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To explore the effects of the rathHC745, hHC1690, humanIn 293T cells, the coagulation activity of FVIII was significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC. The FVIII activity of hHC745+rLC was about 4.6 times higher than that of hHC745+hLC, while hHC1690+rLC was about 2.9 times higher than that of hHC1690+hLC. The data from ELISA showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.1 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 2.8 times compared with hHC1690+hLC. In HA mice administrated with hydrodynamic injection, the FVIII activity of hHC745+rLC and hHC1960+rLC was significantly higher than that of hHC745+hLC and hHC1690+hLC at comparable expression level. The FVIII activity of hHC745+rLC was significantly higher than that of hHC745+hLC, increasing to about 5.1 times, while, that of hHC1690+rLC increasing to about 2.1 times than hHC1690+hLC. ELISA results also showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.2 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 1.8 times compared with hHC1690+hLC. In addition, the activity of hHC1690 combined with rLC was significantly higher than that of hHC745 combined with rLC.rLC can significantly enhance the coagulation activity of FVIII when co-expressed with hHC of different length including hHC745 and hHC1690.腺相关病毒载体血清型8递送的大鼠研究大鼠将hHC745、hHC1690、人在293T细胞中，hHC745和hHC1690与rLC共表达时的FVIII活性显著高于与hLC共表达时的活性，分别提高到约4.6倍和2.9倍；ELISA结果表明hHC745和hHC1690与hLC和rLC共表达时的FVIII抗原表达无统计学差异；根据aPTT测得的FVIII活性与ELISA测得的FVIII抗原表达量计算的比活性结果显示，hHC745与rLC共表达时的比活性提高到与hLC共表达时的比活性的约4.1倍，而hHC1690与rLC共表达时的比活性提高到与hLC共表达时的约2.8倍。在高压注射的HA小鼠中，hHC745和hHC1690与rLC共表达时的FVIII活性显著高于与hLC共表达时的活性，活性分别提高到约5.1倍和2.1倍；ELISA结果同样显示，hHC745和hHC1690与hLC和rLC共表达时FVIII抗原表达无统计学差异；比活性结果表明，hHC745和hHC1690与rLC共表达时的比活性较与hLC共表达时的比活性显著提高，分别提高到约4.2倍和1.8倍；hHC1690与rLC共表达时的FVIII活性显著高于hHC745与rLC共表达时的活性。.rLC可以显著提高不同长度

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset–dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

Lymphadenopathy is an important characteristic of POEMS syndrome, and a Castleman disease (CD)-like pathologic change in the lymph nodes is one of the major diagnostic criteria. However, the characteristics of lymphadenopathy in POEMS still have not been completely elucidated. The lymph node biopsies are available only for a small proportion of patients. A simple and safe way is needed to rule CD in or out. This study aimed to analyse the features of lymphadenopathy and estimate the role of imaging methods, including computed tomography (CT) and positron emission tomography-CT (PET/CT), in the diagnosis of lymphadenopathy in patients with POEMS syndrome. We conducted a retrospective analysis of 23 patients with confirmed POEMS syndrome. All of the patients received chest and abdominal CT scan and/or superficial ultrasound examinations. Four patients underwent PET/CT examinations, and 6 patients received lymph node biopsies. Enlarged lymph nodes (short diameter ≥ 1 cm) were found in 48% (11/23) of patients, but only 1 patient had an enlarged lymph node with a diameter ≥ 2 cm. Lymph nodes with CD-like pathologic changes from 2 patients showed increased maximum standard uptake values (SUV(max)) of (18)F-deoxyglucose ((18)FDG) on PET/CT, while lymph nodes with reactive pathologic changes from 2 other patients showed a normal metabolic PET/CT profile. The extent of lymph node enlargement in patients with POEMS was less than that in patients with CD per se. We draw the conclusion that most of the enlarged lymph nodes had diameters ≤ 2 cm, which is less than that in cases of CD per se and PET/CT may be helpful in determining whether enlarged lymph nodes are characterized by CD-like changes or not.

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.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.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.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.

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).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.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.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.

An efficient and convenient procedure for direct esterification of alkyl and aryl silyl ethers with Ac2O and a catalyst system of perchloric acid immobilized on a silica gel (HClO4–SiO2) has been developed. The silyl protecting groups are directly replaced by acetyls and the protecting groups themselves are transformed into acetates as the sole byproducts, which can be readily recovered and converted back to silylchlorides, the original protecting agents, thus minimizing wastes.

A series of novel 2',3'-diethanethio-2',3',5'-trideoxy-5'-triazoloribonucleosides was synthesized in excellent yields and their antitumor activity was evaluated. These nucleoside analogues with aromatic substituted triazole rings showed significantly improved activity towards a broad range of tumor cell lines and those without arene substitutes were inactive.

Thioacetalization of acyl protected furanosides led to products with an ethanethiol group at C-2 and 3-O-acetyl- 1,2-di-O-isopropylidene-D-furanoses were converted into corresponding thioacetals with two ethanethiol groups at both C-2 and C-3 positions under the standard thioacetalization conditions. All products were characterized by 1H NMR, 13C NMR and HRMS data. X-ray structure analysis indicates that the vicinal acyloxy group is stereoselectively substituted by ethanethiols. The supposed mechanisms for these two kinds of transformations were presented.

Mutational analysis has established that the cytoplasmic tail of the integrin β3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin β3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin β3 and dose-dependently inhibited the purified recombinant β3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the β3 cytoplasmic tyrosines, Y747 and Y759, was inhibited by myr-RGT. These data indicate an important role for β3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with β3 and selective blockade of integrin αIIbβ3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.

The transformation of acetonides into the corresponding diacetates is often required in the synthetic chemistry. An efficient procedure for direct conversion of acetonides into diacetates in the presence of Bi(OTf) 3 · x H 2 O under mild conditions has been developed. Primary hydroxyl-acetonides could be selectively transformed into diacetates in the presence of anomeric acetonides and the anomeric acetonides could be tunably converted into 2-acetoxyisopropyl or diacetate groups.

Aim: The molecular mechanisms of the affinity regulation of αvβ3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of αvβ3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-function within these domains remains unclear. Methods: The extracellular and transmembrane domain of αIIb was fused to the αv integrin cytoplasmic domain, and the chimeric α subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type β3 subunit or with 3 mutant β3 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen. Results: All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric α subunit with the wild-type β3 subunit, but not those with truncated β3, could adhere to and spread on immobilized fibrinogen. Conclusion: The substitution αIIb at the cytoplasmic domain with the αv cytoplasmic sequence rendered the extracellular αIIbβ3 a constitutively activated conformation for ligands without the need of “inside-out” signals. Our results also indicated that the COOH-terminal sequence of β3 might play a key role in integrin αIIb/αvβ3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing αIIb/αvβ3 have enormous potential for facilitating drug screening for antagonists either to αvβ3 intracellular interactions or to αIIbβ3 receptor functions.

OBJLECTIVE: To investigate the effect of integrin β3 cytoplasmic NITY motif on αIIbβ3-mediated cell functions.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.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.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.

To study the effect of interaction of the talin rod domain integrin binding site 2 with integrin β3 on platelet signal transduction.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.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.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.

POEMS syndrome is a rare plasmacyte-associated disease, one of the major diagnostic criteria of which is sclerotic bone lesion. To detect bone lesions in POEMS syndrome, which imaging method should be routinely applied and what characteristics they display are still unconfirmed. We analyzed clinical data and imaging characteristics of bone lesions in 22 patients with POEMS using multimodal methods, including conventional X-ray, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography/computed tomography (PET/CT). Images on X-ray and CT exhibited plaque-like high-density for osteosclerotic lesions and punched-out low-density appearance for osteolytic ones. X-ray had advantage in detecting bone lesions in skull, extremity long bones, clavicle, and scapula, while CT could display sharp outline of lesions and was more sensitive than X-ray in detecting the small lesions. Osteosclerotic lesions on MRI demonstrated decreased signal intensity on both T1 and T2-weighted sequences, while osteolytic lesions or osteolytic part of mixed lesions showed high signal intensity on T2-weighted sequences. MRI had same sensitivity as CT, but with superiority in distinguishing the active osteolytic lesions from the osteosclerotic ones. PET-CT showed (18)F-FDG uptake was normal in the majority of osteosclerotic lesions, and slightly increased in mixed ones, but obviously elevated in osteolytic ones. PET/CT was less sensitive in detecting osteosclerotic lesions than in detecting osteolytic ones. In conclusion, to detect bone lesions in POEMS, conventional X-ray scan should be first performed, further followed by more sensitive CT or MRI. PET-CT is optional when the osteolytic lesions are suspected.

To investigate the binding mechanisms of FVIII Trp1707Ser mutation-associated inhibitor.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.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.97223CG 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.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.

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.

To investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees.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.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.5876TC (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.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.目的 对两个遗传性异常纤维蛋白原血症家系的突变纤维蛋白原（Fg）进行功能研究。方法 常规筛查凝血功能;Fg抗原和活性分别用免疫比浊法和Clauss法测定;抽提DNA,对Fg 3个基因（FGA、 FGB和FGG）以及抗凝血酶基因（AT3）所有外显子及侧翼序列进行PCR扩增、测序及分析;采用常规血栓弹力图（TEG）和功能性Fg TEG检查对家系B先证者及其父亲进行凝血功能的综合评价及血浆功能性Fg评估;应用Western blot 检测血浆Fg肽链分子量;采用Fg动态聚集曲线和纤维蛋白溶解曲线实验检测血浆Fg的功能。结果 2例先证者凝血酶原时间（TT）和爬虫酶凝固时间（RT）明显延长,Fg活性仅为0.5 g/L和0.6 g/L,但其抗原均正常,分别为2.32 g/L和2.66 g/L。两个家系先证者均存在γ链Arg275His杂合突变,家系B先证者的祖父和姑母同时检出AT3 g.5876TC (Ser116Pro)杂合突变。家系B先证者及其父亲TEG检测结果中α值分别接近和低于正常参考值范围下限,但最大波幅（MA值）均为正常;在功能性Fg TEG检测中,MA值明显偏低。Fg动态聚集曲线中先证者和家系患者的起跳时间明显延长、峰值明显降低。纤维蛋白溶解曲线中多数患者的纤维蛋白在特定时间内不能被纤溶酶原完全溶解。结论 首次发现遗传性异常纤维蛋白原血症合并AT缺陷的患者。γ链 Arg275His突变使Fg在纤维蛋白单体聚合以及纤维蛋白溶解方面出现异常。联合应用常规TEG和功能性TEG检测,可以更好地评估异常纤维蛋白原血症患者Fg的功能。

To analyze the phenotype and genotype of two Chinese pedigrees with von Willebrand diseases, and to investigate the molecular pathogenesis.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.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.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.

To analyze the phenotype, genotype and function in four Chinese pedigrees with inherited dysfibrinogenemia.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.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.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.

To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin (AT) deficiency.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.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.13863CA(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.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.

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).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.The proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C1% 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.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.

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.

The fluoride-free one-pot method is environmentally friendly since the silyl protecting groups are transformed into acetates as the sole byproducts, which are readily recoverable and converted back to silyl chlorides as protecting agents by treatment with conc.

To analyze the phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia.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.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.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.

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.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.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.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.

To analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism.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.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.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.

A series of novel triazoloribonucleosides (VII) is synthesized by the route described for the preparation of derivative (VI) and evaluated for antitumor activity.

To identify the clinical phenotype and gene mutation in two kindreds with type I inherited antithrombin (AT) deficiency.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.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.Type I inherited antithrombin deficiency of the two probands were caused by AT gene mutation 3239-3240delCT and 3206A--T (K70Stop).

To identify the phenotype and genotype in four Chinese pedigrees with inherited coagulation factor V (FV) deficiency.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.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).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.

To prepare anti-c-Kit monoclonal antibodies and characterize their specificity of epitope recognition.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.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.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.

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.

The transformation of acetonides into acetates is frequently required in synthetic chemistry. An efficient procedure for direct conversion of acetonides into acetates in the presence of HClO(4)-SiO(2) under mild conditions was developed. The acetonides of primary hydroxy groups are directly converted to diacetates, and the anomeric acetonides of furanosides are stereoselectively transformed into the corresponding acetyl beta-d-furanosides with a 2-acetoxyisopropyl group.

To explore the thrombin generation capacity in patients on warfarin therapy with different prothrombin time international normalized ratio (PT-INR), the capacity in relation to bleeding, and the application of thrombin generation tests to warfarin therapy monitoring.Seventy eight blood samples were taken from patients on warfarin therapy for more than 3 months owing to valve replacement or atrial fibrillation. The patients' case history and PT-INR were collected and thrombin generation tests were performed in all samples.Patients were ranked into three groups according to different PT-INR. There were 23 patients in group I with PT-INR from 1.51 to 2.00, 39 patients in group II with PT-INR from 2.01 to 3.00, and 16 patients in group III with PT-INR from 3.01 to 4.26. There were significant differences between each two of the three groups in lag time, peak, and ttpeak (time to peak) (P0.01). There was a significant difference between group I and group II in endogenous thrombin potential (ETP) (P = 0.0001), but not between group II and group III (P= 0.06). Five patients developed bleeding and their ETP was less than 15% of normal control.In patients on warfarin therapy, when the PT-INR was more than 3.0, increasing the dose of warfarin doesn' t decrease the thrombin generation, but increase bleeding risk. PT-INR combined with ETP may better reflect patient's coagulation status, therefore be of more significance in preventing bleeding.

To study the clinical feature and alpha II b beta 3 gene mutations of three Glanzmann thrombasthenia (GT) pedigrees.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.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.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.

The molecular mechanisms of the affinity regulation of alphavbeta3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of alphavbeta3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-function within these domains remains unclear.The extracellular and transmembrane domain of alphaIIb was fused to the alphav integrin cytoplasmic domain, and the chimeric alpha subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type beta3 subunit or with 3 mutant beta3 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen.All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric alpha subunit with the wild-type beta3 subunit, but not those with truncated beta3, could adhere to and spread on immobilized fibrinogen.The substitution alphaIIb at the cytoplasmic domain with the alphav cytoplasmic sequence rendered the extracellular alphaIIbbeta3 a constitutively activated conformation for ligands without the need of pinside-outq signals. Our results also indicated that the COOH-terminal sequence of beta3 might play a key role in integrin alphaIIb/alphavbeta3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing alphaIIb/alphavbeta3 have enormous potential for facilitating drug screening for antagonists either to alphavbeta3 intracellular interactions or to alphaIIbbeta3 receptor functions.

Overexpression of urokinase-type plasminogen activator receptor (uPAR) on tumor cell surface is essential for invasion and metastasis in a variety of tumor cells. To establish a retroviral-mediated antisense RNA transfer system of uPAR gene for exploring its function on down-regulation of uPAR expression in leukemia cells, the retroviral vector LaCD87SN was constructed by inserting uPAR gene into LXSN vector in an antisense orientation. An uPAR gene antisense RNA transfer system was established by liposome-mediated transfection in combination with cross infection with retrovirus. Human leukemia cells U937 were transduced with aCD87 amphotropic retrovirus, expressing uPAR antisense RNA, and the U937/aCD87 cells was obtained by G418 selection. The integration and expression of antisense uPAR gene were analyzed by polymerase chain reaction (PCR) assay. The cell surface expression of CD87 and the activities of matrix metalloproteinase (MMP) were assayed by flow cytometry (FCM) and gelatin zymography, respectively. The results showed that the amphotropic retroviral producers Am12/aCD87, which expressed antisense RNA of uPAR gene with a titer of 6.3 x 10(5) cfu/ml in supernatants, were obtained by means of transfection and superinfection. U937/aCD87 cells were established by continuative G418 selection after retrovirus infection. In U937/aCD87 cells, the integrated provirus and the overexpression of antisense uPAR gene was confirmed. Compared with U937/NeoR cells, FCM analysis revealed that CD87 expression on U937/aCD87 cell surface was not downregulated significantly. However, MMP-9 secretion was significantly suppressed in U937/aCD87 cells. In conclusion, although the retroviral-mediated antisense RNA transfer could not efficiently suppress uPAR expression on leukemic cell surface, it may interfere the uPAR-MMP interactions.