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1.
Mol Biol Rep ; 49(1): 205-215, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34709571

RESUMO

BACKGROUND: Diabetic nephropathy (DN), is microvascular complication of diabetes causes to kidney dysfunction and renal fibrosis. It is known that hyperglycemia and advanced glycation end products (AGEs) produced by hyperglycemic condition induce myofibroblast differentiation and endothelial-to-mesenchymal transition (EndoMT), and exacerbate fibrosis in DN. Recently, we demonstrated that α2-antiplasmin (α2AP) is associated with inflammatory response and fibrosis progression. METHODS: We investigated the role of α2AP on fibrosis progression in DN using a streptozotocin-induced DN mouse model. RESULTS: α2AP deficiency attenuated EndoMT and fibrosis progression in DN model mice. We also showed that the high glucose condition/AGEs induced α2AP production in fibroblasts (FBs), and the reduction of receptor for AGEs (RAGE) by siRNA attenuated the AGEs-induced α2AP production in FBs. Furthermore, the bloackade of α2AP by the neutralizing antibody attenuated the high glucose condition-induced pro-fibrotic changes in FBs. On the other hand, the hyperglycemic condition/AGEs induced EndoMT in vascular endothelial cells (ECs), the FBs/ECs co-culture promoted the high glucose condition-induced EndoMT compared to ECs mono-culture. Furthermore, α2AP promoted the AGEs-induced EndoMT, and the blockade of α2AP attenuated the FBs/ECs co-culture-promoted EndoMT under the high glucose condition. CONCLUSIONS: The high glucose conditions induced α2AP production, and α2AP is associated with EndoMT and fibrosis progression in DN. These findings provide a basis for clinical strategies to improve DN.


Assuntos
Anticorpos Neutralizantes/farmacologia , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/patologia , Glucose/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , alfa 2-Antiplasmina/genética , Animais , Linhagem Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Células NIH 3T3
2.
Int J Mol Sci ; 22(22)2021 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-34830419

RESUMO

Thrombus formation remains a major cause of morbidity and mortality worldwide. Current antiplatelet and anticoagulant therapies have been effective at reducing vascular events, but at the expense of increased bleeding risk. Targeting proteins that interact with fibrinogen and which are involved in hypofibrinolysis represents a more specific approach for the development of effective and safe therapeutic agents. The antifibrinolytic proteins alpha-2 antiplasmin (α2AP), thrombin activatable fibrinolysis inhibitor (TAFI), complement C3 and plasminogen activator inhibitor-2 (PAI-2), can be incorporated into the fibrin clot by FXIIIa and affect fibrinolysis by different mechanisms. Therefore, these antifibrinolytic proteins are attractive targets for the development of novel therapeutics, both for the modulation of thrombosis risk, but also for potentially improving clot instability in bleeding disorders. This review summarises the main properties of fibrinogen-bound antifibrinolytic proteins, their effect on clot lysis and association with thrombotic or bleeding conditions. The role of these proteins in therapeutic strategies targeting the fibrinolytic system for thrombotic diseases or bleeding disorders is also discussed.


Assuntos
Carboxipeptidase B2/genética , Fibrinogênio/genética , Hemorragia/terapia , alfa 2-Antiplasmina/genética , Anticoagulantes , Complemento C3/genética , Fibrinólise/genética , Hemorragia/genética , Humanos , Inibidor 2 de Ativador de Plasminogênio/genética , Trombose/genética
3.
Mol Biol Rep ; 48(4): 3431-3437, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33913094

RESUMO

Systemic sclerosis (SSc) is characterized by peripheral circulatory disturbance and fibrosis in skin and visceral organs. We recently demonstrated that α2-antiplasmin (α2AP) is elevated in SSc dermal fibroblasts and SSc model mice, and is associated with fibrosis progression and vascular dysfunction. In the present study, we predicted that α2AP could be a target of microRNA-30c (miR-30c) using TargetScan online database, and investigated the effect of miR-30c on the pathogenesis of SSc using a bleomycin-induced SSc model mice. miR-30c attenuated α2AP expression, and prevented the pro-fibrotic changes (increased dermal thickness, collagen deposition, myofibroblast accmulation) and the vascular dysfunction (the reduction of vascular endothelial cells (ECs) and blood flow) in the skin of SSc model mice. Furthermore, miR-30c suppressed pulmonary fibrosis progression in the SSc model mice. miR-30c exerts the anti-fibrotic and anti-angiopathy effects on SSc model mice, and might provide a basis for clinical strategies for SSc.


Assuntos
Escleroderma Sistêmico/metabolismo , Pele/irrigação sanguínea , alfa 2-Antiplasmina/genética , Animais , Bleomicina/toxicidade , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miofibroblastos , Escleroderma Sistêmico/genética , Pele/efeitos dos fármacos , Pele/metabolismo
4.
Int J Mol Sci ; 21(24)2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33317138

RESUMO

OBJECTIVE: Coagulation and fibrinolysis are interrelated with the expression of vascular endothelial growth factor (VEGF), which frequently is increased in axial spondyloarthritis (axSpA). We tested whether (i) α2-antiplasmin (A2AP) Arg6Trp, (ii) fibrinogen, factor XIII A-subunit or B-subunit genotypes are associated with VEGF levels and assessed whether the known association between elevated VEGF and radiographic spinal progression in axSpA depends on genetic background. METHODS: One hundred and eighty-six axSpA patients from the German Spondyloarthritis Inception Cohort were genotyped, characterized for VEGF levels, and statistically analyzed. The association between VEGF and radiographic spinal progression was assessed in dependence on genetic background in stratified analyses. RESULTS: A2AP 6Trp carriage was associated with VEGF elevation (OR: 2.37, 95% CI: 1.06-5.29) and VEGF levels (6Trp, 455 ± 334 pg/mL; 6Arg/Arg, 373 ± 293 pg/mL; p < 0.008). Association between elevated VEGF and radiographic spinal progression in axSpA (OR: 3.11, 95% CI: 1.02-8.82) depended remarkably on the fibrinogen (FGA) genotype. When considering axSpA patients with elevated VEGF, in FGA rs6050A>G wild types, 42.1% of patients (8 of 19) progressed, while in G-allele carriers, no radiographic progression happened (0 of 13) (p < 0.04). CONCLUSIONS: The A2AP Arg6Trp genotype seems to influence VEGF levels in axSpA. The predictive value of VEGF elevations in respect of radiographic spinal progression in axSpA depends on FGA genotypes.


Assuntos
Fibrinogênio/genética , Genótipo , Espondilite Anquilosante/genética , Fator A de Crescimento do Endotélio Vascular/genética , alfa 2-Antiplasmina/genética , Adulto , Fator VIII/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espondilite Anquilosante/diagnóstico por imagem , Espondilite Anquilosante/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Blood ; 134(12): 970-978, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31395599

RESUMO

Stasis of venous blood triggers deep vein thrombosis by activating coagulation, yet its effects on the fibrinolytic system are not fully understood. We examined the relationship between stasis, fibrinolysis, and the development of experimental venous thrombosis. Effects of stasis-induced deep vein thrombosis and fibrinolysis on thrombosis were examined by inferior vena cava ligation in congenic mice with and without α2-antiplasmin (α2AP), the primary inhibitor of plasmin. Venous thrombus weights were measured and thrombus composition was determined by Martius scarlet blue and immunofluorescence staining. Venous thrombi from α2AP+/+ mice contained plasminogen activators, plasminogen activator inhibitor-1, plasminogen, and α2AP, which changed with thrombus age. Normal, α2AP+/+ mice developed large, occlusive thrombi within 5 hours after ligation; thrombi were even larger in plasminogen-deficient mice (P < .001). No significant thrombus formation was seen in α2AP-/- mice (P < .0001) or in α2AP+/+ mice treated with an α2AP-inactivating antibody (P < .001). Venous stasis activated fibrinolysis, measured by D-dimer levels, in α2AP-/- mice vs α2AP+/+ mice (P < .05). Inhibition of fibrinolysis by the indirect plasmin inhibitor ε-aminocaproic acid or by α2AP restored thrombosis in α2AP-/- mice. In addition to its effects on acute thrombosis, thrombus formation was also markedly suppressed in α2AP-/- mice vs α2AP+/+ mice (P < .0001) 1, 7, and 14 days after ligation. We conclude that experimental venous stasis activates the fibrinolytic system to block the development of venous thrombosis. Suppression of fibrinolysis by α2AP appears essential for stasis-induced thrombus development, which suggests that targeting α2AP may prove useful for preventing venous thrombosis.


Assuntos
Fibrinólise/fisiologia , Síndrome Pós-Trombótica/complicações , Trombose Venosa/prevenção & controle , alfa 2-Antiplasmina/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Fibrinólise/genética , Ligadura , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome Pós-Trombótica/sangue , Síndrome Pós-Trombótica/genética , Síndrome Pós-Trombótica/fisiopatologia , Veia Cava Inferior/patologia , Veia Cava Inferior/cirurgia , Trombose Venosa/sangue , Trombose Venosa/genética , Trombose Venosa/fisiopatologia , alfa 2-Antiplasmina/genética
7.
Transfus Apher Sci ; 58(5): 572-577, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31427261

RESUMO

Deficiencies or excessive activation of the fibrinolytic system can result in severe, lifelong bleeding disorders. The most severe clinical phenotype is caused by α2-Antiplasmin (α2-AP) deficiency which results in excess fibrinolysis due to the inability to inhibit plasmin. Another bleeding disorder due to a defect in the fibrinolytic pathway results from Plasminogen activator inhibitor-1 (PAI-1) deficiency causing enhanced fibrinolysis due to the decreased inhibition of plasminogen activators resulting in increased conversion of plasminogen to plasmin. Both these disorders are rare and have an autosomal recessive pattern of inheritance. They can remain undetected as routine coagulation and platelet function tests are normal. A unique gain-of-function defect in fibrinolysis causes the Quebec platelet disorder (QPD) which is characterized by profibrinolytic platelets containing increased urokinase-type plasminogen activator (uPA) in the α-granules. A high index of suspicion based on clinical phenotype along with the availability of specialized hemostasis testing is required for timely and accurate diagnosis. Antifibrinolytic agents, such as tranexamic acid or ε-aminocaproic acid, are the mainstays of treatment which inhibit fibrinolysis by preventing the binding of plasminogen to fibrin and thereby stabilizing the fibrin clot. The purpose of this review is to summarize the pathogenesis, clinical phenotype, approaches to diagnosis and treatment for these three major disorders of fibrinolysis.


Assuntos
Antifibrinolíticos/uso terapêutico , Deficiência do Fator V , Fibrinólise/genética , Transtornos Hemorrágicos , Inibidor 1 de Ativador de Plasminogênio/deficiência , Ácido Tranexâmico/uso terapêutico , alfa 2-Antiplasmina/deficiência , Plaquetas/metabolismo , Plaquetas/patologia , Deficiência do Fator V/tratamento farmacológico , Deficiência do Fator V/genética , Deficiência do Fator V/metabolismo , Deficiência do Fator V/patologia , Transtornos Hemorrágicos/sangue , Transtornos Hemorrágicos/tratamento farmacológico , Transtornos Hemorrágicos/genética , Transtornos Hemorrágicos/metabolismo , Transtornos Hemorrágicos/patologia , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , alfa 2-Antiplasmina/efeitos dos fármacos , alfa 2-Antiplasmina/genética
9.
PLoS One ; 13(5): e0196911, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29723259

RESUMO

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples.


Assuntos
Anticorpos Monoclonais/química , Ensaio de Imunoadsorção Enzimática/normas , alfa 2-Antiplasmina/análise , alfa 2-Antiplasmina/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Arginina/genética , Arginina/imunologia , Clonagem Molecular , Drosophila/citologia , Ensaio de Imunoadsorção Enzimática/métodos , Fibrinólise/efeitos dos fármacos , Expressão Gênica , Humanos , Hibridomas/química , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Proteólise , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Triptofano/genética , Triptofano/imunologia , alfa 2-Antiplasmina/genética , alfa 2-Antiplasmina/farmacologia
11.
Neurol Neurochir Pol ; 52(3): 352-358, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29306602

RESUMO

OBJECTIVE: Genetic background of cryptogenic ischemic stroke (IS) and transient ischemic attack (TIA) remains uncertain. Alpha-2-antiplasmin (α2AP) Arg407Lys polymorphism has been shown to be less common in patients with abdominal aortic aneurysm (AAA) compared with healthy controls. We investigated associations of α2AP Arg407Lys polymorphism with cryptogenic IS and TIA. METHODS: We studied 165 consecutive Caucasian patients who experienced cryptogenic IS (n=123) or TIA (n=42). Neurological outcomes were assessed using the modified Rankin Scale (mRS) in the acute phase of cerebral ischemia and 8 (6-12) months after the index episode. Patients were genotyped for α2AP Arg407Lys polymorphism (rs1057335) using real time PCR technique. RESULTS: The allele frequency of Arg407Lys polymorphism was: 0.82/0.18. The 407Lys allele was more frequent in TIA patients compared to the IS group (0.29 vs. 0.14, p=0.003). In the whole group, as well as in IS and TIA patients analyzed separately, possession of the 407Lys allele was associated with excellent outcome (mRS 0-1) during follow-up (p<0.05) but not in the acute phase of ischemic events both in thrombolyzed and nonthrombolyzed IS patients. The multivariate logistic regression model showed that the excellent outcome (mRS 0-1) assessed after 8 (6-12) months since the index cerebral ischemia was predicted by the occurrence of Lys407 allele (OR 6.18, 95% CI, 2.01-18.98, p=0.001). CONCLUSION: The presence of 407Lys allele is associated with better prognosis in cryptogenic cerebrovascular events. Our findings suggest that the α2AP Arg407Lys polymorphism could be involved in the pathogenesis of cerebral ischemia and its outcomes.


Assuntos
Isquemia Encefálica/genética , Acidente Vascular Cerebral , alfa 2-Antiplasmina/genética , Humanos , Fatores de Risco
12.
Int J Mol Med ; 40(3): 854-858, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28677806

RESUMO

α2-antiplasmin (α2AP) is known to be a physiological inhibitor of plasmin. Previously, we showed that α2AP displays various functions, such as promotion of extracellular matrix production, cell growth, and cell differentiation that are not promoted by its function as a plasmin inhibitor. We herein investigated the role of α2AP in bone formation by examining calcein incorporation after its injection in α2AP-deficient mice. We found that α2AP deficiency enhanced the bone formation rate in mice. We also found that the osteocalcin expression and alkaline phosphatase activity were elevated in the femur and serum of the α2AP-deficient mice. Intriguingly, α2AP deficiency promoted osteoblast (OB) differentiation of primary calvarial OBs. In contrast, α2AP attenuated OB differentiation of mouse osteoblastic the MC3T3-E1 cells. Furthermore, α2AP attenuated Wnt-3a-induced ß-catenin expression and low­density lipoprotein receptor-related protein 6 activation in the MC3T3-E1 cells. These results suggest that α2AP negatively affects OB differentiation and function by inhibiting the Wnt/ß-catenin pathway. These findings provide a basis for clinical strategies to improve various bone disorders.


Assuntos
Diferenciação Celular , Osteoblastos/metabolismo , Osteogênese , Via de Sinalização Wnt , alfa 2-Antiplasmina/metabolismo , Animais , Linhagem Celular , Camundongos , Camundongos Knockout , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , alfa 2-Antiplasmina/genética , beta Catenina/genética , beta Catenina/metabolismo
13.
Circulation ; 135(11): 1011-1020, 2017 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-28028005

RESUMO

BACKGROUND: In patients with hemodynamically significant pulmonary embolism, physiological fibrinolysis fails to dissolve thrombi acutely and r-tPA (recombinant tissue-type plasminogen activator) therapy may be required, despite its bleeding risk. To examine potential mechanisms, we analyzed the expression of key fibrinolytic molecules in experimental pulmonary emboli, assessed the contribution of α2-antiplasmin to fibrinolytic failure, and compared the effects of plasminogen activation and α2-antiplasmin inactivation on experimental thrombus dissolution and bleeding. METHODS: Pulmonary embolism was induced by jugular vein infusion of 125I-fibrin or fluorescein isothiocyanate-fibrin labeled emboli in anesthetized mice. Thrombus site expression of key fibrinolytic molecules was determined by immunofluorescence staining. The effects of r-tPA and α2-antiplasmin inactivation on fibrinolysis and bleeding were examined in a humanized model of pulmonary embolism. RESULTS: The plasminogen activation and plasmin inhibition system assembled at the site of acute pulmonary emboli in vivo. Thrombus dissolution was markedly accelerated in mice with normal α2-antiplasmin levels treated with an α2-antiplasmin-inactivating antibody (P<0.0001). Dissolution of pulmonary emboli by α2-antiplasmin inactivation alone was comparable to 3 mg/kg r-tPA. Low-dose r-tPA alone did not dissolve emboli, but was synergistic with α2-antiplasmin inactivation, causing more embolus dissolution than clinical-dose r-tPA alone (P<0.001) or α2-antiplasmin inactivation alone (P<0.001). Despite greater thrombus dissolution, α2-antiplasmin inactivation alone, or in combination with low-dose r-tPA, did not lead to fibrinogen degradation, did not cause bleeding (versus controls), and caused less bleeding than clinical-dose r-tPA (P<0.001). CONCLUSIONS: Although the fibrinolytic system assembles at the site of pulmonary emboli, thrombus dissolution is halted by α2-antiplasmin. Inactivation of α2-antiplasmin was comparable to pharmacological r-tPA for dissolving thrombi. However, α2-antiplasmin inactivation showed a unique pattern of thrombus specificity, because unlike r-tPA, it did not degrade fibrinogen or enhance experimental bleeding. This suggests that modifying the activity of a key regulator of the fibrinolytic system, like α2-antiplasmin, may have unique therapeutic value in pulmonary embolism.


Assuntos
Plasminogênio/metabolismo , Embolia Pulmonar/patologia , alfa 2-Antiplasmina/metabolismo , Animais , Antifibrinolíticos/uso terapêutico , Modelos Animais de Doenças , Feminino , Fibrinogênio/metabolismo , Fibrinólise , Hemoglobinas/análise , Hemorragia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/metabolismo , Ativador de Plasminogênio Tecidual/uso terapêutico , alfa 2-Antiplasmina/deficiência , alfa 2-Antiplasmina/genética
14.
Blood ; 127(5): 538-45, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26626994

RESUMO

Human α2-antiplasmin (α2AP, also called α2-plasmin inhibitor) is the main physiological inhibitor of the fibrinolytic enzyme plasmin. α2AP inhibits plasmin on the fibrin clot or in the circulation by forming plasmin-antiplasmin complexes. Severely reduced α2AP levels in hereditary α2AP deficiency may lead to bleeding symptoms, whereas increased α2AP levels have been associated with increased thrombotic risk. α2AP is a very heterogeneous protein. In the circulation, α2AP undergoes both amino terminal (N-terminal) and carboxyl terminal (C-terminal) proteolytic modifications that significantly modify its activities. About 70% of α2AP is cleaved at the N terminus by antiplasmin-cleaving enzyme (or soluble fibroblast activation protein), resulting in a 12-amino-acid residue shorter form. The glutamine residue that serves as a substrate for activated factor XIII becomes more efficient after removal of the N terminus, leading to faster crosslinking of α2AP to fibrin and consequently prolonged clot lysis. In approximately 35% of circulating α2AP, the C terminus is absent. This C terminus contains the binding site for plasmin(ogen), the key component necessary for the rapid and efficient inhibitory mechanism of α2AP. Without its C terminus, α2AP can no longer bind to the lysine binding sites of plasmin(ogen) and is only a kinetically slow plasmin inhibitor. Thus, proteolytic modifications of the N and C termini of α2AP constitute major regulatory mechanisms for the inhibitory function of the protein and may therefore have clinical consequences. This review presents recent findings regarding the main aspects of the natural heterogeneity of α2AP with particular focus on the functional and possible clinical implications.


Assuntos
alfa 2-Antiplasmina/química , alfa 2-Antiplasmina/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , alfa 2-Antiplasmina/genética
15.
Infect Immun ; 83(12): 4837-47, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26438794

RESUMO

Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection.


Assuntos
Proteínas de Bactérias/imunologia , Regulação Bacteriana da Expressão Gênica , Pulmão/imunologia , Peste/imunologia , Ativadores de Plasminogênio/imunologia , Inibidores de Serina Proteinase/imunologia , Yersinia pestis/imunologia , alfa 2-Antiplasmina/imunologia , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Progressão da Doença , Interações Hospedeiro-Patógeno , Imunidade Inata , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/patologia , Peste/microbiologia , Peste/mortalidade , Peste/patologia , Ativadores de Plasminogênio/genética , Inibidores de Serina Proteinase/genética , Transdução de Sinais , Análise de Sobrevida , Yersinia pestis/genética , Yersinia pestis/patogenicidade , alfa 2-Antiplasmina/deficiência , alfa 2-Antiplasmina/genética
16.
Blood ; 126(16): 1940-8, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26324704

RESUMO

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.


Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Coagulação Sanguínea/fisiologia , Eritrócitos/metabolismo , gama-Glutamiltransferase/metabolismo , Animais , Fatores de Coagulação Sanguínea/genética , Carboxipeptidase B2/genética , Carboxipeptidase B2/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Transtornos Hemorrágicos/genética , Transtornos Hemorrágicos/metabolismo , Humanos , Camundongos , Camundongos Knockout , alfa 2-Antiplasmina/deficiência , alfa 2-Antiplasmina/genética , alfa 2-Antiplasmina/metabolismo , gama-Glutamiltransferase/genética
17.
Arterioscler Thromb Vasc Biol ; 34(12): 2586-93, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25256235

RESUMO

OBJECTIVE: Ischemic stroke is primarily attributable to thrombotic vascular occlusion. Elevated α2-antiplasmin (a2AP) levels correlate with increased stroke risk, but whether a2AP contributes to the pathogenesis of stroke is unknown. We examined how a2AP affects thrombosis, ischemic brain injury, and survival after experimental cerebral thromboembolism. APPROACH AND RESULTS: We evaluated the effects of a2AP on stroke outcomes in mice with increased, normal, or no circulating a2AP, as well as in mice given an a2AP-inactivating antibody. Higher a2AP levels were correlated with greater ischemic brain injury (rs=0.88, P<0.001), brain swelling (rs=0.82, P<0.001), and reduced middle cerebral artery thrombus dissolution (rs=-0.93, P<0.001). In contrast, a2AP deficiency enhanced thrombus dissolution, increased cerebral blood flow, reduced brain infarction, and decreased brain swelling. By comparison to tissue plasminogen activator (TPA), a2AP inactivation hours after thromboembolism still reduced brain infarction (P<0.001) and hemorrhage (P<0.05). Microvascular thrombosis, a process that enhances brain ischemia, was markedly reduced in a2AP-deficient or a2AP-inactivated mice compared with TPA-treated mice or mice with increased a2AP levels (all P<0.001). Matrix metalloproteinase-9 expression, which contributes to acute brain injury, was profoundly decreased in a2AP-deficient or a2AP-inactivated mice versus TPA-treated mice or mice with increased a2AP levels (all P<0.001). a2AP inactivation markedly reduced stroke mortality versus TPA (P<0.0001). CONCLUSIONS: a2AP has profound, dose-related effects on ischemic brain injury, swelling, hemorrhage, and survival after cerebral thromboembolism. By comparison to TPA, the protective effects of a2AP deficiency or inactivation seem to be mediated through reductions in microvascular thrombosis and matrix metalloproteinase-9 expression.


Assuntos
Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/patologia , alfa 2-Antiplasmina/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Circulação Cerebrovascular , Modelos Animais de Doenças , Fibrinólise , Infarto da Artéria Cerebral Média/enzimologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , alfa 2-Antiplasmina/deficiência , alfa 2-Antiplasmina/genética
18.
Sci Rep ; 4: 5967, 2014 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-25095732

RESUMO

Renal fibrosis is the final common pathway of a wide variety of chronic kidney diseases. Myofibroblast formation via the differentiation of from tissue-resident fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs), and epithelial-to-mesenchymal transition (EMT) is known to play a pivotal role in the development of renal fibrosis. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated the role of alpha 2-antiplasmin (α2AP) in myofibroblast formation and the development of renal fibrosis. We observed the development of renal fibrosis using unilateral ureteral obstruction (UUO). α2AP had accumulated in the UUO-induced obstructed kidneys and α2AP deficiency attenuated UUO-induced renal fibrosis in mice. The degree of myofibroblast formation in the obstructed kidneys of α2AP(-/-) mice was less than that in α2AP(+/+) mice. In vitro, α2AP induced myofibroblast formation in renal tubular epithelial cells (RTECs), renal fibrosblasts, and bone marrow-derived mesenchymal stem cells (MSCs). α2AP also induced the production of TGF-ß, which is known to be a key regulator of myofibroblast formation and fibrosis. α2AP-induced the TGF-ß production was significantly reduced by SP600125, c-Jun N-terminal kinase (JNK) specific inhibitor. Our findings suggest that α2AP induces myofibroblast formation in the obstructed kidneys, and mediates the development of renal fibrosis.


Assuntos
Rim/metabolismo , Miofibroblastos/metabolismo , Insuficiência Renal/genética , Fator de Crescimento Transformador beta/genética , Obstrução Ureteral/genética , alfa 2-Antiplasmina/genética , Animais , Antracenos/farmacologia , Diferenciação Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Regulação da Expressão Gênica , Rim/patologia , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Knockout , Miofibroblastos/patologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Insuficiência Renal/complicações , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Ureter/metabolismo , Ureter/patologia , Obstrução Ureteral/complicações , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , alfa 2-Antiplasmina/deficiência
19.
Thromb Res ; 134(3): 723-8, 2014 09.
Artigo em Inglês | MEDLINE | ID: mdl-25065555

RESUMO

INTRODUCTION: Abdominal Aortic Aneurysm (AAA) involves dilatation of the abdominal aorta, with a natural history of expansion and eventual rupture. We have previously shown that AAA patients form denser clots with smaller pores, which are more resistant to fibrinolysis. The aim of this study was to use functional polymorphisms of the fibrinolytic system to identify how changes to proteins involved in fibrinolysis may play a role in the development of AAA. METHODS: Caucasian subjects ≥ 55 years (602 AAA patients and 490 matched controls) were genotyped for four polymorphisms (α-2-antiplasmin α2AP Arg6Trp and Arg407Lys, Thrombin-activatable fibrinolysis inhibitor TAFI Thr325Ile and tissue plasminogen activator tPA 7351C→T). DNA was extracted from blood, and genotype identified using real time PCR. Fibrin clot structure was analysed by permeation and turbidity in a subset of patients and controls. RESULTS: Genotypes across the study population were in Hardy-Weinberg Equilibrium. The two α2AP polymorphisms, Arg6Trp and Arg407Lys were in linkage disequilibrium (P<0.0001), and possession of a 407Lys allele negatively associated with AAA (odds ratio 0.833, CI95 0.7-0.991, P=0.040). The TAFI Thr325Ile and the tPA 7351C→T polymorphisms were not associated with AAA. The α2AP 407Lys allele was not associated with in-vitro fibrinolysis times in plasma from patients with AAA. CONCLUSION: Possession of the α2AP 407Lys allele was negatively associated with AAA, and thus changes in α2AP may affect aneurysm growth and development. These data indicate that the regulation of plasmin activity (through binding to α2AP), rather than plasmin generation (TAFI, tPA), may play a role in AAA.


Assuntos
Aneurisma da Aorta Abdominal/genética , Polimorfismo de Nucleotídeo Único , alfa 2-Antiplasmina/genética , Idoso , Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/diagnóstico , Aneurisma da Aorta Abdominal/etnologia , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Inglaterra , Feminino , Fibrina/metabolismo , Fibrinólise/genética , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fenótipo , Prevalência , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , População Branca/genética
20.
PLoS One ; 9(5): e97947, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24874880

RESUMO

BACKGROUND: The α2-antiplasmin (α2AP) protein is known to be a principal physiological inhibitor of plasmin, and is expressed in various part of the brain, including the hippocampus, cortex, hypothalamus and cerebellum, thus suggesting a potential role for α2AP in brain functions. However, the involvement of α2AP in brain functions is currently unclear. OBJECTIVES: The goal of this study was to investigate the effects of the deletion of the α2AP gene on the behavior of mice. METHODS: The motor function was examined by the wire hang test and rotarod test. To evaluate the cognitive function, a repeated rotarod test, Y-maze test, Morris water maze test, passive or shuttle avoidance test and fear conditioning test were performed. An open field test, dark/light transition test or tail suspension test was performed to determine the involvement of α2AP in anxiety or depression-like behavior. RESULTS AND CONCLUSIONS: The α2AP knockout (α2AP-/-) mice exhibited impaired motor function compared with α2AP+/+ mice. The α2AP-/- mice also exhibited impairments in motor learning, working memory, spatial memory and fear conditioning memory. Furthermore, the deletion of α2AP induced anxiety-like behavior, and caused an anti-depression-like effect in tail suspension. Therefore, our findings suggest that α2AP is a crucial mediator of motor function, cognitive function, anxiety-like behavior and depression-like behavior, providing new insights into the role of α2AP in the brain functions.


Assuntos
Comportamento Animal , Deleção de Genes , Estudos de Associação Genética , alfa 2-Antiplasmina/genética , Animais , Ansiedade/genética , Cognição , Depressão/genética , Aprendizagem em Labirinto , Memória de Curto Prazo , Camundongos , Camundongos Knockout , Atividade Motora/genética , Memória Espacial
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