Publications by authors named "Kiril Tenekedjiev"

16 Publications

  • Page 1 of 1

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

Human Error in Autonomous Underwater Vehicle Deployment: A System Dynamics Approach.

Risk Anal 2020 06 6;40(6):1258-1278. Epub 2020 Mar 6.

Australian Maritime College, University of Tasmania, Australia.

The use of autonomous underwater vehicles (AUVs) for various applications have grown with maturing technology and improved accessibility. The deployment of AUVs for under-ice marine science research in the Antarctic is one such example. However, a higher risk of AUV loss is present during such endeavors due to the extremities in the Antarctic. A thorough analysis of risks is therefore crucial for formulating effective risk control policies and achieving a lower risk of loss. Existing risk analysis approaches focused predominantly on the technical aspects, as well as identifying static cause and effect relationships in the chain of events leading to AUV loss. Comparatively, the complex interrelationships between risk variables and other aspects of risk such as human errors have received much lesser attention. In this article, a systems-based risk analysis framework facilitated by system dynamics methodology is proposed to overcome existing shortfalls. To demonstrate usefulness of the framework, it is applied on an actual AUV program to examine the occurrence of human error during Antarctic deployment. Simulation of the resultant risk model showed an overall decline in human error incident rate with the increase in experience of the AUV team. Scenario analysis based on the example provided policy recommendations in areas of training, practice runs, recruitment policy, and setting of risk tolerance level. The proposed risk analysis framework is pragmatically useful for risk analysis of future AUV programs to ensure the sustainability of operations, facilitating both better control and monitoring of risk.
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http://dx.doi.org/10.1111/risa.13467DOI Listing
June 2020

Fuzzy System Dynamics Risk Analysis (FuSDRA) of Autonomous Underwater Vehicle Operations in the Antarctic.

Risk Anal 2020 04 4;40(4):818-841. Epub 2019 Dec 4.

Australian Maritime College, University of Tasmania, Tasmania, Australia.

With the maturing of autonomous technology and better accessibility, there has been a growing interest in the use of autonomous underwater vehicles (AUVs). The deployment of AUVs for under-ice marine science research in the Antarctic is one such example. However, a higher risk of AUV loss is present during such endeavors due to the extreme operating environment. To control the risk of loss, existing risk analyses approaches tend to focus more on the AUV's technical aspects and neglect the role of soft factors, such as organizational and human influences. In addition, the dynamic and complex interrelationships of risk variables are also often overlooked due to uncertainties and challenges in quantification. To overcome these shortfalls, a hybrid fuzzy system dynamics risk analysis (FuSDRA) is proposed. In the FuSDRA framework, system dynamics models the interrelationships between risk variables from different dimensions and considers the time-dependent nature of risk while fuzzy logic accounts for uncertainties. To demonstrate its application, an example based on an actual Antarctic AUV program is presented. Focusing on funding and experience of the AUV team, simulation of the FuSDRA risk model shows a declining risk of loss from 0.293 in the early years of the Antarctic AUV program, reaching a minimum of 0.206 before increasing again in later years. Risk control policy recommendations were then derived from the analysis. The example demonstrated how FuSDRA can be applied to inform funding and risk management strategies, or broader application both within the AUV domain and on other complex technological systems.
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http://dx.doi.org/10.1111/risa.13429DOI Listing
April 2020

A Fuzzy-Based Risk Assessment Framework for Autonomous Underwater Vehicle Under-Ice Missions.

Risk Anal 2019 Dec 18;39(12):2744-2765. Epub 2019 Jul 18.

Australian Maritime College, University of Tasmania, Tasmania, Australia.

The use of autonomous underwater vehicles (AUVs) for various scientific, commercial, and military applications has become more common with maturing technology and improved accessibility. One relatively new development lies in the use of AUVs for under-ice marine science research in the Antarctic. The extreme environment, ice cover, and inaccessibility as compared to open-water missions can result in a higher risk of loss. Therefore, having an effective assessment of risks before undertaking any Antarctic under-ice missions is crucial to ensure an AUV's survival. Existing risk assessment approaches predominantly focused on the use of historical fault log data of an AUV and elicitation of experts' opinions for probabilistic quantification. However, an AUV program in its early phases lacks historical data and any assessment of risk may be vague and ambiguous. In this article, a fuzzy-based risk assessment framework is proposed for quantifying the risk of AUV loss under ice. The framework uses the knowledge, prior experience of available subject matter experts, and the widely used semiquantitative risk assessment matrix, albeit in a new form. A well-developed example based on an upcoming mission by an ISE-explorer class AUV is presented to demonstrate the application and effectiveness of the proposed framework. The example demonstrates that the proposed fuzzy-based risk assessment framework is pragmatically useful for future under-ice AUV deployments. Sensitivity analysis demonstrates the validity of the proposed method.
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http://dx.doi.org/10.1111/risa.13376DOI Listing
December 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

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

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

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

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

Uses and misuses of progress curve analysis in enzyme kinetics.

Cent Eur J Biol 2008 Dec;3(4):345-350

Department of Economics and Management, Technical University - Varna, 9010 Varna, Bulgaria.

Progress curve analysis is a convenient tool for the characterization of enzyme action: a single reaction mixture provides multiple experimental measured points for continuously varying amounts of substrates and products with exactly the same enzyme and modulator concentrations. The determination of kinetic parameters from the progress curves, however, requires complex mathematical evaluation of the time-course data. Some freely available programs (e.g. FITSIM, DYNAFIT) are widely applied to fit kinetic parameters to user-defined enzymatic mechanisms, but users often overlook the stringent requirements of the analytic procedures for appropriate design of the input experiments. Flaws in the experimental setup result in unreliable parameters with consequent misinterpretation of the biological phenomenon under study. The present commentary suggests some helpful mathematical tools to improve the analytic procedure in order to diagnose major errors in concept and design of kinetic experiments.
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http://dx.doi.org/10.2478/s11535-008-0035-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701647PMC
December 2008

Suppressed catalytic efficiency of plasmin in the presence of long-chain fatty acids. Identification of kinetic parameters from continuous enzymatic assay with Monte Carlo simulation.

FEBS J 2008 Mar 12;275(6):1274-82. Epub 2008 Feb 12.

Department of Medical Biochemistry, Semmelweis University, Puskin u.9, Budapest, Hungary.

Thrombi, which are dissolved primarily by plasmin (EC 3.4.21.7.), contain up to millimolar concentrations of fatty acids and these are known to affect the action of the protease. In the present study the modulation of plasmin activity was characterized quantitatively in a continuous amidolytic assay based on synthetic plasmin substrate (Spectrozyme-PL). A novel numerical procedure was applied for identification of kinetic parameters and their confidence intervals, with Monte Carlo simulation of the reaction progress curves, providing adequate grounds for discrimination of different models of the enzyme action. All three fatty acids caused a 10-20-fold increase in the Michaelis constant on Spectrozyme-PL (baseline value 5.9 mum). The catalytic constant decreased from 5.8.s(-1) to 2.4-2.8.s(-1) in the presence of arachidonate and oleate, but increased to 14.8.s(-1) in the presence of stearate, implying enhancement of plasmin activity at saturating substrate concentrations. However, based on the ratio of the catalytic and Michaelis constants, all three fatty acids acted as inhibitors of plasmin with various degrees of potency, showing concentration dependence in the range of 10-65 mum for oleate and arachidonate, and 115-230 mum for stearate. The reported effects of the three fatty acids require the presence of kringle 5 in the structure of the protease; miniplasmin (des-kringle 1-4 plasmin) is as sensitive to fatty acids as plasmin, whereas the activity of microplasmin (des-kringle 1-5 plasmin) is not affected.
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http://dx.doi.org/10.1111/j.1742-4658.2008.06288.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447916PMC
March 2008

Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure.

J Biol Chem 2004 Sep 14;279(38):39863-71. Epub 2004 Jul 14.

Department of Medical Biochemistry, Semmelweis University, 1088 Budapest, Hungary.

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.
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http://dx.doi.org/10.1074/jbc.M405172200DOI Listing
September 2004

Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution.

Blood 2003 Jun 23;101(11):4380-6. Epub 2003 Jan 23.

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

Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 microM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 x 10(2) M(-1) x s(-1) and a dissociation rate constant of 3.07 x 10(-4) s(-1) are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosin fibrin clot is relatively resistant to plasmin.
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http://dx.doi.org/10.1182/blood-2002-10-3227DOI Listing
June 2003
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