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1.
Thromb Haemost ; 113(6): 1289-98, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25789443

ABSTRACT

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.


Subject(s)
DNA/blood , Extracellular Traps/metabolism , Fibrin/metabolism , Fibrinolysis , Histones/blood , Neutrophils/metabolism , Plasma/metabolism , Deoxyribonuclease I/metabolism , Fibrin/ultrastructure , Humans , Kinetics , Microscopy, Confocal , Microscopy, Electron, Scanning , Neutrophil Activation , Neutrophils/drug effects , Neutrophils/ultrastructure , Permeability , Plasminogen/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Thrombin/metabolism , Tissue Plasminogen Activator/metabolism
2.
Thromb Res ; 135(4): 760-6, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25686880

ABSTRACT

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.


Subject(s)
Blood Platelets/metabolism , Coronary Artery Disease/diagnostic imaging , Peripheral Arterial Disease/diagnostic imaging , Thrombosis/metabolism , Blood Platelets/cytology , Female , Humans , Male , Thrombosis/blood , Ultrasonography
3.
Biochemistry ; 53(40): 6348-56, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25222106

ABSTRACT

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.


Subject(s)
Fibrin/chemistry , Fibrinolysin/chemistry , Aminocaproic Acid/chemistry , Carboxypeptidase B/chemistry , Fibrin/ultrastructure , Fibrinolysin/ultrastructure , Fractals , Humans , Kinetics , Models, Chemical , Protein Multimerization , Proteolysis
4.
Thromb Res ; 133(1): 80-7, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24094605

ABSTRACT

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.


Subject(s)
Carboxypeptidase B/metabolism , Fibrin/metabolism , Animals , Arginine/chemistry , Arginine/metabolism , Carboxypeptidase B/chemistry , Fibrin/chemistry , Fibrinolysin/metabolism , Fibrinolysis/physiology , Humans , Kinetics , Microscopy, Confocal , Microscopy, Electron, Scanning , Structure-Activity Relationship , Substrate Specificity , Swine , Tissue Plasminogen Activator/metabolism
5.
Thromb Res ; 129(4): e41-6, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22178067

ABSTRACT

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.


Subject(s)
Blood Platelets/physiology , Fibrinolysin/metabolism , Mechanotransduction, Cellular/physiology , Peptide Hydrolases/metabolism , Platelet Adhesiveness/physiology , Thrombin/metabolism , von Willebrand Factor/metabolism , Cells, Cultured , Enzyme Activation , Homeostasis/physiology , Humans , Protein Binding , Proteolysis , Shear Strength , Signal Transduction/physiology , Stress, Mechanical
6.
Arterioscler Thromb Vasc Biol ; 31(10): 2306-13, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21737785

ABSTRACT

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.


Subject(s)
Erythrocytes/metabolism , Fibrin/metabolism , Fibrinolysis , Thrombosis/blood , Eptifibatide , Erythrocytes/drug effects , Fibrin/ultrastructure , Fibrinolysin/metabolism , Fibrinolysis/drug effects , Fibrinolytic Agents/pharmacology , Humans , Kinetics , Microscopy, Atomic Force , Microscopy, Confocal , Peptides/pharmacology , Plasminogen/metabolism , Receptors, Fibrinogen/drug effects , Receptors, Fibrinogen/metabolism , Recombinant Fusion Proteins/metabolism , Tissue Plasminogen Activator/metabolism
7.
Thromb Res ; 126(2): e94-101, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20627279

ABSTRACT

Leukocytes invade newly formed thrombi through interactions with platelets and fibrin and later contribute to the removal of fibrin deposits mainly through the action of neutrophil elastase. The present study attempts to express in quantitative terms the impact of neutrophils on the lytic processes in obliterative thrombi based on the local presence of elastase-specific fibrin degradation products (NE-FDP) in relation to the leukocyte, platelet and fibrin content of thrombi. Immunofluorescent detection of fibrin, NE-FDP and platelet antigens was performed in sections of thrombi from 28 patients subjected to thrombectomy in combination with DNA-staining for identification of nucleated cells. The digitalized fluorescent microscopic images were decomposed according to the color channel of each thrombus constituent. The integrated intensity values for all thrombus constituents were statistically evaluated with correlation, hierarchical agglomerative clustering , Hotelling'sT(2) and F-statistics. Association between NE-FDP and leukocyte content of thrombi is evidenced by a significant Pearson correlation coefficient of 0.71 (p=0.00002). Cluster analysis reveals two classes of thrombi according to NE-FDP, leukocyte and platelet content and also two according to NE-FDP, leukocyte and fibrin content. When NE-FDP, fibrin and platelet content is normalized to the leukocyte count in the same thrombus, clusters with platelet-related thrombolytic resistance (inversely related NE-FDP and platelet content) and advanced cell-dependent thrombolysis (inversely related NE-FDP and fibrin content) are identified. These distinct patterns of thrombus constituents are snapshots of characteristic stages in the cell-dependent thrombolysis, which indicate a clot-stabilizing role for platelets in this process similar to their impact on the plasmin-dependent lysis.


Subject(s)
Blood Platelets/metabolism , Fibrin/metabolism , Leukocyte Elastase/metabolism , Thrombosis/metabolism , Aged , Aged, 80 and over , Antigens, Human Platelet/metabolism , Female , Fibrin Fibrinogen Degradation Products/metabolism , Fibrinolysis , Humans , Leukocytes/cytology , Male , Middle Aged , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Thrombosis/enzymology
8.
FEBS J ; 275(6): 1274-82, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18279394

ABSTRACT

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.


Subject(s)
Arachidonic Acid/chemistry , Fibrinolysin/antagonists & inhibitors , Fibrinolysin/chemistry , Models, Chemical , Oleic Acid/chemistry , Stearates/chemistry , Catalysis , Computer Simulation , Humans , Kinetics , Monte Carlo Method , Oligopeptides/chemistry , Structure-Activity Relationship
9.
Blood Coagul Fibrinolysis ; 19(1): 82-8, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18180621

ABSTRACT

Because both immunoglobulin G (IgG) and phospholipids interfere with fibrinolysis, their combined modulating effects were investigated in experimental models of three consecutive steps of the fibrinolytic process [diffusion of tissue-type plasminogen activator (tPA) into the clot, plasminogen activation on fibrin surface and fibrin dissolution by plasmin] using IgGs isolated from healthy subjects and from patients with antiphospholipid syndrome in combination with mixtures of synthetic dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine. In fibrin clots containing phospholipids the normal IgG enhanced the barrier function of the phospholipids with respect to the diffusion of tPA and plasminogen activation, but did not modify the lysis by plasmin. One of the examined antiphospholipid syndrome-IgGs also restricted the diffusion of tPA, but it accelerated the plasminogen activation on the fibrin surface and slowed down the lysis of fibrin by plasmin. Another antiphospholipid syndrome IgG, which did not affect significantly the tPA penetration into the fibrin gel, did not modify the plasminogen activation on its own, but it partially opposed the inhibiting effect of phospholipids on plasmin formation and accelerated the end-stage lysis of fibrin containing phospholipids. The IgGs from the two examined antiphospholipid syndrome patients did not show consistent deviation from the pattern of normal IgG effects on fibrinolysis in phospholipid environment. Thus, a high degree of heterogeneity with respect to the profibrinolytic or antifibrinolytic effects of the pathological IgGs can be expected in the antiphospholipid syndrome patient population, which may contribute to the variable thrombotic symptoms in this clinical syndrome.


Subject(s)
Antibodies, Antiphospholipid/physiology , Fibrinolysis/physiology , Immunoglobulin G/physiology , Phospholipids/physiology , Antiphospholipid Syndrome/blood , Antiphospholipid Syndrome/physiopathology , Case-Control Studies , Fibrin/physiology , Fibrinolysin/physiology , Humans , Tissue Plasminogen Activator/physiology
10.
Biochem Biophys Res Commun ; 341(3): 736-41, 2006 Mar 17.
Article in English | MEDLINE | ID: mdl-16438933

ABSTRACT

Certain denatured proteins function as cofactors in the activation of plasminogen by tissue-type plasminogen activator. The present study approached the structural requirements for the cofactor activity of a model protein (human serum albumin). Heat denaturation of 100-230 microM albumin (80 degrees C and 60-90 min) reproducibly yielded aggregates with radius in the range of 10-150 nm. The major determinant of the cofactor potency was the size of the aggregates. The increase of particle size correlated with the cofactor activity, and there was a minimal requirement for the size of the cofactor (about 10 nm radius). Similar to other proteins, the molecular aggregates with cofactor function contained a significant amount of antiparallel intermolecular beta-sheets. Plasmin pre-digestion increased the cofactor efficiency (related to C-terminal lysine exposure) and did not affect profoundly the structure of the aggregates, suggesting a long-lasting and even a self-augmenting cofactor function of the denatured protein.


Subject(s)
Plasminogen/metabolism , Serum Albumin/chemistry , Serum Albumin/metabolism , Tissue Plasminogen Activator/metabolism , Benzothiazoles , Electrophoresis, Polyacrylamide Gel , Humans , Particle Size , Protein Denaturation , Thiazoles/pharmacology
11.
Thromb Haemost ; 94(1): 82-7, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16113788

ABSTRACT

Immunoglobulin G (IgG) isolated from the blood plasma of a patient with secondary antiphospholipid syndrome (APS) expresses fibrinogen-clotting and amidolytic activity (the thrombin activity in 20 micromole IgG is equivalent to approximately 5 nmole pure thrombin), and activates factor XIII. Hirudin (1 microM) decreases the intrinsic thrombin activity of the APS IgG by only 25%, whereas it inhibits completely pure thrombin with equivalent activity. Under conditions, when antithrombin inactivates 60% of the thrombin activity in the presence of normal IgG, the APS IgG protects almost completely the added thrombin against inactivation by antithrombin. Heparin, however, partially relieves this protective effect and at the same time it facilitates the inhibition of the intrinsic thrombin activity by antithrombin. The APS IgG reduces the thrombin activity in protein C activation assay by 50% compared to the activity in the presence of normal IgG. All described properties are related to the Fab fragment of the antibody. The IgG preserving the fibrin-generating activity of thrombin with concomitant protection against inhibitors unravels a new aspect of the thrombotic mechanism in APS. This condition is probably rare: only one out of 23 examined patients with primary or secondary APS expresses IgG with the described properties.


Subject(s)
Antiphospholipid Syndrome/drug therapy , Antiphospholipid Syndrome/immunology , Antithrombins/immunology , Antithrombins/pharmacology , Blood Coagulation , Fibrinogen/chemistry , Hirudins/pharmacology , Thrombin/chemistry , Adult , Antigens/chemistry , Antiphospholipid Syndrome/pathology , Blood Coagulation Tests , Blotting, Western , Factor XIII/chemistry , Female , Hirudins/chemistry , Humans , Immunoglobulin G/chemistry , Lupus Erythematosus, Systemic/immunology , Protein C/chemistry , Time Factors
12.
Ann Hematol ; 83(12): 759-63, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15316758

ABSTRACT

The contribution of neutrophil leukocyte elastase (NE) to in vivo thrombolysis is still an open question. The present study examines the impact of variable levels of alpha1-proteinase inhibitor (alpha1-PI) (the major plasma inhibitor of NE) on fibrinolysis within the setting of thromboembolic diseases. Blood samples were taken from 56 patients with pulmonary thromboembolism prior to treatment. alpha1-PI and alpha1-PI-NE complex were measured in the serum and plasma with immunoturbidimetric and enzyme-linked immunosorbent assay (ELISA) methods, respectively. The fibrinolytic potential [spontaneous, tissue-type plasminogen activator (tPA) induced, and plasmin induced] of the plasma was evaluated in vitro with turbidimetric clot lysis assay. Correlation analysis (Pearson product-moment correlation coefficient, r) of the turbidimetric lysis parameters and the blood levels of alpha1-PI and alpha1-PI-NE complex was carried out. Fibrinolysis is slower in clots prepared from plasma containing elevated levels of alpha1-PI and alpha1-PI-NE complex. The maximal turbidity of the plasma clots shows significant correlation with the alpha1-PI level (r=0.39, p=0.003) and the correlation of the maximal turbidity and the tPA-induced lysis time is also significant (r=0.77, p<0.001). The lysis time correlates with the plasma level of alpha1-PI-NE complex, if fibrinolysis is induced with tPA (r=0.37, p=0.02), but not with plasmin (r=0.19, p=0.4). Our study shows that in pulmonary thromboembolism elevated levels of alpha1-PI are associated with suppressed plasma fibrinolytic potential. This effect can be at least partially explained by the coarse fibrin network structure and retarded plasminogen activator-dependent fibrinolysis.


Subject(s)
Leukocyte Elastase/blood , Multiprotein Complexes/blood , Pulmonary Embolism/blood , alpha 1-Antitrypsin/analysis , Adult , Biomarkers/blood , Female , Fibrinolysis , Humans , Male , Middle Aged , Plasminogen/analysis , Plasminogen Activators/blood , Pulmonary Embolism/pathology
13.
J Biol Chem ; 279(38): 39863-71, 2004 Sep 17.
Article in English | MEDLINE | ID: mdl-15254044

ABSTRACT

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.


Subject(s)
Blood Platelets/metabolism , Fibrinolysis/physiology , Phospholipids/chemistry , Phospholipids/metabolism , Thrombosis/metabolism , Anions , Crystallization , Femoral Artery , Fibrinolysin/metabolism , Fibrinolysis/drug effects , Humans , In Vitro Techniques , Plasminogen/metabolism , Tissue Plasminogen Activator/pharmacology
15.
Thromb Haemost ; 90(3): 528-37, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12958623

ABSTRACT

The haemorrhagic transformation in ischemic stroke involves disruption of the integrity of the microvascular beds, partially based on the action of matrix metalloproteinases (MMPs). The objective of the present study was to evaluate the contribution of microvascular endothelial cells from human brain (HBECs) to MMPs' expression and regulation under conditions relevant to brain ischemia. MMPs and their inhibitors were examined with zymography, Western-blotting, ELISA and MMP-activity assay in cultured HBECs. Four-hour hypoxia (pO(2)=60 mmHg) elevated the level of MMP-9 in the supernatant of the HBECs and this early response required collagen-matrix. Active oxygen species sustained the increased MMP-9 activity for at least 24 h. In the post-hypoxic period 20 micro mol/L H(2)O(2) caused a 6-fold increase in the specific activity of MMP-9 over the normoxic cells and a comparable effect was exerted by thrombin (50 nmol/L) and leukocyte elastase (10 nmol/L). The role of NF-kappaB, a redox-state sensitive transcription factor, was evaluated with immunofluorescence confocal microscopy and immunoblotting of nuclear and cytoplasmic extracts. The oxidative stress-dependent MMP-9 induction was accompanied by a significant increase in the NF-kappaB localized in the nuclei and these responses were blunted with a proteasome inhibitor (MG132). Consequently, according to our in vitro data HBECs are a source of MMP-9, which is under the control of triggers relevant to the ischemic/reperfused brain (reactive oxygen species, thrombus and inflammation related proteases) and this regulation is partially based on NF-kappaB activation. The reported regulation of endothelium-derived MMP-9 supports its potential involvement in the post-hypoxic disturbances of the cerebral microcirculation.


Subject(s)
Endothelium, Vascular/enzymology , Endothelium, Vascular/pathology , Hypoxia, Brain/enzymology , Matrix Metalloproteinase 9 , Matrix Metalloproteinase 9/biosynthesis , Brain/blood supply , Capillaries/enzymology , Capillaries/pathology , Cell Hypoxia , Cells, Cultured , Enzyme Induction , Humans , Hypoxia, Brain/pathology , Hypoxia-Ischemia, Brain/enzymology , Hypoxia-Ischemia, Brain/pathology , Matrix Metalloproteinase 9/analysis , NF-kappa B/metabolism , NF-kappa B/physiology , Oxidation-Reduction
16.
Thromb Haemost ; 89(4): 610-21, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12669114

ABSTRACT

The structure of the fibrin network, the hemodynamic environment of the clot, the kinetic properties of the fibrinolytic enzymes and the balance of their formation and inactivation essentially determine the effectiveness of fibrinolysis in vivo. The fibrin structure and the action of proteases, however depend considerably on additional, apparently inert physiological and pathological factors, which are restricted to more or less transient compartments in fluid-solid interface, such as thrombus (fibrin with platelet membrane structures), endothelial cell surface, the environment of polymorphonuclear cells (PMN). In these compartments extreme changes in concentrations and rate enhancements are observed. Components released by endothelial cells, PMNs and platelets or molecules present in circulating blood create a heterogeneous milieu that modulates fibrinolysis. This review summarizes the effects, and where it is possible, explains the mechanism of modulators of the fibrinolytic processes, such as cell membrane and cellular contents of endothelium, PMN and platelets present in thrombi, the action of normal and pathological blood plasma- and extracellular matrix-components.


Subject(s)
Fibrin/metabolism , Fibrinolysis , Animals , Cell Membrane/metabolism , Endothelium/cytology , Extracellular Matrix/metabolism , Fibrin/chemistry , Hemodynamics , Humans , Kinetics , Mice , Mice, Transgenic , Models, Biological , Neutrophils/metabolism , Plasminogen/biosynthesis , Plasminogen Activators/metabolism
17.
Blood ; 101(11): 4380-6, 2003 Jun 01.
Article in English | MEDLINE | ID: mdl-12543859

ABSTRACT

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.


Subject(s)
Fibrin/metabolism , Fibrinolysis , Myosins/physiology , Binding Sites , Blood Proteins/pharmacology , Dimerization , Humans , Kinetics , Myosins/metabolism , Peptide Hydrolases/metabolism , Protein Binding , Surface Plasmon Resonance , Thrombosis , Tissue Plasminogen Activator/pharmacology
18.
J Neurochem ; 80(5): 746-54, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11948237

ABSTRACT

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. We have reported earlier that primary cultures of rat brain capillary endothelial (RBCE) cells express at least two receptors for thrombin: PAR-1 and PAR-3. In the present study we show that PAR-2 activation by trypsin or by the PAR-2 agonist peptide (SLIGRL) evokes [Ca(2+) ](i) signal in RBCE cells. Taking advantage of RBCE cells expressing PAR-1 and PAR-2, we show that trypsin activates both receptors. The relative agonist activity of trypsin and thrombin on PARs of RBCE cells compared with that of SLIGRL were 112% and 48%, respectively, whereas the potency of trypsin was 10(5) -fold higher than that of SLIGRL. Because under pathological conditions other proteases such as plasmin or leukocyte elastase may reach the cells of the blood-brain barrier, we investigated the effect of these proteases on RBCE cells. Elastase evoked a small increase in [Ca(2+) ](i) but preincubation of cells with elastase dose-dependently reduced the trypsin-induced [Ca(2+) ](i) signal. Plasmin had a 30% inhibitory effect on the trypsin-induced response, and reduced the SLIGRL signal by 20%. It is concluded that PAR-2 is functional in brain capillary endothelium, and that the main fibrinolytic proteases, plasmin and elastase, may regulate PAR-2 signalling under pathological conditions.


Subject(s)
Brain/metabolism , Endothelium, Vascular/metabolism , Fibrinolysin/metabolism , Pancreatic Elastase/metabolism , Receptors, Thrombin/metabolism , Animals , Blood-Brain Barrier/physiology , Brain/blood supply , Brain/cytology , Calcium Signaling/drug effects , Calcium Signaling/physiology , Capillaries/cytology , Capillaries/drug effects , Capillaries/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Fibrinolysin/pharmacology , Microcirculation/metabolism , Oligopeptides/metabolism , Pancreatic Elastase/pharmacology , Rats , Receptor, PAR-2 , Receptors, Thrombin/agonists , Signal Transduction/drug effects , Signal Transduction/physiology , Trypsin/metabolism , Trypsin/pharmacology
19.
Thromb Haemost ; 87(3): 502-8, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11916083

ABSTRACT

Immunoglobulin G (IgG) isolated from normal human blood plasma stabilizes the structure of perfused crosslinked fibrin and prolongs the time for its dissolution with plasmin, when the fibrin surface is exposed to 500 s(-1) shear rate flow. The IgG from patients suffering in antiphospholipid syndrome with thrombotic complications exerts even stronger antifibrinolytic effect. A patient, whose IgG does not affect the fibrin dissolution with plasmin, displays a bleeding tendency. The shear stress-induced disassembly of the fibrin clots containing IgGs with antifibrinolytic potency occurs at a much more advanced stage of fibrin digestion, as evidenced by the electrophoretic pattern of the ureatreated samples. The antifibrinolytic effects are also produced under static conditions and these are caused by the variable portion of the IgG molecules (fragment Fab), whereas the constant part (fragment Fc) has no inhibitory effect. The IgGs with antifibrinolytic properties do not affect directly the plasmin activity in amidolytic assay, but the IgGs from APS patients obliterate the competition of the fibrin and the peptidyl-p-nitroanilide for the protease in the same assay system suggesting interference of the IgGs with the plasmin action on the fibrin substrate. Thus, the correlation of the clinical symptoms with the effect of the isolated IgG on the dissolution of perfused fibrin clots supports a physiological and a pathological role of IgG in the fibrinolytic process related to the variability of the cross-reactions of immunoglobulins with fibrin, fibrin degradation products or fibrin-plasmin complexes.


Subject(s)
Antiphospholipid Syndrome/immunology , Fibrinolysis/immunology , Immunoglobulin G/immunology , Adult , Aged , Antibodies, Anticardiolipin/immunology , Antibodies, Anticardiolipin/isolation & purification , Antiphospholipid Syndrome/complications , Autoantibodies/immunology , Autoantibodies/isolation & purification , Case-Control Studies , Cross Reactions , Female , Fibrin/immunology , Fibrin/metabolism , Fibrinolysin/antagonists & inhibitors , Fibrinolysin/immunology , Humans , Immunoglobulin Fab Fragments/pharmacology , Immunoglobulin G/isolation & purification , Kinetics , Male , Middle Aged , Thrombosis/immunology
20.
Orv Hetil ; 143(11): 541-5, 2002 Mar 17.
Article in Hungarian | MEDLINE | ID: mdl-12583322

ABSTRACT

The structure of fibrin network and the properties of the fibrinolytic enzymes (the formation, the efficiency of their activity and the rate of their inactivation) essentially determine the effectiveness of fibrinolysis. The fibrin structure and the action of proteases, however, depend considerably on additional physiological and pathological conditions. These are: cell membrane and components of endothelial cell, elastase of polymorphonuclear cells, actin and myosin in thrombus released by platelets and smooth muscle cells, certain denatured proteins, various immunoglobulins, some metabolites accumulated in diabetes mellitus, the inhibitor system of fibrinolytic enzymes (where and when proteases are available for inhibitors). In this survey, the biochemical basis makes understandable for the medical practitioner why urgent fibrinolytic therapy is required and why it would be ideal to initiate the formation of fibrinolytic enzymes at the site of thrombus.


Subject(s)
Fibrin/metabolism , Fibrinolysis , Plasminogen Activators/metabolism , Plasminogen/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Humans , Protease Inhibitors/metabolism
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