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1.
Scand J Trauma Resusc Emerg Med ; 29(1): 57, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33836790

RESUMO

BACKGROUND: Colloid fluids supplemented with adequate combinations of coagulation factor concentrates with the capability to restore coagulation could be a desirable future treatment component in massive transfusion. METHODS: Starting from a coagulation factor and blood cell-free albumin solution we added Prothrombin Complex Concentrate, Fibrinogen Concentrate and Factor XIII in different combinations and concentrations to analyze their properties to restore thromboelastometry parameters without the use of plasma. Further analysis under the presence of platelets was performed for comparability to whole blood conditions. RESULTS: Albumin solutions enriched with Fibrinogen Concentrate, Factor XIII and Prothrombin Complex Concentrate at optimized concentrations show restoring coagulation potential. Prothrombin Complex Concentrate showed sufficient thrombin formation for inducing fibrinogen polymerization. The combination of Prothrombin Complex Concentrate and Fibrinogen Concentrate led to the formation of a stable in vitro fibrin clot. Fibrinogen and Factor XIII showed excellent capacity to improve fibrin clot firmness expressed as Amplitude at 10 min and Maximal Clot Firmness. Fibrinogen alone, or in combination with Factor XIII, was able to restore normal Amplitude at 10 min and Maximal Clot Firmness values. In the presence of platelets, the thromboelastometry surrogate parameter for thrombin generation (Clotting Time) improves and normalizes when compared to whole blood. CONCLUSIONS: Combinations of coagulation factor concentrates suspended in albumin solutions can restore thromboelastometry parameters in the absence of plasma. This kind of artificial colloid fluids with coagulation-restoring characteristics might offer new treatment alternatives for massive transfusion. TRIAL REGISTRATION: Study registered at the institutional ethic committee "Institut de Recerca, Hospital Santa Creu i Sant Pau, with protocol number IIBSP-CFC-2013-165.


Assuntos
Albuminas/metabolismo , Fatores de Coagulação Sanguínea/metabolismo , Fator XIII/metabolismo , Fibrinogênio/metabolismo , Tromboelastografia/métodos , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Transfusão de Sangue , Humanos
2.
Biomolecules ; 11(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669007

RESUMO

Cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin by activated factor XIII (FXIIIa) is essential for the inhibition of fibrinolysis. Little is known about the factors modifying α2-PI incorporation into the fibrin clot and whether the extent of incorporation has clinical consequences. Herein we calculated the extent of α2-PI incorporation by measuring α2-PI antigen levels from plasma and serum obtained after clotting the plasma by thrombin and Ca2+. The modifying effect of FXIII was studied by spiking of FXIII-A-deficient plasma with purified plasma FXIII. Fibrinogen, FXIII, α2-PI incorporation, in vitro clot-lysis, soluble fibroblast activation protein and α2-PI p.Arg6Trp polymorphism were measured from samples of 57 acute ischemic stroke patients obtained before thrombolysis and of 26 healthy controls. Increasing FXIII levels even at levels above the upper limit of normal increased α2-PI incorporation into the fibrin clot. α2-PI incorporation of controls and patients with good outcomes did not differ significantly (49.4 ± 4.6% vs. 47.4 ± 6.7%, p = 1.000), however it was significantly lower in patients suffering post-lysis intracranial hemorrhage (37.3 ± 14.0%, p = 0.004). In conclusion, increased FXIII levels resulted in elevated incorporation of α2-PI into fibrin clots. In stroke patients undergoing intravenous thrombolysis treatment, α2-PI incorporation shows an association with the outcome of therapy, particularly with thrombolysis-associated intracranial hemorrhage.


Assuntos
Antifibrinolíticos/uso terapêutico , AVC Isquêmico/tratamento farmacológico , Coagulação Sanguínea/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Fator XIII/metabolismo , Fibrinogênio/metabolismo , Fibrinólise/efeitos dos fármacos , Humanos , AVC Isquêmico/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico
3.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540604

RESUMO

Diseases such as myocardial infarction, ischaemic stroke, peripheral vascular disease and venous thromboembolism are major contributors to morbidity and mortality. Procoagulant, anticoagulant and fibrinolytic pathways are finely regulated in healthy individuals and dysregulated procoagulant, anticoagulant and fibrinolytic pathways lead to arterial and venous thrombosis. In this review article, we discuss the (patho)physiological role and laboratory assessment of fibrin, factor XIII and endogenous fibrinolysis, which are key players in the terminal phase of the coagulation cascade and fibrinolysis. Finally, we present the most up-to-date evidence for their involvement in various disease states and assessment of cardiovascular risk.


Assuntos
Fator XIII/fisiologia , Fibrina/fisiologia , Trombose/fisiopatologia , Fator XIII/análise , Fator XIII/metabolismo , Fibrina/análise , Fibrina/metabolismo , Fibrinólise , Humanos , Trombose/sangue , Trombose/metabolismo , Trombose Venosa/sangue , Trombose Venosa/metabolismo , Trombose Venosa/fisiopatologia
4.
Int J Mol Sci ; 22(4)2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562624

RESUMO

Coagulation factor XIII (FXIII) is converted by thrombin into its active form, FXIIIa, which crosslinks fibrin fibers, rendering clots more stable and resistant to degradation. FXIII affects fibrin clot structure and function leading to a more prothrombotic phenotype with denser networks, characterizing patients at risk of venous thromboembolism (VTE). Mechanisms regulating FXIII activation and its impact on fibrin structure in patients with acute VTE encompassing pulmonary embolism (PE) or deep vein thrombosis (DVT) are poorly elucidated. Reduced circulating FXIII levels in acute PE were reported over 20 years ago. Similar observations indicating decreased FXIII plasma activity and antigen levels have been made in acute PE and DVT with their subsequent increase after several weeks since the index event. Plasma fibrin clot proteome analysis confirms that clot-bound FXIII amounts associated with plasma FXIII activity are decreased in acute VTE. Reduced FXIII activity has been associated with impaired clot permeability and hypofibrinolysis in acute PE. The current review presents available studies on the role of FXIII in the modulation of fibrin clot properties during acute PE or DVT and following these events. Better understanding of FXIII's involvement in the pathophysiology of acute VTE might help to improve current therapeutic strategies in patients with acute VTE.


Assuntos
Fator XIII/metabolismo , Fibrina/metabolismo , Tromboembolia Venosa/sangue , Doença Aguda , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/genética , Coagulação Sanguínea/fisiologia , Fator XIII/química , Fator XIII/genética , Fibrina/química , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Fibrinólise/fisiologia , Variação Genética , Humanos , Modelos Cardiovasculares , Tromboembolia Venosa/genética
5.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498248

RESUMO

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.


Assuntos
Automação Laboratorial/métodos , Testes de Coagulação Sanguínea/métodos , Carbono-Nitrogênio Liases/metabolismo , Deficiência do Fator XIII/diagnóstico , Fator XIII/análise , Corantes Fluorescentes/normas , Automação Laboratorial/normas , Bilirrubina/metabolismo , Testes de Coagulação Sanguínea/normas , Compostos Cromogênicos/normas , Fator XIII/metabolismo , Deficiência do Fator XIII/sangue , Fluorometria/métodos , Fluorometria/normas , Hemólise , Humanos , Testes Imunológicos/métodos , Testes Imunológicos/normas , Reprodutibilidade dos Testes , Transglutaminases/metabolismo
6.
Hematology Am Soc Hematol Educ Program ; 2020(1): 67-75, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33275705

RESUMO

Viscoelastic assays (VEAs) that include thromboelastography and rotational thromboelastometry add value to the investigation of coagulopathies and goal-directed management of bleeding by providing a complete picture of clot formation, strength, and lysis in whole blood that includes the contribution of platelets, fibrinogen, and coagulation factors. Conventional coagulation assays have several limitations, such as their lack of correlation with bleeding and hypercoagulability; their inability to reflect the contribution of platelets, factor XIII, and plasmin during clot formation and lysis; and their slow turnaround times. VEA-guided transfusion algorithms may reduce allogeneic blood exposure during and after cardiac surgery and in the emergency management of trauma-induced coagulopathy and hemorrhage. However, the popularity of VEAs for other indications is driven largely by extrapolation of evidence from cardiac surgery, by the drawbacks of conventional coagulation assays, and by institution-specific preferences. Robust diagnostic studies validating and standardizing diagnostic cutoffs for VEA parameters and randomized trials comparing VEA-guided algorithms with standard care on clinical outcomes are urgently needed. Lack of such studies represents the biggest barrier to defining the role and impact of VEA in clinical care.


Assuntos
Algoritmos , Transfusão de Sangue , Procedimentos Cirúrgicos Cardíacos , Hemorragia , Tromboelastografia , Trombofilia , Plaquetas/metabolismo , Fator XIII/metabolismo , Fibrinolisina/metabolismo , Hemorragia/sangue , Hemorragia/diagnóstico , Hemorragia/terapia , Humanos , Trombofilia/sangue , Trombofilia/diagnóstico , Trombofilia/terapia
7.
ACS Appl Mater Interfaces ; 12(50): 55574-55583, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33284021

RESUMO

The development of novel hemostatic agents with distinct modes of action from traditional ones remains a formidable challenge. Self-assembling peptide hydrogels have emerged as a new hemostatic material, not only because of their inherent biocompatibility and biodegradability but also their designability. Especially, rational molecular design can make peptides and their hydrogelation responsive to biological cues. In this study, we demonstrated that transglutaminase-catalyzed reactions not only occurred among designed short peptide I3QGK molecules but also between the peptide and a natural polysaccharide O-carboxymethyl chitosan. Because Factor XIII in the blood can rapidly convert into activated transglutaminase (Factor XIIIa) upon bleeding, these enzymatic reactions, together with the electrostatic attraction between the two hemostatic agents, induced a strong synergetic effect in promoting hydrogelation, blood coagulation, and platelet adhesion, eventually leading to rapid hemostasis. The study presents a promising strategy for developing alternative hemostatic materials and methods.


Assuntos
Materiais Biocompatíveis/química , Quitosana/análogos & derivados , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Materiais Biocompatíveis/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Galinhas , Quitosana/química , Quitosana/metabolismo , Fator XIII/metabolismo , Feminino , Hemólise/efeitos dos fármacos , Humanos , Hidrogéis/química , Masculino , Camundongos , Peptídeos/química , Agregação Plaquetária/efeitos dos fármacos , Reologia
8.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182600

RESUMO

Hemostasis disorder in patients with end-stage renal disease (ESRD) is frequently associated with bleeding diathesis but it may also manifest in thrombotic complications. Analysis of individual coagulation and fibrinolytic factors may shed light on the background of this paradox situation. Here we explored components essential for fibrin formation/stabilization in ESRD patients being on maintenance hemodiafiltration (HDF) or hemodialysis (HD). Pre-dialysis fibrinogen, factor XIII (FXIII) antigen concentrations and FXIII activity were elevated, while α2-plasmin inhibitor (α2PI) activity decreased. The inflammatory status, as characterized by C-reactive protein (CRP) was a key determinant of fibrinogen concentration, but not of FXIII and α2PI levels. During a 4-h course of HDF or HD, fibrinogen concentration and FXIII levels gradually elevated. When compensated for the change in plasma water, i.e., normalized for plasma albumin concentration, only FXIII elevation remained significant. There was no difference between HDF and HD treatments. Individual HDF treatment did not influence α2PI activity, however after normalization it decreased significantly. HD treatment had a different effect, α2PI activities became elevated but the elevation disappeared after normalization. Elevated fibrinogen and FXIII levels in ESRD patients might contribute to the increased thrombosis risk, while decreased α2PI activity might be associated with elevated fibrinolytic potential.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Adolescente , Adulto , Idoso , Coagulação Sanguínea , Proteína C-Reativa/metabolismo , Fator XIII/metabolismo , Feminino , Fibrinogênio/metabolismo , Fibrinólise , Hemodiafiltração , Hemorragia/sangue , Hemorragia/etiologia , Humanos , Falência Renal Crônica/congênito , Masculino , Pessoa de Meia-Idade , Diálise Renal , Fatores de Risco , Trombose/sangue , Trombose/etiologia , Adulto Jovem , alfa 2-Antiplasmina/metabolismo
9.
Sci Rep ; 10(1): 20116, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208779

RESUMO

The adhesion of blood clots to wounds is necessary to seal injured vasculature and achieve hemostasis. However, it has not been specifically tested if adhesive failure of clots is a major contributor to rebleeding and what mechanisms prevent clot delamination. Here, we quantified the contribution of adhesive and cohesive failure to rebleeding in a rat model of femoral artery injury, and identified mechanisms that contribute to the adhesive strength of bulk clots in a lap-shear test in vitro. In the rat bleeding model, the frequency of clot failures correlated positively with blood loss (R = 0.81, p = 0.014) and negatively with survival time (R = - 0.89, p = 0.0030), with adhesive failures accounting for 51 ± 14% of rebleeds. In vitro, adhesion depended on fibrinogen and coagulation factor XIII (FXIII), and supraphysiological FXIII improved adhesive strength. Furthermore, when exogenous FXIII was topically applied into the wound pocket of rats, eleven adhesive failures occurred between eight rats, compared to seventeen adhesive failures between eight untreated rats, whereas the number of cohesive failures remained the same at sixteen in both groups. In conclusion, rebleeding from both adhesive and cohesive failure of clots decreases survival from hemorrhage in vivo. Both endogenous and exogenous FXIII improves the adhesive strength of clots.


Assuntos
Fator XIII/metabolismo , Hemostasia/fisiologia , Trombose/patologia , Administração Tópica , Animais , Plaquetas/citologia , Eritrócitos/citologia , Fator XIII/administração & dosagem , Fator XIII/farmacologia , Artéria Femoral/lesões , Fibrinogênio/metabolismo , Hemorragia/sangue , Hemorragia/mortalidade , Hemorragia/patologia , Hemostasia/efeitos dos fármacos , Humanos , Masculino , Ratos Sprague-Dawley , Ferimentos e Lesões/patologia
10.
J Physiol Pharmacol ; 71(4)2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33214340

RESUMO

Fibrin cross-linking by coagulation factor (F)XIII leads to clot stabilization. Reduced plasma FXIII levels have been reported in acute pulmonary embolism (PE) patients. We investigated the impact of anticoagulant therapy on clot-bound amounts of FXIII and α2-antiplasmin and their associations with fibrin clot properties in patients with PE. Clots generated from plasma of 18 acute symptomatic patients on admission and after a 3-month treatment with rivaroxaban were assessed off anticoagulation using mass spectrometry. Plasma FXIII and α2-antiplasmin activity were determined at the 2 time points along with thrombin generation markers, plasma fibrin clot permeability (Ks), and clot lysis time (CLT). Following anticoagulant therapy, clot-bound FXIII increased from 2.97 (interquartile range, 1.98 - 4.08) to 4.66 (3.5 - 6.9) mg/g protein and α2-antiplasmin from 9.4 (7.2 - 10.6) to 11 (9.5 - 14) mg/g protein (both p < 0.0001). The two parameters showed positive correlation at baseline only (r = 0.63, p = 0.0056). Similarly to clot-bound amounts, plasma FXIII (+25.8%) and α2-antiplasmin activity (+12%) increased at 3 months. Plasma FXIII activity on admission, but not after 3 months since the index PE, was associated with amounts of clot-bound FXIII (r = 0.35, p = 0.043) and α2-antiplasmin (r = 0.47, p = 0.048). At baseline, clot-bound FXIII correlated with plasma F1+2 prothrombin fragments levels (r = 0.51, p = 0.03), while clot-bound α2-antiplasmin correlated with CLT (r = 0.43, p = 0.036). At 3 months associations of clot-bound FXIII and α2-antiplasmin were abolished. This study assessed for the first time changes in the fibrin clot composition following acute PE, suggesting an increase of clot-bound and plasma FXIII and α2-antiplasmin levels after 3 months.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fator XIII/metabolismo , Inibidores do Fator Xa/uso terapêutico , Fibrina/metabolismo , Embolia Pulmonar/tratamento farmacológico , Rivaroxabana/uso terapêutico , alfa 2-Antiplasmina/metabolismo , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Embolia Pulmonar/sangue , Embolia Pulmonar/diagnóstico , Fatores de Tempo , Resultado do Tratamento
11.
Hamostaseologie ; 40(4): 467-471, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32869231

RESUMO

Laboratory diagnosis of congenital and acquired deficiencies of coagulation factor XIII (FXIII) can be challenging. Determination of FXIII function requires specific and sensitive assays which are not always available. This brief review article summarizes currently used FXIII assay methods, their principles and difficulties, and discusses the recommended diagnostic workup in case of a suspected FXIII deficiency. The article also briefly touches on experimental methods used in FXIII research.


Assuntos
Testes de Coagulação Sanguínea/métodos , Fator XIII/metabolismo , Laboratórios/normas , Humanos
13.
Blood ; 136(25): 2946-2954, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-32678423

RESUMO

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity.


Assuntos
Plaquetas/metabolismo , Deficiência do Fator XIII , Fator XIII/metabolismo , Fator XIIIa/metabolismo , Hemorragia/sangue , Animais , Fator XIII/genética , Deficiência do Fator XIII/sangue , Deficiência do Fator XIII/induzido quimicamente , Deficiência do Fator XIII/genética , Fator XIIIa/genética , Técnicas de Silenciamento de Genes , Hemorragia/genética , Camundongos , Camundongos Knockout , Nanopartículas , RNA Interferente Pequeno , Coelhos
14.
Biomolecules ; 10(6)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32560304

RESUMO

The blood coagulation factor XIII (FXIII) plays a critical role in supporting coagulation and fibrinolysis due to both the covalent crosslinking of fibrin polymers, rendering them resistant to plasmin lysis, and the crosslinking of fibrin to proteins of the fibrinolytic system. The hypochlorite-mediated oxidation of the blood coagulation factor XIII (FXIII) at the different stages of its enzymatic activation is studied for the first time in this paper. The consolidated results obtained with the aid of MS/MS, electrophoresis, and colorimetry demonstrate that in the process of FXIII's conversion into FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased as follows: native FXIII < FXIII + Ca2+ << FXIII + Ca2+/thrombin. The modification sites were detected among all the structural regions of the catalytic FXIII-A subunit, except for the activation peptide, and embraced several sushi domains of the FXIII-B subunit. Oxidized amino acid residues belonging to FXIII-A are surface-exposed residues and can perform an antioxidant role. The regulatory FXIII-B subunits additionally contribute to the antioxidant defense of the catalytic center of the FXIII-A subunits. Taken together, the present data along with the data from previous studies demonstrate that the FXIII proenzyme structure is adapted to oxidation.


Assuntos
Fator XIII/metabolismo , Coagulação Sanguínea , Fator XIII/química , Fator XIII/isolamento & purificação , Feminino , Fibrinogênio/química , Fibrinogênio/isolamento & purificação , Fibrinogênio/metabolismo , Humanos , Masculino , Oxirredução
15.
Vasc Health Risk Manag ; 16: 103-110, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32280233

RESUMO

Background: The aim was to evaluate factor XIII activity (FXIIIa) and monocyte-derived microparticles (MDMPs) in cancer patients. Methods: In total, 138 cancer patients (31 malignant lymphomas, 39 multiple myelomas, and 68 lung cancers) were analyzed. We measured various biomarkers including FXIIIa and MDMPs. Results: The values of endothelial activation markers, monocyte chemoattractant peptide (MCP)-1, soluble (s)CD14, and MDMPs were higher in cancer patients than in non-cancerous controls. MCP-1, sCD14, and MDMPs were significantly correlated with FXIIIa in multivariate analysis in cancer patients. In addition, MCP-1, sCD14, and MDMP levels were significantly increased in the high FXIIIa group of patients. Finally, the survival rate of the high FXIIIa group was significantly poor in the Kaplan-Meier analysis. Conclusion: These results suggest that abnormal levels of FXIIIa and MDMPs may offer promise as poor prognostic factors in cancer patients.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Fator XIII/metabolismo , Monócitos/metabolismo , Neoplasias/sangue , Trombose/sangue , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Neoplasias/diagnóstico , Trombose/diagnóstico , Trombose/etiologia
16.
ChemMedChem ; 15(10): 900-905, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32181986

RESUMO

Blood coagulation factor XIII (FXIII, F13) is considered to be a promising target for anticoagulants with reduced bleeding risk because of its unique position in the coagulation cascade downstream of thrombin. However, until now, no potent drug addressing FXIII has been available, indeed no compound has even entered clinical trials yet. In 2013, we published the co-crystal structure of FXIII in the active state (FXIIIa°), thereby providing a detailed map of the active site for the rational design of potent FXIIIa blockers. Here we report, for the first time, a structure-based approach to improving the affinity of FXIIIa inhibitors. FXIII was crystallized in complex with a methyl thiazole moiety to address a novel transient hydrophobic pocket close to the catalytic center. By subsequent structure-based design to rationalize the introduction of an ethyl ester, the potency of the inhibitor was improved significantly compared to that of the parent lead compound. The occupancy of the hydrophobic pocket described here might turn out to be a key step in the development of a potent reversible and orally available FXIIIa blocker.


Assuntos
Inibidores Enzimáticos/farmacologia , Fator XIII/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Fator XIII/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Molecular
17.
J Thromb Haemost ; 18(6): 1302-1309, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32168410

RESUMO

BACKGROUND: The protective/inhibitory B subunits of coagulation factor XIII (FXIII-B) is a ~80 kDa glycoprotein containing two N-glycosylation sites. Neither the structure nor the functional role of the glycans on FXIII-B has been explored. OBJECTIVE: To reveal the glycan structures linked to FXIII-B, to design a method for deglycosylating the native protein, to find out if deglycosylation influences the dimeric structure of FXIII-B and its clearance from the circulation. METHODS: Asparagine-linked carbohydrates were released from human FXIIII-B by PNGase F digestion. The released N-linked oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis. Structural identification utilized glycan database search and exoglycosidase digestion based sequencing. The structure of deglycosylated FXIII-B was investigated by gel filtration. The clearance of deglycosylated and native FXIII-B from plasma was compared in FXIII-B knock out mice. RESULTS: PNGase F completely removed N-glycans from the denatured protein. Deglycosylation of the native protein was achieved by repeated digestion at elevated PNGase F concentration. The total N-glycan profile of FXIII-B featured nine individual structures; three were fucosylated and each structure contained at least one sialic acid. Deglycosylation did not change the native dimeric structure of FXIII-B, but accelerated its clearance from the circulation of FXIII-B knock out mice. CONCLUSION: Characterization of the glycan moieties attached to FXIII-B is reported for the first time. Complete deglycosylation of the native protein was achieved by a deglycosylation workflow. The associated glycan structure is not required for FXIII-B dimer formation, but it very likely prolongs the half-life of FXIII-B in the plasma.


Assuntos
Coagulação Sanguínea , Fator XIII , Testes de Coagulação Sanguínea , Fator XIII/genética , Fator XIII/metabolismo , Glicosídeo Hidrolases , Glicosilação
18.
Thromb Haemost ; 120(3): 392-399, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016928

RESUMO

OBJECTIVE: Craniosynostosis surgery in small children is very often associated with a high blood loss. Tranexamic acid (TXA) reduces blood loss during this procedure, although the potential underlying coagulopathy in these children is not known in detail. Objective was to determine the nature of any coagulopathy found during and after craniosynostosis surgery and to characterize the effect of TXA on fibrin clot formation, clot strength, and fibrinolysis. MATERIALS AND METHODS: Thirty children received either TXA (bolus dose of 10 mg/kg followed by 8 hours continuous infusion of 3 mg/kg/h) or placebo. Dynamic whole blood clot formation assessed by thromboelastometry, platelet count, dynamic thrombin generation/thrombin-antithrombin, clot lysis assay, and fibrinogen/factor XIII (FXIII) levels were measured. Additionally, clot structure was investigated by real-time live confocal microscopy and topical data analysis. RESULTS: Increased ability of thrombin generation was observed together with a tendency toward shortened activated partial thromboplastin time and clotting time. Postoperative maximum clot firmness was higher among children receiving TXA. FXIII decreased significantly during surgery in both groups.Resistance toward tissue plasminogen activator-induced fibrinolysis was higher in children that received TXA, as evidenced by topical data analysis and by a significant longer lysis time. Fibrinogen levels were higher in the TXA group at 24 hours. CONCLUSION: A significant coagulopathy mainly characterized by changes in clot stability and not parameters of thrombin generation was reported. Tranexamic acid improved clot strength and reduced fibrinolysis, thereby avoiding reduction in fibrinogen levels.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Craniossinostoses/cirurgia , Ácido Tranexâmico/uso terapêutico , Criança , Pré-Escolar , Fator XIII/metabolismo , Feminino , Tempo de Lise do Coágulo de Fibrina , Fibrinólise , Hemoglobinas , Hemorragia/sangue , Humanos , Lactente , Coeficiente Internacional Normatizado , Masculino , Microscopia Confocal , Tempo de Tromboplastina Parcial , Contagem de Plaquetas , Trombina/metabolismo , Trombose , Ativador de Plasminogênio Tecidual/uso terapêutico
19.
J Invest Dermatol ; 140(3): 624-635.e7, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31493396

RESUMO

Dermatofibromas are common benign skin lesions, the etiology of which is poorly understood. We identified two unrelated pedigrees in which there was autosomal dominant transmission of multiple dermatofibromas. Whole exome sequencing revealed a rare shared heterozygous missense variant in the F13A1 gene encoding factor XIII subunit A (FXIII-A), a transglutaminase involved in hemostasis, wound healing, tumor growth, and apoptosis. The variant (p.Lys679Met) has an allele frequency of 0.0002 and is predicted to be a damaging mutation. Recombinant human Lys679Met FXIII-A demonstrated reduced fibrin crosslinking activity in vitro. Of note, the treatment of fibroblasts with media containing Lys679Met FXIII-A led to enhanced adhesion, proliferation, and type I collagen synthesis. Immunostaining revealed co-localization between FXIII-A and α4ß1 integrins, more prominently for Lys679Met FXIII-A than the wild type. In addition, both the α4ß1 inhibitors and the mutation of the FXIII-A Isoleucine-Leucine-Aspartate-Threonine (ILDT) motif prevented Lys679Met FXIII-A-dependent proliferation and collagen synthesis of fibroblasts. Our data suggest that the Lys679Met mutation may lead to a conformational change in the FXIII-A protein that enhances α4-integrin binding and provides insight into an unexpected role for FXIII-A in the pathobiology of familial dermatofibroma.


Assuntos
Fator XIII/genética , Fibrina/metabolismo , Histiocitoma Fibroso Benigno/genética , Padrões de Herança , Pele/patologia , Domínio Catalítico/genética , Proliferação de Células/genética , Colágeno Tipo I/biossíntese , Análise Mutacional de DNA , Fator XIII/metabolismo , Feminino , Fibroblastos , Células HEK293 , Histiocitoma Fibroso Benigno/patologia , Humanos , Integrina alfa4/metabolismo , Masculino , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Linhagem , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/citologia , Relação Estrutura-Atividade , Sequenciamento Completo do Exoma
20.
Int J Mol Sci ; 20(23)2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783511

RESUMO

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.


Assuntos
Ceratócitos da Córnea/metabolismo , Substância Própria/metabolismo , Fator XIII/metabolismo , Transglutaminases/metabolismo , Testes de Coagulação Sanguínea/métodos , Lesões da Córnea/metabolismo , Humanos , RNA Mensageiro/metabolismo , Cicatrização/fisiologia
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