible to estimate the initiating place of the thrombus as well as the direction of the growth. Each thrombus could be divided into parts formed mainly either by platelets or by red blood cells. It seems that thrombus develops a regional architecture defined by the extent of platelet activation and packing density. It has been reported that in contracted clots and thrombi, erythrocytes are compressed to close-packed polyhedral structures with platelets and fibrin on the surface demonstrating how contracted clots form an impermeable barrier important for hemostasis and wound healing (D Cines, T Lebedeva, J Weisel et al, Blood 2014; 123:1596). Our investigation of the composition of the in vivo formed thrombi supports these results and helps to explain how fibrinolysis is greatly retarded as clots grow and contract. We have found that on the surfaces of late-comers thrombi fibrin thick fibrils were present. It has been shown that the association of soluble fibrinogen with the fibrin clot results in the reduced adhesiveness of such fibrinogen/fibrin matrices toward leukocytes and platelets (VK Lishko, T Burke, T Ugarova, Blood 2007; 109:1541). Fibrinopeptides A are less accessible for thrombin in surface bound fibrinogen which thus provides additional level of protection of thrombi from premature dissolution (T Riedel, L Medved, JE Dyr, Blood 2011; 117:1700). These findings may have great impact on our knowledge of pathophysiology of the thrombus growth and possible therapeutic consequences related to the time of symptom onset. Disclosures No relevant conflicts of interest to declare.