RESUMO

Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban, edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies. The interaction of active compounds with human serum albumin (HSA), the most abundant protein in blood plasma, is a key research area and provides crucial information about drugs' pharmacokinetics and pharmacodynamic properties. This research focuses on the study of the interactions between HSA and four commercially available direct oral FXa inhibitors, applying methodologies including steady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and molecular dynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and the complex formation in the ground states affects the fluorescence of HSA, with a moderate binding constant of 104 M-1. However, the ITC studies reported significantly different binding constants (103 M-1) compared with the results obtained through spectrophotometric methods. The suspected binding mode is supported by molecular dynamics simulations, where the predominant interactions were hydrogen bonds and hydrophobic interactions (mainly π-π stacking interactions between the phenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications of the obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.

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RESUMO

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Assuntos

RESUMO

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

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Assuntos

RESUMO

Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents extensive sequence homology to TFPI. It binds to FXa or FX as scaffolds and inhibits tissue factor/FVIIa complex (extrinsic Xnase). Differently from TFPI, ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. In this report, we show that recombinant (125)I-ixolaris interacts with rat and human FX in plasma and prolongs the prothrombin time (PT) and activated partial thromboplastin time (aPTT) in vitro. We have also investigated the effects of ixolaris in vivo, using a venous thrombosis model. Subcutaneous (s.c.) or intravenous (i.v.) administration of ixolaris in rats caused a dose-dependent reduction in thrombus formation, with complete inhibition attained at 20 microg/kg and 10 microg/kg, respectively. Antithrombotic effects were observed 3 h after s.c. administration of ixolaris and lasted for 24 h thereafter. Ex vivo experiments showed that ixolaris (up to 100 microg/kg) did not affect the aPTT, while the PT was increased by approximately 0.4-fold at the highest ixolaris concentration. Remarkably, effective antithrombotic doses of ixolaris (20 microg/kg) was not associated with bleeding which was significant only at higher doses of the anticoagulant (40 microg/kg). Our experiments demonstrate that ixolaris is an effective and possibly safe antithrombotic agent in vivo.

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RESUMO

Physiological secretions from some invertebrates have toxic effects on mammalian blood coagulation and fibrinolytic systems. Some of these effects occur because the substances contained in the secretions resemble the components of the hemostatic system. Some of the substances have been characterized, and have been found to have similar molecular weights or sequences, which may indicate a common ancestry. The components can be divided into five groups: antithrombic agents (group I); inhibitors and activators of the prothrombinase complex (group II); substances that affect platelet function (group III); substances that affect the fibrinolytic mechanism (group IV); and a group of miscellaneous agents whose activities are difficult to group together (group V). In group I special mention of the antithrombin agents in Hirudo medicinalis should be made. In group II, the agents affecting the prothrombinase complex are antistasin from Haementeria officinalis, ghilanten from Haementeria Ghiliani and the tick anticoagulant protein from Ornithodoros moubata, a factor V activator/inhibitor from Lonomia achelous and factor II and factor X activators from L. achelous and Lonomia obliqua. Examples of factors which affect platelet function (group III) are glossina from the black fly Glossina morsitans, calin from H. medicinalis, decorsin (a desintegrin) from Macrobdella decorsa, and FAGA from Stichopus japonicus selenka. The first three of these are inhibitors of platelet aggregation, and the last is an inducer. The plasminogen activators (group IV) from the L. achelous caterpillar and Eutriatoma maculata trigger the fibrinolytic system, whereas hementin from H. officinalis and hementerin from Haementeria depressa are directly fibrinolytic. The last group of substances (group V) include those with factor-XIIa-like activity from D. farinae, kallikrein-like activity and a factor XIII degrading enzyme from L. achelous, destabilase from H. medicinalis and prolixin S (nitroforin 2, or anti-factor-IXa) from Rhodnius prolixus. Some of these components have been well characterized, cloned and prepared in recombinant form, and seem to be very promising from the therapeutic point of view.

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