A novel GATA1 variant p.G229D causing the defect of procoagulant platelet formation.

INTRODUCTION: GATA1 is one of the master transcription factors in hematopoietic lineages development which is crucial for megakaryocytic differentiation and maturation. Previous studies have shown that distinct GATA1 variants are associated with varying severities of macrothrombocytopenia and platelet dysfunction. OBJECTIVE: To determine the underlying pathological mechanisms of a novel GATA1 variant (c. 686G > A, p. G229D) in a patient with recurrent traumatic muscle hematomas. METHODS: Comprehensive phenotypic analysis of the patient platelets was performed. Procoagulant platelet formation and function were detected using flow cytometry assay and thrombin generation test (TGT), respectively. The ANO6 expression was measured by qPCR and western blot. The intracellular supramaximal calcium flux was detected by Fluo-5N fluorescent assay. RESULTS: The patient displayed mild macrothrombocytopenia with defects of platelet granules, aggregation, and integrin αIIbß3 activation. The percentage of the procoagulant platelet formation of the patient upon the stimulation of thrombin plus collagen was lower than that of the healthy controls (40.9 % vs 49.0 % ± 5.1 %). The patient platelets exhibited a marked reduction of thrombin generation in platelet rich plasma TGT compared to the healthy controls (peak value: ∼70 % of the healthy controls; the endogenous thrombin potential: ∼40 % of the healthy controls). The expression of ANO6 and intracellular calcium flux were impaired, which together with abnormal granules of the patient platelets might contribute to defect of procoagulant platelet function. CONCLUSIONS: The G229D variant could lead to a novel platelet phenotype characterized by defective procoagulant platelet formation and function, which extended the range of GATA1 variants associated platelet disorders.

A case-report of the unprovoked thrombotic event in a patient with thymoma and severe FVII deficiency.

BACKGROUND: Factor VII deficiency is a rare bleeding disorder caused by a deficiency of clotting factor VII. However, there have been some case reports of venous thrombosis in patients with factor VII deficiency, especially underlying the prothrombotic risk factors exposure. Patients with factor VII deficiency require special considerations before undergoing surgery to minimize the risk of bleeding or thrombogenesis. CASE PRESENTATION: Here, we described a patient with early-stage thymoma and severe factor VII deficiency who experienced an unprovoked thrombotic episode before thymectomy and a fatal thrombotic event after surgery. By adopting gene screening, a reported homozygous F7 mutation (p.His408Gln) and a novel heterozygous PROS1 mutation (p.Pro147Ala) were identified. The former resulted in severe factor VII deficiency but did not protect against thrombosis, and the latter was correlated with normal expression and cofactor activities of protein S through the thrombin generation test. The perioperative infusion of recombinant factor VII concentrate and the absence of antithrombotic prophylaxis may collectively contribute to her fatal thrombotic event after surgery. CONCLUSIONS: For the patients with severe factor VII deficiency undergoing surgery, uniform replacement therapy may not be recommended, and antithrombotic prophylaxis should be used in the case with thrombotic history to minimize the risk of bleeding and thrombogenesis.

Congenital (hypo-)dysfibrinogenemia and bleeding: A systematic literature review.

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.

Integrin ß3 Deficiency Results in Hypertriglyceridemia via Disrupting LPL (Lipoprotein Lipase) Secretion.

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.

The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X.

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.

Paradoxical bleeding and thrombotic episodes of dysprothrombinaemia due to a homozygous Arg382His mutation.

We have characterised the pathogenic basis of dysprothrombinaemia in a patient exhibiting paradoxical bleeding and thrombotic defects during pregnancy and postpartum. Genetic analysis revealed that the proband is homozygous for the prothrombin Arg382His mutation, possessing only ~1 % clotting activity. The proband experienced severe bleeding episodes during her pregnancy, which required treatment with prothrombin complex concentrates, and then pulmonary embolism and deep-vein thrombosis at 28 days postpartum, which required treatment with LMWH and fresh frozen plasma. Analysis of haemostatic parameters revealed that the subject had elevated FDP and DD and decreased fibrinogen levels, indicating the presence of hyperfibrinolysis. Thrombin generation and clotting assays with the proband's plasma in the presence of soluble thrombomodulin and tissue-type plasminogen activator indicated a defect in activation of both protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Unlike normal plasma, no TAFI activation could be detected in the patient's plasma. The expression and characterisation of recombinant prothrombin Arg382His indicated that zymogen activation by prothrombinase was markedly impaired and the activation of protein C and TAFI by thrombin-Arg382His was impaired 600-fold and 2500-fold, respectively. The recombinant thrombin mutant exhibited impaired catalytic activity toward both fibrinogen and PAR1 as determined by clotting and signalling assays. However, the mutant activated factor XI normally in both the absence and presence of polyphosphates. Arg382 is a key residue on (pro)exosite-1 of prothrombin and kinetic analysis of substrate activation suggested that the poor zymogenic activity of the mutant is due to its inability to bind factor Va in the prothrombinase complex.

[Correlation of Thrombosis and Prothrombotic State with Coagulation Factor V Gene Polymorphism and APCR. HHcy].

OBJECTIVE: To investigate the correlation of patients with thrombosis or prothrombotic status with hyperhomocysteinemia (HHcy), activated protein C-resistance(APCR) and gene polymorphism of coagulation factor V. METHODS: Three hundred healthy voluteers were selected as controls, 223 cases of thrombosis (80 cases of cerebral infarction of CT, the MI of 82 cases of myocardial infarction, venous thrombosis of VTE 61 cases), 270 cases of patients with prothrombotic state (76 cases of pregnancy disease of PIH, 62 cases of chronic obstructive pulmonary disease (COPD), 60 cases of diabetes(DM) and 72 cases of cancer) were enrolled in this study. The plasma APCR and hyperhomocysteinemia were detected by APTT coagulation method and cycling enzyme method respectively, and restriction fragment length polymorphism(RFLP) were was used to detect the gene polymorphism of FV G1691-A, G1091-C and A1090-G in the patient and control groups. RESULTS: APCR positive rate was 62.29% and 7.33%, and the positive hyperhomocysteinemia accounted for 68.42% and 10.00% respectively in the group of the patients with venous thrombosis and the normal control group. 3 cases of heterozygous FV gene mutations were found in the APCR-positive patients with venous thrombosis. CONCLUSION: HHcy possitive rate of patients with venous thrombosis is signiticantly higher than that in control, the HHcy is one of the important causes resulting in thrombosis, the patients with venous thrombosis have proved to be with APCR, and the possitive APCR may be related with the coagulation factor V gene polymorphism.

Clinical manifestations and mutation spectrum of 57 subjects with congenital factor XI deficiency in China.

Congenital factor XI (FXI) deficiency is a rare bleeding disorder with unpredictable bleeding tendency. Few studies in a large cohort have been reported regarding associations between FXI activity (FXI:C) or genotypes and bleeding symptoms currently. This study characterized clinical manifestations and mutation spectrum of 57 subjects with FXI deficiency in China. Clinical data were collected and mutations were identified by direct sequencing and determined by mRNA analysis. The result revealed bleeding symptoms were only found in 12 patients (12/57, 21.1%) with severely reduced FXI:C, and prolonged bleeding post injury/surgery as well as easy bruising were the commonest bleeding manifestations presented in respective 5 cases (5/12, 41.7%). A total number of 37 mutations were identified including 19 missense mutations, 9 nonsense mutations, 6 splice site mutations and 3 small deletions. Among them, 4 missense mutations, 5 splice mutations, 3 small deletions and a nonsense mutation were newly detected. W228*, G400V, Q263* and c.1136-4delGTTG with a total frequency of 48.3% were the most four common mutations in Chinese patients. RT-PCR analysis was carried out and confirmed that both c.596-8T>A and c.1136-4delGTTG were pathogenic due to frameshift resulting in respective truncated proteins. Our findings suggested clinical manifestations had little to do with FXI:C or genotypes, which required further study. This study, the largest investigation of FXI deficiency in China revealed that the F11 mutation spectrum of Chinese population was distinct from those of other populations earlier established.

A novel factor X gene mutation Val (GTC) 384Ala (GCC) in a Chinese family resulting in congenital factor X deficiency.

FX is a vitamin K-dependent coagulation protease critically essential for the coagulation cascade. FXD (congenital deficiency of factor X) is a rare coagulation disorder that inherited as an autosomal recessive trait. Here we reported a patient with bleeding diathesis from infant. The proband with pseudotumor in cerebral articular and cavity were identified as encapsulated hematocele ultimately. FX sequence analysis revealed that the patient carried a novel homozygous missense mutation that resulted in the Val384Ala substitution. Further investigation of the novel mutation would deepen our understanding of the bleeding mechanism involved in FXD.

A unique feature of iron loss via close adhesion of Helicobacter pylori to host erythrocytes.

Iron deficiency anemia is an extra-stomach disease experienced in H. pylori carriers. Individuals with type A blood are more prone to suffering from H. pylori infection than other individuals. To clarify the molecular mechanisms underlying H. pylori-associated anemia, we collected erythrocytes from A, B, O, and AB blood donors and analyzed morphology, the number of erythrocytes with H. pylori colonies attached to them, and iron contents in erythrocytes and H. pylori (NCTC11637 and SS1 strains) by means of optical microscopy, scanning electron microscopy, and synchrotron radiation soft X-ray imaging. The number of type A erythrocytes with H. pylori attached to them was significantly higher than that of other erythrocytes (P<0.05). Far more iron distribution was observed in H. pylori bacteria using dual energy analysis near the iron L2, 3 edges by soft X-ray imaging. Iron content was significantly reduced in host erythrocytes after 4 hours of exposure to H. pylori. H. pylori are able to adhere more strongly to type A erythrocytes, and this is related to iron shift from the host to the bacteria. This may explain the reasons for refractory iron deficiency anemia and elevated susceptibility to H. pylori infection in individuals with type A blood.

Impact of polymorphisms in genes involved in autoimmune disease on inhibitor development in Chinese patients with haemophilia A.

One of the most severe and important complication in the treatment of haemophilia A (HA) patients is the formation of inhibitors. The mechanism that leads to factor (F)VIII inhibitor formation is complicated. Both genetic and environmental factors have been mentioned to play decisive roles. Recently, polymorphisms in the genes encoding interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-α), cytotoxic T-lymphocyte antigen-4 (CTLA-4), have been suggested to be contributing determinants of the inhibitor risk. In order to investigate the influence of the single nucleotide polymorphisms (SNPs) in the genes encoding for cytokines to the inhibitors development in Chinese HA patients, we genotyped 10 SNPs with high heterozygote rates in Chinese and a CA repeat microsatellite at the gene loci IL-10 as well in a separate, unrelated case-controlled cohort of 122 affected HA patients; 63 with inhibitors and 59 without inhibitors. The particular SNPs included in this study were as follows: -592C/A and -819C/T in IL-10, -590C/T in IL-4, -318C/T and 49A/G in CTLA-4, -827C/T in TNF-α, -1112C/T and 2044G/A in IL-13, 874A/T in interferon (IFN)-γ and -295T/C in IL-16. Our results demonstrated that -819T and -592A alleles in IL-10 were observed more frequently in patients with inhibitors. This indicated that -819C/T and -592A/C in IL-10 may influence the inhibitors development in HA patients. Furthermore, we concluded that the haplotype in IL-10 (TA, CA, CC at positions -819 and -582, respectively) may predispose FVIII inhibitor development in HA patients. In conclusion, the data reported in our study clearly highlight the participation of IL-10 in inhibitors formation in Chinese HA patients.

[Molecular mechanisms of recurrent venous thrombosis in two pedigrees with type I antithrombin deficiency].

OBJECTIVE: To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin (AT) deficiency. METHODS: The routine coagulation screening tests were detected, thrombin generation tests was performed to evaluate the hypercoagulation. Anticardiolipin antibody (ACA) and lupus anticoagulant (LA) were detected with enzyme-linked immunosorbent assay (ELISA) and diluted viper venom time assay (DVVT), respectively. The activities of protein C, protein S and AT (PC:A, PS:A, AT:A) were tested with chromogenic substrate assay or clotting method. The antigen of AT (AT:Ag) was performed with immunoturbidimetry methods. Western blot was used to analyze the molecular weight (MW) and the plasma levels of AT:Ag. All 7 exons and the flanking sequences were amplified by PCR. The mutation of AT gene and thrombophilia associated gene polymorphisms were analyzed by direct DNA sequencing. The expression plasmid of Ala404Asp mutant was constructed with site-directed mutagenesis method based on the wild-type (WT) AT cDNA contained in pcDNA 3.1 vector, and transiently expression of AT WT and the Ala404Asp mutant was performed using HEK293T cells. Cultured supernatant and cell lysates were collected and measured for AT:Ag by ELISA and Western blot. RESULTS: The results of routine coagulation tests in two probands were normal, thrombin generation tests indicated that proband 1 presented hypercoagulable state with 2.8 and 1.5 times higher of the endogenous thrombin potential (ETP) and peak height compared with that of normal, respectively. The levels of PC:A, PS:A, ACA and LA were normal. AT:A in proband 1 and proband 2 were 45% and 32%, and AT:Ag were almost half of the normal (121 mg/L and 158 mg/L), respectively. The results of Western blot showed that both probands' plasma levels of AT:Ag were lower than the normal pooled plasma and MW was normal. Two heterozygous mutations of g.3291C→T(Thr98Ile), g.13863C > A(Ala404Asp) were identified in the probands, respectively. No proband had venous thrombosis associated gene polymorphisms. Expression in vitro showed that AT:Ag in culture media and lysates of Ala404Asp are 4.8% and 60.6% of that of WT, respectively. CONCLUSION: Thr98Ile and Ala404Asp mutation of AT gene significantly correlate with recurrent venous thrombosis in the two probands, respectively. Ala404Asp has not been described before. The mutant Ala404Asp protein can not be expressed due to impaired secretion and increased intracellular degradation, resulting in type I AT deficiency.

Genetic analysis of a pedigree with combined factor XII and factor XI deficiency.

The objective of the present study was to identify the gene mutations of factor XI (FXI) and factor XII (FXII) in a Chinese pedigree with combined congenital FXI and FXII deficiencies. The proband was a 40-year-old woman with deficiency in both FXI (49%) and FXII (0%) activities. Blood samples from 10 other members of her family were collected and used for detection of FXI, FXII activities (FXI: C, FXII: C) and antigen levels. Genetic analysis to detect mutations in FXI, FXII genes was also performed. The proband's mother, three brothers, two sisters, her son and her daughter all have lowered FXII: C. Furthermore, her mother and one of her brothers also have lowered FXI: C. Gene sequencing for FXI in affected members revealed a heterozygous C23179T point mutation in exon 11 resulting in substitution of arginine 396 by cysteine. Gene sequencing for FXII revealed a C46T in the promoter region and a deletion mutation of two nucleotides CA at position 9160 and 9161 in exon 5. The deletion mutation can lead to frameshift mutation and premature termination of transcription in exon 6. We found a new heterozygous missense mutation in the FXI gene and a new nonsense mutation of two nucleotides deletion which caused frameshift mutation and premature termination of transcription in the FXII gene in a Chinese family with combined FXI and FXII deficiencies.

PML/RARalpha fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association.

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.

[Preparation and characterization of a monoclonal antibody against human c-Kit].

AIM: To prepare anti-c-Kit monoclonal antibodies and characterize their specificity of epitope recognition. METHODS: cDNA encoding human c-Kit extracellular domain was constructed into a procaryotic expression vector pQE30 and the correctness of the reconstructed plasmid pQE30-KitD4-5 was verified by sequencing. The plasmid was transformed into E.coli M15 strain. Recombinant 6 x His pQE30-KitD4-5 was expressed after induction by IPTG for 4 h. Then SDS-PAGE results suggested that the products mainly formed inclusion bodies. The fusion protein was further purified with Ni-NTA-His affinity chromatography and then used to immunize BALB/c mice. The hybridomas were achieved by fusing the immunized spleen cells with the Sp2/0 myeloma cell line. The positive clones were screened by FCM with CHO-hKit cells. Hybidoma clones secreting anti-c-Kit antibodies were further subcloned and investigated for their biological activities by Western blot, rapid isotyping analysis and FCM. RESULTS: Recombinant human c-Kit fusion proteins were in vitro expressed and purified to be used as immunogen. One stable hybridoma cell line, which continuously secrets specific anti-c-Kit monoclonal antibody ((SRJ1)) was established. The biological activity studies showed that the monoclonal antibody recognized the natural c-Kit expressed on the Kasumi leukemia cell line, but failed to bind to the normal human peripheral blood cells. Interestingly, this monoclonal antibody failed to recognize a subpopulation of Kasumi cells that is reactive with the commercial anti-c-Kit mAb Ab81 suggesting that the c-Kit expressed by this subpopulation contains some sequencial and/or structural aberrations that are distinguishable by mAb SRJ1. CONCLUSION: With an immunization procedure using purified recombinant human c-Kit fusion proteins. a hybridoma cell line continuously and stably secreting anti-c-Kit monoclonal antibody has been established. The monoclonal antibody SRJ1 specifically recognizes human c-Kit expressed on the leukemia cells, and may provide a novel approach to analyze the possible structural variations of c-Kit expressed by different cells.

[Molecular mechanisms of Glanzmann thrombasthenia caused by alphaII b P126H mutation: an in vitro experiment].

OBJECTIVE: To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha IIb P126H mutation. METHODS: Eukaryotic vector of alpha IIb P126H was constructed by PCR site-directed mutagenesis and then co-transfected with eukaryotic vector PCDM8 II a expressing the subunit beta3 into human renal epithelial cells of the line 293& and Chinese hamster ovarian cancer cells of the line CHO after sequencing. The membrane expression of alpha IIb P126H mutant was analyzed by flow cytometry and the whole expression was confirmed by Western blotting. The alpha IIb P126H mutant subcellular localization was determined by laser confocal scanning microscopy. RESULTS: The 293T cells cotransfected with cDNAs of mutated alpha IIb and wild-type beta3 failed to express alpha II bbeta3 on the cell surface as shown by FACS. Western blotting of the cell lysate showed no detectable mature alpha IIb. Immunofluorescence studies demonstrated proa II bbeta3 complex colocalized with an endoplasmic reticulum (ER) marker, but showed minimal colocalization with an Golgi marker. CONCLUSION: The P126H mutation in alpha IIb prevents transport of the pro-alpha II bbeta3 complex from ER to the Golgi body, thus hindering its maturation and surface expression. The impaired alpha II bp33 transport is responsible for the thrombasthenia.

Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette.

Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene.

Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10-100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.

[Antithrombin deficiency due to heterozygous antithrombin gene mutation and a pedigree study].

OBJECTIVE: To identify the antithrombin (AT) phenotype and gene mutation of a kindred with hereditary antithrombin deficiency. METHODS: Plasma AT activity and AT antigen level of the propositus and his kindred members were determined with chromogenic substrate method and immunoassay, respectively. All the seven exons and intron-exon boundaries of antithrombin gene were analyzed by PCR and direct sequencing of amplified PCR products from the propositus. RESULTS: The propositus AT antigen level was normal but his AT activity was only 65% of normal value suggesting that he had type II AT deficiency. A heterozygous G13830A mutation in exon 6 resulting in Arg393His missense mutation in his AT polypeptide was identified in the propositus. The same phenotype and gene mutation were found in other 3 kindred members. CONCLUSION: The type II AT deficiency found in this kindred is caused by heterozygous G13830A mutation in AT gene.