OBJECTIVES: To investigate the application value of thromboelastography (TEG) in assessing coagulation function in children with severe hemophilia A (HA) after emicizumab (EMI) therapy. METHODS: A retrospective analysis was performed on the activated partial thromboplastin time (APTT) and TEG testing results of 17 children with severe HA before and after EMI treatment at Shenzhen Children's Hospital from January 2023 to July 2024. Correlation analysis was conducted between coagulation factor VIII (FVIII) equivalent activity and reaction time (R value) measured by TEG. RESULTS: After EMI treatment, the mean bleeding rate for children with severe HA was 1.6 events per year, with 15 children (88%) without spontaneous bleeding or joint bleeding. The children with severe HA showed a significant reduction in APTT after EMI treatment (P<0.05), with a significantly shorter APTT than the normal control group (P<0.05). There was no correlation between APTT and FVIII equivalent activity after treatment (P>0.05). After EMI treatment, TEG parameters, including R value, kinetic time, alpha angle (α), maximum amplitude, clot strength, and coagulation index, shifted from a hypocoagulable state before treatment to a nearly normal state after treatment (P<0.05). The R value demonstrated a strong negative correlation with FVIII equivalent activity (r=-0.758, P<0.05). CONCLUSIONS The bleeding condition of children with severe HA can be effectively controlled after EMI treatment. Routine APTT testing cannot reflect true coagulation function, whereas TEG testing is clinically valuable in assessing the coagulation function of children with severe HA undergoing EMI treatment.
Humans ; Thrombelastography ; Hemophilia A/physiopathology* ; Male ; Child ; Antibodies, Bispecific/therapeutic use* ; Antibodies, Monoclonal, Humanized/therapeutic use* ; Blood Coagulation/drug effects* ; Child, Preschool ; Retrospective Studies ; Female ; Partial Thromboplastin Time ; Adolescent ; Infant

OBJECTIVE: To analyze two families with inherited dysfibrinogenemia, and explore the molecular pathogenic mechanisms. METHODS: The coagulation indexes of the probands and their family members were detected. The FGA, FGB, and FGG exons and their flanking sequences were amplified by PCR, and the mutation sites were identified by sequencing. SIFT, PolyPhen2, LRT, ReVe, MutationTaster, phyloP, and phastCons bioinformatics software were used to predict the functional impact of the mutation sites. Protein structure and amino acid conservation analysis of the variant were conducted using PyMOL and Clustal X software. RESULTS: The thrombin time (TT) of the proband in family 1 was prolonged to 37.00 s, and Fg∶C decreased to 0.52 g/L. The TT of the proband in family 2 was 20.30 s, and Fg∶C was 1.00 g/L, which was lower than the normal range. Genetic analysis revealed that the proband in family 1 had a heterozygous mutation c.80T>C in FGA, resulting in the substitution of phenylalanine 27 with serine (Phe27Ser). The proband in family 2 had a heterozygous mutation c.1007T>A in FGG, resulting in the substitution of methionine 336 with lysine (Met336Lys). Bioinformatics software prediction analysis indicated that both mutations were deleterious variants. PyMOL mutation models revealed that the Aα chain mutation (Phe27Ser) in family 1 and γ chain mutation (Met336Lys) in family 2 resulted in alterations in spatial structure and reduced protein stability. Clustal X results showed that both Aα Phe27 and γMet336 were highly conserved across homologous species. CONCLUSION Heterozygous mutations of FGA gene c.80T>C and FGG gene c.1007T>A are both pathogenic variants, causing inherited dysfibrinogenemia.
Female ; Humans ; Male ; Afibrinogenemia/genetics* ; Fibrinogen/genetics* ; Heterozygote ; Mutation ; Pedigree

OBJECTIVE: To investigate a family with hereditary coagulation factor XI (FXI) deficiency, identify its possible genetic etiology, analyze the bleeding risk of the proband, and provide a blood transfusion regimen. METHODS: The blood samples from the family members were collected, and the coagulation parameters of the proband and her family members were detected. Whole-exome sequencing was performed on the blood samples of the proband to identify gene variants, and validate the variants in the family using Sanger sequencing. Bioinformatics softwares were used to analyze the conservation of amino acid variant sites and the impact of the variations on protein function. The pathogenicity of the variant sites was analyzed according to the genetic variation classification criteria and guidelines of the American College of Medical Genetics and Genomics (ACMG). Thromboelastography (TEG) was used to assess the coagulation function of the family members and evaluate the transfusion regimen and its efficacy in the proband. RESULTS: The activated partial thromboplastin time (APTT) of the proband was significantly prolonged to 96.7 seconds, and FXI activity (FXI: C) and FXI antigen (FXI: Ag) decreased to 1.3% and 1%, respectively, both of which were extremely reduced. The FXI: C of the proband's father was also significantly lower than the normal value. The TEG results showed that the coagulation function of the proband was reduced, while the coagulation function of other family members was normal. The F11 gene of the proband exhibited compound heterozygous variants of c.738G>A (p.Trp246 *) and c.1288G>A (p.Ala430Thr). The proband's father carried a heterozygous missense variant of c.1288G>A (p.Ala430Thr), while her mother, her eldest daughter, and her youngest daughter carried a heterozygous nonsense variant of c.738G>A (p.Trp246 *). According to the ACMG genetic variation classification criteria and guidelines, c.738G>A (p.Trp246 *) is classified as a pathogenic variant (PVS1+PS3-Moderate+PP4), and c.1288G>A (p.Ala430Thr) is classified as a possible pathogenic variant (PS3-Moderate+PM1+PM3_Srong+PP4). p.Trp246 and p.Ala430 are highly conserved across different species. Swiss PdbViewer software analysis showed that p.Ala430Thr variant caused a change in the number of hydrogen bonds in FXI protein, affecting protein function. The following transfusion regimen was determined through TEG evaluation in vitro: 600 ml of fresh frozen plasma (FFP) was administered 24 hours before surgery to prevent bleeding. And there was no significant bleeding during or after the surgery. CONCLUSION The heterozygous nonsense variant ofc.738G>A (p.Trp246 *) and the heterozygous missense variant of c.1288G>A (p.Ala430Thr) in the F11 gene are the pathogenic factors of this hereditary FXI deficiency family.
Humans ; Factor XI Deficiency/therapy* ; Factor XI/genetics* ; Blood Transfusion ; Pedigree ; Female ; Male ; Mutation ; Thrombelastography ; Partial Thromboplastin Time ; Blood Coagulation ; Adult

OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

OBJECTIVE: To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). METHODS: The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. RESULTS: In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). CONCLUSION Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.
Abietanes/therapeutic use* ; Osteosarcoma/enzymology* ; Cisplatin/therapeutic use* ; Humans ; Cell Line, Tumor ; Animals ; Apoptosis/drug effects* ; Mice, Nude ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MAP Kinase Signaling System/drug effects* ; Bone Neoplasms/enzymology* ; Cell Cycle/drug effects* ; Xenograft Model Antitumor Assays ; Mice ; Drug Resistance, Neoplasm/drug effects* ; Neoplasm Invasiveness ; Mice, Inbred BALB C

Acquired hemophilia A (AHA) is a rare autoimmune bleeding disorder. Its occurrence secondary to hepatobiliary malignancies is even rarer, and without timely diagnosis and treatment, the mortality rate is extremely high. There is a need to raise awareness of this disease. This report describes a case of a 70-year-old female patient diagnosed with AHA 2 months after surgery for cholangiocarcinoma, admitted to the Second Affiliated Hospital of Bengbu Medical College in October 2022. The patient presented with subcutaneous hematoma in both lower limbs. Coagulation function tests showed a markedly prolonged activated partial thromboplastin time (APTT) of 74.5 seconds, with no correction in the APTT mixing test. Coagulation factor assays revealed a severely reduced coagulation factor VIII activity (FVIII:C) of 0.3%, and an inhibitor titer of 25.6 BU/mL was detected. After ruling out other potential causes, the patient was diagnosed with cholangiocarcinoma-associated AHA. With chemotherapy to control the primary tumor, alongside hemostatic and immunosuppressive therapy for inhibitor eradication, AHA was brought under control. The patient had no further coagulation abnormalities or bleeding, enabling timely and full-course chemotherapy for cholangiocarcinoma and significantly improving survival and quality of life. Therefore, in patients with malignancies who present with spontaneous bleeding or unusual bleeding following surgery, trauma, or invasive procedures, clinicians should be alert to the possibility of secondary AHA. Timely diagnosis and treatment can significantly improve prognosis.
Humans ; Cholangiocarcinoma/surgery* ; Female ; Hemophilia A/drug therapy* ; Aged ; Bile Duct Neoplasms/surgery* ; Factor VIII

Humans ; Ankle ; Ankle Joint/surgery* ; Arthritis ; Arthrodesis ; Lower Extremity ; Hemophilia A/complications*

OBJECTIVE: To analyze the sequence of the F12 gene and molecular mechanism for 20 patients with coagulation factor Ⅻ (FⅫ) deficiency. METHODS: The patients were selected from the outpatient department of the Second Hospital of Shanxi Medical University from July 2020 to January 2022. The activity of coagulation factor Ⅷ (FⅧ:C), factor Ⅸ (FⅨ:C), factor Ⅺ (FⅪ:C) and factor Ⅻ (FⅫ:C) were determined by using a one-stage clotting assay. All exons and 5' and 3' UTR of the F12 gene were analyzed by Sanger sequencing to detect the potential variants. Bioinformatic software was used to predict the pathogenicity of the variants, conservation of amino acids, and protein models. RESULTS: The FⅫ:C of the 20 patients has ranged from 0.07% to 20.10%, which was far below the reference values, whilst the other coagulation indexes were all normal. Sanger sequencing has identified genetic variants in 10 patients, including 4 with missense variants [c.820C>T (p.Arg274Cys), c.1561G>A (p.Glu521Lys), c.181T>C (p.Cys61Arg) and c.566.G>C (p.Cys189Ser)], 4 deletional variants c.303_304delCA(p.His101GlnfsX36), 1 insertional variant c.1093_1094insC (p.Lys365GlnfsX69) and 1 nonsense variant c.1763C>A (p.Ser588*). The remaining 10 patients only harbored the 46C/T variant. The heterozygous c.820C>T(p.Arg274Cys) missense variant in patient 1 and the homozygous c.1763C>A (p.Ser588*) nonsense variant in patient 2 were not included in the ClinVar and the Human Gene Mutation Database. Bioinformatic analysis predicted that both variants were pathogenic, and the corresponding amino acids are highly conserved. The protein prediction models suggested that the c.820C>T (p.Arg274Cys) variant may affect the stability of the secondary structure of FⅫ protein by disrupting the original hydrogen bonding force and truncating the side chain, leading to changes in the vital domain. c.1763C>A (p.Ser588*) may produce a truncated C-terminus which may alter the spatial conformation of the protein domain and affect the serine protease cleavage site, resulting in extremely reduced FⅫ:C. CONCLUSION Among individuals with low low FⅫ:C detected by one-stage clotting assay, 50% have harbored variants of the F12 gene, among which the c.820C>T and c.1763C>A were novel variants underlying the reduced coagulating factor FⅫ.
Humans ; Factor XII/genetics* ; Pedigree ; Mutation ; Mutation, Missense ; Heterozygote ; Factor XII Deficiency/genetics*

OBJECTIVE: To explore the clinical characteristics and genetic etiology of a consanguineous Chinese pedigree affected with Congenital coagulation factor XII (XII) deficiency. METHODS: Members of the pedigree who had visited Ruian People's Hospital on July 12, 2021 were selected as the study subjects. Clinical data of the pedigree were reviewed. Peripheral venous blood samples were taken from the subjects. Blood coagulation index and genetic testing were carried out. Candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: This pedigree has comprised 6 individuals from 3 generations, including the proband, his father, mother, wife, sister and son. The proband was a 51-year-old male with kidney stones. Blood coagulation test showed that his activated partial thromboplastin time (APTT) was significantly prolonged, whilst the FXII activity (FXII:C) and FXII antigen (FXII:Ag) were extremely reduced. The FXII:C and FXII:Ag of proband's father, mother, sister and son have all reduced to about half of the lower limit of reference range. Genetic testing revealed that the proband has harbored homozygous missense variant of c.1A>G (p.Arg2Tyr) of the start codon in exon 1 of the F12 gene. Sanger sequencing confirmed that his father, mother, sister and son were all heterozygous for the variant, whilst his wife was of the wild type. By bioinformatic analysis, the variant has not been included in the HGMD database. Prediction with SIFT online software suggested the variant is harmful. Simulation with Swiss-Pbd Viewer v4.0.1 software suggested that the variant has a great impact on the structure of FXII protein. Based on the Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics (ACMG), the variant was rated as likely pathogenic. CONCLUSION The c.1A>G (p.Arg2Tyr) variant of the F12 gene probably underlay the Congenital FXII deficiency in this pedigree. Above finding has further expanded the spectrum of F12 gene variants and provided a reference for clinical diagnosis and genetic counseling for this pedigree.
Male ; Female ; Humans ; Middle Aged ; Factor XII/genetics* ; Pedigree ; Codon, Initiator ; East Asian People ; Mothers ; Factor XII Deficiency/genetics* ; Mutation

OBJECTIVE: To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia. METHODS: Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene. RESULTS: The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups. CONCLUSION After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.
Humans ; Transduction, Genetic ; Genetic Vectors ; Hemophilia A/genetics* ; Transfection ; Blood Coagulation Factors/genetics* ; Lentivirus/genetics*