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1.
Anal Chem ; 93(44): 14802-14809, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34694784

RESUMO

Fragment-based lead discovery is a usual strategy in drug discovery to identify innovative lead compounds. The success of this approach strongly relies on the capacity to detect weak binders and characterize their binding site. NMR and X-ray crystallography are the conventional technologies used to tackle this challenge. However, their large protein consumption and the cost of equipment reduce their accessibility. Here, an affinity capillary electrophoresis methodology was developed that enables the detection of mM binders, the determination of dissociation constants, and the characterization of the fragment binding site. On the basis of multiple equilibrium theory, dissociation constants in the µM-mM range were determined, and a new methodology is proposed to establish graphically if two fragments bind the same protein pocket. The applicability of this methodology was demonstrated experimentally on coagulation factor XIIa by evaluating pairs of fragments with expected behavior. This study reinforces the significance of using affinity capillary electrophoresis to gather valuable information for medicinal chemistry projects.


Assuntos
Descoberta de Drogas , Fator XIIa , Sítios de Ligação , Cristalografia por Raios X , Eletroforese Capilar , Ligação Proteica
2.
Nat Commun ; 12(1): 5596, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552086

RESUMO

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.


Assuntos
Coagulação Sanguínea , Fator XII/química , Fator XII/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Fator XII/genética , Fator XII/imunologia , Fator XIIa/metabolismo , Camundongos , Mutação , Tempo de Tromboplastina Parcial/normas , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Trombose/diagnóstico , Trombose/genética , Trombose/metabolismo
3.
J Thromb Haemost ; 19(9): 2235-2247, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34060720

RESUMO

BACKGROUND: Factor XII (FXII) is a serine protease that participates in the intrinsic coagulation pathway. Several studies have shown that plasma FXII exerts a deleterious role in cerebral ischemia and traumatic brain injury by promoting thrombo-inflammation. Nevertheless, the impact of FXII on neuronal cell fate remains unknown. OBJECTIVES: We investigated the role of FXII and FXIIa in neuronal injury and apoptotic cell death. METHODS: We tested the neuroprotective roles of FXII and FXIIa in an experimental model of neuronal injury induced by stereotaxic intracerebral injection of N-methyl-D-aspartic acid (NMDA) in vivo and in a model of apoptotic death of murine primary neuronal cultures through serum deprivation in vitro. RESULTS: Here, we found that exogenous FXII and FXIIa reduce brain lesions induced by NMDA injection in vivo. Furthermore, FXII protects cultured neurons from apoptosis through a growth factor--like effect. This mechanism was triggered by direct interaction with epidermal growth factor (EGF) receptor and subsequent activation of this receptor. Interestingly, the "proteolytically" active and two-chain form of FXII, FXIIa, exerts its protective effects by an alternative signaling pathway. FXIIa activates the pro-form of hepatocyte growth factor (HGF) into HGF, which in turn activated the HGF receptor (HGFR) pathway. CONCLUSION: This study describes two novel mechanisms of action of FXII and identifies neurons as target cells for the protective effects of single and two-chain forms of FXII. Therefore, inhibition of FXII in neurological disorders may have deleterious effects by preventing its beneficial effects on neuronal survival.


Assuntos
Fator XII , Proteínas Proto-Oncogênicas c-met , Animais , Apoptose , Coagulação Sanguínea , Fator XIIa , Camundongos , Neurônios
4.
Front Immunol ; 12: 649122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34177896

RESUMO

Thromboplasminflammation in coronavirus disease 2019 (COVID-19) coagulopathy consists of angiotensin II (Ang II)-induced coagulopathy, activated factor XII (FXIIa)- and kallikrein, kinin system-enhanced fibrinolysis, and disseminated intravascular coagulation (DIC). All three conditions induce systemic inflammation via each pathomechanism-developed production of inflammatory cytokines. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) downregulates angiotensin-converting enzyme 2, leading to an increase in Ang II levels. Ang II-induced coagulopathy comprising platelet activation, thrombin generation, plasminogen activator inhibitor-1 expression and endothelial injury causes thrombosis via the angiotensin II type 1 receptor. SARS-CoV-2 RNA and neutrophil extracellular trap (NET) DNA activate FXII, resulting in plasmin generation through FXIIa- and kallikrein-mediated plasminogen conversion to plasmin and bradykinin-induced tissue-type plasminogen activator release from the endothelium via the kinin B2 receptor. NETs induce immunothrombosis at the site of infection (lungs), through histone- and DNA-mediated thrombin generation, insufficient anticoagulation control, and inhibition of fibrinolysis. However, if the infection is sufficiently severe, immunothrombosis disseminates into the systemic circulation, and DIC, which is associated with the endothelial injury, occurs. Inflammation, and serine protease networks of coagulation and fibrinolysis, militate each other through complement pathways, which exacerbates three pathologies of COVID-19 coagulopathy. COVID-19 coagulopathy causes microvascular thrombosis and bleeding, resulting in multiple organ dysfunction and death in critically ill patients. Treatment targets for improving the prognosis of COVID-19 coagulopathy include thrombin, plasmin, and inflammation, and SARS-CoV-2 infection. Several drugs are candidates for controlling these conditions; however, further advances are required to establish robust treatments based on a clear understanding of molecular mechanisms of COVID-19 coagulopathy.


Assuntos
Transtornos da Coagulação Sanguínea/metabolismo , COVID-19/metabolismo , SARS-CoV-2/fisiologia , Angiotensina II/metabolismo , Animais , Transtornos da Coagulação Sanguínea/imunologia , COVID-19/imunologia , Citocinas/metabolismo , Fator XIIa/metabolismo , Humanos , Inflamação , Mediadores da Inflamação/metabolismo
5.
Expert Opin Ther Pat ; 31(12): 1155-1176, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34142629

RESUMO

Introduction: Blood coagulation factor XII (FXII) is an emerging and potentially safe drug target, which dysregulation is associated with thrombosis, hereditary angioedema, and (neuro)inflammation. At the same time, FXII-deficiency is practically asymptomatic. Industrial and academic institutions have developed a number of potential therapeutic agents targeting either FXII zymogen or its active form FXIIa for the treatment of thrombotic and inflammatory conditions associated with the activity of this enzyme.Areas covered: A short overview of the FXII(a) structure and function, underlining its suitability as a drug target, is given. The article reviews patents reported over the last three decades on FXII(a)-targeting therapeutic agents. These agents include small molecules, proteins, peptides, oligonucleotides, siRNAs, and monoclonal antibodies.Expert opinion: The performed analysis of patents revealed that many FXII(a) inhibitors are in the early preclinical stage, while several already showed efficacy in vivo animal models of thrombosis, sepsis, hereditary angioedema, and multiple sclerosis. Two anti-FXIIa agents namely tick protein Ir-CPI and monoclonal antibody CSL312 are currently in human clinical trials. The results of these trials and further studies of FXII(a) pathophysiological functions will encourage the development of new FXII(a) inhibitors.


Assuntos
Fator XII/antagonistas & inibidores , Fator XIIa/antagonistas & inibidores , Angioedemas Hereditários/tratamento farmacológico , Angioedemas Hereditários/patologia , Animais , Desenvolvimento de Medicamentos , Fator XII/metabolismo , Fator XIIa/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Patentes como Assunto , Trombose/tratamento farmacológico , Trombose/patologia
6.
J Med Chem ; 64(11): 7853-7876, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34044534

RESUMO

The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.


Assuntos
Fator XIIa/antagonistas & inibidores , Peptídeos Cíclicos/química , RNA Mensageiro/metabolismo , Inibidores de Serino Proteinase/química , Sítios de Ligação , Fator XIIa/metabolismo , Biblioteca Gênica , Código Genético , Humanos , Concentração Inibidora 50 , Calicreínas/química , Calicreínas/metabolismo , Simulação de Dinâmica Molecular , Tempo de Tromboplastina Parcial , Peptídeos Cíclicos/metabolismo , Estabilidade Proteica , Tempo de Protrombina , Puromicina/química , RNA Mensageiro/química , Inibidores de Serino Proteinase/metabolismo , Relação Estrutura-Atividade
7.
Vox Sang ; 116(1): 99-105, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32986885

RESUMO

BACKGROUND: Prekallikrein activator (PKA) is a contaminating enzyme found in therapeutic albumin and immunoglobulin products. The level is commonly measured using methods such as that defined by the European Pharmacopoeia (Ph Eur) with traceability to the WHO International Standard for PKA. This method generally works well, but problems are sometimes observed. MATERIALS AND METHODS: A simplified one-step method has been developed to replace the existing Ph Eur two-step method which consists of kallikrein generation followed by kallikrein measurement using a chromogenic substrate. Analysis of data from the one-stage method is simplified by the use of a dedicated online app. RESULTS: The one-stage method was validated against the current Ph Eur method using batches of albumin and immunoglobulins. Problem batches of immunoglobulins were investigated using the one-stage method. Improved methodology using true initial rate determinations and use of acid-treated prekallikrein substrate (PKS) helped understand and reduce artefactual results. CONCLUSIONS: The one-stage method and associated app streamline real-time determination of PKA and promote good principles of enzyme assays to limit substrate depletion, while also conserving expensive PKS. Blanking steps and reproducibility are simplified.


Assuntos
Albuminas , Fator XIIa/análise , Imunoglobulinas , Fator XIIa/metabolismo , Humanos , Pré-Calicreína/metabolismo , Reprodutibilidade dos Testes
8.
J Thromb Haemost ; 19(2): 323-329, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33047454

RESUMO

Clinical practice shows that a critical unmet need in the field of medical device-associated thrombosis prevention is the availability of an anticoagulant therapy without hemorrhagic risk. In the quest for new drugs that are at least as effective as those currently available, while avoiding bleeding complications, molecules that target nearly every step of the coagulation pathway have been developed. Among these molecules, inhibitors of factor XII (FXII) or factor XI (FXI) are promising alternatives as deficiencies in these factors protect against thrombosis without causing spontaneous hemorrhage, as revealed by epidemiological and preclinical data. Ixodes ricinus-contact phase inhibitor (Ir-CPI), a new anticoagulant candidate with an innovative mechanism of action could be this ideal anticoagulant agent for safe prevention from clotting on medical devices. This protein, which selectively binds to FXIIa, FXIa, and plasma kallikrein and inhibits the reciprocal activation of FXII, prekallikrein, and FXI in human plasma, was shown to prevent thrombosis in an ovine cardiopulmonary bypass system associated with cardiac surgeries. Furthermore, as opposed to unfractionated heparin, Ir-CPI appears to be devoid of bleeding risk. This review outlines the rationale for targeting upstream coagulation factors in order to prevent medical device-associated thrombosis; examines the novel approaches under development; and focuses on Ir-CPI, which shows promising properties in the field of thrombosis prevention.


Assuntos
Fator XIIa , Fator XIa , Trombose/prevenção & controle , Animais , Coagulação Sanguínea , Fator XI , Fator XII , Fator XIIa/antagonistas & inibidores , Fator XIa/antagonistas & inibidores , Heparina , Humanos , Ovinos
9.
Blood Adv ; 4(24): 6135-6147, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33351111

RESUMO

Factor XI (FXI) is the zymogen of a plasma protease (FXIa) that contributes to hemostasis by activating factor IX (FIX). In the original cascade model of coagulation, FXI is converted to FXIa by factor XIIa (FXIIa), a component, along with prekallikrein and high-molecular-weight kininogen (HK), of the plasma kallikrein-kinin system (KKS). More recent coagulation models emphasize thrombin as a FXI activator, bypassing the need for FXIIa and the KKS. We took an evolutionary approach to better understand the relationship of FXI to the KKS and thrombin generation. BLAST searches were conducted for FXI, FXII, prekallikrein, and HK using genomes for multiple vertebrate species. The analysis shows the KKS appeared in lobe-finned fish, the ancestors of all land vertebrates. FXI arose later from a duplication of the prekallikrein gene early in mammalian evolution. Features of FXI that facilitate efficient FIX activation are present in all living mammals, including primitive egg-laying monotremes, and may represent enhancement of FIX-activating activity inherent in prekallikrein. FXI activation by thrombin is a more recent acquisition, appearing in placental mammals. These findings suggest FXI activation by FXIIa may be more important to hemostasis in primitive mammals than in placental mammals. FXI activation by thrombin places FXI partially under control of the vitamin K-dependent coagulation mechanism, reducing the importance of the KKS in blood coagulation. This would explain why humans with FXI deficiency have a bleeding abnormality, whereas those lacking components of the KKS do not.


Assuntos
Deficiência do Fator XI , Fator XI , Animais , Fator XI/genética , Fator XI/metabolismo , Deficiência do Fator XI/genética , Fator XIIa/metabolismo , Feminino , Humanos , Sistema Calicreína-Cinina , Gravidez , Pré-Calicreína/genética , Pré-Calicreína/metabolismo
10.
Orv Hetil ; 161(50): 2099-2103, 2020 12 13.
Artigo em Húngaro | MEDLINE | ID: mdl-33310922

RESUMO

Összefoglaló. Bevezetés: Egy új, számítógép által segített betegminta-asszociációs analízis eredménye szerint a COVID-19 tüneteinek kialakításában kiemelt tényezoként jelenik meg a bradikinin. Eszerint a bradikinin lebontása lelassul az angiotenzinkonvertáló enzim aktivitásának csökkenése miatt, ami jelentosen megemelkedo bradikininszinthez vezet a tüdoben. Nem merült fel azonban a véralvadási faktorok lehetséges szerepe a "bradikininviharban", annak ellenére, hogy az idosebb cardiovascularis betegekben aktiválódó XII-es faktor és a C1-észteráz-inhibitor (C1INH) alacsony szintje nagy mennyiségu bradikinin képzodéséhez vezethet. Módszer: Átfogó irodalmi áttekintés. Eredmények: 1) A vírus által fertozött, sérült endotheliumsejtek felülete az a hely, amellyel érintkezve elindulhat a XII-es véralvadási faktor aktivációja - ez serkenti a prekallikrein/kallikrein/kinin rendszert, és bradikininképzodést okoz. Ez a folyamat megtörténik a súlyos vese- és tüdokárosodást okozó hantavírus-fertozésekben. 2) Idos betegekben az atherosclerosis miatt többszörösen sérült, merev, "stiff" erek endotheliumfelszínein jóval magasabb lehet a XII-es faktor kontakt úton történo aktivációja, mint a fiatal egyének ereiben. Ez a tény egyik oka lehet az idos, cardiovascularis betegek körében tapasztalt magasabb halálozásnak. Következtetés: Az aktivált XII-es véralvadási faktor célzott gátlása újabb gyógyítási lehetoség lehet a SARS-CoV-2-fertozött idos betegekben. Jelenleg már hatásosnak bizonyult a bradikininképzést gátló C1INH-nak, továbbá a bradikininreceptor-gátlóknak az adása is. Orv Hetil. 2020; 161(50): 2099-2103. INTRODUCTION: Bradykinin was implicated in a new complex model of pathomechanism leading to the symptoms of COVID-19 created by a computer-assisted association analysis. According to this model, the decrease in angiotensin-converting enzyme expression leads to impaired bradykinin elimination and subsequent enrichment in the lungs. However, there is no mentioning of the importance of blood coagulation factor XII in increased bradykinin production, in spite of its age-dependent activation and the lower level of C1-esterase inhibitor (C1INH). Activated factor XII may be an important contributor to the "bradykinin storm" in elder cardiovascular patients. METHOD: Literature review. RESULTS: 1) Activation of the coagulation factor XII on the surface of SARS-CoV-2 infected endothelial cells may trigger the prekallikrein/kallikrein/kinin system producing bradykinin. Such process is taking place in hantavirus infections causing severe lung and kidney damages. 2) The endothelial system is dysregulated in elderly patients, resulting in potentially higher factor XII activities on the surface of damaged endothelial cells in the stiffened arteries. This can contribute to the higher mortality rates in the elderly. CONCLUSION: The targeted inhibition of activated blood coagulation factor XII may represent a new therapeutic target for COVID-19, especially for elder patients. Recently, beneficial results have already been observed by the clinical applications of recombinant C1INH and bradykinin receptor antagonists. Orv Hetil. 2020; 161(50): 2099-2103.


Assuntos
Betacoronavirus , Bradicinina , Fator XIIa , Fatores Etários , Idoso , Inibidores da Enzima Conversora de Angiotensina , COVID-19 , Proteína Inibidora do Complemento C1 , Células Endoteliais , Humanos , SARS-CoV-2
11.
J Med Chem ; 63(21): 13159-13186, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33089691

RESUMO

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.


Assuntos
Aminas/química , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator XIIa/metabolismo , Trombina/metabolismo , Aminas/síntese química , Aminas/metabolismo , Anticoagulantes/síntese química , Anticoagulantes/metabolismo , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fator XIIa/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Agregação Plaquetária/efeitos dos fármacos , Relação Estrutura-Atividade , Trombina/antagonistas & inibidores , Triazóis/química
12.
Eur J Med Chem ; 208: 112753, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32883641

RESUMO

Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its in vivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review.


Assuntos
Anticoagulantes/farmacologia , Fator XII/antagonistas & inibidores , Fator XIIa/antagonistas & inibidores , Inibidores de Serino Proteinase/farmacologia , Animais , Anticoagulantes/química , Descoberta de Drogas , Fator XII/química , Fator XIIa/química , Humanos , Inibidores de Serino Proteinase/química
13.
Sci Rep ; 10(1): 14160, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843685

RESUMO

Immediate hypersensitivity reaction (IHR) can be divided into allergic- and non-allergic-mediated, while "anaphylaxis" is reserved for severe IHR. Clinically, true penicillin allergy is rare and most reported penicillin allergy is "spurious". Penicillin-initiated anaphylaxis is possible to occur in skin test- and specific IgE-negative patients. The contact system is a plasma protease cascade initiated by activation of factor XII (FXII). Many agents with negative ion surface can activate FXII to drive contact system. Our data showed that penicillin significantly induced hypothermia in propranolol- or pertussis toxin-pretreated mice. It also caused a rapid and reversible drop in rat blood pressure, which did not overlap with IgE-mediated hypotension. These effects could be countered by a bradykinin-B2 receptor antagonist icatibant, and consistently, penicillin indeed increased rat plasma bradykinin. Moreover, penicillin not only directly activated contact system FXII-dependently, but also promoted bradykinin release in plasma incubated-human umbilical vein endothelial cells. In fact, besides penicillin, other beta-lactams also activated the contact system in vitro. Since the autoactivation of FXII can be affected by multiple-factors, plasma from different healthy individuals showed vastly different amidolytic activity in response to penicillin, suggesting the necessity of determining the potency of penicillin to induce individual plasma FXII activation. These results clarify that penicillin-initiated non-allergic anaphylaxis is attributed to contact system activation, which might bring more effective diagnosis options for predicting penicillin-induced fatal risk and avoiding costly and inappropriate treatment clinically.


Assuntos
Anafilaxia/induzido quimicamente , Coagulação Sanguínea/efeitos dos fármacos , Fator XIIa/metabolismo , Sistema Calicreína-Cinina/efeitos dos fármacos , Penicilina G/toxicidade , Anafilaxia/imunologia , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Bradicinina/fisiologia , Antagonistas dos Receptores da Bradicinina/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipotermia/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Penicilina G/efeitos adversos , Toxina Pertussis/toxicidade , Propranolol/toxicidade , Ratos , Ratos Sprague-Dawley , Receptor B2 da Bradicinina/efeitos dos fármacos , Receptor B2 da Bradicinina/fisiologia , beta-Lactamas/toxicidade
14.
Nat Commun ; 11(1): 3890, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753636

RESUMO

Inhibiting thrombosis without generating bleeding risks is a major challenge in medicine. A promising solution may be the inhibition of coagulation factor XII (FXII), because its knock-out or inhibition in animals reduced thrombosis without causing abnormal bleeding. Herein, we have engineered a macrocyclic peptide inhibitor of activated FXII (FXIIa) with sub-nanomolar activity (Ki = 370 ± 40 pM) and a high stability (t1/2 > 5 days in plasma), allowing for the preclinical evaluation of a first synthetic FXIIa inhibitor. This 1899 Da molecule, termed FXII900, efficiently blocks FXIIa in mice, rabbits, and pigs. We found that it reduces ferric-chloride-induced experimental thrombosis in mice and suppresses blood coagulation in an extracorporeal membrane oxygenation (ECMO) setting in rabbits, all without increasing the bleeding risk. This shows that FXIIa activity is controllable in vivo with a synthetic inhibitor, and that the inhibitor FXII900 is a promising candidate for safe thromboprotection in acute medical conditions.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator XIIa/antagonistas & inibidores , Peptídeos Cíclicos/efeitos dos fármacos , Trombose/prevenção & controle , Animais , Cloretos/efeitos adversos , Clonagem Molecular , Modelos Animais de Doenças , Descoberta de Drogas , Oxigenação por Membrana Extracorpórea/métodos , Fator XII/antagonistas & inibidores , Feminino , Compostos Férricos/efeitos adversos , Humanos , Pulmão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coelhos , Proteínas Recombinantes/farmacologia , Suínos
15.
Front Immunol ; 11: 2014, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32849666

RESUMO

To date the pathophysiology of COVID-19 remains unclear: this represents a factor determining the current lack of effective treatments. In this paper, we hypothesized a complex host response to SARS-CoV-2, with the Contact System (CS) playing a pivotal role in innate immune response. CS is linked with different proteolytic defense systems operating in human vasculature: the Kallikrein-Kinin (KKS), the Coagulation/Fibrinolysis and the Renin-Angiotensin (RAS) Systems. We investigated the role of the mediators involved. CS consists of Factor XII (FXII) and plasma prekallikrein (complexed to high-molecular-weight kininogen-HK). Autoactivation of FXII by contact with SARS-CoV-2 could lead to activation of intrinsic coagulation, with fibrin formation (microthrombosis), and fibrinolysis, resulting in increased D-dimer levels. Activation of kallikrein by activated FXII leads to production of bradykinin (BK) from HK. BK binds to B2-receptors, mediating vascular permeability, vasodilation and edema. B1-receptors, binding the metabolite [des-Arg9]-BK (DABK), are up-regulated during infections and mediate lung inflammatory responses. BK could play a relevant role in COVID-19 as already described for other viral models. Angiotensin-Converting-Enzyme (ACE) 2 displays lung protective effects: it inactivates DABK and converts Angiotensin II (Ang II) into Angiotensin-(1-7) and Angiotensin I into Angiotensin-(1-9). SARS-CoV-2 binds to ACE2 for cell entry, downregulating it: an impaired DABK inactivation could lead to an enhanced activity of B1-receptors, and the accumulation of Ang II, through a negative feedback loop, may result in decreased ACE activity, with consequent increase of BK. Therapies targeting the CS, the KKS and action of BK could be effective for the treatment of COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/fisiopatologia , Fibrinólise/imunologia , Sistema Calicreína-Cinina/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/fisiopatologia , Sistema Renina-Angiotensina/imunologia , Enzima de Conversão de Angiotensina 2 , Bradicinina/metabolismo , COVID-19 , Permeabilidade Capilar , Proteína Inibidora do Complemento C1 , Infecções por Coronavirus/virologia , Fator XIIa/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Cininogênio de Alto Peso Molecular/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Calicreína Plasmática/metabolismo , Pneumonia Viral/virologia , Pré-Calicreína/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , SARS-CoV-2 , Vasodilatação
16.
Cardiovasc Eng Technol ; 11(4): 448-455, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32607901

RESUMO

PURPOSE: Crosslinked poly(vinyl alcohol) (PVA) is a biomaterial that can be used for multiple cardiovascular applications. The success of implanted biomaterials is contingent on the properties of the material. A crucial consideration for blood-contacting devices is their potential to incite thrombus formation, which is dependent on the material surface properties. The goal of this study was to quantify the effect of different crosslinking methods of PVA hydrogels on in vitro thrombogenicity. METHODS: PVA was manufactured using three different crosslinking methods: 30% sodium trimetaphosphate (STMP), three 24 h freeze-thaw cycles (FT), and 2% glutaraldehyde-crosslinked (GA) to produce STMP-PVA, FT-PVA and GA-PVA, respectively. Expanded polytetrafluoroethylene (ePTFE) was used as a clinical control. As markers of thrombus formation, the degree of coagulation factor (F) XII activation, fibrin formation, and platelet adhesion were measured. RESULTS: The GA-PVA material increased FXII activation in the presence of cofactors compared to vehicle and increase platelet adhesion compared to other PVA surfaces. The STMP-PVA and FT-PVA materials had equivalent degrees of FXII activation, fibrin formation and platelet adhesion. CONCLUSION: This work supports crosslinker dependent thrombogenicity of PVA hydrogels and advances our understanding of how the manufacturing of a PVA hydrogel affects its hemocompatibility.


Assuntos
Reagentes para Ligações Cruzadas/química , Congelamento , Glutaral/química , Polifosfatos/química , Álcool de Polivinil/química , Trombose/prevenção & controle , Materiais Biocompatíveis , Coagulação Sanguínea , Prótese Vascular , Reagentes para Ligações Cruzadas/toxicidade , Fator XIIa/metabolismo , Fibrinólise , Congelamento/efeitos adversos , Glutaral/toxicidade , Oclusão de Enxerto Vascular/sangue , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/prevenção & controle , Humanos , Hidrogéis , Teste de Materiais , Adesividade Plaquetária , Polifosfatos/toxicidade , Álcool de Polivinil/toxicidade , Desenho de Prótese , Propriedades de Superfície , Trombose/sangue , Trombose/etiologia
17.
Biomolecules ; 10(7)2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32668719

RESUMO

The limited hemocompatibility of currently used oxygenator membranes prevents long-term use of artificial lungs in patients with lung failure. To improve hemocompatibility, we developed a novel covalent C1-esterase inhibitor (C1-INH) coating. Besides complement inhibition, C1-INH also prevents FXII activation, a very early event of contact phase activation at the crossroads of coagulation and inflammation. Covalently coated heparin, as the current anticoagulation gold standard, served as control. Additionally, a combination of both coatings (C1-INH/heparin) was established. The coatings were tested for their hemocompatibility by dynamic incubation with freshly drawn human whole blood. The analysis of various blood and plasma parameters revealed that C1-INH-containing coatings were able to markedly reduce FXIIa activity compared to heparin coating. Combined C1-INH/heparin coatings yielded similarly low levels of thrombin-antithrombin III complex formation as heparin coating. In particular, adhesion of monocytes and platelets as well as the diminished formation of fibrin networks were observed for combined coatings. We could show for the first time that a covalent coating with complement inhibitor C1-INH was able to ameliorate hemocompatibility. Thus, the early inhibition of the coagulation cascade is likely to have far-reaching consequences for the other cross-reacting plasma protein pathways.


Assuntos
Proteína Inibidora do Complemento C1/farmacologia , Fator XII/efeitos dos fármacos , Fator XIIa/efeitos dos fármacos , Heparina/farmacologia , Anticoagulantes , Antitrombina III/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Adesão Celular/efeitos dos fármacos , Proteína Inibidora do Complemento C1/química , Heparina/química , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Oxigenadores de Membrana , Trombina/metabolismo
18.
Comput Math Methods Med ; 2020: 2852051, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32549905

RESUMO

Human coagulation factor XIIa (FXIIa) is a trypsin-like serine protease that is involved in pathologic thrombosis. As a potential target for designing safe anticoagulants, FXIIa has received a great deal of interest in recent years. In the present study, we employed virtual high-throughput screening of 500,064 compounds within Enamine database to acquire the most potential inhibitors of FXIIa. Subsequently, 18 compounds with significant binding energy (from -65.195 to -15.726 kcal/mol) were selected, and their ADMET properties were predicted to select representative inhibitors. Three compounds (Z1225120358, Z432246974, and Z146790068) exhibited excellent binding affinity and druggability. MD simulation for FXIIa-ligand complexes was carried out to reveal the stability and inhibition mechanism of these three compounds. Through the inhibition of activated factor XIIa assay, we tested the activity of five compounds Z1225120358, Z432246974, Z45287215, Z30974175, and Z146790068, with pIC50 values of 9.3∗10-7, 3.0∗10-5, 7.8∗10-7, 8.7∗10-7, and 1.3∗10-6 M, respectively; the AMDET properties of Z45287215 and Z30974175 show not well but have better inhibition activity. We also found that compounds Z1225120358, Z45287215, Z30974175, and Z146790068 could be more inhibition of FXIIa than Z432246974. Collectively, compounds Z1225120358, Z45287215, Z30974175, and Z146790068 were anticipated to be promising drug candidates for inhibition of FXIIa.


Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Fator XIIa/antagonistas & inibidores , Fator XIIa/química , Sítios de Ligação , Biologia Computacional , Bases de Dados de Produtos Farmacêuticos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Fator XIIa/metabolismo , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Interface Usuário-Computador
19.
Circ Res ; 127(5): 651-663, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32450779

RESUMO

RATIONALE: Epidemiological studies have identified an associate between iron deficiency (ID) and the use of oral contraceptives (CC) and ischemic stroke (IS). To date, however, the underlying mechanism remains poorly understood. Both ID and CC have been demonstrated to upregulate the level and iron-binding ability of Tf (transferrin), with our recent study showing that this upregulation can induce hypercoagulability by potentiating FXIIa/thrombin and blocking antithrombin-coagulation proteases interactions. OBJECTIVE: To investigate whether Tf mediates IS associated with ID or CC and the underlying mechanisms. METHODS AND RESULTS: Tf levels were assayed in the plasma of IS patients with a history of ID anemia, ID anemia patients, venous thromboembolism patients using CC, and ID mice, and in the cerebrospinal fluid of some IS patients. Effects of ID and estrogen administration on Tf expression and coagulability and the underlying mechanisms were studied in vivo and in vitro. High levels of Tf and Tf-thrombin/FXIIa complexes were found in patients and ID mice. Both ID and estrogen upregulated Tf through hypoxia and estrogen response elements located in the Tf gene enhancer and promoter regions, respectively. In addition, ID, administration of exogenous Tf or estrogen, and Tf overexpression promoted platelet-based thrombin generation and hypercoagulability and thus aggravated IS. In contrast, anti-Tf antibodies, Tf knockdown, and peptide inhibitors of Tf-thrombin/FXIIa interaction exerted anti-IS effects in vivo. CONCLUSIONS: Our findings revealed that certain factors (ie, ID and CC) upregulating Tf are risk factors of thromboembolic diseases decipher a previously unrecognized mechanistic association among ID, CC, and IS and provide a novel strategy for the development of anti-IS medicine by interfering with Tf-thrombin/FXIIa interactions.


Assuntos
Anemia Ferropriva/complicações , Coagulação Sanguínea , Anticoncepcionais Orais Hormonais/efeitos adversos , Estrogênios/toxicidade , AVC Isquêmico/etiologia , Trombofilia/etiologia , Transferrina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Ferropriva/sangue , Anemia Ferropriva/diagnóstico , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Linhagem Celular , Modelos Animais de Doenças , Fator XIIa/metabolismo , Feminino , Humanos , AVC Isquêmico/sangue , AVC Isquêmico/diagnóstico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Trombina/metabolismo , Trombofilia/sangue , Trombofilia/diagnóstico , Regulação para Cima , Adulto Jovem
20.
J Thromb Haemost ; 18(6): 1357-1369, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32145147

RESUMO

BACKGROUND: Factor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood. OBJECTIVE: To test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities. METHODS: Recombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain. RESULTS AND CONCLUSIONS: We found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide-induced plasma coagulation. Wild-type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide-induced auto-activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+ -binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.


Assuntos
Fator XII , Sulfoglicoesfingolipídeos , Anexina A4 , Coagulação Sanguínea , Fator XII/metabolismo , Fator XIIa/metabolismo , Humanos
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