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.

[Increased NF-κB and iNOS Expression in Keratoconus Keratocytes - Hints for an Inflammatory Component?] / Erhöhte NF-κB- und iNOS-Expression in Keratozyten von Keratokonuspatienten ­ Hinweise auf eine entzündliche Komponente?

PURPOSE: In recent years, there has been increasing evidence of an inflammatory component in keratoconus. A key gene in inflammatory processes is the nuclear factor kappa B (NF-κB). NF-κB is a transcription factor for the enzyme nitric oxide synthase (NOS), which is involved with the competing enzyme arginase (Arg) in inflammatory processes. The aim of this study was to analyze the isotypes of NOS and arginase, the expression of NF-κB, NOS and arginase, and the regulatory mechanism of NOS and arginase in keratocytes of keratoconus patients using the inhibitor 1400W in vitro. METHODS: Human keratocytes were isolated from surgically removed corneas of 8 KC patients and 8 normal human corneal buttons and were cultured to confluence, in vitro. Quantitative PCR and Western blot analysis were performed to examine NF-κB, NOS and arginase expression in keratocytes. Nitrite and urea concentrations in the supernatant of the cells were analyzed using 0 - 40 µM 1400W iNOS inhibitor concentrations. RESULTS: Only the isotypes iNOS and Arg-II were detected in the keratocytes. The mRNA expression of NF-κB and iNOS were higher in KC keratocytes than in normal cells (p = 0.0135 and p = 0.0001), whereas no differences were measurable in Arg-II expression. In the WB, a higher band intensity was measurable in NF-κB (p = 0.0012), and in iNOS, no differences in band intensity could be detected. In the supernatant of the KC keratocytes, lower concentrations of nitrite and urea were measured after the addition of the inhibitor 1400W (p ≤ 0.014), but not in normal cells (p ≥ 0.178). CONCLUSION: Due to the increased expression of NF-κB and iNOS, an inflammatory component in keratoconus must be assumed. The different regulation of the KC keratocytes by the iNOS inhibitor 1400W suggests an altered metabolic activity which can be caused by inflammatory processes.

Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors.

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.

Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes.

PURPOSE: Keratoconus (KC) is a disease characterized by thinning and deformation of the cornea, but its etiology remains unknown. Seventy percent of the corneal stroma consists of collagen, which is composed of three intertwined polypeptide chains with glycine-hydroxyproline-proline repeats along their sequence. Arginase is a cytoplasmatic enzyme and catalyzes the conversion of arginine to urea and ornithine, which serves as a precursor for the endogenous synthesis of proline and hydroxyproline. The purpose of this study was to analyze arginase activity, as well as collagen and urea formation in normal and KC-keratocytes and to determine the impact of urea on keratocyte viability and proliferation in vitro. METHODS: Primary human keratocytes were isolated by digestion in collagenase (1.0 mg/mL) from surgically removed corneas of eight keratoconus patients and eight normal human corneal buttons and cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. Arginase activity and urea concentration were measured in cell-lysates, hydroxyproline concentration in supernatant of cultured keratocytes using colorimetric assay. Cell viability and cell proliferation of cultured keratocytes were assessed after treatment with urea at concentrations up to10 mM for 24 h using assays for metabolic activity and DNA replication. RESULTS: Arginase activity and urea concentration in KC-keratocytes decreased by about 50 % compared to normal keratocytes (p = 0.003 and p = 0.008). Hydroxyproline synthesized by cultured KC-keratocytes was also approximately 50 % less compared to normal keratocytes (p = 0.02) and this difference decreased following treatment with 5.0 or 10.0 mM urea (p = 0.02; 0.03), without any change in cell viability (p > 0.09). However, the urea treatment increased modestly (by 20 %) the proliferation rate of KC-keratocytes (p = 0.04; 0.04; 0.04), without any effect on normal cultured keratocytes (p > 0.09). CONCLUSIONS: We identified suppressed arginase activity in the metabolic program of cultured keratoconus keratocytes. The level of urea, as one product of the enzyme arginase was also decreased. This results in impaired collagen synthesis, evidenced in the culture by reduced hydroxyproline concentration. In addition, our data showed that the other product of the arginase reaction, urea supports the proliferation of KC-keratocytes, without changes in their viability. The metabolic reprogramming of keratoconus keratocytes and its impact on development of a clinically detectable keratoconus disease has to be further analyzed.

Bleeding related to disturbed fibrinolysis.

The components and reactions of the fibrinolysis system are well understood. The pathway has fewer reactants and interactions than coagulation, but the generation of a complete quantitative model is complicated by the need to work at the solid-liquid interface of fibrin. Diagnostic tools to detect disease states due to malfunctions in the fibrinolysis pathway are also not so well developed as is the case with coagulation. However, there are clearly a number of inherited or acquired pathologies where hyperfibrinolysis is a serious, potentially life-threatening problem and a number of antifibrinolytc drugs are available to treat hyperfibrinolysis. These topics will be covered in the following review.

Evaluation of the mechanical properties of the anterior lens capsule following femtosecond laser capsulotomy at different pulse energy settings.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule opening performed with femtosecond laser capsulotomy at different energy settings in ex vivo porcine anterior lens capsule specimens. METHODS: Twenty-five fresh porcine eyes per group were included in the study. Femtosecond laser capsulotomy was performed with three different pulse energy levels: 2 µJ (low energy group), 5 µJ (intermediate energy group), and 10 µJ (high energy group). The capsule openings were stretched with universal testing equipment until they ruptured. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy. RESULTS: The high energy group had significantly lower rupture force (108 ± 14 mN) compared to the intermediate energy group (118 ± 10 mN) (P < .05) and low energy group (119 ± 11 mN) (P < .05), but the difference between the intermediate energy and low energy groups was not significant (P = .9479). The high energy group had significantly lower circumference stretching ratio (144% ± 3%) compared to the intermediate energy group (148% ± 3%) (P < .05) and low energy group (148% ± 3%) (P < .05), but the difference between the intermediate energy group and low energy group was not significant (P = .9985). Scanning electron microscopy images showed that the edge was only serrated with low and intermediate energy, but additional signs of collagen melting and denaturation were observed at high energy. CONCLUSIONS: Anterior capsule openings created at a high energy level were slightly weaker and less extensible than those created at low or intermediate levels, possibly due to the increased thermal effect of photo-disruption.

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.

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.

Comparison of the mechanical properties of the anterior lens capsule following manual capsulorhexis and femtosecond laser capsulotomy.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule openings performed with the continuous curvilinear capsulorhexis (CCC) technique and femtosecond laser capsulotomy (FLC) in ex vivo porcine lens capsule specimens. METHODS: Fresh porcine eyes were included in the study (CCC group, n = 50; FLC group, n = 30). The capsule openings were stretched with universal testing equipment until they ruptured. The rupture force and circumference stretching ratio were evaluated. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy (SEM). RESULTS: The average rupture force was higher in the CCC group (median: 155 mN; interquartile range [IQR]: 129 to 201 mN; range: 71 to 294 mN) than in the FLC group (median: 119 mN; IQR: 108 to 128 mN; range: 91 to 142 mN) (P < .01, Mann-Whitney U test). The average circumference stretching ratio in the CCC group was greater (median: 150%; IQR: 146% to 156%; range: 136% to 161%) than in the FLC group (median: 148%; IQR: 145% to 150%; range: 141% to 154%) (P = .0468, Mann-Whitney U test). When less than 71 mN, no capsular tear occurred in either group. When less than 91 mN, no capsular tear occurred in the FLC group, whereas at 91 mN, the probability of capsular tears was 9% for the CCC group. SEM examination found that the CCC group had smooth edges, whereas those of the FLC group were gently serrated. CONCLUSIONS: According to the current results in a porcine eye model, FLC had less average resistance to capsule tear than CCC, but the weakest openings were seen in the CCC group.

Synergism of red blood cells and tranexamic acid in the inhibition of fibrinolysis.

BACKGROUND: Postpartum hemorrhage (PPH) is the leading cause of maternal death worldwide. The World Maternal Antifibrinolytic trial showed that antifibrinolytic tranexamic acid (TXA) reduces PPH deaths. Maternal anemia increases the risk of PPH. The World Maternal Antifibrinolytic-2 trial is now assessing whether TXA can prevent PPH in women with anemia. Low red blood cell (RBC) counts promote fibrinolysis by altering fibrin structure and plasminogen activation. OBJECTIVES: We explored interactions between RBCs and TXA in inhibiting fibrinolysis. METHODS: We used global fibrinolytic assays (ball sedimentation and viscoelasticity) to monitor the lysis of fibrin containing plasminogen and tissue-type plasminogen activator. We applied a fluorogenic kinetic assay to measure plasmin generation in fibrin clots and scanning electron microscopy to study fibrin structure. RESULTS: According to parallel-line bioassay analysis of the fibrin lysis-time data, the antifibrinolytic potency of 4-128 µM TXA was increased in the presence of 10% to 40% (v/v) RBCs. Global fibrinolysis assays showed that the joint effect of RBCs and TXA was about 15% larger than the sum of their individual effects in the inhibition of fibrinolysis. In plasminogen activation, TXA added the same increment of inhibition to the effect of RBCs at any cell count in the fibrin clot. Regarding fibrin structure, TXA thickened fibrin fibers, which impaired plasminogen activation, whereas RBCs promoted fine fibers that were more resistant to plasmin. CONCLUSIONS: The antifibrinolytic potency of TXA is enhanced in fibrin formed in the presence of RBCs through inhibition of plasminogen activation and fibrin lysis, which correlates with modifications of fibrin structures.

Coagulation and platelet biology at the intersection of health and disease: illustrated capsules of the 11th Symposium on Hemostasis at the University of North Carolina.

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field. This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks.

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia.

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies.

Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.

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.

Lytic resistance of fibrin containing red blood cells.

OBJECTIVE: Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. METHODS AND RESULTS: If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs. CONCLUSIONS: RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.

Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates.

INTRODUCTION: Beyond the three-dimensional fibrin network, the mechanical and lytic stability of thrombi is supported by the matrix of neutrophil extracellular traps (NETs) composed of polyanionic DNA meshwork with attached proteins including polycationic histones. Polyphosphates represent another type of polyanions, which in their linear form are known to enhance the fibrin stabilizing effects of DNA and histones. However, in vivo polyphosphates are also present in the form of nanoparticles (PolyP-NP), the interference of which with the fibrin/NET matrix is poorly characterized. AIMS: To compare the effects of linear and nanoparticulate polyphosphates, and their combinations with relevant NET components (DNA, histone H3) on fibrin formation, structure, and lysis in in vitro assays focusing on histone-polyphosphate interactions. METHODS: Transmission electron microscopy and dynamic light scattering for stability of the PolyP-NP preparations. Turbidimetry for kinetics of fibrinogen clotting by thrombin and fibrin dissolution by tissue-type plasminogen activator/plasminogen. Scanning electron microscopy for fibrin structure. Surface plasmon resonance for strength of histone-PolyP interactions. RESULTS: Both linear PolyP and PolyP-NP accelerated the fibrin formation and slowed down its dissolution and these effects were strongly dependent on the number of individual PolyP particles and not on their size. Addition of DNA did not modify significantly the PolyP-NP effects on fibrin formation and lysis. Both linear and nanoparticulate PolyP counteracted the effect of histone in the acceleration of fibrinogen clotting by thrombin. PolyP-NP, but not linear PolyP enhanced the prolongation of lysis time in fibrin containing histone and caused more pronounced thickening of the fibrin fibers than the linear form. Finally, PolyP-NP bound weaker to histone than the linear form. CONCLUSIONS: The interaction of PolyP with histone was a stronger modulator of fibrin formation and lysis than its interaction with DNA. In addition, the PolyP nanoparticles enhanced the thrombus stabilizing effects of histone more effectively than linear PolyP.

Fibrin to von Willebrand factor ratio in arterial thrombi is associated with plasma levels of inflammatory biomarkers and local abundance of extracellular DNA.

INTRODUCTION: The composition of thrombi determines their structure, mechanical stability, susceptibility to lysis, and consequently, the clinical outcome in coronary artery disease (CAD), acute ischemic stroke (AIS), and peripheral artery disease (PAD). Fibrin forms the primary matrix of thrombi intertwined with DNA, derived from neutrophil extracellular traps (NETs), and von Willebrand factor (VWF) bridging DNA and platelets. Here we examined the relative content of fibrin, DNA and VWF in thrombi and analyzed their interrelations and quantitative associations with systemic biomarkers of inflammation and clinical characteristics of the patients. PATIENTS, METHODS: Thrombi extracted from AIS (n = 17), CAD (n = 18) or PAD (n = 19) patients were processed for scanning electron microscopy, (immune)stained for fibrin, VWF and extracellular DNA. Fibrin fiber diameter, cellular components, fibrin/DNA and fibrin/VWF ratios were measured. RESULTS: Patients' age presented as a strong explanatory factor for a linear decline trend of the VWF content relative to fibrin in thrombi from CAD (adjusted-R2 = 0.43) and male AIS (adjusted-R2 = 0.66) patients. In a subgroup of CAD and PAD patients with dyslipidemia and high (above 80%) prevalence of atherothrombosis a significant correlation was observed between the VWF and DNA content in thrombi (adjusted-R2 = 0.40), whereas a 3.7-fold lower linear regression coefficient was seen in AIS patients, in whom the fraction of thrombi of atherosclerotic origin was 57%. Independently of anatomical location, in patients with atherosclerosis the VWF in thrombi correlated with the plasma C-reactive protein levels. CONCLUSIONS: The observed interrelations between thrombus constituents and systemic inflammatory biomarkers suggest an intricate interplay along the VWF/NET/fibrin axis in arterial thrombosis.

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.

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.