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1.
Thromb Res ; 217: 36-47, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35853369

RESUMO

Ranging from bleeding to thrombosis, the clinical features of congenital fibrinogen qualitative disorders, including dysfibrinogenemia and hypodysfibrinogenemia, are highly heterogeneous. Although the associations between some specific fibrinogen mutations and the thrombotic phenotypes have been well elucidated, the underlying mechanism between fibrinogen variants and bleeding events remains underestimated. After systematically reviewing the literature of (hypo-)dysfibrinogenemia patients with bleeding phenotypes, we identified several well-characterized bleeding-related fibrinogen variants in those patients. Several possible pathomechanisms are proposed to explain the genotype-phenotype associations: 1, mutations in the NH2-terminal portion of the Aα chain hamper fibrinogen fitting into the active site cleft of thrombin and drastically slow the conversion of fibrinogen into monomeric fibrin; 2, mutations adding new N-linked glycosylation sites introduce bulky and negatively charged carbohydrate side chains and undermine the alignment of fibrin monomers during polymerization; 3, mutations generating unpaired cysteine form extra disulfide bonds between the abnormal fibrinogen chains and produce highly branched and fragile fibrin networks; 4, truncation mutations in the fibrinogen αC regions impair the lateral fibril aggregation, as well as factor XIII crosslinking, endothelial cell and platelet binding. These established relationships between specific variants and the bleeding tendency will help manage (hypo-)dysfibrinogenemia patients to avoid adverse bleeding outcomes.


Assuntos
Afibrinogenemia , Fibrinogênios Anormais , Trombose , Afibrinogenemia/genética , Testes de Coagulação Sanguínea , Fibrina/metabolismo , Fibrinogênio/metabolismo , Fibrinogênios Anormais/genética , Hemorragia/genética , Humanos , Trombose/genética
2.
Front Mol Biosci ; 9: 877170, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35601826

RESUMO

A novel coagulation factor X (FX) Tyr319Cys mutation (Y99C as chymotrypsin numbering) was identified in a patient with severe bleeding. Unlike the earlier reported Y99A mutant, this mutant can bind and cleave its specific chromogenetic substrate at a normal level, suggesting an intact binding pocket. Here, using molecular dynamics simulations and MM-PBSA calculations on a FX-rivaroxaban (RIV) complex, we confirmed a much stronger binding of RIV in Y99C than in Y99A on a molecular level, which is actually the average result of multiple binding poses in dynamics. Detailed structural analyses also indicated the moderate flexibility of the 99-loop and the importance of the flexible side chain of Trp215 in the different binding poses. This case again emphasizes that binding of ligands may not only be a dynamic process but also a dynamic state, which is often neglected in drug design and screening based on static X-ray structures. In addition, the computational results somewhat confirmed our hypothesis on the activated Tyr319Cys FX (Y99C FXa) with an impaired procoagulant function to bind inhibitors of FXa and to be developed into a potential reversal agent for novel oral anticoagulants (NOAC).

3.
Hum Mutat ; 43(7): 928-939, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35391506

RESUMO

There is growing evidence that synonymous codon variants (SCVs) can cause disease through the disruption of different processes of protein production. The aim of the study is to investigate whether the 14 SCVs reported in the F9 variant database were the pathogenic causes of hemophilia B. The impacts of SCVs on splicing and protein expression were detected using a combination of in silico prediction, in vitro minigene splicing assay and cell expression detection. The splicing transcripts were identified and quantified by co-amplification fluorescent PCR. The mechanism of splicing was verified by a modified pU1snRNA and pU7snRNA approach. Aberrant splicing patterns were found in eight SCVs. Five of the 8 SCVs produced almost all aberrant splicing isoforms, which were expected to truncate protein, three of them presented a partial defect on both splicing and protein secretion, the overall effects were consistent with the residual Factor IX activity of the affected cases. Neither the pre-messenger RNA (mRNA) splicing process nor the protein function was impaired in the rest six SCVs. In conclusion, our study firstly revealed the pathogenic mechanism of the 14 F9 SCVs and highlighted the importance of performing mRNA splicing analysis and protein expression studies of SCVs in inherited disorders.


Assuntos
Fator IX/genética , Hemofilia B , Splicing de RNA , Mutação Silenciosa , Códon , Hemofilia B/genética , Humanos , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Hum Mutat ; 43(2): 215-227, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34882887

RESUMO

Approximately 10% of von Willebrand factor (VWF) gene variants are suspected to disrupt messenger RNA (mRNA) processing, the number of which might be underestimated due to the lack of transcript assays. In the present study, we provided a detailed strategy to evaluate the effects of nine putative splice site variants (PSSVs) of VWF on mRNA processing as well as protein properties and establish their genotype-phenotype relationships. Eight of nine PSSVs affected VWF splicing: c.322A>T, c.1534-13_1551delinsCA, and c.8116-2del caused exon skipping; c.221-2A>C, c.323+1G>T, and c.2547-13T>A resulted in the activation of cryptic splice sites; c.2684A>G led to exon skipping and activation of a cryptic splice site; c.2968-14A>G created a new splice site. The remaining c.5171-9del was likely benign. The efficiency of nonsense-mediated mRNA decay (NMD) was much higher in platelets compared to leukocytes, impairing the identification of aberrant transcripts in 4 of 8 PSSVs. The nonsense variant c.322A>T partially impaired mRNA processing, leaking a small amount of correct transcripts with c.322T (p.Arg108*), while the missense variant c.2684A>G totally disrupted normal splicing of VWF, rather than produced mutant protein with the substitution of Gln895Arg. The results of this study would certainly add novel insights into the molecular events behind von Willebrand disease.


Assuntos
Sítios de Splice de RNA , Doenças de von Willebrand , Fator de von Willebrand , Humanos , Splicing de RNA , RNA Mensageiro/genética , Doenças de von Willebrand/genética , Fator de von Willebrand/genética
5.
Thromb Haemost ; 122(5): 679-691, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34256393

RESUMO

A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient's plasma clotting time indicated that both activated partial thromboplastin time (52.6 seconds) and prothrombin time (19.4 seconds) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired approximately fivefold. Thrombin generation assay using patient's plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased; however, the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with approximately 12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased by approximately 10-fold; however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated approximately 20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient's normal hemostatic phenotype.


Assuntos
Protrombina , Trombomodulina , Antitrombina III , Antitrombinas , Transtornos Herdados da Coagulação Sanguínea , Humanos , Proteína C/metabolismo , Protrombina/metabolismo , Trombina/metabolismo , Trombomodulina/metabolismo
7.
Clin Appl Thromb Hemost ; 26: 1076029620944471, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886527

RESUMO

The changes in the coagulation, fibrinolytic, and endothelial functions are correlated with the pathophysiology of the thromboembolic diseases during acute illness. However, these changes in patients with hereditary thrombophilia who were not in the acute stage of venous thromboembolism (VTE) are unclear. A panel of 4 biomarkers, including thrombin-antithrombin complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC), tissue-type plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAIC), and soluble thrombomodulin (sTM), were assayed in 100 healthy controls and 100 patients with thrombophilia. Although significantly higher concentrations of TAT, PIC, t-PAIC, and sTM were observed in patients with thrombophilia than in healthy controls, 70 patients showed absolutely normal levels of the above 4 biomarkers. Among the other 30 patients who had at least 1 biomarker out of the corresponding reference interval, 26 of them presented elevated PIC with or without increased TAT. Except for sTM, other 3 biomarkers did not show significant differences in patients with previous VTE compared to those without. Patients with single episode of VTE had obviously lower t-PAIC than those with multiple episodes of VTE, whereas the levels of TAT, PIC, and sTM were unassociated with the number of thrombosis episodes. Most thrombophilia patients who were not in the acute stage of VTE showed normal coagulation, fibrinolytic, and endothelial functions. Thus, we were unable to show that the one-time response of this panel was clinically helpful in determining thrombosis risk in thrombophilia individuals. Future studies should focus on the dynamic monitoring during the chronic phase of VTE to offer special advantages for patients with thrombophilia.


Assuntos
Biomarcadores/sangue , Trombofilia/complicações , Tromboembolia Venosa/etiologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trombofilia/patologia
9.
Thromb Haemost ; 120(7): 1045-1055, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32422680

RESUMO

Antithrombin (AT) is a serine protease inhibitor that regulates the activity of coagulation proteases of both intrinsic and extrinsic pathways. We identified an AT-deficient patient with a heterozygous Thr90Ser (T90S) mutation who experiences recurrent venous thrombosis. To understand the molecular basis of the clotting defect, we expressed AT-T90S in mammalian cells, purified it to homogeneity, and characterized its properties in established kinetics, binding, and coagulation assays. The possible effect of mutation on the AT structure was also evaluated by molecular modeling. Results demonstrate the inhibitory activity of AT-T90S toward thrombin and factor Xa has been impaired three- to fivefold in both the absence and presence of heparin. The affinity of heparin for AT-T90S has been decreased by four- to fivefold. Kinetic analysis revealed the stoichiometry of AT-T90S inhibition of both thrombin and factor Xa has been elevated by three- to fourfold in both the absence and presence of heparin, suggesting that the reactivity of coagulation proteases with AT-T90S has been elevated in the substrate pathway. The anticoagulant activity of AT-T90S has been significantly impaired as analyzed in the AT-deficient plasma supplemented with AT-T90S. The anti-inflammatory effect of AT-T90S was also decreased. Structural analysis predicts the shorter side-chain of Ser in AT-T90S has a destabilizing effect on the structure of AT and/or the AT-protease complex, possibly increasing the size of an internal cavity and altering a hydrogen-bonding network that modulates conformations of the allosterically linked heparin-binding site and reactive center loop of the serpin. This mutational effect increases the reactivity of AT-T90S with coagulation proteases in the substrate pathway.


Assuntos
Deficiência de Antitrombina III/genética , Antitrombina III/genética , Coagulação Sanguínea/genética , Heterozigoto , Mutação , Trombose Venosa/genética , Adulto , Antitrombina III/metabolismo , Deficiência de Antitrombina III/sangue , Deficiência de Antitrombina III/diagnóstico , Fator Xa/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Fenótipo , Conformação Proteica , Recidiva , Relação Estrutura-Atividade , Trombina/metabolismo , Trombose Venosa/sangue , Trombose Venosa/diagnóstico
10.
Arterioscler Thromb Vasc Biol ; 40(5): 1296-1310, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32237906

RESUMO

OBJECTIVE: Integrin ß3 is implicated in numerous biological processes such as its relevance to blood triglyceride, yet whether ß3 deficiency affects this metabolic process remains unknown. Approach and Results: We showed that the Chinese patients with ß3-deficient Glanzmann thrombasthenia had a 2-fold higher serum triglyceride level together with a lower serum LPL (lipoprotein lipase) level than those with an αIIb deficiency or healthy subjects. The ß3 knockout mice recapitulated these phenotypic features. The elevated plasma triglyceride level was due to impaired LPL-mediated triglyceride clearance caused by a disrupted LPL secretion. Further analysis revealed that ß3 directly bound LPL via a juxtamembrane TIH (threonine isoleucine histidine)720-722 motif in its cytoplasmic domain and functioned as an adaptor protein by interacting with LPL and PKD (protein kinase D) to form the PKD/ß3/LPL complex that is required for ß3-mediated LPL secretion. Furthermore, the impaired triglyceride clearance in ß3 knockout mice could be corrected by adeno-associated virus serotype 9 (AAV9)-mediated delivery of wild-type but not TIH720-722-mutated ß3 genes. CONCLUSIONS: This study reveals a hypertriglyceridemia in both ß3-deficient Chinese patients and mice and provides novel insights into the molecular mechanisms of the significant roles of ß3 in LPL secretion and triglyceride metabolism, drawing attention to the metabolic consequences in patients with ß3-deficient Glanzmann thrombasthenia.


Assuntos
Hipertrigliceridemia/etiologia , Cadeias beta de Integrinas/metabolismo , Integrina beta3/metabolismo , Lipase Lipoproteica/sangue , Trombastenia/complicações , Triglicerídeos/sangue , Adolescente , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , China , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/diagnóstico , Hipertrigliceridemia/enzimologia , Cadeias beta de Integrinas/genética , Integrina beta3/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Fatores de Risco , Trombastenia/sangue , Trombastenia/diagnóstico , Trombastenia/genética
11.
J Thromb Haemost ; 18(5): 1141-1153, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32078247

RESUMO

BACKGROUND: Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein C-deficient patients both of whom carry a heterozygous Gly197 to Arg (G197R) mutation in PROC and experience venous thrombosis. OBJECTIVE: The objective of this study was to determine the molecular basis of the clotting defect in patients carrying the G197R mutation. METHODS: We expressed protein C-G197R in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems. RESULTS: The activation of protein C-G197R by thrombin was improved ~10-fold; however, its activation by thrombin was not promoted by thrombomodulin (TM). In a tissue factor-mediated thrombin generation assay, the addition of soluble TM to protein C-deficient plasma, supplemented with protein C-G197R, did not have a significant inhibitory effect on thrombin generation parameters. APC-G197R did not exhibit a significant anticoagulant activity in either purified or plasma-based assay systems. APC-G197R was essentially inactive because it showed no activity in an aPTT assay. Anti-inflammatory activity of APC-G197R was also dramatically impaired as determined by an endothelial cell permeability assay. Structural modeling predicted that the side-chain of Arg cannot be accommodated at this site of APC without a major distortion of the local structure that appears to propagate and adversely affect the reactivity/folding of the catalytic pocket. CONCLUSION: The G197R mutation in patients appears to be functionally equivalent to a heterozygous protein C knockout with half of the protein having no significant activity and thus causing thrombosis.


Assuntos
Proteína C , Trombose , Animais , Testes de Coagulação Sanguínea , Heterozigoto , Humanos , Mutação , Proteína C/genética , Trombina , Trombose/genética
12.
Arterioscler Thromb Vasc Biol ; 40(2): 483-494, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31875702

RESUMO

OBJECTIVE: Defective PC (protein C) pathway predisposes patients to venous thromboembolism (VTE) and is mostly, but not exclusively, attributed to hereditary PC or PS (protein S) deficiencies and activated PC resistance caused by factor V Leiden mutation. Approach and Results: In a patient with acute mesenteric venous thrombosis and positive family history of VTE associated with the impaired PC pathway function determined by thrombin generation test, we identified a novel heterozygous prothrombin mutation p.Arg541Trp. Two more patients with positive family history of VTE carrying the same mutation were identified in a cohort of another 373 unrelated patients, making an overall prevalence of 0.8%. Family investigation revealed 11 individuals in the 3 pedigrees harboring the heterozygous prothrombin p.Arg541Trp mutation, and 8 of them (72%) had experienced episodes of VTE. Functional studies indicated the mutation moderately decreased procoagulant activity of prothrombin and had mild impact on the inactivation of thrombin by its inhibitor antithrombin. However, the amino acid residue substitution significantly compromised PC activation by thrombin, both in the absence and presence of soluble thrombomodulin, and thus rendered prothrombin function procoagulant biased. CONCLUSIONS: In summary, the prothrombin p.Arg541Trp mutation constitutes a new genetic risk factor of VTE by impairing function of PC pathway and tilting thrombin's procoagulant activity over anticoagulant function.


Assuntos
DNA/genética , Predisposição Genética para Doença , Isquemia Mesentérica/genética , Mutação , Proteína C/metabolismo , Protrombina/genética , Adulto , Análise Mutacional de DNA , Feminino , Heterozigoto , Humanos , Isquemia Mesentérica/sangue , Pessoa de Meia-Idade , Linhagem , Protrombina/metabolismo , Risco
13.
Haematologica ; 105(6): 1712-1722, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31399531

RESUMO

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.


Assuntos
Fator de Crescimento Epidérmico , Trombose , Glicosilação , Humanos , Mutação , Proteína C/genética , Proteína C/metabolismo , Trombina/metabolismo , Trombose/genética
14.
Blood ; 134(20): 1745-1754, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31558466

RESUMO

Combined factor V (FV) and FVIII deficiency (F5F8D) is a rare autosomal-recessive bleeding disorder caused by mutations in lectin mannose binding-1 (LMAN1) and multiple coagulation factor deficiency-2 (MCFD2). Six causative homozygous mutations (5 in LMAN1 and 1 in MCFD2) were identified in 6 patients with F5F8D. A thrombin-generation assay, triggered with tissue factor (1 pM) in F5F8D plasma, paradoxically exhibited enhanced thrombin generation compared with normal plasma. Significantly lower free tissue factor pathway inhibitor (fTFPI) was found in F5F8D patients compared with healthy controls (P < .01). Normalizing tissue factor pathway inhibitor α (TFPIα) in F5F8D plasma greatly delayed and reduced thrombin generation. Increasing FV concentrations by adding plasma FV to F5F8D plasma only caused a gradual decrease in thrombin generation, suggesting that low levels of TFPIα and FV cocontributed to the elevated thrombin generation by reducing anticoagulant effects. On the contrary, thrombin generation in F5F8D platelet-rich plasma (PRP) was significantly lower than in normal controls (P < .05); however, it was fully corrected by normalizing FVIII or after 1-deamino-8-d-arginine vasopressin (DDAVP) infusion, indicating that the hypocoagulable state of F5F8D patients is associated with low FVIII levels. In addition, plasma and platelet FV in F5F8D PRP were sufficient to support normal thrombin generation, and low TFPIα may have no effect on thrombin generation. DDAVP infusion induced a complete response in 5 F5F8D patients and a partial response in the remaining patient. Based on our findings, we suggest that DDAVP may be considered a potential substitute for FVIII concentrates, and fresh-frozen plasma (FFP) infusion may not be necessary for F5F8D patients with minor bleeding challenges.


Assuntos
Deficiência do Fator V/sangue , Fator V/análise , Hemofilia A/sangue , Hemorragia/sangue , Adulto , Deficiência do Fator V/complicações , Feminino , Hemofilia A/complicações , Hemorragia/etiologia , Hemostasia , Humanos , Masculino , Pessoa de Meia-Idade , Trombina/análise , Adulto Jovem
15.
Orphanet J Rare Dis ; 14(1): 182, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340840

RESUMO

BACKGROUND: Inherited Factor XIII deficiency (FXIIID) is one of the most severe and under-diagnosed rare bleeding disorders. Only 5 large deletions involving one or more exons in F13A1 have been reported, and lacking of multiplex ligation-dependent probe amplification (MLPA) assay might underestimate the copy number variations (CNVs) in F13A1 and F13B. We had characterized the clinical presentation of two unrelated severe FXIIID probands and explored the pathogenic mechanisms. RESULTS: Both probands experienced several episodes of fatal bleeding and delayed wound healings prior to diagnosis. FXIII activity was measured by the ammonia release assay, and FXIII-A and FXIII-B antigens were determined by ELISA. All the exons including exon-intron boundaries and promoter regions of F13A1 and F13B were amplified and directly sequenced. Copy number variations (CNVs) of F13A1 and F13B were detected by the CNVplex® method. Breakpoints of the F13A1 large deletion were identified by quantitative primer walking combined long-range PCR (LR-PCR) strategies. Proband 1 was found to have compound heterozygous mutations of a novel small deletion (c.1147del) and a missense mutation p.Arg383Ser. Proband 2 was compound heterozygous for a novel large deletion (g.[77815_112815del;112837_116628del]) and a missense mutation p.Arg716Gly in F13A1. Bioinformatics analysis of the large deletion breakpoints predicted that two fork stalling and template switching and/or microhomology-mediated break-induced replication (FoSTeS/MMBIR) events with two homologies of TCT and C might be responsible for the complex rearrangement. Prophylactic replacement therapy was immediately administered for the two probands upon establishment of the diagnosis. CONCLUSIONS: We detected two type I FXIIID pedigrees and adopted CNVplex® method to detect CNVs of F13A1 and F13B for the first time. A large heterozygous deletion of g.[77815_112815del;112837_116628del] in F13A1, mediated by two FoSTeS/MMBIR events, was identified.


Assuntos
Deficiência do Fator XIII/genética , Fator XIII/genética , Adolescente , Biologia Computacional , Variações do Número de Cópias de DNA/genética , Éxons/genética , Feminino , Genótipo , Humanos , Íntrons , Mutação/genética , Linhagem , Fenótipo , Deleção de Sequência/genética
16.
Chem Biol Drug Des ; 94(3): 1664-1671, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31108011

RESUMO

Rivaroxaban (RIV) is a direct oral anticoagulant (DOAC) targeting activated coagulation factor X (FXa). An earlier study reported the F174A mutant of FXa resistant to a RIV-like inhibitor, Apixaban. In current study, the detailed molecular mechanism of the resistance has been explored by molecular dynamics simulations on the impaired interactions between RIV and FXa in the damaged S4 pocket of F174A mutant. Besides, an unexpected relative stable binding mode of S1'S1 was revealed, which required dynamic motions of Gln192 and Gln61 to allow the morpholinone moiety of RIV to shift into the S1' pocket and form strong interactions. These dynamic motions of RIV and critical residues might be important in drug design for direct inhibitors of coagulation factors.


Assuntos
Anticoagulantes/química , Fator X/antagonistas & inibidores , Inibidores do Fator Xa/química , Simulação de Dinâmica Molecular , Proteínas Mutantes/antagonistas & inibidores , Pirazóis/química , Piridonas/química , Rivaroxabana/química , Sequência de Aminoácidos , Sítios de Ligação , Desenho de Fármacos , Fator X/genética , Humanos , Proteínas Mutantes/genética , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
17.
Haemophilia ; 25(3): 475-483, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30866119

RESUMO

INTRODUCTION: Only two large duplications of F9 causing haemophilia B (HB) have been reported. AIM: To analyse the pathogenic mechanisms of large F9 duplications. METHODS: We have identified two large duplications of F9 (dup ex 1-6 and dup ex 4-6) associated with mild and severe HB in probands A and B, respectively. Here, we localized the breakpoints of the two duplications using long-range PCR and genome walking combined with quantitative primer walking strategies. We traced the origin of dup ex 4-6 by haplotype analysis then performed somatic mosaicism detection in sporadic pedigree B and detected the effect of chimeric intron derived from the duplication on transcription by minigene assay. RESULTS: Mechanisms of fork stalling and template switching and/or microhomology-mediated break-induced replication (FoSTeS/MMBIR) might be responsible for the formation of two tandem direct duplications. The dup ex 4-6 was traced to maternal grandmother of proband B, who was both somatic mosaicism and germline mosaic and the duplication might be formed during mitosis of her early embryonic cells. Minigene assay demonstrated that chimeric intron generated three transcripts, one minor transcript produced an in-frame protein adding duplicated 143 amino acids into the normal FIX, explaining the small amount of larger FIX shown in Western blot. The inter-F9 dup ex 1-6 adjacent to the original F9 copy created two identical promoters, and promoter competition might be the pathogenic mechanism of the duplication causing mild HB. CONCLUSIONS: This study highlights that duplications can be associated with diseases by complicated pathogenic mechanisms.


Assuntos
Fator IX/genética , Duplicação Gênica , Hemofilia B/genética , Sequência de Bases , Criança , Biologia Computacional , Feminino , Avós , Hemofilia B/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Transcrição Genética
18.
Thromb Haemost ; 119(6): 871-881, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30919383

RESUMO

The Cys22-Cys27 disulfide bond of factor X (FX) protease domain is not conserved among coagulation factors and its contribution to the physiological haemostasis and implication in the pathogenesis of haemostatic and thrombotic disorders remain to be elucidated. Mutation p.Cys27Ser was identified in a pedigree of congenital FX deficiency and fluorescence labelling study of transiently transfected HEK293 cells showed accumulation of FX p.Cys27Ser within cell, indicating incompetent secretion partially responsible for the FX deficiency. The clotting activity of FX p.Cys27Ser was decreased to about 90% of wild-type, while amidolytic and pro-thrombinase activities (kcat/Km) determined with recombinant FXa mutant were 1.33- and 4.77-fold lower. Molecular dynamic simulations revealed no major change in global structure between FXa p.Cys27Ser and wild-type FXa; however, without the Cys22-Cys27 disulfide bond, the insertion of newly formed N terminal of catalytic domain after the activation cleavage is hindered, perturbing the conformation transition from zymogen to enzyme. The crystal structure of FXa shows that this disulfide bond is solvent accessible, indicating that its stability might be subject to the oxidation/reduction balance. As demonstrated with FX p.Cys27Ser here, Cys22-Cys27 disulfide bond may modulate FX clotting activity, with reduced FX pertaining less pro-coagulant activity.


Assuntos
Deficiência do Fator X/genética , Fator X/metabolismo , Mutação/genética , Coagulação Sanguínea , Cristalização , Cisteína/genética , Dissulfetos/química , Ativação Enzimática/genética , Fator X/química , Fator X/genética , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos/genética , Proteólise , Relação Estrutura-Atividade
19.
Thromb Haemost ; 119(3): 449-460, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30669159

RESUMO

Protein S (PS) deficiency is associated with a 10-fold increased risk of venous thromboembolism (VTE), but its diagnosis is quite difficult and complicated. In this study, we identified 53 unrelated pedigrees with PS deficiency in China. Data of their clinical characteristics and laboratory examinations were collected. Genetic analysis of PROS1 including direct sequencing, copy number variant detection and messenger ribonucleic acid analysis was performed in probands and related family members. Of these 53 probands, 52.8% (28/53) experienced multi-site and/or recurrent thrombotic episodes, mainly manifested as deep venous thrombosis and/or pulmonary embolism (82.7%). Additional risk factors of VTE were observed in 39.6% (21/53) probands who exhibited a significantly higher rate of recurrent VTE compared with those not, in which 7 probands were complicated by anti-phospholipid syndrome. Most probands and family members exhibited quantitative PS deficiency with impairment of both activated protein C and tissue factor pathway inhibitor cofactor activities. Note that 87.2% (34/39) PROS1 detectable mutation rate was obtained through comprehensive phenotypic and genetic analysis. A total of 36 PROS1 causative mutations including 16 novel mutations were identified in 48 probands, whereas no PROS1 mutations were detected in the other 5 probands. Three hotspot mutations (Glu67Ala, Arg561Trp and Tyr560*) were identified in the Chinese population for the first time. This article provides a framework for correlating the clinical pathogenesis of PS deficiency to genetic backgrounds in the Chinese population.


Assuntos
Proteínas Sanguíneas/genética , Mutação , Deficiência de Proteína S/genética , Proteína S/genética , Embolia Pulmonar/genética , Tromboembolia Venosa/genética , Trombose Venosa/genética , Adolescente , Adulto , China/epidemiologia , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Deficiência de Proteína S/sangue , Deficiência de Proteína S/diagnóstico , Deficiência de Proteína S/etnologia , Embolia Pulmonar/sangue , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/etnologia , Recidiva , Fatores de Risco , Tromboembolia Venosa/sangue , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/etnologia , Trombose Venosa/sangue , Trombose Venosa/diagnóstico , Trombose Venosa/etnologia , Adulto Jovem
20.
Haemophilia ; 25(2): 316-323, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30648777

RESUMO

INTRODUCTION: Sporadic haemophilia B (HB) without obvious familial history poses challenges for genetic diagnosis and counselling. AIM: To identify the F9 variants in sporadic HB patients and probe the origin of these de novo mutations. METHOD: A total of 294 unrelated HB pedigrees sought genetic diagnosis were analysed in this single-centre study. The F9 gene was analysed by direct sequencing, and AccuCopy technique was adopted to screen for gene copy number variations. Six short tandem repeats approximal or within F9 gene were applied for linkage analysis. Mosaicism of sequence variant was determined by ddNTP Primer Extension method. RESULTS: Sporadic HB patients constituted 36% (61/294) of cases enrolled in current study. The sporadic and familial HB patients shared similar spectrum of F9 variants, with single nucleotide substitution as predominant form of disease-causing mutation and no mutation prone hotspot sites, including CpG dinucleotide sequences, had been identified. Majority of the mothers of sporadic HB patients were F9 mutation carriers (70%, 43/61), and most of them (95%, 41/43) had the inherited bleeding trait traced back to maternal grandfathers. Although most de novo mutations occur in germ cells, 2 maternal grandfathers, who had somatic mosaic mutations of F9, were also revealed to be the source of genetic variations identified in patients. In our cohort, FIX inhibitor incidence was 1%, developed only in patients carrying null mutations. CONCLUSION: The diversity of F9 genetic variants and possible mosaicism of de novo mutation demand extensive study and more cautious in genetic counselling of sporadic HB.


Assuntos
Fator IX/genética , Hemofilia B/genética , China , Códon sem Sentido , Variações do Número de Cópias de DNA , Éxons , Hemofilia B/diagnóstico , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Polimorfismo Genético , Splicing de RNA
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