Optimized AAV package and experimental application of recombinant AAV8/hFⅧ for gene therapy on hemophilia A mice / 中华血液学杂志

Objective: To evaluate the effects of adeno-associated virus (AAV) carrying hFⅧ by serotype 8 (AAV8/hFⅧ) on hemophilia A (HA) mice by gene therapy strategy. Methods: pAAV-CB-EGFP, pH22 (serotype 2) and pfΔ6 (adenovirus helper) were used to package AAV into HEK-293 cells in different conditions (ratios of cells to plasmids). The efficiency of transfection and infection were evaluated using immunofluorescence microscope to seek an optimized package condition. pAAV-TTR-hFⅧ, pH 28 (serotype 8) and pfΔ6 were applied to package AAV8/hFⅧ in HEK-293 cells using the optimized package condition. The purified AAV8/hFⅧ were intravenously injected into HA mice and the effects of gene therapy were estimated. Results: The efficiency of package was evaluated according to the amount and intensity of enhanced green fluorescent protein (EGFP) under immunofluorescence microscope. Four package conditions including 10 cm-dish to transfect 10 μg plasmids, 20 cm-dish to 20 μg, 30 μg and 40 μg plasmids were employed, and the condition of 20 cm-dish to transfect 20 μg plasmids reached the highest transfection efficiency at 24 h, 48 h and 72 h after transfection. The small scale AAV-EGFP was packaged using the optimized condition and an AAV crude extract was harvested by a freeze-thaw method. HEK-293 and 16095 cells were infected by the AAV crude extract, and the preferential infection efficiency was recognized in 16095 cells under immunofluorescence microscope. Then, AAV8/hFⅧ was packaged and purified based on the optimized transfection condition, and the high purity of AAV8/hFⅧ was detected by Western blot. Fractions of AAV8/hFⅧ at the dose of 8×10(12) vg/kg were injected into HA mice through tail vein, an eye-bleeding was performed at every two weeks, and the activity of FⅧ was measured by aPTT assay. Results showed that the activity of FⅧ maintained at the therapeutic level and lasted up to 12 weeks after injection. Conclusion: The purified AAV8/hFⅧ based on the optimized package condition could play a role in HA mice gene therapy, and the long-term therapeutic effects of AAV8/hFⅧ were observed in vivo.

Effect of Amino Acid Motifs in Integrin β3 Cytoplasmic Tail on αⅡbβ3-Mediated Cell function in 293T cell models / 中国实验血液学杂志

OBJECTIVE@#To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function.@*METHODS@#293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested.@*RESULTS@#293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen.@*CONCLUSION@#To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.

Role and Mechanism of the Interaction of BCR-ABL with E3 Ligase c-CBL in Targeting Therapy of Chronic Myelogenous Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the interaction domains between BCR-ABL and E3 liagase c-CBL, so as to reveal the structure-basis for the arsenic to treat chronic myelogenous leukemia(CML).</p><p><b>METHODS</b>The interactional interface of BCR-ABL and c-CBL was simulated and analyzed according to the available structure model. Based on the structural information, the WT and mutant Migr1-BCR-ABL-GFP (ΔSH2,ΔTyrKC,ΔSH2/TyrKC (S/H) and pFlag-c-CBL (ΔRF) were constructed and co-transfected into the 293T and HeLa cells. The co-immunoprecipitation (Co-IP) was performed by using M2 beads (anti-Flag), anti-GFP antibody and protein A beads, and the interaction was identified by using GFP and M2 antibody, respectively. Moreover, the colocalization of BCR-ABL and c-CBL was further evaluated by using immunofluorescent(IF) assay in transfected HeLa cells.</p><p><b>RESULTS</b>Co-IP demonstrated that the TyrKC domain of BCR-ABL was primarily involved in the interaction with c-CBL, while both the SH2 domain of BCR-ABL and the RF domain of c-CBL also participated in the interaction to a certain degree, which were consistent with the structure-based simulation. IF elucidated that the colocalization of BCR-ABL and c-CBL was almost entirely vanished when the deleted TyrKC domain of BCR-ABL was co-transfected with c-CBL, which were elegantly coincident with the results from Co-IP.</p><p><b>CONCLUSION</b>The TyrKC domain of BCR-ABL is sufficient and necessary to mediate the interaction between BCR-ABL and c-CBL, the SH2 domain of BCR-ABL and the RF domain of c-CBL are also involved in the association between the two proteins. It suggests that the association of BCR-ABL and c-CBL can modulate the stability and degradation of BCR-ABL, thus illustrating the molecular mechanisms of the targeting therapy for CML by arsenic.</p>

Myr-RKEFAK Peptide Selectively Regulates Outside-in Signaling Transduction-related Functions in Human Platelets / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the effect of interaction of the talin rod domain integrin binding site 2 with integrin β3 on platelet signal transduction.</p><p><b>METHODS</b>A peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin β3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested.</p><p><b>RESULTS</b>myr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation.</p><p><b>CONCLUSION</b>The cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin β3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.</p>

Effect of the Integrin β3 Cytoplasmic NITY Motif on α II bβ3-Mediated Cell Functions in CHO Cell Model / 中国实验血液学杂志

<p><b>UNLABELLED</b>OBJLECTIVE: To investigate the effect of integrin β3 cytoplasmic NITY motif on αIIbβ3-mediated cell functions.</p><p><b>METHODS</b>Stable Chinese hamster ovary (CHO) cell lines that co-express human wild type integrin αIIb and wild type β3 or mutant β3ΔNITY (β3 deleting cytoplasmic NITY motif) were established. Expression of αIIb and β3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions.</p><p><b>RESULTS</b>CHO-αIIbβ3, CHO-αIIbβ3ΔNITY cells were successfully established. The CHO cells transfected with wild type αIIbβ3 had the ability of adhesion and spreading. Compared with CHO-αIIbβ3 cells, CHO-αIIbβ3ΔNITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered cells. The co-immunoprecipitation showed that kindlin-2 associated with wild type integrin αIIbβ3. The β3ΔNITY mutation substantially reduced kindlin-2 association.</p><p><b>CONCLUSION</b>Deletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin β3, thereby partially inhibit the integrin β3 signaling.</p>

Functional study of abnormal fibrinogen caused by Arg275His mutation in fibrinogen γ chain / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees.</p><p><b>METHODS</b>Routine coagulation tests were conducted in the probands and related family members. The antigen and activity levels of fibrinogen were detected by immunoturbidimetry assay and clauss assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes and antithrombin gene（AT3）were analyzed by PCR amplification and direct sequencing. Routine thrombelastography (TEG) test and functional fibrinogen TEG test were both used to make a comprehensive evaluation of coagulation status and functional fibrinogen level in patients. The molecular weights of the three peptides from fibrinogen were measured by Western blot. The function of abnormal fibrinogen was assessed by fibrinogen dynamic polymerization and fibrinolysis velocity.</p><p><b>RESULTS</b>The coagulation routine tests were normal in two probands except for prolonged thrombin time (TT) and reptilase time (RT), as well as reduced activity levels of 0.5 g/L and 0.6 g/L fibrinogen, respectively. The antigen levels of fibrinogen were 2.32 g/L and 2.66 g/L in two probands, which were in the normal reference range. The genotype analysis showed that Arg275His in fibrinogen γ chain (γ Arg275His) existed in both probands and patients in these two pedigrees. Meanwhile, proband B's grandfather and aunt also carried heterozygote g.5876T>C (Ser116Pro) mutation in AT3. The results of routine TEG test demonstrated that the α values of proband B and his father were close to and lower than the lower limit of reference range, respectively, while the MA values were normal in both of them. However, functional fibrinogen TEG test revealed obviously reduced MA value. All the probands and patients demonstrated prolonged lag-off time and reduced peak value in fibrinogen dynamic polymerization tests. Meanwhile, most of fibrin formed from the patients' plasma could not be dissolved completely by plasminogen (PLG) and urokinase-typeplasminogenactivator (u-PA) at a certain time.</p><p><b>CONCLUSION</b>We first reported cases of inherited dysgibrinogenemia associated with inherited AT deficiency. γArg275His mutation caused the abnormal fibrinogen in terms of fibrin mono polymerization and possibly in fibrinolysis. Combined use of routine TEG test and functional fibrinogen TEG test with comprehensive analyses of the parameters in both tests could better evaluate the level of functional fibrinogen and predict the risk of hemorrhage and thrombosis in patients with inherited dysfibrinogenemia.</p>

Transgenic mouse models of the truncated platelet integrin β3 cytoplasmic tail established by stem cell transplantation / 中国实验血液学杂志

This study was purpose to establish the transgenic mouse models of the truncated platelet integrin β3 by retrovirus-infected hematopoietic stem cells (HSCs) transplantation and to provide the basis for further study of the role of integrin β3 cytoplasmic domain in platelet bi-directional signaling pathways. Wild-type β3, β3-Δ759 (R(760) GT(762) truncated β3) and β3-Δ754 (T(755) NITYRGT(762) truncated β3) cDNAs were subcloned into MSCV MigR1 retroviral vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the β3 deficient mice were infected by the retroviruses, and transplanted into lethally-irradiated wild type C57BL/6 mice. GFP positive rate and surface β3 expression of the recipients' platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaluate the transgenic efficiency. The results showed that four kinds of transgenic mouse models including vector, wild-type β3, β3-Δ759 and β3-Δ754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the β3 expression of transgenic mouse reached heterozygote (β3(+/-) level of mouse). It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs transplantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin β3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.

The binding mechanisms of F VIII Trp1707Ser mutation-associated inhibitor / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the binding mechanisms of FVIII Trp1707Ser mutation-associated inhibitor.</p><p><b>METHODS</b>The APPT, PT, TT, Fg and FVIII:C were detected to make phenotypic diagnosis of haemophilia A. Inhibitors titer were measured by Bethesda method. Long distance-PCR (LD-PCR) and sequence-specific PCR were adopted for screening the intron 22 and intron 1 inversions respectively. FVIII coding and boundary sequences were analyzed by direct DNA sequencing. Inhibitor was reacted with different segments of FVIII, including heavy chain and its components A1 and A2, light chain and its components A3, C1 and C2. Corrected test was used to measure the remaining F VIII:C (% ) by adding pooled normal plasmas. After labeling purified inhibitors with biotin, western blot was performed to further confirm the binding reactions between inhibitors and segments.</p><p><b>RESULTS</b>The haemophilia A patient had mild deficiency of FVIII:C (1.1%) and had high FVIII inhibitor titer of 18.4 BU. A mutation c.97223C>G in exon 14 of F8 gene resulted to p.Trp1707Ser was identified by DNA sequencing. Corrected test showed that the remaining F VIII:C was increased when inhibitors reacted with heavy chain and light chain, especially with heavy chain. The remaining FVIII:C was also increased in the A2 and C2 domain reactions. No significant differences were seen in the A1, A3 and C1 domain reactions. Antigen-antibody reaction bands were confirmed by western blots when degenerated B-domain deleted recombinant FVIII, A2 and C2 were used as antigens.</p><p><b>CONCLUSION</b>The binding sites of FVIIITrp1707Ser mutation inhibitor were the A2 domain of heavy chain and C2 domain of light chain. The binding reaction with heavy chain was more intense.</p>

Phenotype and genotype analysis of two Chinese pedigrees with type 3 von Willebrand diseases / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype of two Chinese pedigrees with von Willebrand diseases, and to investigate the molecular pathogenesis.</p><p><b>METHODS</b>Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor-ristocetin cofactor (vWF:Rco), von Willebrand factor antigen (vWF:Ag), von Willebrand factor activity (vWF:A), von Willebrand factor collagen binding assay (vWF:CB) and multimer analysis were used for phenotype diagnosis. DNA was extracted. All of the 52 exons and exon-intron bounda ries of the VWF gene were amplified with polymerase chain reaction(PCR) and analyzed by direct sequencing.</p><p><b>RESULTS</b>APTT and BT were prolonged. Plasma RIPA, vWF:Rco, vWF:Ag, vWF:A and vWF:CB was significantly decreased. No VWF multimer can be found by plasma VWF multimer analysis. Homozygous insertional mutation g.82888_82889insCATG in exon 17 was found in proband A. Compound heterozygous mutations g.94865 G to A (Trp856stop) in exon 20 and g.110698_110699delinsG in exon 28 were found in proband B.</p><p><b>CONCLUSION</b>Homozygous insertional mutation g.82888_82889insCATG and compound heterozygous mutations g.94865G to A(Trp856X) and g.110698_110699delinsG probably have respectively induced type 3 von Willebrand diseases in the two probands.</p>

Genotype and function analyses of four inherited dysfibrinogenemia pedigree caused by Arg16 amino acid substitution in fibrinogen Aα chain / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype, genotype and function in four Chinese pedigrees with inherited dysfibrinogenemia.</p><p><b>METHODS</b>Routing tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), the activities of antithrombin (AT), protein C (PC) and protein S (PS) were detected in four pedigrees. The activity and antigen of plasma fibrinogen were analyzed by Clauss and immunoturbidimetry methods, respectively. The molecular weight of fibrinogen of four probands was assessed by Western blot. The function of abnormal fibrinogen was evaluated by fibrinogen clottability, fibrinogen dynamic polymerization and fibrinolysis velocity, respectively. The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>Four probands had prolonged TT and RT, reduced plasma fibrinogen activity levels and normal antigen levels. The assays of Western blot showed no abnormal molecular weight of fibrinogen. Function tests revealed reduced fibrinogen clottability, delayed and decreased fibrinogen dynamic polymerization and reduced fibrinolysis velocity. Aα chain Arg16His and Arg16Cys mutations were identified in the four probands, respectively.</p><p><b>CONCLUSION</b>The four probands with dysfibrinogenemia were caused by the mutations of Aα chain Arg16His or Arg16Cys. Mutation of the fibrinogen induced dysfunction of plasma fibrinogen.</p>

Analysis of phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia.</p><p><b>METHODS</b>Laboratory tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), and the activities of antithrombin (AT:C), protein C (PC:C) and protein S(PS:C) were detected in three pedigrees. The activity and antigen of plasma fibrinogen (Fg) were analyzed by Clauss and immunoturbidimetry methods, respectively. The Fg of three probands was assessed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of all the exons and exon-intron boundaries of the three Fg genes FGA, GFB and FGG were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>Three probands had normal APTT, PT, PC:C, PS:C and AT:C, but prolonged TT and RT. The activity levels of the 3 probands's plasma Fg were reduced, but antigen levels were normal. Western blot and SDS-PAGE showed no abnormal molecular weight of Fg. The 3 heterozygous mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys were identified in the 3 probands, respectively.</p><p><b>CONCLUSION</b>The three probands with dysfibrinogenemia were caused by the mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys, respectively. Both Aα Pro18Leu and Aα Arg16Cys were first reported in Chinese population.</p>

Two novel mutations in one pedigree with hereditary Factor VII deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mutations of coagulation factor VII (FVII) gene in one pedigree with hereditary FVII deficiency, and to investigate the molecular mechanisms of FVII deficiency.</p><p><b>METHODS</b>FVII gene mutations were analysed in the pedigree by direct DNA sequencing. The mutated DNA fragments were cloned into pMD19-T simple TA vector, and sequenced to confirm their distribution on chromosome. The plasma activity of FVII of the probands and their family members was detected with coagulation assay. The antigen of FVII were identified with ELISA.</p><p><b>RESULTS</b>Three gene mutations were detected in the pedigree: A/G to C at 15386 resulting in Arg353Pro/Gln353Pro, A to T at 15274 resulting in Lys316Stop, all three mutations were heterozygotes. Three kinds of polymorphisms were identified in his father: A to G transition at position 15386 resulting in Arg353Gln, heterozygotic deletion of 2050 - 2059 cctatatcct in promoter and G to A mutation in intron 1a, the same polymorphisms were found in his grandfather. The three polymorphisms were located in the same chromosome of his father.</p><p><b>CONCLUSION</b>Two mutations were found in the pedigree with hereditary FVII deficiency. One is nonsense mutation (Lys316Stop), the other is missense one (Gln353Pro). Gln353Pro and Lys316Stop might be the molecular mechanisms of FVII deficiency. The two novel mutations were reported for the first time in the literature.</p>

Molecular analysis of a patient with hemophilia A caused by FVIII His99Arg mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FVIII activity (FVIII: C).</p><p><b>METHODS</b>FVIII: C was detected by chromogenic and one-stage methods, and FVIII: Ag by ELISA. The APTT corrected test was used to screen the FVIII inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FVIII mutant expression plasmids His99Arg and His99Ala (pRC/RS V - BDhFVIIIcDNA) were constructed and transfected into HEK293T transiently. FVIII: Ag and FVIII: C of the expression products were assayed.</p><p><b>RESULTS</b>The proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C <1% and no FVIII inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive (CRM+) hemophilia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H (His) to R (Arg) missense mutation and the same heterozygous was identified in his mother. In vitro expression of FVIII: Ag and FVIII: C of His99Arg were 180.0% and 5.8% , respectively, while FVIII: Ag and FVIII: C of His99Ala were 45.0% and 20.0% of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively.</p><p><b>CONCLUSION</b>Both the two F VIII mutations could express FVIII protein. However, CRM His99Arg mutant protein has little FVIII procoagulant activity and His99Ala has reduced FVIII function by routine methods.</p>

A single E726Q mutation in the membrane proximal α-helix of integrin β3 subunit induces membrane blebbing by disrupting the membrane-actin cortex interaction / 中国实验血液学杂志

The membrane proximal α helix of integrin β subunit cytoplasmic tails plays an important functional role by interacting with various intracellular proteins, namely talin, α-actinin or skelemin. This study was designed to investigate the functional role of 5 highly conserved charged amino acids (R(724), K(725), E(726), E(731), E(733)) within this α helix by site-directed mutagenesis. The result showed that CHO cells expressing the αIIbβ3E726Q mutant had the most prominent phenotype and characterized by defective cell spreading on immobilized fibrinogen. In addition, this E726Q mutation induced membrane blebbing in cells adherent on fibrinogen, and this blebbing could be inhibited by the myosin light chain ATPase inhibitor blebbistatin. It is concluded that the membrane proximal α-helix of integrin β3 subunit is important in linking the phospholipid membrane to the submembraneous actin cortex.

Molecular mechanisms of recurrent venous thrombosis in two pedigrees with type I antithrombin deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin (AT) deficiency.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Phenotype and genotype analysis of three Chinese pedigrees with von Willebrand disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism.</p><p><b>METHODS</b>Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor (vWF): ristocetin cofactor (RCof) (vWF:RCof), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB), vWF and Factor VIII (FVIII) binding assay (vWF:FVIII:B) and multimer analysis were used for phenotype diagnosis. Genomic DNA was extracted from the peripheral blood (PB). All the 52 exons and flanking sequences of the probands' vWF gene were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>APTT were prolonged in all three probands, while BT were normal excepting for proband 3. Plasma RIPA, vWF:RCo, vWF:Ag, vWF:A and vWF:CB were decreased in different extents. In multimer analysis, proband 3 lost the large and intermediate molecular weight multimers, while proband 1 and 2 were normal. Gene analysis in the three probands revealed three heterozygous missense mutations of 144067 G→A (R2287Q) in exon 39, 110374G→A (R1374H) and 110770C→T (S1506L) in exon 28 and heterozygous polymorphism 110667G→A (D1472H) in exon 28, respectively.</p><p><b>CONCLUSION</b>The three heterozygous mutations (R2287Q, R1374H and S1506L) and an heterozygous polymorphism (D1472H) are genetic defects of the hereditary vWD of the three pedigrees respectively. R2287Q is a novel mutation reported for the first time in the literature.</p>

Two new mutations of AT gene in type I inherited antithrombin deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the clinical phenotype and gene mutation in two kindreds with type I inherited antithrombin (AT) deficiency.</p><p><b>METHODS</b>The coagulation and anticoagulation testing and thrombophilia screening were used for phenotypic diagnosis and immunonephelometry and chromogenic assay for plasma level of AT antigen (AT:Ag) and AT activity (AT:A), respectively. All of the seven exons and intron-exon boundaries and untranslation regions of AT gene were amplified by PCR, and the PCR products analysis was by direct sequencing. The corresponding gene sites of the two family members and healthy individuals were detected according to the gene mutation sites.</p><p><b>RESULTS</b>The plasma levels of AT:Ag of proband 1 and proband 2 were 126 mg/L and 117 mg/L, and AT:A was 49% and 48%, respectively. Heterozygotic deletion of 3239-3240delCT in proband 1 and nonsense mutation 3206A-->T (K70Stop) in proband 2 were rchaacterized in exon 2 of AT gene. And some of their family members were also detected with the heterozygotic gene mutation.</p><p><b>CONCLUSION</b>Type I inherited antithrombin deficiency of the two probands were caused by AT gene mutation 3239-3240delCT and 3206A-->T (K70Stop).</p>

Analysis of phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V (FV) deficiency.</p><p><b>METHODS</b>The tests of activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) were used for phenotype diagnosis. All the exons and exon-intron boundaries of F5 gene were amplified by PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>The APTT and PT in each of the four probands were obviously prolonged, and both activity and antigen of FV in the four probands were extremely lower compared with that of normal mixed plasma. Sequencing of F5 gene in proband 1 identified a heterozygous mutation, G16088C (Asp68His), and four polymorphisms, T35788C (Met385Thr), A47295G (His1299Arg), A58668G (Met1736Val) and A74083G (Asp2194Gly), which were located in the same chromosome; proband 2 was homozygous for two mutations, C46253T (Arg952Cys) and C46724T(Gln1109stop); the F5 gene of proband 3 showed a homozygous missense mutation, C67793G(Pro2006Ala); and proband 4 was homozygous for one missense mutation, C74022T (Arg2174Cys).</p><p><b>CONCLUSION</b>Five mutations (Asp68His, Arg952Cys, Gln1109stop, Pro2006Ala and Arg2174Cys) and four polymorphisms (Met385Thr, His1299Arg, Met1736Val and Asp2194Gly) may lead to type I inherited FV deficiency for these four probands, respectively. Gln1109stop, Pro2006Ala and Arg2174Cys haven't been identified before.</p>

Study of signal transduction mediated by integrin alphaIIbbeta3 using a dominant negative model / 中国实验血液学杂志

This study was purposed to investigate the role of integrin beta3 cytoplasmic domain in signal transduction mediated by integrin alphaIIbbeta3 and to explore the effect of integrin beta3 on signal transduction and specificity in condition without alphaIIb subunit. The fusion protein (Tac/beta3) was stably expressed in CHO cell line expressing GPIbIX, integrin alphaIIbbeta3 (IbIX/IIbIIIa-CHO cell line) by combining extracellular and transmembrane domains (Tac) of IL-2 receptor with integrin beta3 cytoplasmic domain (beta3) for formation of fusion protein (Tac/beta3). Then a series of tests were performed, including spreading and stable adhesion of IbIX/IIbIIIa-CHO cell line in solid phase fibrinogen (Fg), fibrin clot restriction and soluble fibrinogen binding, which represent outside-in and inside-out signal transduction events. The results showed that the bidirectional signal transduction mediated by alphaIIbbeta3 in IbIX/IIbIIIa-CHO/Tac-762 cells stably expressing Tac/beta3 was seriously inhibited. It is concluded that the Tac/beta3 can play a significant role in IbIX/IIbIIIa-CHO/Tac-762 cells through a dominant negative mode, the independent presence of beta3 subunit cytoplasmic domain can regulate the bidirectional signal transduction mediated by integrin alphaIIbbeta3.

Analysis of clinical features and genotype in three Chinese pedigrees with Glanzmann thrombasthenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the clinical feature and alpha II b beta 3 gene mutations of three Glanzmann thrombasthenia (GT) pedigrees.</p><p><b>METHODS</b>Platelet counts (BPC), blood film, bleeding time, platelet aggregation and flow cytometry were used for phenotype diagnosis of all the patients. All the exons of alpha II b and beta 3 genes were amplified by polymerase chain reaction (PCR) and direct sequencing was performed for mutational screening. One hundred and three healthy blood donors were as normal controls.</p><p><b>RESULTS</b>Three probands showed normal BPC, defective platelets aggregation, prolonged bleeding time and significantly reduced platelet aggregation to ADP, epinephrine, and collagen, while relatively normal aggregation to ristocetin. Flow cytometry showed platelet surface expressed alpha II b beta 3 was strongly reduced in proband 1 and proband 3 and mildly reduced in the amount of surface expressed alpha II b beta 3 (63%) in proband 2. Sequencing results showed that proband 1 had a G10A homozygous mutation in alpha II b, and a G1412T homozygous mutation in beta3. Compound heterozygous mutations in beta3, G1199A and 1525delC were identified in proband 2. No mutations in alpha II b beta 3 gene were identified in proband 3.</p><p><b>CONCLUSIONS</b>Compound homozygous mutations, GI0A in alpha II b and G1412T in beta3, lead to GT in proband 1. Compound heterozygous mutations in beta3, G1199A and 1525delC, lead to GT in proband 2. The mutations of G10A, G1412T and 1525delC were reported for the first time in GT patients.</p>