Citrullinated fibrinogen forms densely packed clots with decreased permeability.

BACKGROUND: Fibrin, the main scaffold of thrombi, is susceptible to citrullination by PAD (peptidyl arginine deiminase) 4, secreted from neutrophils during the formation of neutrophil extracellular traps. Citrullinated fibrinogen (citFg) has been detected in human plasma as well as in murine venous thrombi, and it decreases the lysability and mechanical resistance of fibrin clots. OBJECTIVE: To investigate the effect of fibrinogen citrullination on the structure of fibrin clots. METHODS: Fibrinogen was citrullinated with PAD4 and clotted with thrombin. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to measure fiber thickness, fiber height/width ratio, and fiber persistence length in clots containing citFg. Fiber density was measured with laser scanning microscopy (LSM) and permeability measurements were carried out to estimate the porosity of the clots. The intra-fiber structure of fibrin was analyzed with small-angle X-ray scattering (SAXS). RESULTS: SEM images revealed a decrease in the median fiber diameter that correlated with the fraction of citFg in the clot, while the fiber width/length ratio remained unchanged according to AFM. With SAXS we observed that citrullination resulted in the formation of denser clots in line with increased fiber density shown by LSM. The permeability constant of citrullinated fibrin decreased more than 3-fold indicating significantly decreased porosity. SAXS also showed largely preserved periodicity in the longitudinal assembly of fibrin monomers. CONCLUSION: The current observations of thin fibers combined with dense packing and low porosity in the presence of citFg can provide a structural framework for the mechanical fragility and lytic resistance of citrullinated fibrin.

Networks that stop the flow: A fresh look at fibrin and neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are DNA and histone-based networks enriched with granule-derived proteins cast out by neutrophils in response to various inflammatory stimuli. Another molecular network, fibrin is the primary protein scaffold that holds both physiological blood clots and pathological thrombi together. There is mounting evidence that NETs and fibrin form a composite network within thrombi: in the past 10 years, a variety of molecular pathways have been revealed that help elucidate the nature of the NET-fibrin interaction. Besides discussing the effects of various NET components on hemostasis, this review takes a closer look at the interaction of these individual effects, with novel perspectives on how the NET and fibrin networks stabilize each other. Similarities and molecular connections are also outlined between the processes responsible for the degradation (fibrinolysis and NET lysis) as well as elimination of these networks. In addition, the complex relationship of pathogens with the NET-fibrin network is discussed, with a particular focus on the role of peptidyl-arginyl deiminases (PADs) in NET formation as well as in pathogen intrusion, where PADs act as a virulence factor expressed by bacteria -an aspect that is currently left out from discussions in the field.

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin.

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as 'strong' (e.g. thrombin and collagen) or 'mild' (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since 'strong' stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to 'mild' stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD.

Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems.

Neutrophil extracellular traps (NETs) composed primarily of DNA and histones are a link between infection, inflammation and coagulation. NETs promote coagulation and approaches to destabilise NETs have been explored to reduce thrombosis and treat sepsis. Heparinoids bind histones and we report quantitative studies in plasma and purified systems to better understand physiological consequences. Unfractionated heparin (UFH) was investigated by activated partial thromboplastin time (APTT) and alongside low-molecular-weight heparins (LMWH) in purified systems with thrombin or factor Xa (FXa) and antithrombin (AT) to measure the sensitivity of UFH or LMWH to histones. A method was developed to assess the effectiveness of DNA and non-anticoagulant heparinoids as anti-histones. Histones effectively neutralised UFH, the IC50 value for neutralisation of 0.2 IU/ml UFH was 1.8 µg/ml histones in APTT and 4.6 µg/ml against 0.6 IU/ml UFH in a purified system. Histones also inhibited the activities of LMWHs with thrombin (IC50 6.1 and 11.0 µg/ml histones, for different LMWHs) or FXa (IC50 7.8 and 7.0 µg/ml histones). Direct interactions of UFH and LMWH with DNA and histones were explored by surface plasmon resonance, while rheology studies showed complex effects of histones, UFH and LMWH on clot resilience. A conclusion from these studies is that anticoagulation by UFH and LMWH will be compromised by high affinity binding to circulating histones even in the presence of DNA. A complete understanding of the effects of histones, DNA and heparins on the haemostatic system must include an appreciation of direct effects on fibrin and clot structure.

DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment.

In response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10(-9) cm(2). Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.

Fractal kinetic behavior of plasmin on the surface of fibrin meshwork.

Intravascular fibrin clots are resolved by plasmin acting at the interface of gel phasesubstrate and fluid-borne enzyme. The classic Michaelis.Menten kinetic scheme cannot describe satisfactorily this heterogeneous-phase proteolysis because it assumes homogeneous well-mixed conditions. A more suitable model for these spatial constraints,known as fractal kinetics, includes a time-dependence of the Michaelis coefficient Km(F) = Km0F (1+ t)h, where h is a fractal exponent of time, t. The aim of the present study was to build up and experimentally validate a mathematical model for surface-acting plasmin that can contribute to a better understanding of the factors that influence fibrinolytic rates. The kinetic model was fitted to turbidimetric data for fibrinolysis under various conditions. The model predicted Km0(F) = 1.98 µM and h = 0.25 for fibrin composed of thin fibers and Km0(F) = 5.01 µM and h = 0.16 for thick fibers in line with a slower macroscale lytic rate (due to a stronger clustering trend reflected in the h value) despite faster cleavage of individual thin fibers (seen as lower Km0(F) ). ε-Aminocaproic acid at 1 mM or 8 U/mL carboxypeptidase-B eliminated the time-dependence of Km F and increased the lysis rate suggesting a role of C-terminal lysines in the progressive clustering of plasmin. This fractal kinetic concept gained structural support from imaging techniques. Atomic force microscopy revealed significant changes in plasmin distribution on a patterned fibrinogen surface in line with the time-dependent clustering of fluorescent plasminogen in confocal laser microscopy. These data from complementary approaches support a mechanism for loss of plasmin activity resulting from C-terminal lysine-dependent redistribution of enzyme molecules on the fibrin surface.

[Short and long term prognosis of patients with myocardial infarction. Hungarian Myocardial Infarction Registry]. / Szívinfarktus miatt kezelt betegek korai és késoi prognózisa. Magyar Infarctus Regiszter Vizsgálat.

INTRODUCTION: Mortality data of patients with acute myocardial infarction are incomplete in Hungary. AIM: The aim of the authors was to analyse the data of 8582 myocardial infarction patients (4981 with ST-elevation myocardial infarction) registered in the Hungarian Myocardial Infarction Register in order to define the hospital, 30-day, and 1-year mortality. To evaluate the prehospital mortality of myocardial infarction, all myocardial infarction and sudden death were registered in five districts of Budapest. METHOD: Multivariate logistic regression was performed to define risk factors of mortality and the model were assessed using c statistics. RESULTS: The hospital, 30-day and 1-year mortality of patients with ST elevation myocardial infarction were 3.7%, 9.5% and 16.5%, respectively. In patients without ST elevation myocardial infarction these figures were 4%, 9.8% and 21.7%, respectively. The 1-year mortality of patients without ST elevation was higher than those of with ST elevation and the difference was statistically significant. Age, Killip class, diabetes mellitus, history of stroke and myocardial infarction were independent predictors of death. Coronary intervention improved the prognosis of patients with myocardial infarction significantly. CONCLUSIONS: The rate of pre-hospital mortality was considerably high; 72.5% of 30 day mortality occurred before admission to hospital.

Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones.

Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nM dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator.

[Myocardial Infarction Registry--2010. Experience and first results in Hungary]. / Myocardialis Infarctus Regiszter--2010. Tapasztalatok--elso eredmények.

Authors present the methodology and first data of Hungarian Myocardial Infarction Register Pilot Study started 1st of January, 2010. The aim of the study is to collect epidemiological data on myocardial infarction, to examine the natural history of the disease and to investigate the main characteristics on patient care in the pilot area. The program is using standardized diagnostic criteria and predefined electronic data record forms (eCRF). The pilot area consists of 5 districts in the capital, and Szabolcs-Szatmár-Bereg county. The area has 997 324 inhabitants. Eight cardiology departments, 5 with heart catheterization facility (C) in Budapest, four hospitals with one C in Szabolcs-Szatmar-Bereg county have been responsible of the patients' care. After starting the program 16 other hospitals joined the program from different parts of Hungary. Between 1st of January 2010 and 1st of May 2011 4293 patients were registered, among them 52.1% with ST segment elevation myocardial infarction (STEMI), 42.1% with non-ST segment elevation myocardial infarction (NSTEMI), while 3% of the patients had unstable angina, and 2.8% of the cases had other diagnosis or the hospital diagnosis was missing in the eCRF. Authors compare the patients care with STEMI in five districts of Budapest and Szabolcs-Szatmár-Bereg county. In Budapest 79.7% of the 301 STEMI patients were treated in C and 84.6% of them were treated with primary percutaneous intervention (pPCI). In Szabolcs-Szatmár-Bereg county 402 patients were registered with STEMI, 62.9% of them were treated in C, where 77% of them were treated with pPCI. The drugs (beta blockers, ACE inhibitors, statins) important for secondary prevention were given more often to patients treated in the capital, however no difference was found in the platelet aggregation inhibitors therapy. Hospital mortality of STEMI patients was 8% in the capital, and 10% in Szabolcs- Szatmár-Bereg county. Authors conclude that the web based myocardial infarction register is feasible and important to have reliable data on patient care and a necessary quality control tool. Authors propose to broaden this pilot program and to start a nationwide myocardial infarction register.