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Krasimir Kolev  - - - 
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Krasimir Kolev

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Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

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(1995 - 2018)
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Article 2 Reads 0 Citations Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin Imre Varjú, Veronika Judit Farkas, László Kohidai, Laszlo Sz... Published: 29 March 2018
Scientific Reports, doi: 10.1038/s41598-018-23725-4
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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.
Article 4 Reads 0 Citations Hyaluronic acid decreases the mechanical stability, but increases the lytic resistance of fibrin matrices Erzsébet Komorowicz, Nóra Balázs, Zoltán Varga, László Szabó... Published: 01 November 2017
Matrix Biology, doi: 10.1016/j.matbio.2016.12.008
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Article 4 Reads 0 Citations Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes Anna Tanka-Salamon, Attila Bóta, András Wacha, Judith Mihály... Published: 01 January 2017
BioMed Research International, doi: 10.1155/2017/5130495
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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 () and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-based 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 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. 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 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.1. IntroductionThrombolysis based on enzymatic dissolution of fibrin is currently the first-line treatment of ischemic stroke as well as certain selected cases of acute myocardial infarction [1, 2]. Most of the fibrinolytic agents are plasminogen activators, which can be classified as “indirect fibrinolytics,” because their enzymatic action is directed towards plasminogen, while, in contrast, fibrinolytics such as plasmin and its derivatives degrade fibrin without any intermediate step of plasminogen activation and are therefore designated as “direct fibrinolytics.” Systemic (intravenous) administration of indirect fibrinolytics is accompanied by frequent bleeding side effects related to the large excess of activator at its therapeutic dose over the inhibitor capacity of blood plasma [3]. In contrast, locally administered plasmin exerts its fibrinolytic action in fibrin-bound form being protected from its main inhibitor (-plasmin inhibitor) but is immediately inactivated when entering the circulation, thus preventing bleeding at remote sites of vascular injury (reviewed in [4]). However, catheter directed thrombolysis combined with systemic thrombolysis may be associated with higher risk of pulmonary embolism or intracranial haemorrhage compared to systemic thrombolysis alone [5].Nanomedicine offers approaches for noninvasive and rapid thrombolytic treatment, with the hope of further reducing the morbidity and mortality of occlusive cardiovascular events. Being encapsulated into liposomes, drugs are preserved from metabolization prior to reaching target tissues, and simultaneously they minimize exposure of healthy tissue to the encapsulated drug during its circulation in the blood. The possibility to target liposomes helps in localizing sufficient quantities of thrombolytic agents to the desired thrombus. In vivo results show strong evidence that external targeting is superior to passive targeting of highly stable long-circulating drug formulations. A promising alternative for external targeting is achieved by temperature-triggered, localized intravascular drug release from thermosensitive liposomes with focused heating ([6], reviewed in [7]).Trypsin could be a novel candidate for being a liposome-encapsulated thrombolytic drug because of its high fibrinolytic efficiency [8] and about 3.5 times lower molecular mass compared to that of plasmin allowing higher encapsulated enzyme concentration. If administered in a plasma environment, encapsulated trypsin is protected against plasma inhibitors, the most abundant of which is -PI (-protease inhibitor). However, in contrast to plasmin and tPA, trypsin lacks any structural domains for recognition and specific binding to fibrin. Thus, an alternative targeting strategy is required to allow for a local proteolytic action of trypsin in thrombi. Such an option is offered by the temperature-dependent release of the enzyme from the thermosensitive liposomes developed and characterized in the current study, which in vivo could be achieved with focused ultrasound thermal effects. Following the local release of trypsin in thrombi, the availability of the fibrin substrate will protect the enzyme from inactivation by -PI, similarly to the fibrin-mediated protection of other proteases (plasmin and PMN-elastase) against plasma protease inhibitors [9].The purpose of our work was to prepare trypsin-loaded PEGylated liposomes and characterize them in terms of their structure and proteolytic efficiency in pure fibrin, as well as in plasma environment.2. Materials and Methods2.1. Proteins and ReagentsIf not otherwise indicated, experiments were performed in HEPES buffered saline (HBS, 10 mM HEPES, 150 mM NaCl, pH 7.4). Porcine trypsin and bovine thrombin were purchased from Serva Electrophoresis GmbH (Heidelberg, Germany) and the latter was further purified as described in [10] yielding a preparation with specific activity of 2100 IU/mg. Thrombin activity of 1 IU/mL was considered equivalent to approximately 10.7 nM by active site titration [11]. Fibrinogen (human, plasminogen-depleted) was from Calbiochem (San Diego, CA, USA). Hydrogenated soybean phosphatidylcholine, cholesterol, distearoyl-phosphatidylethanolamine PEG2000, and cholesterol sulphate were acquired from Avanti (Birmingham, AL, USA). DPH (1,6-diphenyl-1,3,5-hexatriene) and fluorescamine were from Sigma-Aldrich Kft. (Budapest, Hungary) and Spectrozyme-PL (H-D norleucylhexahydrotyrosyl-lysine-p-nitroanilide) was from Sekisui Diagnostics (Pfungstadt, Germany). Citrated, fresh frozen plasma (fibrinogen concentration: 2.35 g/l) was obtained from the Hungarian Blood Supply Service (Budapest, Hungary).2.2. Preparation of Trypsin-Loaded LiposomesTrypsin-loaded () and empty (SSL) sterically stabilized liposomes consisting of hydrogenated soybean phosphatidylcholine, cholesterol, distearoyl phosphatidylethanolamine PEG2000, and cholesterol sulphate at a molar ratio of 15 : 4 : 1 : 1 were prepared by thin-layer evaporation method as follows. Constituents were mixed and dissolved in a chloroform/methanol mixture (95 : 5 volume ratio) at a total phospholipid concentration of 34 mg/ml. Evaporation of the organic solvent was facilitated by a vacuum pump (20 mbar, at 24°C, overnight). The lipid film was hydrated at room temperature with HBS for preparing empty liposomes or 0.1 mM HCl in distilled water containing 10 mg/ml porcine trypsin for trypsin-loaded liposomes and stirred for 10 min at 1200 rpm. Formation of unilamellar vesicles was promoted by sonication (three cycles of 30 s at 50 watt, 20 kHz in a Branson Sonifier 250, Branson Ultrasonics Corp., Danbury, CT, USA) followed by ten freeze-thaw cycles (−78°C; +42°C). was then extruded through a 100 nm pore diameter polycarbonate filter in a LiposoFast mini-extruder (Avestin Inc., Ottawa, Canada). Nonencapsulated trypsin and lipid debris were removed from supernatant by centrifugation (3 times for 15 min at 133,000) with a Beckman Airfuge 340401 ultracentrifuge. The sediment was resuspended in HBS yielding a liposome preparation of neutral pH, with trypsin encapsulated in its inactive form due to the low pH inside of the enzyme loaded vesicles. The concentration of phospholipids in the suspension was determined with the fluorescent probe DPH [12] yielding a concentration of about 30 mg/ml. Changes in enzyme activity of the encapsulated trypsin were monitored daily on the chromogenic small peptide substrate Spectrozyme-PL (SpPL) at 405 nm after lysing liposomes by stirring together with 2.5 v/v% Triton X-100 for ten seconds at 1200 rpm. was stored at 4°C until use.2.3. Dynamic, Permeation-Driven Lysis of Fibrin and Plasma ClotsIn order to examine the fibrinolytic activity of our liposome preparation under dynamic conditions, fibrin and plasma permeation studies were performed at 24 and 45°C. The inner surfaces of 5 ml pipette tips (Finntip, Thermo Scientific, Budapest, Hungary) were precoated with 1 g/l fibrinogen for 3 h and then air-dried [13]. Fibrin clots were prepared from fibrinogen at 7.5 μM clotted with 16 nM thrombin in the fibrinogen-coated pipette tips. Plasma clots were prepared from citrated, fresh frozen plasma supplemented with fibrinogen to 7.5 μM final concentration and CaCl2 to 12.5 mM and filled in the tips. After 70 min of incubation at 37°C, stable clots were washed thoroughly with HBS to remove unclotted fibrinogen and other plasma proteins. Trypsin-loaded liposomes (50 μl) containing phospholipid at 5 mg/ml and enzyme activity corresponding to that of 300 nM free trypsin, empty liposomes, or HBS were layered over clots of 200 μl volume. After the entry of the liposomal suspension into the clots, HBS was added and continuously supplemented to keep a constant hydrostatic pressure over the clots. The pipette tips were kept at 24 or 45°C during the lytic process. Consecutive fractions of 50 μl were then collected and their protein concentration was determined by the fluorescamine method [14] and plotted against the eluted volume. Fractions with the highest protein content were analyzed with SDS polyacrylamide gel electrophoresis (SDS PAGE) in 4–15% gradient gels under nonreducing conditions, followed by visualization of protein bands with silver staining.2.4. Lysis of Fibrin and Plasma Clots in a Turbidimetric AssayTwo different experimental setups were designed to measure the fibrinolytic effectiveness of our liposomes under static conditions. First, fibrinogen at 7.5 μM or plasma supplemented with fibrinogen (7.5 μM
Article 2 Reads 1 Citation Free Fatty Acids Modulate Thrombin Mediated Fibrin Generation Resulting in Less Stable Clots Anna Tanka-Salamon, Erzsébet Komorowicz, Laszlo Szabo, Kiril... Published: 12 December 2016
PLOS ONE, doi: 10.1371/journal.pone.0167806
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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.
Article 4 Reads 1 Citation Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes Tanja Stachon, Krasimir Kolev, Zsuzsa Flaskó, Berthold Seitz... Published: 25 October 2016
Graefe's Archive for Clinical and Experimental Ophthalmology, doi: 10.1007/s00417-016-3520-x
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Article 3 Reads 1 Citation Decreased fibrinolytic potential and morphological changes of fibrin structure in dermatitis herpetiformis Anna Görög, Krisztián Németh, László Szabó, Balázs Mayer, Pá... Published: 01 October 2016
Journal of Dermatological Science, doi: 10.1016/j.jdermsci.2016.07.005
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