一个重症vWD家系临床与基因分析

目的：研究重症血管性血友病（ｖＷＤ）患者家系成员临床表型特征，并分析血管性血友病因子（ｖＷＦ）基因缺陷的传递规律。方法：应用ｖＷＦ多聚体分析和ＥＬＩＳＡ法检测重症ｖＷＤ患者父母家系四代２６位成员临床表型。在此基础上针对ｖＷＦ基因内多个ＲＦＬＰ和ＶＮＴＲ位点，对所有家系成员进行缺陷基因遗传分析。结果：患者血浆ｖＷＦ∶Ａｇ＜２％，检测不到多聚体条带。基因分析表明两条ｖＷＦ等位基因分别来自父方和母方家系，且均有缺陷。部分家系成员携有一条缺陷基因，ｖＷＦ水平正常或有轻度下降，多聚体图谱正常。结论：①先证者可能为复合杂合子型的３型ｖＷＤ患者，携带者的缺陷基因可被正常基因弥补。②应用ｖＷＦ基因多种多态性标志行ＤＮＡ分析对重症ｖＷＤ家系遗传咨询有实用意义。     

人血浆凝血因子V双抗体夹心ELISA测定

目的 建立一种新的双抗体夹心酶联免疫吸附试验（ELISA），以测定人血浆凝血因子V（FV）抗原含量。方法 采用兔抗人FV抗体为包被抗体，FV单抗为检测抗体，首次对中国正常人（n=26)和一个遗传性FV缺乏家系的有关成员血浆中的FV抗原进行了检测。结果 所建方法线性范围广、特异性强、灵敏度高、重复性好，与FV活性测定有很好的相关性；正常人血浆FV抗原呈偏态分布；FV缺乏家系纯合子血浆FV抗原量只有正常人的2％，证实该家系为I型遗传性FV缺乏症。结论 该法是一种较好的FV蛋白定量测定法，可以对FV缺乏症进行辅助诊断和分型。     

凝血因子V缺陷：血栓形成与出血转换的分子基础

第Ｖ因子（ＦＶ）是分子量为３３０ＫＤ的单链糖蛋白,与第Ⅷ因子（ＦⅧ）及铜蓝蛋白之间在结构上有许多相似之处,ＦＶ参与凝血过程中的凝血酶的生成,促进凝血,同时,活化的蛋白Ｃ（ＡＰＣ）通过灭活ＦＶ来抑制凝血,ＦＶ陷根据产生机制不同可导致对ＡＰＣ的抵抗（ＡＰＣＲＥ）引起血栓病或遗传性ＦＶ缺乏症,导致出血,目前已证实部分ＡＰＣＲ和遗传性ＥＦＶ缺乏症均存在分子缺陷,本文主要对ＦＶ的结构,功能,活化蛋白Ｃ抵抗及     

蛋白免疫印迹法分析纤维连接蛋白分子降解状态

为了解蛋白免疫印迹法在纤维连接蛋白（Ｆｎ）分子降解状态分析中应用效果，观察了８６例慢性阻塞性肺疾病（ＣＯＰＤ）患者血浆Ｆｎ和５３例急性白血病、１２例淋巴瘤患者脑脊髓液（ＣＳＦ）－Ｆｎ分子状态。发现ＣＯＰＤ患者血浆Ｆｎ降解程度增加，并与ＣＯＰＤ病程和病情相关；急性白血病和淋巴瘤浸润中枢神经系统者，ＣＳＦ－Ｆｎ增高系Ｆｎ片段产生所致。表明蛋白免疫印迹法是一项适合研究体液中Ｆｎ降解片段特性和发现新片段的方法。     

蛋白免疫印迹法分析纤维连接蛋白分子降解状态

为了解蛋白免疫印迹法在纤维连接蛋白（Ｆｎ）分子降解状态分析中应用效果，观察了８６便慢性阻塞性肺疾病（ＣＯＰＤ）患者血浆Ｆｎ和５３例急性白血病、１２例淋巴瘤患者脑脊髓液（ＣＳＦ）－Ｆｎ分子状态。发现ＣＯＰＤ患者血浆Ｆｎ降解程度增加，并与ＣＯＰＤ病程和病情相关；急性白血病和淋巴瘤浸润中枢神经系统者，ＣＳＦ－Ｆｎ增高系Ｆｎ片段所致，表明蛋白免疫印迹法是一项适合研究体液中Ｆｎ降解片段特性和发现新片段的方法     

人血浆凝血因子V双抗体夹心ELISA测定

目的建立一种新的双抗体夹心酶联免疫吸附试验(ELISA),以测定人血浆凝血因子V(FV)抗原含量.方法采用兔抗人FV抗体为包被抗体,FV单抗为检测抗体,首次对中国正常人(n=26)和一个遗传性FV缺乏家系的有关成员血浆中的FV抗原进行了检测.结果所建方法线性范围广、特异性强、灵敏度高、重复性好,与FV活性测定有很好的相关性;正常人血浆FV抗原呈偏态分布;FV缺乏家系纯合子血浆FV抗原量只有正常人的2%,证实该家系为Ⅰ型遗传性FV缺乏症.结论该法是一种较好的FV蛋白定量测定法,可以对FV缺乏症进行辅助诊断和分型.     

遗传性第Ⅴ因子缺乏症血小板第Ⅴ因子活性分析

凝血第 Ｖ因子（ＦＶ）是分子质量为 ３３０ｕ的单链糖蛋白，正常人约７５％存在于血浆中，其余的２５％存在于血小板α颗粒中。活化的第Ｖ因子（ＦＶａ）作为辅因子，与活化的第Ｘ因子（ＦＸａ）、钙离子及磷脂共同构成凝血酶原酶复合物，参与凝血酶原的活化，是一种重要的凝血因子［１］。遗传性 ＦＶ缺乏症是一种罕见的常染色体隐性遗传性出血性疾病，近年发现，其出血症状的严重性与血浆中ＦＶ水平并不平行，而与血小板中 ＦＶ活性高低有更密切的关系［１］。有关血小板中ＦＶ的测定，国内尚未见报道。我们采用发色底物法，对一个遗传性…     

一例凝血因子Ⅷ1680A→G点突变

为了研究在无内含子２２倒位的甲型血友病中的ＦⅧ基因的其他遗传性点突变。应用变性梯度凝胶电泳（ＤＧＧＥ）、单链构象多态性（ＳＳＣＰ）对５１例无内含子２２倒位的甲型血友病患者的ＦⅧ基因外显子１４３′侧进行研究。结果发现一例遗传性点突变，密码子１６８０ＴＡＴ（酪）→ＴＧＴ（半胱）的突变。阐明甲型血友病的点突变有助于了解ＦⅧ蛋白各功能域的精细功能，为甲型血友病家系携带者检测和产前诊断提供一种有价值的方法     

一例凝血因子Ⅷ1680A→C点突变

为了研究在无内含子２２倒位的甲因友病中的ＦⅧ基因的基他遗传性点突变。应用变性梯度凝胶电泳（ＤＧＧＥ）、单链构象多态性（ＳＳＣＰ）对５１例无内含子２２倒位的甲型血友病患者的ＦⅧ基因外显子１４３＇侧进行研究。结果发现一例遗传性点突变，密码子１６８０ＴＡ（酷）→ＴＧＴ（半胱）的突变。阐明甲型血友病的点突变有助于了解ＦⅧ蛋白各功能域的精细功能，为甲型血友病家系携带者检测和产前诊断提供一种有价值的方法。     

因子Ⅷ基因倒位的检测与血友病甲的分子诊断

应用Ｓｏｕｔｈｅｒｎ印迹杂交技术，分析了３６个无亲缘关系的血友病甲家系共９８名成员的凝血因子ＶＩＩＩ（ＦＶＩＩＩ）基因倒位。１２个（３３．３％）家系显示内含子２２倒位，其中１０个为远端倒位，２个为近端倒位。此１２例皆为重型患者，占全部２３例重型患者的５２．２％，证实ＦＶＩＩＩ基因倒位是中国人重型血友病甲最重要的分子缺陷。ＦＶＩＩＩ基因倒位分析既可以揭示血友病甲患者的分子缺陷，又可以直接用于血友病甲的分子诊断。     

The factor V Glu1608Lys mutation is recurrent in familial thrombophilia

BACKGROUND: Co-inheritance of heterozygous factor V deficiency with FV Leiden enhances the activated protein C resistance (APCR) associated with this mutation, resulting in pseudo-homozygous APCR. The role of FV deficiency in modulating thrombotic risk in this rare condition is poorly understood. METHODS AND RESULTS: We have identified in thrombophilic patients with FV deficiency a novel FV gene mutation (c. 4996G>A), predicting the Glu1608Lys substitution in the A3 domain. The heterozygous mutation was detected in three unrelated patients, two carriers of the FV Leiden mutation, and one of the FVHR2 haplotype. The Glu1608Lys change was also present in two subjects with mild FV deficiency, and absent in 200 controls. The FV1608Lys carriers showed reduced mean FV activity (42% +/- 12%) and antigen (53% +/- 18%) levels and, in Western blot analysis, reduced amounts of intact platelet FV. The restriction fragment length polymorphism (RFLP) study identified two haplotypes underlying the mutation, which suggests that it is recurrent. In heterozygous subjects the amount of FV1608Lys mRNA in white blood cells was similar to that produced by the counterpart alleles (FVWt or FVHR2). Recombinant FV1608Lys (rFV1608Lys), detected by Western blot in the conditioned medium, was indistinguishable from rFVWt and FV antigen and activity were found to be respectively 44% +/- 20% and 13% +/- 4% of rFVWt. CONCLUSIONS: Our data indicate that FVGlu1608Lys predicts a CRM (plasma)/CRMred (cell culture) FV deficiency, and may contribute to thrombophilia in carriers of FV Leiden and FVHR2 haplotype via a pseudo-homozygosity mechanism. Our findings help to define the molecular bases of FV deficiency and thrombophilia.     

Characterization of an immunologic polymorphism (D79H) in the heavy chain of factor V.

BACKGROUND: During the study of a family with hereditary factor (F)V deficiency (FV Amersfoort, 1102 A > T in exon 7) we identified an individual with 5% FV heavy chain antigen (FV(HC)) and 50% FV light chain antigen (FV(LC)). Further testing revealed that apart from the FV Amersfoort allele a second variant FV allele was segregating in this family, which encodes for a FV molecule with a reduced affinity for mAb V-23 used in the FV heavy chain ELISA (ELISA(HC)). OBJECTIVE: Identification and characterization of the molecular basis responsible for the reduced affinity of the variant FV for mAb V-23. METHODS: Family members of the proband were screened for mutations in the exons coding for the heavy chain of FV, after which the recombinant variant FV could be generated and characterized. Next, the cases and controls of the Leiden Thrombophilia Study (LETS) were genotyped for carriership of the variant FV. RESULTS: In the variant FV allele a polymorphism in exon 3 (409G > C) was identified, which predicts the replacement of aspartic acid 79 by histidin (D79H). Introduction of this mutation in recombinant FV confirmed that it reduces the affinity for binding to mAb V-23. The substitution has no effect on FV(a) stability and Xa-cofactor activity. In Caucasians the frequency of the FV-79H allele is approximately 5%. Analysis of the LETS revealed that the FV-79H allele is not associated with FV levels (FV(LC)), activated protein C sensitivity (using an activated partial thromboplastin time-based test) or risk of venous thrombosis (OR 1.07, CI 95: 0.7-1.7). CONCLUSION: The D79H substitution in FV should be considered as a neutral polymorphism. The monoclonal antibody V-23, which has a strongly reduced affinity for FV-79H, is not suitable for application in diagnostic tests.     

获得性凝血因子Ⅴ缺乏症的临床诊治

目的:总结获得性凝血因子Ⅴ缺乏症(acquired factorⅤdeficiency,AFⅤD)的临床诊治经验。方法:回顾性分析10例获得性凝血因子Ⅴ缺乏症患者的临床资料,分析临床特点,总结临床诊治经验。结果:10例患者中男性7例,女性3例,年龄51~71岁,中位年龄60岁,均无遗传性凝血因子缺乏病史及家族史,临床表现为牙龈出血、鼻衄、血尿、黑便,甚至颅内出血。10例患者均有凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)显著延长,凝血因子Ⅴ活性降低,抑制物定性或定量试验阳性,均诊断为获得性凝血因子Ⅴ缺乏症。10例患者均输注新鲜冰冻血浆控制出血症状,并采用糖皮质激素或联合环磷酰胺、硫唑嘌呤免疫抑制治疗,其中5例患者(5/10)缓解,未再出现出血症状;3例患者(3/10)应用糖皮质激素及环磷酰胺治疗效果差,随后加用利妥昔单抗治疗均有效,其中2例经治疗后凝血因子Ⅴ(factorⅤ,FⅤ)浓度恢复正常,未再出现出血症状,1例患者需用小剂量利妥昔单抗维持治疗;2例患者(2/10)死于颅内出血。结论:AFⅤD是一种罕见疾病,临床出血症状轻重不一,经糖皮质激素及环磷酰胺、利妥昔单抗治疗有助于缓解症状     

Anti-factor V auto-antibody in the plasma and platelets of a patient with repeated gastrointestinal bleeding

Summary.  Development of autoantibody against coagulation factor V (FV) is a rare clinical condition with hemorrhagic complications of varying severity. The aim of this study was to establish the pathomechanism of an acquired FV deficiency and characterize the FV inhibitor responsible for the clinical symptoms. A 78-year-old female was admitted to hospital with severe gastrointestinal bleeding. General clotting tests and determination of clotting factors were performed by standard methods. FV antigen and FV containing immune complexes were measured by ELISA. The FV molecule was investigated by Western blotting and by sequencing the f5 gene. The binding of patient's IgG to FV and activated FV (FVa) was demonstrated in an ELISA system and its effect on the procoagulant activity of FVa was tested in clotting tests and in a chromogenic prothrombinase assay. Localization of the epitope for the antibody was performed by blocking ELISA. FV activity was severely suppressed both in plasma and platelets. FV antigen levels were normal by ELISA using polyclonal anti-FV antibody or monoclonal antibody against the connecting region of FV, but depressed when HV1 monoclonal antibody against the C2 domain in the FV light-chain was used as capture antibody. The FV molecule was found intact. An IgG reacting with both FV and FVa was present in the patient's plasma and its binding to FV was inhibited by HV1 antibody. FV-containing immune complexes were detected in the patient's plasma and platelet lysate. The patient's IgG inhibited the procoagulant function of FVa. An anti-FV IgG was present in the patient's plasma and platelets. The autoantibody reacted with an epitope in the C2 domain of FV light chain and neutralized the procoagulant function of FVa.     

一例遗传性凝血因子V缺乏症发病机制研究

目的 研究遗传性凝血因子V（FV）缺乏症的发病机制。方法 通过免疫学方法和发色底物法检测血浆和血小板中的FV含量,采用PCR产物直接测序和限制性酶切分析FV基因,应用分子模建对所鉴定突变的生物结构病理学进行研究。结果 先证才血浆和血小板中FV均缺乏。先证者FV基因第1763位核苷酸存在A→C突变（1763A→C,EMBL AJ297254）。模建分析表明,该突变将导致分子内部形成空洞,并可能破坏Tyr530与Glu330之间形成的氢键。结论 1763A→C突变将导致FV稳定性降低,是致病的重要原因。     

一种新的突变引起遗传性凝血因子Ⅴ缺乏症

目的 研究一个遗传性凝血因子Ⅴ(FⅤ)缺乏症家系的基因缺陷。方法 采用比浊法测定凝血酶原时间、凝血酶时间、活化的部分凝血活酶时间；采用凝血因子活性测定法(一步法)和BAELISA法对先证及其家系成员血浆FⅤ活性和抗原进行测定；采用PCR及DNA序列测定技术，对FⅤ基因组DNA中25个外显子和5′非翻译端的序列进行了：PCR扩增，PCR产物回收纯化后直接测序，并经T／A克隆测序对所发现突变进行证实。结果 先证血浆FⅤ严重缺乏，FⅤ活性为1％，FⅤ抗原为1．54％。基因研究显示为复合杂合子，其基因组DNA第4外显子675位核苷酸发生单个碱基A缺失，呈杂合状态，该致病基因来自先证母亲，与来自父方的另外一条等位基因的未知缺陷，共同导致先证血浆FⅤ严重缺乏。结论发现一种新的突变675delA，该缺失引起移码，导致转录提前终止，引起遗传性FⅤ缺乏症。     

Does activated protein C‐resistant factor V contribute to thrombin generation in hemophilic plasma?

In this study we assessed the role of factor V (FV) inactivation in hemophilic plasma with particular reference to the activated protein C (APC)-resistant variants FV-R506Q (FV Leiden) and FV-R306T (FV Cambridge). Purified recombinant full-length FV carrying these single substitutions and FV-R306T/R506Q were used in thrombin generation experiments. Plasma was first immunodepleted of FV, and subsequently of factors VIII, IX, or combinations thereof. Thrombin generation was initiated by low concentrations of recombinant tissue factor. Recombinant soluble thrombomodulin (TM) was used to trigger the APC system. Surprisingly, TM concentrations that reduced thrombin generation in normal plasma by no more than 50% virtually abolished thrombin formation in plasma deficient in the factor VIII/IX complex. This was already apparent at TM levels as low as 0.1 nmol L(-1). By varying the concentrations of purified (activated) protein C to plasma that was additionally depleted of protein C, we confirmed that impaired thrombin generation indeed was the result of the action of APC. In contrast, this did not occur when FV-depleted plasma had been reconstituted with FV-R306T/R506Q. Addition of FV-R306T or FV-R506Q partially reduced prothrombin activation, demonstrating the involvement of both APC cleavage sites. FV inactivation also occurred on the surface of human microvascular endothelial cells. Apparently, these cells express sufficient TM to down-regulate thrombin production via the APC pathway. We further conclude that in hemophilic plasma this pathway can induce a secondary defect because of premature FV inactivation. It therefore seems conceivable that APC-resistant FV has the potential of alleviating hemophilic bleeding.     

Homozygous F5 deep‐intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet‐rich plasma

Summary. Background: Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet‐rich plasma (PRP). Therefore, we hypothesized that FV‐deficient patients with severe bleeding manifestations may lack platelet FV. Objectives: To characterize a FV‐deficient patient with a severe bleeding diathesis. Patients/Methods: We performed FV mutation screening and functional studies in a 31‐year‐old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. Results: The proband was homozygous for a deep‐intronic mutation (F5 IVS8 +268A→G) causing the inclusion of a pseudo‐exon with an in‐frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the proband’s plasma or platelets with a prothrombinase‐based assay. Moreover, no thrombin generation was observed in PRP triggered with 1–50 pm tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the proband’s whole blood, as assessed by thromboelastometry, was markedly delayed but not abolished. Conclusions: This is the first report of a pathogenic deep‐intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.