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.

Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor

2017 ◽  
Vol 117 (04) ◽  
pp. 682-690 ◽  
Author(s):  
Tomasz Brzoska ◽  
Yuko Suzuki ◽  
Hideto Sano ◽  
Seiichirou Suzuki ◽  
Martyna Tomczyk ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Network Formation ◽  
Laser Scanning Microscopy ◽  
Tissue Type ◽  
Confocal Laser ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Activated Platelets ◽  
Fibrin Network ◽  
Fibrinolysis Inhibitor ◽  
Coagulation And Fibrinolysis

SummaryUsing intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-de- pendent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets’ surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).Supplementary Material to this article is available online at www.thrombosis-online.com.

Identification and characterisation of monoclonal antibodies that impair the activation of human thrombin activatable fibrinolysis inhibitor through different mechanisms

2011 ◽  
Vol 106 (07) ◽  
pp. 90-101 ◽  
Author(s):  
Niraj Mishra ◽  
Ellen Vercauteren ◽  
Jan Develter ◽  
Riet Bammens ◽  
Paul J. Declerck ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Lysis Time ◽  
Human Thrombin ◽  
Fibrinolysis Inhibitor ◽  
Dose Dependent ◽  
Clot Lysis Time

SummaryThrombin activatable fibrinolysis inhibitor (TAFI) forms a molecular link between coagulation and fibrinolysis and is a putative target to develop profibrinolytic drugs. Out of a panel of monoclonal antibodies (MA) raised against TAFI-ACIIYQ, we selected MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4, which revealed high affinity towards human TAFITI- wt. MA-TCK11A9 was able to inhibit mainly plasmin-mediated TAFI activation, MA-TCK22G2 inhibited plasmin- and thrombin-mediated TAFI activation and MA-TCK27A4 inhibited TAFI activation by plasmin, thrombin and thrombin/thrombomodulin (T/TM) in a dose-dependent manner. These MA did not interfere with TAFIa activity. Using an eightfold molar excess of MA over TAFI, all three MA were able to reduce clot lysis time significantly, i.e. in the presence of exogenous TM, MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 47 ± 9.1%, 80 ± 8.6% and 92 ± 14%, respectively, compared to PTCI. This effect was even more pronounced in the absence of TM i.e. MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 90 ± 14%, 140 ± 12% and 147 ± 29%, respectively, compared to PTCI. Mutagenesis analysis revealed that residues at position 268, 272 and 276 are involved in the binding of MA-TCK11A9, residues 147 and 148 in the binding of MA-TCK22G2 and residue 113 in the binding of MATCK27A4. The present study identified three MA, with distinct epitopes, that impair the activation of human TAFI and demonstrated that MATCK11A9 which mainly impairs plasmin-mediated TAFI activation can also reduce significantly clot lysis time in vitro.

Plasma TAFI Levels Influence the Clot Lysis Time in Healthy Individuals in the Presence of an Intact Intrinsic Pathway of Coagulation

1998 ◽  
Vol 80 (11) ◽  
pp. 829-835 ◽  
Author(s):  
Peter von dem Borne ◽  
Joost Meijers ◽  
Bonno Bouma ◽  
Laurent Mosnier
Keyword(s):  
Tissue Type ◽  
Clot Lysis ◽  
Healthy Individuals ◽  
Intrinsic Pathway ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Factor Xi ◽  
Lysis Time ◽  
Fibrinolysis Inhibitor ◽  
High Concentrations ◽  
Clot Lysis Time

SummaryThrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently identified fibrinolysis inhibitor in plasma, that when converted to an enzyme potently attenuates fibrinolysis. It is activated by relatively high concentrations of thrombin that exceed the thrombin concentration required for fibrin formation. These high concentrations of thrombin are generated by the intrinsic pathway via activation of factor XI by thrombin. The down regulation of fibrinolysis by TAFI can be measured in a clot lysis assay. When the clot lysis times of healthy individuals were determined, large inter-individual differences were observed. To determine if differences in concentration of TAFI explain the variation in clot lysis between individuals, specific assays were developed for the measurement of TAFI antigen and activity in plasma. In normal plasma, there was a dose-dependent relationship between TAFI antigen and TAFI activity. There was also a correlation between clot lysis time and plasma TAFI antigen, indicating that the amount of TAFI that is activated during the clot lysis assay, is dependent on the concentration of TAFI. In the plasmas of 20 healthy individuals, clot lysis times, TAFI antigen and TAFI activity were determined. Both TAFI antigen and TAFI activity showed a significant correlation with the clot lysis time. No correlation between TAFI antigen and clot lysis time was found when the clot lysis time was determined in the presence of an antibody blocking the factor XI feedback loop. These results indicate that plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.Abbreviations: The abbreviations used are: TAFI, Thrombin Activatable Fibrinolysis Inhibitor; TAFIa, activated TAFI; CPI, carboxypeptidase inhibitor from potato tubers; CPN, carboxypeptidase N; tPA, tissue-type plasminogen activator.

Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site

2011 ◽  
Vol 392 (4) ◽  
Author(s):  
Jin-Hyung Ahn ◽  
Ho-Jeong Lee ◽  
Eun-Kyoung Lee ◽  
Hyun-Kyung Yu ◽  
Tae-Ho Lee ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Safety Concern ◽  
Tissue Type ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Kringle Domains ◽  
Apolipoprotein A

AbstractMany proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effectsin vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV9-IV10-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic processin vitroand analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical signi-ficance of these findings will require further validationin vivo.

Regulation of Fibrinolysis in Plasma by TAFI and Protein C Is Dependent on the Concentration of Thrombomodulin

2001 ◽  
Vol 85 (01) ◽  
pp. 5-11 ◽  
Author(s):  
Joost Meijers ◽  
Bonno Bouma ◽  
Laurent Mosnier
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
The Body ◽  
Tissue Type ◽  
Clot Lysis ◽  
Microvascular Endothelial Cell ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Functional Protein ◽  
Low Concentrations ◽  
Fibrinolysis Inhibitor

SummaryThrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme, that after activation down regulates fibrinolysis. TAFI is activated by thrombin in the presence of the cofactor thrombomodulin (TM). By stimulation of TAFI activation TM down regulates fibrinolysis, however TM is also a cofactor in the activation of protein C. Activated protein C (APC) can up regulate fibrinolysis by limiting the activation of TAFI via the attenuation of thrombin production. We studied these counteracting fibrinolytic properties of TM in plasma by measuring the activation of TAFI during tissue factor induced coagulation. TAFI activation was stimulated at low concentrations of TM but decreased at higher concentrations of TM. Similarly, the clot lysis times increased at low concentrations of TM but decreased at higher concentrations of TM. The reduction of TAFI activation at high TM concentrations was found to be dependent on a functional protein C pathway. The concentration of TM is therefore an important factor in the regulation of TAFI activation and in the regulation of fibrinolysis. High concentrations of TM result in up regulation of fibrinolysis, whereas low concentrations of TM have a down regulatory effect on fibrinolysis. These results suggest that fibrinolysis might be differentially regulated by TM in different parts of the body depending on the local TM concentration in the vasculature. Abbreviations: TAFI, thrombin activatable fibrinolysis inhibitor; TAFIa, activated TAFI; CPI, carboxypeptidase inhibitor from potato tubers; tPA, tissue-type plasminogen activator; TM, thrombo-modulin; TM4-6, Thrombomodulin fragment containing EGF domains 4 to 6; HMEC, human microvascular endothelial cell.

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.

Effects of fibrinogen concentrate, factor XIII, and thrombin-activatable fibrinolysis inhibitor on clot firmness and fibrinolytic resistance in the model of hyperfibrinolysis

2017 ◽  
pp. 44-50
Author(s):  
И.А. Будник ◽  
О.Л. Морозова ◽  
А.А. Цымбал ◽  
Б. Шенкман ◽  
Ю. Эйнав
Keyword(s):  
Factor Xiii ◽  
Onset Time ◽  
Clot Formation ◽  
Clot Lysis ◽  
Fibrinogen Concentrate ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Antifibrinolytic Agents ◽  
Fibrinolysis Inhibitor ◽  
Vitro Model

Цель исследования - изучение возможности коррекции формирования кровяного сгустка и его фибринолитической устойчивости с помощью концентратов фибриногена, фактора XIII и активируемого тромбином ингибитора фибринолиза (TAFI) в модели гиперфибринолиза in vitro . Методика. В образцы цитратной крови, полученной от 24 взрослых здоровых добровольцев, добавляли концентрат фибриногена, фактора XIII и/или TAFI. Фибринолиз индуцировали добавлением тканевого активатора плазминогена. Свертывание крови индуцировали рекальцификацией и добавлением препарата тканевого фактора. Формирование и лизис сгустка изучали методом ротационной тромбоэластометрии. Результаты. Индукция фибринолиза не влияла на время свертывания и скорость формирования сгустка, но значительно уменьшала максимальную плотность сгустка и вызывала его лизис. Концентрат фибриногена замедлял скорость лизиса сгустка; концентрат фактора XIII усиливал механическую прочность сгустка и замедлял скорость его лизиса, не влияя при этом на время начала лизиса; TAFI усиливал механическую прочность и значительно отдалял время начала лизиса, оказывая тем самым наибольший корригирующий эффект. Заключение. Полученные данные демонстрируют потенциальную возможность коррекции гемостатического потенциала крови при гиперфибринолизе с помощью концентратов фибриногена, фактора XIII и TAFI, которые могут стать альтернативой традиционным антифибринолитикам. Aim. To investigate effects of fibrinogen concentrate, factor XIII, and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and fibrinolytic resistance using an in vitro model of hyperfibrinolysis. Methods. Citrated whole blood from 24 adult healthy volunteers was supplemented with fibrinogen concentrate, factor XIII, and/or TAFI. Fibrinolysis was induced by tissue plasminogen activator. Clotting was induced by recalcification and addition of tissue factor and monitored using rotation thromboelastometry. Results. Induction of fibrinolysis did not affect clotting time and the rate of clot formation but significantly reduced the maximum clot firmness and caused lysis of a clot. Addition of fibrinogen concentrate to blood reduced the rate of clot lysis without affecting clot firmness or lysis onset time; addition of factor XIII improved clot firmness and reduced clot lysis rate without affecting lysis onset time; TAFI improved clot firmness and considerably delayed the onset of clot lysis thereby providing the greatest antifibrinolytic effect. Conclusion. Fibrinogen concentrate, factor XIII, and TAFI may potentially serve as an alternative to traditional antifibrinolytic agents and be beneficial for the treatment of patients with hyperfibrinolysis.

Characterization of Mouse Thrombin-activatable Fibrinolysis Inhibitor

2000 ◽  
Vol 83 (02) ◽  
pp. 297-303 ◽  
Author(s):  
Gerry Wagenaar ◽  
Arie Reijerkerk ◽  
Margriet Tiekstra ◽  
Agnes van Rossum ◽  
Martijn Gebbink ◽  
...  
Keyword(s):  
Genetically Modified ◽  
Physiological Role ◽  
Tissue Type ◽  
Clot Lysis ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Specific Expression ◽  
Endogenous Expression ◽  
Fibrinolysis Inhibitor

SummaryBased on in vitro studies, thrombin-activatable fibrinolysis inhibitor (TAFI) has been hypothesized as a link between coagulation and fibrinolysis, but the physiological role of TAFI in vivo has not yet been established. To anticipate on the availability of genetically modified mouse models, we studied the endogenous expression of TAFI in mice. Functional TAFI was found in mouse plasma. TAFI mRNA was only detectable in the liver, showing a hepatocyte-specific expression with a pericentral lobular distribution pattern. The murine TAFI cDNA was cloned and sequenced. The deduced amino acid sequence revealed that murine TAFI is highly identical to human TAFI. The murine cDNA was stably expressed and the activated recombinant protein was functionally active; it converted the substrate hippuryl-arginine, and prolonged the clot lysis time of TAFI depleted plasma. We conclude that mice have functional TAFI in plasma, which is highly similar to human TAFI. Therefore, genetically modified mice may provide useful models to study the role of TAFI in vivo. Abbreviations: TAFI, thrombin-activatable fibrinolysis inhibitor; CPI, carboxypeptidase inhibitor from potato tubers; CPN, carboxypeptidase N; t-PA, tissue-type plasminogen activator; PPACK, H-D-Phe-Pro-Arg-chloromethylketone.

Abstract 34: Impact of Thrombomodulin and Thrombin-activatable Fibrinolysis Inhibitor on the Anti-coagulant and Pro-fibrinolytic Effects of Rivaroxaban

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Justin J Garabon ◽  
Michael B Boffa
Keyword(s):  
Formation Rate ◽  
Anticoagulation Therapy ◽  
Clot Formation ◽  
Clot Lysis ◽  
Anatomical Location ◽  
Wild Type ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Plasma Clot ◽  
Fibrinolysis Inhibitor

Rivaroxaban is a novel oral anticoagulant that directly inhibits factor Xa and has recently been implemented as a favorable alternative to warfarin in anticoagulation therapy. Rivaroxaban effectively reduces the generation of thrombin, which plays a major role in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) to TAFIa. Activated TAFI functions by downregulating fibrinolysis, and this effect is highly dependent on the availability of thrombomodulin (TM), as the thrombin-TM complex activates TAFI with a 1250-fold greater efficiency than thrombin alone. Additionally, a naturally occurring SNP in the gene encoding TAFI gives rise to a Thr325Ile polymorphism that increases the stability and antifibrinolytic potential of TAFIa. Since inhibition of thrombin generation could lead to decreased TAFIa formation, we hypothesized that these parameters could influence the pharmacodynamics and pharmacogenomics of rivaroxaban. To assess this, effects on coagulation and fibrinolysis were measured using an in vitro plasma clot lysis assay. Rivaroxaban and TM were titrated into TAFI-deficient plasma in the presence or absence of 10 nM wild-type or T325I recombinant TAFI. With increasing concentrations of rivaroxaban, clot formation latency was accordingly increased. TM further delayed clot formation, and this effect was equal in the absence or presence of either TAFI variant. Rivaroxaban was also shown to decrease the rate of coagulation, which was decreased further at higher concentrations of TM. The presence of either TAFI variant also seemed to decrease clot formation rate at higher levels of rivaroxaban and TM. At all concentrations of TM, TAFI-dependent resistance to fibrinolysis was attenuated by rivaroxaban. The effect of rivaroxaban was, however, greater for wild-type TAFI than for the T325I variant. In conclusion, rivaroxaban exhibits TAFI-dependent profibrinolytic effects that are influenced by the levels of TM and the by intrinsic stability of TAFIa. TM also affected the dynamics of coagulation. These findings suggest a role for the anatomical location of a procoagulant stimulus and plasma TM-altering disease phenotypes in the pharmacodynamics and a role for the T325I polymorphism in the pharmacogenomics of rivaroxaban.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

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