The Kinetics of Activated Thrombin-Activatable Fibrinolysis Inhibitor with Respect to Plasminogen Binding Site Removal From Fibrin Degradation Products.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3185-3185
Author(s):  
Jonathan H. Foley ◽  
Michael E. Nesheim
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Degradation Products ◽  
Catalytic Efficiency ◽  
Plasminogen Activation ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Fibrin Degradation Products ◽  
Fibrinolysis Inhibitor ◽  
Arginine Residues ◽  
Kinetics Of

Abstract Abstract 3185 Poster Board III-122 TAFI (thrombin activatable fibrinolysis inhibitor, or carboxypeptidase U) is a plasma zymogen that can be activated by thrombin, thrombin-thrombomodulin or plasmin. When activated, TAFIa cleaves C-terminal lysine and arginine residues from plasmin modified fibrin (Fn'). Fn' as a cofactor increases the rate of plasminogen activation by 3-fold over intact fibrin and 3000-fold compared to in the absence of fibrin. Upon extensive treatment with TAFIa, the cofactor activity of TAFIa modified fibrin decreases by approximately 97%. Determining the kinetics of TAFIa will give insight into how much TAFIa is required to efficiently inhibit plasminogen activation and fibrinolysis. The kinetics of TAFIa on its primary physiological substrate were measured by exploiting the binding of plasminogen to fibrin degradation products (FDPs). Fluorescently labeled plasminogen (5IAF-Pg) was equilibrated with FDPs labeled with a quencher, QSY C5-maleimide (QSY-FDP). When 5IAF-Pg is bound to QSY-FDP a baseline fluorescence reading is obtained. When treated with TAFIa, plasminogen binding sites are removed from the QSY-FDP and the fluorescence increases. A model was used to convert the rate of fluorescence increase into the rate of Plasminogen binding site removal. The model includes two distinct binding sites on QSY-FDPs (C-terminal and internal lysines), only one of which is susceptible to removal by TAFIa (C-terminal lysine). 5IAF-Glu-Pg (fluorescent native plasminogen) binds to QSY-FDP with a Kd of 176nM and when QSY-FDP are treated with TAFIa the Kd increases to 1.06μM. It appears that 5IAF-Glu-Pg has the ability to weakly bind TAFIa-treated QSY-FDP, however, the capacity is greatly reduced. Similar binding constants were obtained for 5IAF-Lys-Pg (fluorescent plasmin-cleaved plasminogen) (Kd=92nM; Kd (+TAFIa)=1.55μM). The increase in Kd upon treatment of the QSY-FDP with TAFIa is similar to that observed with 5IAF-Glu-Pg, however, the capacity of the FDPs to bind 5IAF-Lys-Pg is relatively unchanged. The calculated rate of 5IAF-Glu-Pg binding site removal by TAFIa was determined at various QSY-FDP concentrations (0-2 μM). The data are hyperbolic in nature and when fit using the Michaelis-Menten model the kcat and Km of plasminogen binding site removal were 2.34 s-1 and 142.6nM, respectively, implying a catalytic efficiency of 16.41 μM-1s-1. The rate is sensitive to the TAFIa concentration with all TAFIa concentrations (50, 75 and 100pM) yielding similar kinetic parameters. The data described here suggest that TAFIa is very efficient in removing plasminogen binding sites. The catalytic efficiency of TAFIa toward QSY-FDP is 60-fold higher than reported for bradykinin, which was previously the best known substrate of TAFIa. This increased catalytic efficiency is due to a much lower Km (0.146 μM compared to 70.6 μM). These data are reflective of plasminogen site removal and not every C-terminal lysine or arginine cleaved by TAFIa is expected to be involved in plasminogen binding. Therefore, the catalytic efficiency of TAFIa reported here (16.41 μM-1s-1) is likely a lower limit for the true value. Disclosures No relevant conflicts of interest to declare.

scholarly journals Kinetics of Activated Thrombin-activatable Fibrinolysis Inhibitor (TAFIa)-catalyzed Cleavage of C-terminal Lysine Residues of Fibrin Degradation Products and Removal of Plasminogen-binding Sites

2011 ◽  
Vol 286 (22) ◽  
pp. 19280-19286 ◽  
Author(s):  
Jonathan H. Foley ◽  
Paul F. Cook ◽  
Michael E. Nesheim
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Degradation Products ◽  
Catalytic Efficiency ◽  
Tissue Type ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Fibrin Degradation Products ◽  
Lysine Residues ◽  
Fibrinolysis Inhibitor ◽  
Kinetics Of

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the kcat and Km of Glu1-plasminogen (Glu-Pg)-binding site removal are 2.34 s−1 and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm−1 s−1. The corresponding values of Lys77/Lys78-plasminogen (Lys-Pg)-binding site removal are 0.89 s−1 and 96 nm implying a catalytic efficiency of 9.23 μm−1 s−1. These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm−1 s−1. Interestingly, this value increases to 3.85 μm−1 s−1 in the presence of Glu-Pg. These changes are due to a decrease in Km. This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.

scholarly journals Thrombin-thrombomodulin connects coagulation and fibrinolysis: more than an in vitro phenomenon

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3168-3175 ◽  
Author(s):  
Tanya M. Binette ◽  
Fletcher B. Taylor ◽  
Glenn Peer ◽  
Laszlo Bajzar
Keyword(s):  
Degradation Products ◽  
Lethal Dose ◽  
Catalytic Efficiency ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
E Coli ◽  
Fibrin Degradation Products ◽  
Baboon Model ◽  
Fibrinolysis Inhibitor

Abstract Thrombin activatable fibrinolysis inhibitor (TAFI), when activated, forms a basic carboxypeptidase that can inhibit fibrinolysis. Potential physiologic activators include both thrombin and plasmin. In vitro, thrombomodulin and glycosaminoglycans increase the catalytic efficiency of TAFI activation by thrombin and plasmin, respectively. The most relevant (patho-) physiologic activator of TAFI has not been disclosed. Our purpose was to identify the physiologic activator of TAFI in vivo. Activation of protein C (a thrombin-thrombomodulin–dependent reaction), prothrombin, and plasminogen occurs during sepsis. Thus, a baboon model of Escherichia coli–induced sepsis, where multiple potential activators of TAFI are elaborated, was used to study TAFI activation. A monoclonal antibody (mAbTAFI/TM#16) specifically inhibiting thrombin-thrombomodulin–dependent activation of TAFI was used to assess the contribution of thrombin-thrombomodulin in TAFI activation in vivo. Coinfusion of mAbTAFI/TM#16 with a lethal dose of E coli prevented the complete consumption of TAFI observed without mAbTAFI/TM#16. The rate of fibrin degradation products formation is enhanced in septic baboons treated with the mAbTAFI/TM#16; therefore, TAFI activation appears to play a key role in the extent of fibrin(ogen) consumption during E coli challenge, and thrombin-thrombomodulin, in a baboon model of E coli–induced sepsis, appears to be the predominant activator of TAFI.

Abstract 363: Thrombin Activatable Fibrinolysis Inhibitor is a Regulator of Angiogenic Potential in Endothelial Cells

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Zainab A Bazzi ◽  
Jennifer L Balun ◽  
Michael B Boffa
Keyword(s):  
Endothelial Cells ◽  
Cancer Metastasis ◽  
Tube Formation ◽  
Collagen Degradation ◽  
Matrix Metalloproteases ◽  
Soluble Form ◽  
Plasminogen Activation ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Fibrinolysis Inhibitor ◽  
Arginine Residues

Angiogenesis, the sprouting of new blood vessels from existing vessels, is a process that is fundamental to both normal physiology, as well as disease processes such as atherosclerosis and cancer metastasis. A key regulator of the angiogenesis is the elaboration of pericellular proteolytic activity including plasmin and the matrix metalloproteases (MMPs). Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that can be cleaved by thrombin, plasmin, or thrombin in complex with thrombomodulin (TM) to form activated TAFI (TAFIa). TAFIa cleaves carboxyl terminal lysine and arginine residues from substrates, including plasminogen-binding sites on cell surface receptors. TAFIa is thus a regulator of pericellular plasminogen activation. Plasmin regulates angiogenesis through MMP activation and degradation of the extracellular matrix (ECM). Previous studies have shown that TAFIa downregulates endothelial tube formation in a fibrin matrix, likely through inhibition of fibrin degradation. The current study was undertaken to evaluate if TAFIa is capable of modulating angiogenic responses of endothelial cells in vitro in the context of reconstituted basement membrane. We found that treatment of primary human umbilical vein endothelial cells (HUVECs) with a specific inhibitor of TAFIa, potato tuber carboxypeptidase inhibitor, resulted in an increase in proliferation, invasion, tube formation, and collagen degradation. On the other hand, treatment with a stable variant of TAFIa (TAFIa-CIIYQ) with a 150-fold increased half-life at 37°C resulted in a decrease in HUVEC tube formation and collagen degradation of HUVECs. Furthermore, a mutant soluble form of TM that supports TAFI, but not protein C, activation also had an inhibitory effect on HUVEC tube formation and collagen degradation. Finally, we assessed the effect of TAFIa on plasminogen activation. Treatment of HUVECs with various concentrations of TAFIa resulted in a decrease in uPA-mediated plasminogen activation on the surface of HUVECs. Together these experiments provide direct evidence of an anti-angiogenic effect of TAFIa. Our results suggest that promotion of TAFI activation or TAFIa stability may be viable therapeutic approaches for inhibition of angiogenesis.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

scholarly journals Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system

2020 ◽  
Vol 4 (21) ◽  
pp. 5501-5511
Author(s):  
Yuko Suzuki ◽  
Hideto Sano ◽  
Liina Mochizuki ◽  
Naoki Honkura ◽  
Tetsumei Urano
Keyword(s):  
Binding Site ◽  
Neutralizing Antibody ◽  
Laser Scanning Microscopy ◽  
Clot Formation ◽  
Tissue Type ◽  
Clot Lysis ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Fibrinolysis Inhibitor

Abstract Our previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their procoagulant functions, platelets enhanced profibrinolytic potential by augmenting the accumulation of tissue-type plasminogen activator (tPA) and plasminogen, in vivo in a murine microthrombus model, and in vitro in a platelet-containing plasma clot model. To clarify the role of thrombin-activatable fibrinolysis inhibitor (TAFI), which regulates coagulation-dependent anti-fibrinolytic potential, we analyzed tPA-induced clot lysis times in platelet-containing plasma. Platelets prolonged clot lysis times in a concentration-dependent manner, which were successfully abolished by a thrombomodulin-neutralizing antibody or an activated TAFI inhibitor (TAFIaI). The results obtained using TAFI- or factor XIII–deficient plasma suggested that TAFI in plasma, but not in platelets, was essential for this prolongation, though its cross-linkage with fibrin was not necessary. Confocal laser scanning microscopy revealed that fluorescence-labeled plasminogen accumulated on activated platelet surfaces and propagated to the periphery, similar to the propagation of fibrinolysis. Plasminogen accumulation and propagation were both enhanced by TAFIaI, but only accumulation was enhanced by thrombomodulin-neutralizing antibody. Labeled TAFI also accumulated on both fibrin fibers and activated platelet surfaces, which were Lys-binding-site-dependent and Lys-binding-site-independent, respectively. Finally, TAFIaI significantly prolonged the occlusion times of tPA-containing whole blood in a microchip-based flow chamber system, suggesting that TAFI attenuated the tPA-dependent prolongation of clot formation under flow. Thus, activated platelet surfaces are targeted by plasma TAFI, to attenuate plasminogen accumulation and fibrinolysis, which may contribute to thrombogenicity under flow.

Low Levels of Thrombin Activatable Fibrinolysis Inhibitor (TAFI) in Patients with Chronic Liver Disease

2001 ◽  
Vol 85 (04) ◽  
pp. 667-670 ◽  
Author(s):  
Magdalene George ◽  
Jawed Fareed ◽  
David Van Thiel
Keyword(s):  
Liver Disease ◽  
Chronic Liver Disease ◽  
Degradation Products ◽  
Chronic Liver ◽  
Low Grade ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Disseminated Intravascular Coagulopathy ◽  
Fibrinolysis Inhibitor ◽  
Intravascular Coagulopathy ◽  
Low Levels

SummaryThrombin Activatable Fibrinolysis Inhibitor (TAFI) is a 60 κD glycoprotein present in plasma that regulates fibrinolysis by limiting the amount of fibrin available for fibrinolysis by tissue plasminogen activator (t-PA). Chronic liver disease is well-known to be associated with a low-grade fibrinolytic syndrome that under the appropriate stimulus proceeds to an overt disseminated intravascular coagulopathy (DIC) with demonstrable bleeding. In the present study, TAFI activity was measured in the plasma of 74 patients with advanced liver disease, and the levels of TAFI were related to those of other important coagulation and fibrinolytic factors. TAFI levels were very low and essentially undetectable in the plasma of patients with advanced hepatocellular liver disease. No relationship with the degradation products of fibrin was evident.

Plasminogen Activation In Normal Individuals And Patients With Thrombotic Tendency

1981 ◽  
Author(s):  
S Schulman ◽  
A Holmgren
Keyword(s):  
Catalytic Efficiency ◽  
Plasminogen Activation ◽  
Zymogen Activation ◽  
Normal Individuals ◽  
Maximal Activation ◽  
Km Value ◽  
History Of ◽  
Human Plasminogen ◽  
Kinetics Of ◽  
Thrombotic Tendency

Following the principles described by Wohl, Summaria and Robbins we have studied the kinetics of human plasminogen activation by streptokinase. Plasma plasminogen activation kinetic parameters were measured in the following way: Citrated plasma was mixed with a Tris/NaCl buffer, pH 7.4, and increasing amounts of streptokinase were added. After incubation at 37°C for 5 min, synthetic substrate, H-D-val- -L-leu-L-lys-p-nitroanilide was added and initial plasmin rates were recorded. We also determined the Michaelis-Man- ten parameters for the plasmin of the plasma samples following maximal activation of the plasminogen with streptokinase. This was done by incubating citrated plasma (diluted 1:200) with streptokinase (5000 U/ml) for 10 min at 37°C.In the normal individuals the zymogen.streptokinase complex gave a mean catalytic efficiency, kcat/KM, of 0.15 μM-1S-1. For the streptokinase activator species the average catalytic efficiency, kplg/Kplg, was 26 μM-1min-1, Iμ(95%) = {8.4; 44.5} μM-lmin-1.So far we have studied eight patients with a history of thrombosis. With the exception of one patient, all showed values within the range mentioned above. The deviating patient had a significantly lower concentration of plasminogen, 6.7 mg/dl, as measured by immunodiffusion, and a significantly higher kplg/Kplg-value, 54 μM-1min-1, but a similar kcat/KM-value, 0.2 μM-1S-1. The lower plasminogen concentration is thus matched by an apparent increase of streptokinase activation. We do not know if the observed effect is caused by a mutation in the enzyme. An explanation for the low concentration of plasminogen could be chronic fibrinolysis of thrombi.We will investigate this further and also screen more patients with a history of thrombosis for possible variants with lowered catalytic efficiency of zymogen activation.

The uptake of atropine and related drugs by intestinal smooth muscle of the guinea-pig in relation to acetylcholine receptors

1965 ◽  
Vol 163 (990) ◽  
pp. 1-44 ◽  
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Equilibrium Constant ◽  
Rate Constant ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Kinetic Properties ◽  
Kinetic Behaviour ◽  
Kinetics Of ◽  
Bound Drug

In an attempt to study the properties of acetylcholine receptors in intestinal smooth muscle, measurements have been made of the uptake of tritium-labelled atropine and methylatropinium, and of 14 C-labelled methylfurmethide by the longitudinal muscle of guinea-pig small intestine in vitro . Substantial amounts of atropine were taken up from very dilute solutions, a clearance of 160 ml. per g tissue (wet weight) being achieved at the lowest concentration tested (1.5 × 10 -10 M). Analysis of the curve relating atropine uptake at equilibrium to the bath concentration, which was explored over a concentration range 1.5 × 10 -10 M to 2.5 × 10 -3 M, enabled three components to be distinguished: (1) A binding site with a capacity of 180 pmoles/g, and equilibrium constant 1.1 × 10 -9 M. (2) A binding site of capacity about 1000 pmoles/g and equilibrium constant about 5 × 10 -7 M. (3) A compartment with a clearance of 4.7 ml./g (nonsaturable). The equilibrium constant of the first binding site agreed exactly with that measured for acetylcholine antagonism in the same tissue. Methylatropinium was taken up in rather smaller amounts than atropine, and analysis of the uptake curve showed a binding site of capacity about 90 pmoles/g with an equilibrium constant 6.5 × 10 -10 M, an ill-defined series of binding sites with much higher equilibrium constants, and a constant clearance of about 0.4 ml. /g. Analysis of this curve was much less clear cut than that of atropine. The equilibrium constant for blockade of acetylcholine receptors by methylatropinium was 4.7 × 10 -10 M. Atropine was not taken up appreciably by striated muscle, nerve or tendon of the guineapig; hydrolysed atropine was not taken up by smooth muscle (and lacks atropinic activity); cocaine and d -tubocurarine in high concentrations did not affect atropine uptake; lachesine and benzhexol blocked atropine uptake competitively at low concentrations, and with lachesine the equilibrium constant for this interaction agreed with that measured for acetylcholine antagonism (1.4 × 10 -9 M). These findings suggested that the atropine taken up could be related to receptor-bound drug. The kinetics of atropine uptake and washout were studied over the concentration range 0.5-5 × 10 -9 M. Uptake and washout took place approximately exponentially between 2½ and 50 min, and the rate constant was 4.5-5 × 10 -4 s -1 for both uptake and washout. The uptake rate constant did not increase with concentration. This contrasted with the kinetics of receptor blockade, which took place much faster, with a rate constant which increased linearly with concentration, in accordance with the theoretical kinetic behaviour of a single binding site. This finding precluded a simple identification of atropine taken up with receptor-bound drug. Studies with various metabolic inhibitors suggested that no metabolic energy was required for the accumulation of atropine, and by dialysis experiments, the atropine taken up was shown to be bound in homogenized tissue. A theoretical study, using an analogue computer, was made of the kinetic properties of three passive binding systems, in order to see whether the observed kinetic behaviour could be simulated. It was found that a system of four binding sites in series, with only one communicating directly with the surrounding medium, could show these kinetic properties, and the outermost binding site could still show the kinetic behaviour of receptors. Experimental testing of this model demands more accurate kinetic measurements than can be made by the method used in this study. The acetylcholine-like stimulant, methylfurmethide, was taken up very slowly (taking more than 24 h to reach equilibrium), reaching a clearance of about 5 ml. /g after 6 h. This uptake was unaffected by atropine in a concentration sufficient to block 80% of acetylcholine receptors, but was blocked by depolarization in high potassium solution, suggesting that it was behaving passively as a slowly permeant cation. No uptake referable to acetylcholine receptors was detected. These findings are discussed in relation to the abundance and chemical behaviour of acetylcholine receptors in smooth muscle, and in relation to current theories of drug action.

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