DNA and histones impair the mechanical stability and lytic susceptibility of fibrin formed by staphylocoagulase.

Background: Staphylocoagulase (SCG) is a virulence factor of Staphylococcus aureus, one of the most lethal pathogens of our times. The complex of SCG with prothrombin (SCG/ProT) can clot fibrinogen, and SCG/ProT-induced fibrin and plasma clots have been described to show decreased mechanical and lytic resistance, which may contribute to septic emboli from infected cardiac vegetations. At infection sites, neutrophils can release DNA and histones, as parts of neutrophil extracellular traps (NETs), which in turn favor thrombosis, inhibit fibrinolysis and strengthen clot structure. Objectives: To characterize the combined effects of major NET-components (DNA, histone H1 and H3) on SCG/ProT-induced clot structure, mechanical and lytic stability. Methods: Recombinant SCG was used to clot purified fibrinogen and plasma. The kinetics of formation and lysis of fibrin and plasma clots containing H1 or core histones+/-DNA were followed by turbidimetry. Fibrin structure and mechanical stability were characterized with scanning electron microscopy, pressure-driven permeation, and oscillation rheometry. Results: Histones and DNA favored the formation of thicker fibrin fibers and a more heterogeneous clot structure including high porosity with H1 histone, whereas low porosity with core histones and DNA. As opposed to previous observations with thrombin-induced clots, SCG/ProT-induced fibrin was not mechanically stabilized by histones. Similarly to thrombin-induced clots, the DNA-histone complexes prolonged fibrinolysis with tissue-type plasminogen activator (up to 2-fold). The anti-fibrinolytic effect of the DNA and DNA-H3 complex was observed in plasma clots too. Heparin (low molecular weight) accelerated the lysis of SCG/ProT-clots from plasma, even if DNA and histones were also present. Conclusions: In the interplay of NETs and fibrin formed by SCG, DNA and histones promote structural heterogeneity in the clots, and fail to stabilize them against mechanical stress. The DNA-histone complexes render the SCG-fibrin more resistant to lysis and thereby less prone to embolization.

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.

Fibrin structure, viscoelasticity and lysis face the interplay of biorelevant polyions.

PURPOSE OF REVIEW: In the past 5 decades, heparins have been widely used as anticoagulants in the prevention and treatment of thrombosis. Subsequent development of heparin variants of various size and charge facilitated the discovery of their multiple biological actions and nonanticoagulant benefits. Platelet-derived or microbial polyphosphates, as well as DNA released in the course of neutrophil extracellular trap-formation are additional polyanions, which can modulate the development and stability of thrombi associated with cancer or inflammation. In this review, we focus on the size-dependent and electric charge-dependent modulatory effects of the three polyanions of different chemical structure. RECENT FINDINGS: The polycationic histones have been recognized as potential biomarkers and therapeutic targets in several diseases related to inflammation and thrombosis. Since combating histones with activated protein C or heparin could cause unwanted bleeding, the quest for nonanticoagulant histone-neutralizing agents is ongoing. Polyanions may neutralize or exaggerate certain histone-mediated effects depending on their electric charge, size and histone effects under investigation. Several prothrombotic effects of polyphosphates and DNA are also size-dependent. SUMMARY: The efficiency of future therapeutics targeting prothrombotic polyanions or histones is not a simple matter of electric charge, but may rely on a delicate combination of size, charge and chemical composition.

3D culturing of human pluripotent stem cells-derived endothelial cells for vascular regeneration.

Rationale: Human induced pluripotent stem cell-derived endothelial cells can be candidates for engineering therapeutic vascular grafts. Methods: Here, we studied the role of three-dimensional culture on their characteristics and function both in vitro and in vivo. Results: We found that differentiated hPSC-EC can re-populate decellularized biomatrices; they remain viable, undergo maturation and arterial/venous specification. Human PSC-EC develop antifibrotic, vasoactive and anti-inflammatory properties during recellularization. In vivo, a robust increase in perfusion was detected at the engraftment sites after subcutaneous implantation of an hPSC-EC-laden hydrogel in rats. Histology confirmed survival and formation of capillary-like structures, suggesting the incorporation of hPSC-EC into host microvasculature. In a canine model, hiPSC-EC-seeded onto decellularised vascular segments were functional as aortic grafts. Similarly, we showed the retention and maturation of hiPSC-EC and dynamic remodelling of the vessel wall with good maintenance of vascular patency. Conclusions: A combination of hPSC-EC and biomatrices may be a promising approach to repair ischemic tissues.

Cryopreservation moderates the thrombogenicity of arterial allografts during storage.

INTRODUCTION: Management of vascular infections represents a major challenge in vascular surgery. The use of cryopreserved vascular allografts could be a feasible therapeutic option, but the optimal conditions for their production and use are not precisely defined. AIMS: To evaluate the effects of cryopreservation and the duration of storage on the thrombogenicity of femoral artery allografts. METHODS: In our prospective study, eleven multi-organ-donation-harvested human femoral arteries were examined at five time points during storage at -80°C: before cryopreservation as a fresh native sample and immediately, one, twelve and twenty-four weeks after the cryopreservation. Cross-sections of allografts were perfused with heparin-anticoagulated blood at shear-rates relevant to medium-sized arteries. The deposited platelets and fibrin were immunostained. The thrombogenicity of the intima, media and adventitia layers of the artery grafts was assessed quantitatively from the relative area covered by fibrin- and platelet-related fluorescent signal in the confocal micrographs. RESULTS: Regression analysis of the fibrin and platelet coverage in the course of the 24-week storage excluded the possibility for increase in the graft thrombogenicity in the course of time and supported the hypothesis for a descending trend in fibrin generation and platelet deposition on the arterial wall. The fibrin deposition in the cryopreserved samples did not exceed the level detected in any of the three layers of the native graft. However, an early (up to week 12) shift above the native sample level was observed in the platelet adhesion to the media. CONCLUSIONS: The hemostatic potential of cryopreserved arterial allografts was retained, whereas their thrombogenic potential declined during the 6-month storage. The only transient prothrombotic change was observed in the media layer, where the platelet deposition exceeded that of the fresh native grafts in the initial twelve weeks after cryopreservation, suggesting a potential clinical benefit from antiplatelet therapy in this time-window.

Size- and charge-dependent modulation of the lytic susceptibility and mechanical stability of fibrin-histone clots by heparin and polyphosphate variants.

BACKGROUND: Neutrophil extracellular traps (NETs) containing DNA and histones are expelled from neutrophils in infection and thrombosis. Heparins, anticoagulant polyanions, can neutralize histones with a potential therapeutic advantage in sepsis. Polyphosphates, procoagulant polyanions, are released by platelets and microorganisms. OBJECTIVES: To characterize the combined effects of NET components and polyanions on clot structure, mechanical properties and lytic susceptibility. METHODS: Scanning electron microscopy, pressure-driven permeation, turbidimetry, and oscillation rheometry were used for the characterization of the structure, viscoelasticity, and kinetics of formation and lysis of fibrin and plasma clots containing histones+/-DNA in combination with unfractionated heparin, its desulfated derivatives, low molecular weight heparin (LMWH), pentasaccharide, and polyphosphates of different sizes. RESULTS: Histones and DNA inhibited fibrin lysis by plasmin, but this behavior was not neutralized by negatively charged heparins or short polyphosphates. Rather, fibrin lysis was further inhibited by added polyanions. Histones inhibited plasma clot lysis by tissue plasminogen activator and the response to added heparin was size dependent. Unfractionated heparin, LMWH, and pentasaccharide had no effect, exacerbated, or reversed histone inhibition, respectively. Histones increased the mechanical strength of fibrin, which was exacerbated by smaller heparin and polyphosphate molecules. Histones increased fibrin diameter and pore size of fibrin clots and this effect was neutralized by all heparin variants but enhanced by polyphosphates. CONCLUSIONS: Despite their common polyanionic character, heparins and polyphosphates exert distinct effects on fibrin mechanical and fibrinolytic stability. Anti-fibrinolytic effects of histones were more often enhanced by polyanions not counteracted. Careful selection of anti-histone strategies is required if they are to be combined with thrombolytic therapy.

Biorelevant polyanions stabilize fibrin against mechanical and proteolytic decomposition: Effects of polymer size and electric charge.

The release of neutrophil extracellular traps (NETs) containing DNA and histones is an essential mechanism in the neutrophil-mediated innate immunity. In thrombi the polyanionic DNA confers mechanical and lytic resistance to fibrin and heparins interfere with the effects of NET components. Heparins are polyanions used not only as therapeutic agents, but they are also released by mast cells at entry sites of pathogens. Platelets and microorganisms release a different type of polyanions (polyphosphates) of various size (in the range 60-1000 phosphate monomers). With the current study we aimed to evaluate if the stability of fibrin is influenced by the type of polyanion, its molecular size or relative electric charge. Fibrin structure was approached with scanning electron microscopy (SEM) and pressure-driven permeation. An oscillation rheometer was used to investigate viscoelastic properties. Kinetic turbidimetric assays for the generation and dissolution of composite fibrin clots containing unfractionated heparin (UFH), and its partially or fully desulfated derivatives, as well as low molecular-weight heparin (LMWH), pentasaccharide (S5), and polyphosphates composed of 45 (P45), 100 (P100) or 700 (P700) monomers at average. The smaller polyanions P45, P100, LMWH, and S5 accelerated, whereas P700 and UFH retarded clot formation. All polyanions altered the fibrin structure: SEM and clot permeation showed thicker fibers with smaller (LMWH, S5, P700) or larger (UFH, P100) pores. All polyanions stabilized the clots mechanically, but the smaller P45, P100 and LMWH decreased the deformability of fibrin, whereas the large UFH and P700 increased the maximal bearable deformation of clots. Despite the size-dependent structural changes, all heparins caused a 10-15% prolongation of lysis-times with plasmin, and UFH-effects depended on sulfation patterns. The 20-35% prolongation of lysis-times caused by all polyphosphates was a kringle-dependent phenomenon, and was dampened in the presence of 6-aminohexanoate blocking the lysine-binding sites of plasmin. In summary, we found that polyanions of different chemical structure stabilize fibrin clots via size-dependent modulation of fibrin structure and kringle-dependent inhibition of plasmin-mediated fibrinolysis.

Hyaluronic acid decreases the mechanical stability, but increases the lytic resistance of fibrin matrices.

Hyaluronic acid (HA) is a large, non-sulfated glucosaminoglycan abundantly present at sites where fibrin is also formed (during wound healing, in arterial restenotic lesions and eroded atherosclerotic plaques). The aim of the present study was to characterize the structure of composite fibrin-HA clots with scanning electron microscopy (SEM), pressure-driven permeation and small-angle X-ray scattering (SAXS) and their viscoelastic properties with an oscillation rheometer. In addition the efficiency of fibrinolysis in these clots was investigated by kinetic turbidimetric and chromogenic assays for dissolution of fibrin and plasminogen activation by tissue-type plasminogen activator (tPA). Fibrin formed in the presence of native (1500kDa) HA and its 500kDa fragments had thicker fibers and larger pores according to the SEM and clot permeation data, whereas the 25kDa HA fragments had only minor effects. SAXS evidenced a mild disarrangement of protofibrils. These structural alterations suggest that HA modifies the pattern of fibrin polymerization favouring lateral association of protofibrils over formation of branching points. Rheometer data showed softer fibrin structures formed with 1500kDa and 500kDa HA and these clots presented with lower dynamic viscosity values and lower critical stress values at gel/fluid transition. tPA-catalysed plasminogen activation was markedly inhibited by HA, both in free solution and on the surface of fibrin clots, in the presence and in the absence of 6-aminohexanoate suggesting a kringle-independent mechanism. HA of 1500 and 500kDa size prolonged clot lysis with both plasmin and tPA and this inhibition was kringle-mediated, because it was abolished by 6-aminohexanoate and was not observed with des-(kringle1-4)-plasmin. Our data suggest that HA size-dependently modifies the pattern of fibrin polymerization with consequent inhibition of fibrinolysis. At sites of tissue injury and inflammation, HA could stabilize fibrin through modification of its structure and lysibility.

Free Fatty Acids Modulate Thrombin Mediated Fibrin Generation Resulting in Less Stable Clots.

Upon platelet activation, free fatty acids are released at the stage of thrombus formation, but their effects on fibrin formation are largely unexplored. Our objective was to characterize the kinetic effects of fatty acids on thrombin activity, as well as the structural and mechanical properties of the resultant fibrin clots. Thrombin activity on fibrinogen was followed by turbidimetry and detailed kinetic characterization was performed using a fluorogenic short peptide substrate. The viscoelastic properties of fibrin were measured with rotatory oscillation rheometer, whereas its structure was analyzed with scanning electron microscopy (SEM). In turbidimetric assays of fibrin generation, oleate and stearate at physiologically relevant concentrations (60-600 µM) produced a bell-shaped inhibitory dose response, increasing 10- to 30-fold the time to half-maximal clotting. Oleate inhibited thrombin activity on a short peptide substrate according to a mixed-type inhibitor pattern (a 9-fold increase of the Michaelis constant, Km and a 20% decrease of the catalytic constant), whereas stearate resulted in only a minor (15%) drop in the catalytic constant without any change in the Km. Morphometric analysis of SEM images showed a 73% increase in the median fiber diameter in the presence of stearate and a 20% decrease in the presence of oleate. Concerning the viscoelastic parameters of the clots, storage and loss moduli, maximal viscosity and critical shear stress decreased by 32-65% in the presence of oleate or stearate, but loss tangent did not change indicating decreased rigidity, higher deformability and decreased internal resistance to shear stress. Our study provides evidence that free fatty acids (at concentrations comparable to those reported in thrombi) reduce the mechanical stability of fibrin through modulation of thrombin activity and the pattern of fibrin assembly.

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.

Ultrastructure and composition of thrombi in coronary and peripheral artery disease: correlations with clinical and laboratory findings.

INTRODUCTION: Fibrin structure and cellular composition of thrombi profoundly affect the clinical outcomes in ischemic coronary and peripheral artery disease. Our study addressed the interrelations of structural features of thrombi and routinely measured laboratory parameters. MATERIALS AND METHODS: Thrombi removed by thromboaspiration following acute myocardial infarction (n=101) or thrombendarterectomy of peripheral arteries (n=50) were processed by scanning electron microscopy and immunostaining for fibrin and platelet antigen GPIIb/IIIa to determine fibrin fibre diameter and relative occupancy by fibrin and cells. Correlations between the structural characteristics and selected clinical parameters (age, sex, vascular localization, blood cell counts, ECG findings, antiplatelet medication, accompanying diseases, smoking) were assessed. RESULTS: We observed significant differences in mean fibre diameter (122 vs. 135 nm), fibrin content (70.5% vs. 83.9%), fluorescent fibrin/platelet coverage ratio (0.18 vs. 1.06) between coronary and peripheral thrombi. Coronary thrombi from smokers contained more fibrin than non-smokers (78.1% vs. 62.2% mean occupancy). In the initial 24 h, fibrin content of coronary thrombi decreased with time, whereas in peripheral thrombi platelet content increased in the first 7 days. In coronaries, higher platelet content and smaller vessel diameter were associated with thinner fibrin fibres, whereas hematocrit higher than 0.35 correlated with larger intrathrombotic platelet occupancy. Smoking and dyslipidaemia strengthened the dependence of clot platelet content on systemic platelet count (the adjusted determination coefficient increased from 0.33 to 0.43 and 0.65, respectively). CONCLUSION: Easily accessible clinical parameters could be identified as significant determinants of ultrastructure and composition of coronary and peripheral thrombi.

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.

Physical and enzymatic perturbation of the architecture of the Tunica media destroys its inherent thromboresistance.

The media layer of the arterial cryo-cross sections, is defective for vWf-dependent platelet adhesion. Exposure of the same layer by stripping off the most inner portions of the vessel wall results in a highly thrombogenic surface. Stripping or balloon dilation was applied to porcine arteries prior to functional assays. Cryosections of treated or untreated arteries were perfused with porcine blood at 3,350 s(-1) and platelet deposition was detected by indirect immunofluorescence. Following balloon dilation, vWf-dependent platelet deposition increased; covering 9.08 +/- 1.36% of the total media surface area, this value for untreated vessels was 0.88 +/- 0.14%. A 10-fold increase was also found in the binding of vWf-coated fluorescent beads to the media. In addition to mechanical procedures, treatment by serine-proteases like trypsin, chymotrypsin and proteinase 3, or by chondroitinase ABC, but not by heparitinase also resulted in a 7-10-fold increase in platelet coverage over the media. Collagen in the media may be complexed with another vessel wall component shielding the vWf-binding sites. Mechanical or biochemical processes unmask these sites, and increase the thrombogenicity of the vessel wall.

Influence of anatomical location on arterial thrombosis.

Atherosclerosis manifests as a systemic disease with near global involvement of the named segments of the arterial tree. Acute thrombotic arterial occlusion, however, is not equally distributed. To evaluate intra-individual regional differences in arterial thrombogenicity, we compared (111)In-platelet deposition in porcine carotid and femoral arteries after a standardized crush injury. Within the unidirectional flow conditions of elastic carotid arteries, platelet deposition was more than 3-fold higher compared with predominantly muscular femoral arteries with triphasic arterial flow. To determine the influence of rheology on platelet deposition after crush injury, carotid arteries were transplanted into the femoral position and compared with the paired native carotid and femoral arteries. Similarly, femoral arteries transposed to the carotid position were compared with the paired native carotid artery. In each of these experiments, arterial transposition to a new anatomic location imparts a predilection for platelet deposition indigenous to the new location. In the controlled environment of two high-shear thrombin-independent and -dependent flow chambers, porcine carotid and femoral arterial substrates were indistinguishable from one another with respect to platelet deposition. Regional differences in arterial hemodynamics may account for substantial differences in thrombosis arising from deep arterial injury.