Objective To analyze the screening results of von Willebrand factor among patients before blood transfusion in Ruijin Hospital and discuss von Willebrand factor in ABO blood group and the relationship between age and gender,refine the classification of vWF antigen and activity by reference factors. Methods The von Willebrand factor among 247 cases of patients before blood transfusion in Ruijin Hospital with no clinical manifestations of abnormal blood clots and routine coagulation as laboratory tests for normal surgical patients. The vWF: Ag and vWF: Act were measured by immune turbidimetric method and ABO blood group was identified by blood type serology.Furthermore,the differences between A,B,O,AB different blood groups,sex and high( ≥40 years) and low age group( <40 years) were compared by statistical methods. Results The levels of vWF: Ag in different blood groups were as follows: A blood type: 98. 5-142. 00,B blood type: 97. 90-160. 30,O blood type: 82. 13-125. 45,AB blood type: 103. 00-135. 80.The levels of vWF: Act in different blood groups were as follows: A blood type: 76-130. 14,B blood type: 78. 06-144. 3,O blood type: 60. 89-116. 11,AB blood type: 88. 99-124. 09.O blood type vWF: Ag and vWF: Act were lower significantly( P<0. 05) than non-O blood type,the difference was.Besides,young vWF: Ag and vWF: Act were lower significantly than in the elderly. There was no significant difference in vWF: Ag and vWF: Act levels between male and female groups. At last,the reference range of four groups of vWF activity( antigen) was obtained.Conclusion Plasma vWF antigen and activity levels were significantly affected by ABO blood type and age,and the refined reference range established for these influencing factors was beneficial for more detailed diagnosis of VWD and predicting vWF levels associated with bleeding and thrombosis risk.

Objective To study the procoagulant property of tissue factor-positive microparticles(TF+MP) generated in vitro through the culture and stimulation of cell line. Methods Human acute monocytic leukemia cell line(THP-1) was stimulated by different concentrations of lipopolysaccharide(LPS). Microparticle(MP)was separated by gradient centrifugation. Thrombin generation abilities of tissue factor(TF) and phosphatidylserine(PS) were determined by thrombin generation test by adding MP into MP-depleted plasma. ZYMUPHEN MP-TF kit was performed to determine TF procoagulant property. The 0.45 and 0.65 μm filters were selected to determine MP procoagulant properties(< 0.45 and < 0.65 μm). Results Both thrombin generation test and chromogenic substrate test demonstrated a procoagulant property of MP generated in vitro. Thrombin generation test further showed that the procoagulant property of MP increased by the addition of LPS in a concentration-dependent manner. MP > 0.65 μm had a strong procoagulant property. Conclusions MP generated in vitro through the culture and stimulation of THP-1 demonstrates certain procoagulant property.

Objective To attempt to use thromboelastography (TEG)reaction time (R)to monitor the clinical use of low molecular weight heparin (LMWH).Methods A total of 20 hospitalized patients from nephrology department with nephritic syndrome were enrolled.Blood samples were collected at 4 h after both firstly and secondly receiving dose in order to perform TEG and anti-Xa activity test.TEG R and anti-Xa activity were determined.The associations between TEG R and anti-Xa activity,between the dose of taking drugs at first time and anti-Xa activity,and between the dose of taking drugs at first time and TEG R,were analyzd.The comparative analysis of anti-Xa activity with different doses of taking drugs was performed.The comparative analysis of TEG R with different doses of taking drugs was performed.Results There was no association between TEG R and anti-Xa activity (P=0.54,R=-0.1 44 9).There was no association between anti-Xa activity and the dose of taking drugs at first time (P=0.26,R=-0.263 6).There was obvious association between TEG R and the dose of taking drugs at first time (P<0.000 1 ,R=0.875).There was difference in anti-Xa activity between the 2 doses of taking drugs (P=0.000 5,t =4.152).There was significant difference in TEG R between the 2 doses of taking drugs (P<0.000 1,t=15.19).Conclusions TEG R reflects patients′condition of using LMWH much more accurately than anti-Xa activity.

目的 :评价STA-Liatest纤维蛋白（原）降解产物[fibrin（ogen）degradation products,FDP]试剂在STA-R Evolution全自动血凝仪上的性能。方法 :选择质控血浆、患者血浆及健康成人血浆样本总共360份,在全国范围内按不同地域分布选取了6家医院进行多中心同步实验检测。分别使用STA-Liatest FDP和N-FDP,在STA-R Evolution全自动血凝仪上行FDP检测,检测结果参考美国临床实验室修正法规1988（CLIA88）标准要求进行统计分析。结果:STA-Liatest FDP多中心总的批内重复性,变异系数分别为8.0%（质控水平1）和4.7%（质控水平2）;与N-FDP的相关系数为0.718（P＜0.05,FDP＜5μg/m L）和0.983（P＜0.01,FDP≥5μg/m L）;稳定性验证中,同一瓶试剂连续使用3 d的重复性,变异系数≤11.5%。结论 :STA-Liatest FDP的上述性能指标符合临床实验室的质量要求,可为临床提供可靠的检测结果。同时,为保证检测系统的溯源性,建议实验室尽可能使用配套试剂,以最大限度保证检验结果的可靠性。

<正>目的对5个遗传性高分子量激肽原(HMWK)缺陷家系进行表型诊断及基因型分析,探讨其分子发病机制。方法测先证者及家系成员活化部分凝血活酶时间(APTT),1期法检测内源途径相应凝血因子(FⅧ、FⅨ、FⅪ、FⅫ)活性,vWF活性。用1例携带Null突变的高分子量激肽原患者的

Objective To disclose the molecular mechanisms of large deletion in the F8 gene in a sever haemophilia A patient and to screen the potential carriers.Methods Coagulation assay was performed to establish the phenotype.LD-PCR and sequence-specific PCR were adopted for detecting intron 22 and intron 1 inversion of F8 gene.All the exons of F8 gene and its flanking sequences were amplified and sequenced directly.Deletion breakpoints were identified by primer walking strategy.Breakpoint-specific assays were performed for the identification of carriers.Multiplex fluorescent PCR was used to detect the polymorphism of six STR locus (F8Up226,F8Up146,F8Int13,F8Int25,F8Down48 and DXS1073) for gene linkage analysis.Results The proband had a significantly reduced activity of factor Ⅷ (＜ 1％).Inversion of intron 22 and intron l showed negative results in the proband.Deletion of exons 4-7 in the F8 gene in the proband was suspected by the repeated failure to amplify the corresponding exons by PCR.Sequencing the products of prime walking showed that the patient had a deletion of 27 685 bp comprising exons 4-7,a 2-bp microhomology (AT) was observed at the breakpoint junction.The results of breakpoint-specific PCR showed that the fetal and the proband's daughter were deletion carriers,consistent with the results of the genetic linkage analysis.Conclusions The deletion of exons 4-7 in the F8 gene results in a sever haemophilia A.Microhomology-mediated end-joining mediates the formation of detected deletion.The breakpoint-specific PCR provids a reliable diagnostic tool for identification of carrier status.

<正>目的探讨高同型半胱氨酸血症伴其他血栓危险因素血栓患者的发病机制。方法详细调查患者血栓发生史及家系情况,对6例患者及家系成员进行常规凝血项目及各项易栓症指标筛查。其中易栓症筛查指标包括有抗凝血酶(AT)、蛋白C(PC)、蛋白S(PS)、抗心磷脂抗体(ACA)、狼疮抗凝物质(LA)、凝血因子Ⅷ活性(FⅧ:C)、α2纤溶酶抑制物(α2-AP)及同型半胱氨酸(Hcy)等。同时通过化学发光法检测血清中Hcy代谢相关辅因子叶酸(FA)和VitB12含量,以

Objective To investigate the molecular mechanism of super-instability of FⅧ and discrepancy of onestage and two-stage FⅧ∶ C assay in a mild hemophilia A patient with His99Arg mutation. Methods Coagulation function was assayed for establishing phenotype diagnosis. FⅧ∶ C was detected by one stage method and chromogenic method. Patient's plasma was incubated at routine storage temperature( 37℃,25℃,4℃,- 20℃,- 80℃) and aliquots were taken to assess the FⅧ∶ C at the indicated times. One-stage method and ELISA were applied to measure activity and amount of remaining FⅧ when the plasma was incubated at 56℃ at different time. LD-PCR and sequence specific PCR were adopted for detecting intron 22 and intron 1 inversion of F8 gene. All the exons of F8 gene and its flanking sequences were amplified and sequenced directly. Three-dimensional structure analysis was performed by PyMOL program to analyze the impact of His99Arg mutation on the spatial structure of FⅧ. The B-domainless FⅧ mutant expression plasmid His99Arg was constructed and transfected into HEK293T,the stability of the stable expression products were assayed. Results The FⅧ∶ C of the proband was 14. 7% detected by one-stage method but just 1. 2% measured by chromogenic method. The activity of mutant FⅧ exhibited significantly increased rates of loss of activity when the plasma incubated at routine storage temperature. Furthermore,the activity of FⅧ His99Arg was less stable following heat denaturation analysis,this reduced stability appeared to result from an about 300% increase in the dissociation rate judged by ELISA. Gene analysis revealed a A30716G substitution in exon 3 resulting in a His99Arg missense mutation. Three-dimensional structural analysis showed that His99 residue closed to His1957( 3. 49) and Ser1959( 3.42) of A3 subunit,when His99 mutated into Arg99,the distance changed into 4. 19 and 2. 39,respectively. The FⅧ activity of stable expression products showed similar decrease trend in accordance with that of patient' s FⅧ. Conclusion His99Arg mutation caused changes in the spatial structure of FⅧ and increased the dissociation rate of the heavy chain and light chain. His99Arg mutation resulted in instability of FⅧ∶ C and discrepancy of one-stage and two-stage FⅧ∶ C assay.

易栓症包括遗传性和获得性.获得性易栓症继发于手术创伤、制动、口服避孕药、母体免疫性疾病、恶性肿瘤等,遗传性易栓症主要是由蛋白C(PC)、蛋白S(PS)和抗凝血酶(AT)缺陷导致.AT缺陷的发病率为1/5000～1/500,病情较为严重.遗传性PC缺陷为常染色体显性遗传病,杂合子型PC缺陷症患者多在30～40岁发病,有很大一部分杂合子型不发病,纯合子和复合杂合较罕见.PS缺陷为常染色体显性遗传,杂合子型60％ ～ 80％患有深静脉血栓(VTE),发病年龄多在40～45岁[1].本研究对我院2010至2012年收治的6例AT、PC和PS缺陷的患者及其家系进行血栓发生及相关研究.

Objective: To investigate the clinical phenotype,genotype and molecular mechanisms of a Chinese pedigree with type Ⅰ antithrombin（AT） deficiency.Methods Routine coagulation screening tests were performed,and thromboelastogram test was done to evaluate the hypercoagulation.Anticardiolipin antibody（ACA） and anti-β2 glycoprotein Ⅰ（anti-β2GPI） were detected with enzyme-linked immunosorbent assay（ELISA）,and lupus anticoagulant（LA） was assayed by diluted viper venom time assay（DVVT）.The activities of antithrombin（AT:A） and protein C（PC:A） were detected using the chromomeric substrate method,and the activity of protein S（PS:A） was assayed by a clotting method.Total homocysteine（Hcy） levels were determined by fluorescence polarization immunoassay（FPIA）.AT antigen（AT:Ag） was measured with immunoturbidimetry method and Western blot analysis was used to analyze the molecular weight（MW） and plasma level of AT:Ag.Polymerase chain reaction（PCR） amplification of all exons and intron-exon junction regions of AT gene followed by direct sequencing was performed for the proband.Ectopic transcription study was performed for novel splice site mutation.Total mRNA was isolated from peripheral leukocytes of the proband and the ectopic transcripts were analyzed by nested polymerase chain reaction after reverse transcription（RT-PCR）.Results The results of routine coagulation tests were normal,thromboelastogram test indicated hypercoagulable state in the proband.The levels of ACA,anti-β2GPI,LA,PC:A,PS:A,and Hcy were normal.The AT:A and AT:Ag were decreased equally,being 45% and 145 mg/L respectively.The results of Western blot showed that the proband′s AT MW was normal but antigen level was lower than normal.Heterozygous mutation of g.13776;3777insAGGT was identified in the 3′ splice site of intron 5.The aberrant transcripts from the g.13776;3777insAGGT mutant allele were detected by ectopic transcription study.TA cloning analysis revealed that the ratio of aberrant transcripts to normal transcripts was 1∶5.Conclusions Splice site mutation of g.13776;3777insAGGT produces aberrant mRNA and affects the normal synthesis and secretion of AT proteins,which is responsible for the type Ⅰ AT deficiency in this pedigree.AT g.13776;3777insAGGT mutation is first reported internationally.

Objective To analysis frameshift mutations in poly A stretches of exon 14 in factor Ⅷ(FⅧ) gene.Methods The APTT,PT,TT,Fg,FⅧ:C,vWF:Ag,vWF:Act were tested to make phenotypic diagnosis.LD-PCR was used to detect FⅧ gene intron 22 inversion and two sets sequence specific PCR was adopted to detect F Ⅷ gene intron 1 inversion.The promoter and all 26 extrons of FⅧ gene and its flank sequences were directly sequenced.The AccuCopy method was employed to investigate copy number variations(CNVs).The multiplex PCR was performed to analysis a novel panel of the 6 short tandem repeats(FⅧUp226,FⅧUp146,FⅧInt13,FⅧInt25,FⅧDown48 and DXS1073) and amelogenin locus(the sex-typing marker) for genetic counseling.Results In all the 343 HA families,32 were frameshift mutations in poly A stretches of exon 14 in FⅧ gene(9.33%),which account for 42.11% of small deletions/insertion.In the 32 cases,21 HA families were sporadic,and 10 unrelated hemophilia families were due to de novo mutations.Interestingly,part of the hemophilia A patients manifested as mild to moderately severe phenotype,with the clotting activities of F Ⅷ(FⅧ:C) more than 2%.Conclusions Frameshift mutations in poly A stretches at position g.96418_96425(A8) and g.95674_95683(A9) of exon 14 in FⅧ gene are proved to be the mutation hot spots of HA.Potential mechanism of the high mutability of these regions may be due to polymerase errors or slippage during replication.At the same time,errors in DNA replication or RNA transcription/translation may result in a partial restoration of the correct reading frame,and ameliorate an expected severe phenotype.

Objective To diagnosis a Gilbert′s syndrome patient on the basis of gene,testing her first degree relatives for the relative gene and making an analysis of heredity.Methods By ruling out other related diseases and in accordance with the result of low calorie diet test and characteristic history,a case of Gilbert′s syndrome was diagnosed.Blood samples were collected from the patient and her first degree relatives and the genomic DNA was extracted.Amplified the relative gene UGT1A1 by polymerase chain reaction(PCR) and mutations were identified by direct sequencing.Results The patient′s unconjugated bilirubin(UCB) is higher than normal and lead to a high total bilirubin.The low calorie diet test showed that after taking the low calorie diet,the patient′s UCB was two times higher than before.The patient and her sister had a TA insertion in TATA box of the promoter region as A(TA)7TAA.Their parents are both heterozygote individuals with A(TA)6/(TA)7TAA.Conclusions The TA insertion in the promoter region of the UGT1A1 gene affects the bilirubin metabolism of the patient.The A(TA)7TAA is essential for the pathogenesis of Gilbert′s syndrome and is inherited in an autosomal recessive manner.

Objective: To investigate the clinical phenotype,genotype and molecular mechanisms of a Chinese pedigree with type Ⅰ antithrombin(AT) deficiency.Methods Routine coagulation screening tests were performed,and thromboelastogram test was done to evaluate the hypercoagulation.Anticardiolipin antibody(ACA) and anti-β2 glycoprotein Ⅰ(anti-β2GPI) were detected with enzyme-linked immunosorbent assay(ELISA),and lupus anticoagulant(LA) was assayed by diluted viper venom time assay(DVVT).The activities of antithrombin(AT:A) and protein C(PC:A) were detected using the chromomeric substrate method,and the activity of protein S(PS:A) was assayed by a clotting method.Total homocysteine(Hcy) levels were determined by fluorescence polarization immunoassay(FPIA).AT antigen(AT:Ag) was measured with immunoturbidimetry method and Western blot analysis was used to analyze the molecular weight(MW) and plasma level of AT:Ag.Polymerase chain reaction(PCR) amplification of all exons and intron-exon junction regions of AT gene followed by direct sequencing was performed for the proband.Ectopic transcription study was performed for novel splice site mutation.Total mRNA was isolated from peripheral leukocytes of the proband and the ectopic transcripts were analyzed by nested polymerase chain reaction after reverse transcription(RT-PCR).Results The results of routine coagulation tests were normal,thromboelastogram test indicated hypercoagulable state in the proband.The levels of ACA,anti-β2GPI,LA,PC:A,PS:A,and Hcy were normal.The AT:A and AT:Ag were decreased equally,being 45% and 145 mg/L respectively.The results of Western blot showed that the proband′s AT MW was normal but antigen level was lower than normal.Heterozygous mutation of g.13776_13777insAGGT was identified in the 3′ splice site of intron 5.The aberrant transcripts from the g.13776_13777insAGGT mutant allele were detected by ectopic transcription study.TA cloning analysis revealed that the ratio of aberrant transcripts to normal transcripts was 1∶5.Conclusions Splice site mutation of g.13776_13777insAGGT produces aberrant mRNA and affects the normal synthesis and secretion of AT proteins,which is responsible for the type Ⅰ AT deficiency in this pedigree.AT g.13776_13777insAGGT mutation is first reported internationally.

目的 分析3例遗传性血管性血友病（vWD）患者的表型和基因型，探讨其分子发病机制。方法 采用出血时间（BT）、APTT、瑞斯托霉素诱导的血小板聚集试验（RIPA）、血管性血友病因子（vWF）瑞斯托霉素辅因子活性（vWF∶Rco）、vWF抗原（vWF∶Ag）、vWF活性（vWF∶A）、vWF胶原结合试验（vWF∶CB）和多聚体分析对3例vWD患者进行表型诊断，通过血栓弹力图检测分析全血凝固功能。提取外周血基因组DNA，测序分析vWF基因突变情况。结果 3例先证者APTT、BT均延长，vWF∶Rco、vWF∶Ag、vWF∶A和vWF∶CB均不同程度减低，除先证者A外，先证者B、C血浆RIPA明显减低，多聚体分析结果显示先证者A和C血浆多聚体分布基本正常，先证者B大相对分子质量多聚体缺失。先证者C血栓弹力图检测全血呈明显低凝状态。基因测序发现，先证者A、B和C分别存在26号外显子g.106782G>T（Cys1130Phe）、28号外显子g.110988G>A（Gly1579Arg）和28号外显子g.110373C>T（Arg1374Cys）的杂合错义突变。结论 3例先证者表型诊断分别为1型、2A型和2M型vWD，Cys1130Phe、Gly1579Arg和Arg1374Cys的杂合错义突变分别为3例先证者的致病基础。

Objective To analyze the phenotype and genotype of a Chinese family with inherited hypofibrinogenemia,and to investigate its molecular mechanism.Methods Peripheral blood was collected from seven people of this family and then plasma was separated.Activated partial thromboplastin time ( APTT),prothrombin time ( PT),thrombin time ( TT),reptilase time ( RT),the activities of antithrombin( AT∶ A ),protein C ( PC ∶ A ) and protein S ( PS ∶ A ) were tested.The activity and antigen of plasma fibrinogen were analyzed by Clauss method and immunoturbidimetry method,respectively.The fibrinogen peptide chain of the proband was semiquantitatively assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Thrombin generation test was performed by calibrated automated thromhogram.The dynamic process of blood coagulation was evaluated by the thrombelastography (TEG).Genomic DNA was extracted from the peripheral blood.The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes FGA,FGB and FGG were amplified by polymerase chain reaction ( PCR ) and analyzed by direct sequen(c)ing.Results The activity and the antigen levels of the proband' s plasma fibrinogen were reduced to 0.48 g/L and 0.68 g/L,respectively.TT prolonged to 29.2 s and RT prolonged to 75.8 s.The assays of SDS-PAGE showed no abnormal molecular weight of fibrinogen.Peak height of thrombin generation was reduced to 249.93 nmol/L and endogenous thrombin potential was reduced to 1007.0 nmol · L-1 · min.Hypocoagulability state of the whole blood was found by TEG test.The coagulation index was - 8.6.The proband was diagnosed as inherited hypofibrinogenemia by phenotype analysis.Two mutations (Gln143Pro and g.4642delC) were found in the proband's fibrinogen Aa-chain gene,Gln143Pro came from her mother and g.4642delC came form her father.Conclusion Compound Heterozygous Mutations (Gln143Pro and g.4642delC ) of fibrinogen Aa-chain causes the proband congenital hypofibrinogenemia.

Objective To investigate the relationship between phenotypes and genotypes in three Chinese pedigrees with inherited antithrombin deficiency.Methods The routine coagulation tests including activated partial thromboplastin time(APTT),prothrombin time(PT),fibrinogen(Fg) and thrombin time(TT) were performed.The activities of protein C(PC:A),protein S(PS:A),and antithrombin(AT:A) were tested with chromogenic substrate assay.The antigen of AT(AT:Ag) was tested with immunoturbidimetry methods.The molecular weight(MW) and amount of AT was assessed by Western blot.The sequences of all the exons and exon-intron boundaries of the AT gene were amplified by PCR and analyzed by direct sequencing.The novel mutation in AT gene was tested in 100 normal persons to exclude polymorphism.The heterozygous deletion was checked and identified by sequencing the PCR products constructed in TA cloning vector.Results Three probands had recurrent venous thrombosis.The levels of APTT,PT,Fg,TT,PC:A and PS:A were normal,AT:A in the plasma was 60%,52% and 60% of the normal levels in each of the 3 probands,with respective AT:Ag of 16.9 mg/dL,14.1 mg/dL and 11.4 mg/dL.Western blot showed that their AT MW was normal(58 kD) but the antigen levels were lower than normal.Three heterozygous mutations of Leu340Phe,Phe122del and Phe123Cys were identified in the 3 probands.In each of the 3 pedigrees,the same genotypes(heterozygous mutations) were identified in the family members with similar low level of AT as the probands.However,only the proband′s father in pedigree 2 had venous thrombosis once,there were no other family members having had thrombosis in both pedigree 1 and 3.Conclusion The three probands with inherited antithrombin deficiency were each caused by mutations of Leu340Phe,Phe122del and Phe123Cys.Both Leu340Phe and Phe123Cys were internationally first reported.

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

Objective To identify the clinical features, the molecular diagnosis and the molecular mechanism of three unrelated factor X deficiency families. Methods Three probands were male and the diagnosis was validated by coagulant parameters. The F X coagulation activity ( F X∶ C ) and antigen (FX∶ Ag) were tested by clotting test and ELISA method. The cross-corrected test was used to rule out the inhibitor of FX in plasma. Thrombin generation test was evaluated. The antigen and the molecule weight of the FX in plasma were measured with western blotting. Gene mutations were analyzed in the probands and their family members with PCR and DNA sequencing. FX expression plasmids were constructed and transientby being transfected into 293T cells. FX: C and FX: Ag of the expression products were tested. Results APTT and PT in proband 1 were obviously prolonged, 113.4 s and 62.3 s, respectively. And there was no inhibitor in plasma. The thrombin generation was lower compared to normal reference. APTT and PT in proband 2 were 56. 5 s and 28.7 s. There was no inhibitor in the plasma. The thrombin generation was 1 101.5 nmol · min. APTT and PT in proband 3 were 117.3 s and 44. 3 s. The thrombin generation was 782.5 nmol · min. FX∶ C and FX∶ Ag in proband 1 were 1.4% and 3.6%, with a homozygous mutation in FX gene (Ser425→Pro). In vitro expression of the mutation showed a normal synthesis in the cell but secretion dysfuntion. In proband 2 F X: C and F X: Ag were 2. 2% and 5. 5%, with two heterozygous mutations in FX gene (Ala-29→Pro and Phe324→Leu). The Ala-29 → Pro mutation led to significantly reduced expressions of FX in both cell lysate and cell culture supernatants compared to wild-type plasmid,(41.32 ±5.21 )% and(6. 30 ± 1.84)% respectively. However Phe324→Leu mutation almost did not affect the FX synthesis. FX: C and FX: Ag in proband 3 were 2. 2% and 35%, with two heterozygous mutations in FX gene( Ala235→Thr and Arg347→Cys). The expressions of these two mutant FX proteins in cell lysate were similar to those of wild-type but obviously lower in the supernatant. Conclusions Five mutations of F X gene are found in this study. These mutations (Ser425Pro, Phe324Leu, Ala235Thr and Arg347Cys)can not affect F X protein synthesis. However Ala-29Pro mutation can reduce F X protein synthesis and cause secretion dysfunction.

Objective To investigate the clinical phenotype and genotype in three probands with antithmmbin(AT)deficiency and their families,and to identify the molecular mechanism of AT deficiency.Methods Chromogenic substrate method and immunoturbidimetry assay was used to detect the plasma levels of AT:A and AT:Ag,respectively.Genomic DNA was extracted from the peripheral blood.All 7 exons and the flanking sequences were amplified by PCR.and the abnormal mutant genes were analyzed by direct sequencing.Western blot was used to detect the AT levels and thrombin generation tests were used to detect coagulation status.Results The plasma levels of AT:A and AT:Ag of the three probands declined by 50%.G7386C(Trp225Cys)mutation in exon 4,C2591G(Ser36stop)in exon 2 and C9819T(Arg359stop)in exon 5 were characterized in the three prebands and they could result in W(Trp)225C(Cys)missense mutation,S(Set)36X(stop)nonsense mutation and R(Arg)359X(stop)nonsense mutation respectively,The testing results of phenotype and genotype from some of their family members showed consistent with results from the probands.Western blot results indicated that the Icyels of PC:Ag were lower compared with the normal pooled plasma.The hypercoagulative status was present in the probands using thrombin generation tests.Conclusions Type Ⅰ hereditary AT deficiency was found in these three families.The 3 heterozygous mutations.W225C,S36X and R359X are genetic defects of hereditary AT deficiency.W225C and S36X have not been described before.

Objective To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia. Methods Venous blood samples were collected from the proband and his siblings,and routine coagulation tests were conducted.The activity and antigen of fibrinogen in plasma were measured by functional and immunoturbldlmetry assay,respectively.All the exons and exon-intron boundaries of the three fibrinogen genes were analyzed by direct sequencing.Results The proband had normal activated partial thromboplastin time (aPTT)and prothrombin time(PT),but prolonged thrombin time(28.10 s).His plasma fibrinogen antigen was normal,but fibrinogen activity level was reduced(0.7g/L),which was also found in his father.The sequencing results of the proband revealed heterozygous A1211G in the exon 2 of FGA gene originating from his father,which caused Arg19Gly missense mutation.Conclusion Dysfibrinogenemia in the family is caused by Arg19Gly in theαchain of fibrinogen and it is the second report on a Chinese family with inherited dysfibrinogenemia.