Publications by authors named "Krasimir Kolev"

44 Publications

Cryopreservation moderates the thrombogenicity of arterial allografts during storage.

PLoS One 2021 22;16(7):e0255114. Epub 2021 Jul 22.

Department of Vascular and Endovascular Surgery, Heart and Vascular Center, Semmelweis University, Budapest, Hungary.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0255114PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297765PMC
July 2021

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

J Thromb Haemost 2021 05 20;19(5):1307-1318. Epub 2021 Feb 20.

Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary.

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.
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http://dx.doi.org/10.1111/jth.15258DOI Listing
May 2021

Editorial: Fibrinolysis in Immunity.

Front Immunol 2020 31;11:582. Epub 2020 Mar 31.

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia.

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http://dx.doi.org/10.3389/fimmu.2020.00582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137896PMC
March 2021

Structure, Mechanical, and Lytic Stability of Fibrin and Plasma Coagulum Generated by Staphylocoagulase From .

Front Immunol 2019 20;10:2967. Epub 2019 Dec 20.

Haemostasis Section, Biotherapeutics Group, National Institute for Standards and Control, Potters Bar, United Kingdom.

causes localized infections or invasive diseases (abscesses or endocarditis). One of its virulence factors is staphylocoagulase (SCG), which binds prothrombin to form a complex with thrombin-like proteolytic activity and leads to uncontrolled fibrin generation at sites of bacterial inoculation. The aim of this study was to characterize the formation, structure, mechanical properties and lysis of SCG-generated clots. Recombinant SCG was expressed in , purified and the amidolytic activity of its complexes with human prothrombin (SCG-PT) and thrombin (SCG-T) was determined using human thrombin as a reference. Fibrin clots were prepared from purified fibrinogen and human plasma using thrombin, SCG-PT or SCG-T as a coagulase. The kinetics of clot formation and lysis by tissue-type plasminogen activator (tPA) were monitored with turbidimetric assays. Fibrin ultrastructure was examined with scanning electron microscopy and small-angle X-ray scattering (SAXS). Fibrin clot porosity was characterized with fluid permeation assays, whereas the viscoelastic properties and mechanical stability were evaluated with oscillation rheometry. Compared to thrombin, the amidolytic and clotting activity of SCG-PT was 1.6- to 2.5-fold lower on a molar basis. SCG-T had equivalent amidolytic, but reduced clotting activity both on pure fibrinogen (1.6-fold), and in plasma (1.3-fold). The SCG-PT and SCG-T generated fibrin with thicker fibers (10-60% increase in median diameter) than thrombin due to increased number of fibrin protofibrils per fiber cross-section. According to the fluid permeability of the clots SCG-PT and SCG-T promoted the formation of more porous structures. The shear stress resistance in the pure fibrin and plasma clots generated by SCG-PT was significantly lower than in the thrombin clots (243.8 ± 22.0 Pa shear stress was sufficient for disassembly of SCG-PT fibrin vs. 937.3 ± 65.6 Pa in thrombin clots). The tPA-mediated lysis of both pure fibrin and plasma clots produced by SCG-PT or SCG-T was accelerated compared to thrombin, resulting in up to a 2.1-fold increase in tPA potency. Our results indicate that SCG generates a thrombus scaffold with a structure characterized by impaired mechanical stability and increased lytic susceptibility. This proneness to clot disintegration could have implications in the septic embolism from endocardial bacterial vegetation.
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http://dx.doi.org/10.3389/fimmu.2019.02967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933771PMC
November 2020

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

J Mech Behav Biomed Mater 2020 02 28;102:103459. Epub 2019 Sep 28.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmbbm.2019.103459DOI Listing
February 2020

[Increased NF-κB and iNOS Expression in Keratoconus Keratocytes - Hints for an Inflammatory Component?]

Klin Monbl Augenheilkd 2021 Sep 10;238(9):1010-1017. Epub 2019 Oct 10.

Klinik für Augenheilkunde, Universitätsklinikum des Saarlandes UKS, Homburg/Saar.

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.
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http://dx.doi.org/10.1055/a-1002-0100DOI Listing
September 2021

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

Thromb Res 2019 Oct 7;182:1-11. Epub 2019 Aug 7.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. Electronic address:

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.
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http://dx.doi.org/10.1016/j.thromres.2019.08.003DOI Listing
October 2019

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

Haematologica 2020 01 2;105(1):218-225. Epub 2019 May 2.

Department of Medicine, Division of Hematology and Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

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.
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http://dx.doi.org/10.3324/haematol.2019.217083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939515PMC
January 2020

Neutrophil extracellular traps in thrombi retrieved during interventional treatment of ischemic arterial diseases.

Thromb Res 2019 Mar 15;175:46-52. Epub 2019 Jan 15.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. Electronic address:

Introduction: The ultrastructure and cellular composition of thrombi has a profound effect on the outcome of acute ischemic stroke (AIS), coronary (CAD) and peripheral artery disease (PAD). Activated neutrophils release a web-like structure composed mainly of DNA and citrullinated histones, called neutrophil extracellular traps (NET) that modify the stability and lysability of fibrin. Here, we investigated the NET-related structural features of thrombi retrieved from different arterial localizations and their interrelations with routinely available clinical data.

Patients And Methods: Thrombi extracted from AIS (n = 78), CAD (n = 66) or PAD (n = 64) patients were processed for scanning electron microscopy, (immune)stained for fibrin, citrullinated histone H3 (cH3) and extracellular DNA. Fibrin fiber diameter, cellular components, DNA and cH3 were measured and analyzed in relation to clinical parameters.

Results: DNA was least present in AIS thrombi showing a 2.5-fold lower DNA/fibrin ratio than PAD, whereas cH3 antigen was unvaryingly present at all locations. The NET content of thrombi correlated parabolically with systemic inflammatory markers and positively with patients' age. The median platelet content was lower in PAD (2.2%) than in either AIS (3.9%) or CAD (3.1%) and thrombi from smokers contained less platelets than non-smokers. Fibrin fibers were significantly thicker in male patients with CAD (median fiber diameter 76.3 nm) compared to AIS (64.1 nm) or PAD (62.1 nm) and their diameter correlated parabolically with systemic inflammatory markers.

Conclusions: The observed NET-related variations in thrombus structure shed light on novel determinants of thrombus stability that eventually affect both the spontaneous progress and therapeutic outcome of ischemic arterial diseases.
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http://dx.doi.org/10.1016/j.thromres.2019.01.006DOI Listing
March 2019

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

Sci Rep 2018 03 29;8(1):5366. Epub 2018 Mar 29.

Department of Medical Biochemistry, Semmelweis University, Budapest, 1094, Hungary.

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.
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http://dx.doi.org/10.1038/s41598-018-23725-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876378PMC
March 2018

Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes.

Biomed Res Int 2017 3;2017:5130495. Epub 2017 Jul 3.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically stabilized trypsin-loaded liposomes (SSL) and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-based SSL were prepared and their structure was studied by transmission electron microscopy combined with freeze fracture (FF-TEM), Fourier transform infrared spectroscopy (FT-IR), and small-angle X-ray scattering (SAXS). Fibrinolytic activity was examined at 45, 37, or 24°C on fibrin or plasma clots with turbidimetric and permeation-driven lysis assays. Trypsin was shown to be attached to the inner surface of vesicles (SAXS and FF-TEM) close to the lipid hydrophilic/hydrophobic interface (FT-IR). The thermosensitivity of SSL was evidenced by enhanced fibrinolysis at 45°C: time to reduce the maximal turbidity to 20% decreased by 8.6% compared to 37°C and fibrin degradation product concentration in the permeation lysis assay was 2-fold to 5-fold higher than that at 24°C. SSL exerted its fibrinolytic action on fibrin clots under both static and dynamic conditions, whereas plasma clot dissolution was observed only in the permeation-driven assay. The improved fibrinolytic efficiency of SSL under dynamic conditions suggests that they may serve as a novel therapeutic candidate for dissolution of intravascular thrombi, which are typically exposed to permeation forces.
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http://dx.doi.org/10.1155/2017/5130495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512056PMC
April 2018

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

Matrix Biol 2017 11 19;63:55-68. Epub 2016 Dec 19.

Department of Medical Biochemistry, Semmelweis University, 1094 Budapest, Tűzoltó utca 37-47, Hungary. Electronic address:

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.
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http://dx.doi.org/10.1016/j.matbio.2016.12.008DOI Listing
November 2017

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

PLoS One 2016 12;11(12):e0167806. Epub 2016 Dec 12.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167806PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5152833PMC
June 2017

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

Graefes Arch Clin Exp Ophthalmol 2017 Jan 25;255(1):91-97. Epub 2016 Oct 25.

Department of Ophthalmology, Saarland University Medical Center, Kirrberger Str. 100, D-66424, Homburg, Saar, Germany.

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.
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http://dx.doi.org/10.1007/s00417-016-3520-xDOI Listing
January 2017

Bleeding related to disturbed fibrinolysis.

Br J Haematol 2016 10 1;175(1):12-23. Epub 2016 Aug 1.

Biotherapeutics Group, National Institute for Biological Standards and Control, South Mimms, UK.

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.
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http://dx.doi.org/10.1111/bjh.14255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096260PMC
October 2016

Decreased fibrinolytic potential and morphological changes of fibrin structure in dermatitis herpetiformis.

J Dermatol Sci 2016 Oct 6;84(1):17-23. Epub 2016 Jul 6.

Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest, Hungary. Electronic address:

Background: Recently, high prevalence of cryofibrinogenaemia has been observed in plasma of untreated dermatitis herpetiformis (DH) patients, and the pathological IgA and TG3 deposits in the papillary dermis were found to co-localize with fibrin and fibrinogen.

Objective: To study the fibrinolytic potential in plasma of untreated, dapsone and or/gluten-free diet treated DH patients as well as the in vitro effect of dapsone on the fibrinolytic profile.

Method: Plasma samples of 23 DH patients, 19 healthy subjects and 5 pemphigus vulgaris patients were investigated by a turbidimetric-clot lysis assay. Out of them 5 DH plasma samples representing different fibrinolytic parameters, and 3 healthy controls were selected for parallel fibrin clot preparation. The clot fibrin structure was examined by scanning electron microscopy (SEM), and the diameters of 900 fibrin fibres were determined in each clot.

Results: A significantly prolonged clot lysis time was detected in untreated DH patients. The turbidity values of DH plasma clots indicated an altered fibrin structure that was also confirmed by SEM: significantly thicker fibrin fibers were observed in untreated, TG3 antibody positive DH patients compared to healthy controls, whereas the fiber diameters of dapsone-treated patients were similar or thinner than the control values. In line with the structural changes of fibrin, the fibrinolytic profile of 5 DH patients under dapsone treatment approached the control values.

Conclusion: This study revealed that the fibrinolytic potential was impaired in the plasma of untreated DH patients, whereas dapsone corrected the fibrinolytic defect. These data suggest a pathogenic role for plasma-derived factors in the development of skin symptoms and add a new aspect to the long-known beneficial, symptomatic effect of dapsone in active DH.
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http://dx.doi.org/10.1016/j.jdermsci.2016.07.005DOI Listing
October 2016

Circulating Transglutaminase 3-Immunoglobulin A Immune Complexes in Dermatitis Herpetiformis.

J Invest Dermatol 2016 08 20;136(8):1729-1731. Epub 2016 Apr 20.

Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest, Hungary. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2016.03.039DOI Listing
August 2016

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

Thromb Haemost 2016 Mar 3;115(3):591-9. Epub 2015 Dec 3.

Colin Longstaff, Biotherapeutics Group, National Institute for Biological Standards and Control, S Mimms, Herts, EN6 3QG, UK, Tel.: +44 1707 641253, Fax: +44 1707 641050, E-Mail:

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.
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http://dx.doi.org/10.1160/TH15-03-0214DOI Listing
March 2016

Femtosecond laser-assisted cataract surgery in Alport syndrome with anterior lenticonus.

Eur J Ophthalmol 2015 Nov-Dec;25(6):507-11. Epub 2015 Apr 9.

Department of Ophthalmology, Semmelweis University, Budapest - Hungary.

Purpose: To report the surgical treatment of 3 eyes of 2 patients with bilateral anterior lenticonus due to Alport syndrome using femtosecond laser-assisted cataract surgery (FLACS).

Methods: Two patients with Alport syndrome presented to our department due to anterior lenticonus in both eyes. We performed FLACS with posterior chamber lens implantation in both eyes of one patient and in one eye of the other patient. Anterior segment morphologic changes were visualized with a Scheimpflug camera, and anterior segment optical coherence tomography preoperatively and 3 months after surgery. Ultrastructure of the cut capsule edges was observed with scanning electron microscopy and compared to the edge of femtosecond laser capsulotomy performed on an otherwise healthy patient with cataract (control).

Results: The intraocular lens (IOL) postoperative positioning parameters met the international requirements of aspherical and wavefront customized IOLs (tilt <10 degree, decentration <800 µm). Scanning electron microscopy revealed the same characteristics of the cut capsule edges in the Alport and in the control eyes.

Conclusions: Femtosecond laser cataract surgery can be a safe and successful method for optical rehabilitation of anterior lenticonus in patients with Alport syndrome.
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http://dx.doi.org/10.5301/ejo.5000603DOI Listing
February 2016

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

Thromb Haemost 2015 Jun 19;113(6):1289-98. Epub 2015 Mar 19.

Krasimir Kolev, Semmelweis University, Department of Medical Biochemistry, Tűzoltó utca 37-47., 1094 Budapest, Hungary, Tel.: +36 1 4591500/60035, Fax: +36 1 2670031, E-mail:

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.
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http://dx.doi.org/10.1160/TH14-08-0669DOI Listing
June 2015

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

J Refract Surg 2015 Mar;31(3):153-7

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.
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http://dx.doi.org/10.3928/1081597X-20150220-02DOI Listing
March 2015

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

Thromb Res 2015 Apr 9;135(4):760-6. Epub 2015 Feb 9.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. Electronic address:

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.
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http://dx.doi.org/10.1016/j.thromres.2015.02.004DOI Listing
April 2015

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

J Refract Surg 2014 Oct;30(10):660-4

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.
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http://dx.doi.org/10.3928/1081597X-20140903-08DOI Listing
October 2014

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

Biochemistry 2014 Oct 26;53(40):6348-56. Epub 2014 Sep 26.

Department of Medical Biochemistry, Semmelweis University , 1094 Budapest, Hungary.

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.
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http://dx.doi.org/10.1021/bi500661mDOI Listing
October 2014

Ambivalent roles of carboxypeptidase B in the lytic susceptibility of fibrin.

Thromb Res 2014 Jan 21;133(1):80-7. Epub 2013 Sep 21.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. Electronic address:

Background: Removal of C-terminal lysine residues that are continuously exposed in lysing fibrin is an established anti-fibrinolytic mechanism dependent on the plasma carboxypeptidase TAFIa, which also removes arginines that are exposed at the time of fibrinogen clotting by thrombin.

Objective: To evaluate the impact of alterations in fibrin structure mediated by constitutive carboxypeptidase activity on the function of fibrin as a template for tissue plasminogen activator-(tPA) induced plasminogen activation and its susceptibility to digestion by plasmin.

Methods And Results: We used the stable carboxypeptidase B (CPB), which shows the same substrate specificity as TAFIa. If 1.5 - 6μM fibrinogen was clotted in the presence of 8U/mL CPB, a denser fibrin network was formed with thinner fibers (the median fiber diameter decreased from 138 - 144nm to 89 - 109nm as established with scanning electron microscopy). If clotting was initiated in the presence of 5 - 10μM arginine, a similar decrease in fiber diameter (82 -95nm) was measured. The fine structure of arginine-treated fibrin enhanced plasminogen activation by tPA, but slowed down lysis monitored using fluorescent tPA and confocal laser microscopy. However, if lysis was initiated with plasmin in CPB-treated fibrin, the rate of dissolution increased to a degree corresponding to doubling of the plasmin concentration.

Conclusion: The present data evidence that CPB activity generates fine-mesh fibrin which is more difficult to lyse by tPA, but conversely, CPB and plasmin together can stimulate fibrinolysis, possibly by enhancing plasmin diffusion.
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http://dx.doi.org/10.1016/j.thromres.2013.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891004PMC
January 2014

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

J Biol Chem 2013 Mar 4;288(10):6946-56. Epub 2013 Jan 4.

Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Potters Bar EN6 3QG, United Kingdom.

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.
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http://dx.doi.org/10.1074/jbc.M112.404301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591605PMC
March 2013

Modulation of the von Willebrand factor-dependent platelet adhesion through alternative proteolytic pathways.

Thromb Res 2012 Apr 15;129(4):e41-6. Epub 2011 Dec 15.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

Introduction: Platelet adhesion to collagen under high shear rates depends on the optimal size of the von Willebrand factor (VWF) multimers, which is determined by their limited proteolysis. The present study attempts to identify the role of hemostatic-fibrinolytic enzymes (thrombin, plasmin) and leukocyte-derived proteases (matrix metalloproteinase (MMP)-8, MMP-9, neutrophil elastase) in the cleavage of VWF and to characterize the effect of flow and platelets on this proteolysis and its functional consequences on platelet adhesion. Methods and results According to VWF immunoblots, plasmin, neutrophil elastase and thrombin at concentrations of in vivo relevance resulted in extensive degradation of VWF within several minutes. Platelets protected VWF against this proteolysis under static conditions, whereas perfusion of the proteases at 3350s(-1) shear rate over VWF immobilized on artery cross sections enhanced its degradation and blocked the protective effect of platelets. In parallel with VWF digestion, the examined proteases impaired the VWF-dependent platelet adhesion as reflected in the decreased surface-bound GpIIb/IIIa immunoreactivity following perfusion of collagen-coated surfaces or artery sections with blood and plasmin, neutrophil elastase or thrombin. Within the time frame of minutes no VWF cleavage could be detected under static or flow conditions after exposure to MMP-8 and MMP-9 at concentrations relevant to physiological neutrophil counts.

Conclusion: Our results indicate a shear- and platelet-dependent role for several proteases in the local modulation of the VWF function.
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http://dx.doi.org/10.1016/j.thromres.2011.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323834PMC
April 2012

Lytic resistance of fibrin containing red blood cells.

Arterioscler Thromb Vasc Biol 2011 Oct 7;31(10):2306-13. Epub 2011 Jul 7.

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

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.
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http://dx.doi.org/10.1161/ATVBAHA.111.229088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3339800PMC
October 2011
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