一例2N型遗传性血管性血友病家系的表型诊断和基因型分析

目的：探讨1个遗传性血管性血友病（von Willebrand disease,vWD）2N型家系的表型诊断和基因型分析结果,明确患者的发病机制。方法：对该家系的先证者和家系成员进行出血时间、活化部分凝血活酶时间、瑞斯托霉素诱导的血小板聚集（ristocetin induced platelet aggregation,RIPA）试验、血管性血友病因子（von Willebrand factor,vWF）瑞斯托霉素辅因子、vWF抗原、vWF活性测定、vWF胶原结合试验、凝血因子Ⅷ（coagulation factor Ⅷ,FⅧ）活性、vWF与FⅧ结合试验检测,并作出表型诊断。提取先证者的外周血基因组DNA,用PCR法扩增VWF基因和F8基因的所有外显子及侧翼序列,通过直接测序分析VWF基因和F8基因变异。结果：vWD家系先证者的活化部分凝血活酶时间和出血时间明显延长,血浆瑞RIPA试验、vWF瑞斯托霉素辅因子、vWF抗原、vWF活性和vWF胶原结合试验检测结果均正常,FⅧ活性下降,vWF与FⅧ的结合能力降低。对先证者进行基因测序,发现其VWF基因19号外显子存在c.2446C>T（p.Arg816Trp）错义突变,其儿子在该位点为杂合突变,而先证者及家系成员的F8基因未发现突变。结论：c.2446C>T（p.Arg816Trp）错义突变是导致该家系先证者发生2N型遗传性vWD的原因。     

遗传性血管性血友病7例实验诊断和分子发病机制研究

目的:建立系统的血管性血友病(vWD)实验诊断和研究的方法,对7个遗传性vWD家系进行系列的实验诊断和分子发病机制研究。方法:自行建立瑞斯托霉素诱导的血小板凝集试验(RIPA)、血管性血友病因子(vWF)瑞斯托霉素辅因子(vWF:RCo)、vWF抗原(vWF:Ag)、vWF胶原结合试验(vWF:CB)和多聚体分析方法,对vWD患者及家系成员进行测定。采用PCR产物测序以鉴定vWF基因缺陷。采用定点突变、剪接位点分析、序列复杂性分析和核基质结合区评分等生物信息学方法,研究基因突变对蛋白功能的影响。结果:明确诊断vWD患者1型1例,2A型2例,2B型1例,2M型1例及3型2例,其中1型、2M型和2B型vWD为国内首次报道。鉴定到10个基因突变,其中6个为国际上首次发现。体外表达实验显示,R1374S突变引起细胞分泌下降;R1308C突变导致蛋白功能降低;C2327S突变影响多聚体形成。结论:本研究建立的方法和推荐的组合实验适合于绝大多数vWD患者的诊断和分型需要。R1374S、R1308C和C2327S突变分别导致患者出现1型、2B型和2A型vWD表型。插入、缺失和无义突变诱发无义介导的mRNA衰变(NMD),导致vWF合成减少,是本研究中3型患者的发病机制。     

血管性血友病的实验诊断

血管性血友病因子(von Willebrand factor,vWF)是血管内皮细胞和骨髓巨核细胞合成的一种糖蛋白,正常情况下存在于血浆和血小板(占全血15%)中。vWF 可与血小板膜糖蛋白 GP Ⅰb/Ⅸ/Ⅴ结合,激活 GPⅡb/Ⅲa 并桥接血小板与vWF,从而促使血小板聚集;vWF 还可介导血小板黏附到内皮细胞损伤后暴露出的胶原表面,使血小板聚集,形成血小板血栓,在一期止血中起重要作用。此外,血浆中的 vWF 还与 FⅧ形成复合物以稳定 FⅧ,在二期止血中发挥重要作用。vWF 缺乏将导致患者出现血管性血友病(von Willebrand     

血管性血友病诊断与分型方法的建立和临床应用

目的 建立系列的血管性血友病（vWD）筛查、诊断和分型的方法。方法 以Ⅲ型胶原包被微孔板，以辣根过氧化物酶（HRP）-兔抗人血管性血友病因子（vWF）IgG为检测抗体，建立vWF胶原结合试验（vWF：CB）；通过垂直式琼脂糖电泳、电转印和化学发光法对vWF多聚体进行分析。对可疑vWD患者测定血小板、出血时间、活化部分凝血活酶时间（Am）、血小板聚集试验（RIPA）、FVⅢ：C、vWF：RCo、vWF：Ag、vWF：CB，并进行多聚体分析。结果 所建vWF：CB线性范围广，灵敏度高，重复性好；所建多聚体琼脂糖电泳分析方法安全、敏感、解像度高。10例可疑病例血小板均正常，大部分出血时间延长、APTY延长、RIPA反应低下、FVⅢ：C降低；vWF：RCo、vWF：Ag含量和vWF：CB均不同程度降低；部分患者vWF：Ag／vWF：CB比值〉2，中、高分子量vWF显著缺如。8例患者可明确诊断为vWD，其中1型2例、2A型4例和3型2例。结论 本研究建立的方法和推荐的组合实验适合于绝大多数vWD患者的诊断和分型需要。     

四个遗传性凝血因子V缺陷症家系临床表型和基因型变化的研究

目的 研究4个遗传性凝血因子V(FV)缺陷症家系的临床表型和基因突变.方法 测定家系成员活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)及FV促凝活性(FV:C)和FV抗原(FV:Ag)含量进行表型诊断;用PCR法扩增先证者F5基因的25个外显子及其侧翼序列,PCR产物纯化后直接测序,检测其基因突变.结果 4例先证者APTT、PT明显延长,血浆FV:C和FV:Ag含量均显著降低.基因分析发现,先证者1的F5基因存在G16088C(Asp68His)杂合错义突变及4种位于同一条染色体上的杂合多态性T35788C(Met385Thr)、A47295G(Hisl299Arg)、A58668G(Metl736Val)和A74083G(Asp2194Gly);先证者2的F5基因存在CA6253T(Arg952Cys)和CA6724T (Glnl 109stop)两种纯合突变;先证者3的F5基因存在C67793G(Pr02006Ala)纯合错义突变;先证者4的F5基因存在C74022T(Arg2174Cys)纯合错义突变.结论 Asp68His、Arg952Cys、Glnl109stop、Pro2006Ala和Arg2174Cys这5种突变,及Met385Thr、Hisl299Arg、Metl736Val和Asp2194Gly这4种多态性是导致相应先证者I型遗传性FV缺陷症的原因.其中,Glnl109stop、Pro2006Ala和Arg2174Cys是国际七首次报道的新突变.     

血管性血友病因子研究进展

血管性血友病因子在止血和血栓形成过程中发挥重要作用,井与心、脑血管痍病及血管新生密切相关.研究血管性血友病因子的结构和功能,将为血管性血友病及其他相关疾病的诊断和治疗提供可靠理论基础.     

血管性血友病因子的研究进展

血管性血友病因子（von Willebrand Factor,vWF）在止血和血栓形成过程中起重要作用，并与心、脑血管疾病及血管新生密切相关，因此研究vWF的生物学特性和功能具有重要的意义。本文就vWF的生物合成、结构特点有生物学功能的研究进展作一综述。     

月经过多患者血浆血管性血友病因子水平分析

目的观察不明原因月经过多(HMB)患者血浆血管性血友病因子(vWF)的水平。方法选择不明原因HMB患者126例及健康女性73例,比较血浆凝血酶原时间(PT)、国际标准化比值(INR)、活化部分凝血活酶时间(APTT)、FⅧ凝血活性(FⅧ:C)、血管性血友病因子抗原(vWF:Ag)及瑞斯托霉素辅因子活性(vWF:RCo)及vWF:RCo/vWF:Ag比值的差异;按HMB类型分成周期规则HMB组、周期不规则HMB组、青春期HMB组,按Hb值分成轻度贫血组、中重度贫血组,分别比较各组间vWF水平的差异。结果 HMB组、不同类型HMB组血浆PT、INR、APTT、FⅧ:C、vWF:Ag、vWF:RCo及vWF:RCo/vWF:Ag水平与对照组比较,差异均无统计学意义(P均0.05)。Hb正常组、轻度贫血组、中重度贫血组与对照组血浆vWF:Ag、vWF:RCo及vWF:RCo/vWF:Ag水平差异均有统计学意义(P均0.05);中重度贫血组血浆vWF:Ag、vWF:Rco水平及vWF:RCo/vWF:Ag比值与对照组比较差异均有统计学意义(P均0.01)。结论不明原因HMB伴中重度贫血女性人群血浆vWF水平与健康人差异明显,针对该人群血浆vWF的检测有助于检出血管性血友病(vWD)。     

A1500E突变对血管性血友病因子的ADAMTS13依赖性水解的影响

目的 研究血管性血友病因子(VWF) A1500E突变体对金属蛋白酶ADAMTS13敏感性的改变,为VWF-A1500E突变导致2A型血管性血友病(VWD)的发病机制提供直接依据.方法 将野生型VWF质粒和A1500E突变体VWF质粒分别瞬时转染HeLa细胞,收集并浓缩培养上清,分别用重组人ADAMTS13( rADAMTS13)进行水解,然后通过十二烷基硫酸钠-琼脂糖凝胶电泳进行VWF多聚体分析,观察与野生型VWF相比,A1500E突变体VWF对ADAMTS13的敏感性有无改变.结果 体外表达研究结果显示WT-VWF和A1500E突变体VWF的表达上清中VWF:Ag的平均含量分别为1.10 U/ml和0.78 U/ml,突变体细胞裂解液中的VWF:Ag表达量为野生型的90.6％,两者差异均无统计学意义(P＞0.05).VWF多聚体电泳显示突变体VWF与WT-VWF的多聚体分布亦无明显差别.在无尿素和盐酸胍等变性剂的静态条件下,rADAMTS13即可对A1500E突变体VWF进行有效地水解,VWF多聚体分析显示大中分子量VWF多聚体明显减少和小分子量VWF多聚体明显增多;相反,野生型VWF在非变性条件下则无被rADAMTS13水解的证据.结论 A1500E突变导致突变体VWF对ADAMTS13的敏感性异常性增高,符合第二组2A型VWD的突变特点.     

血管性血友病因子的研究进展

血管性血友病因子（von Willebrand factor,vWF）是由血管内皮细胞和骨髓巨核细胞合成的一种多结构域、多功能糖蛋白,在1期和2期止血中发挥重要作用。vWF缺陷将导致血管性血友病（vWD）等出血性疾病,而在静脉栓塞、血栓性血小板减少性紫癜（TTP）、中风等血栓性疾病中,其活性水平可明显增高。血浆vWF水平与多种影响因素有关。随着近年来对vWF的结构、功能以及活性水平调控机制的了解,人们对于出血与血栓性疾病的病理生理,诊断和治疗有了全面的认识。本文将就血浆vWF活性水平调控与上述疾病关系的研究进展作一综述。     

A comparative analysis of different automated von Willebrand factor glycoprotein Ib‐binding activity assays in well typed von Willebrand disease patients

Essentials

• Von Willebrand ristocetin cofactor activity (VWF:RCo) is not a completely reliable assay.
• Three automated VWF activity assays were compared within a von Willebrand disease (VWD) cohort.
• Raw values for all three assays were virtually the same.
• An overall problem within type 2A/IIE VWD using VWF:GPIb‐binding activity/VWF:Ag was observed.

Summary

Background

von Willebrand disease (VWD) is an inherited bleeding disorder caused by quantitative (type 1 and 3) or qualitative (type 2) von Willebrand factor (VWF) defect. VWD diagnosis and classification require numerous laboratory tests. VWF: glycoprotein Ib (GPIb)‐binding activity assays are used to distinguish type 1 from type 2 VWD.

Objectives

Three different automated VWF:GPIb‐binding activity assays were compared.

Patients and methods

BC‐VWF:RCo (Siemens Healthcare Diagnostics), HemosIL^® VWF:RCo (Instrumentation Laboratory) and INNOVANCE^® VWF:Ac (Siemens Healthcare Diagnostics) were performed in a well typed VWD cohort (n = 142).

Results

Based on the three most used VWD parameters (FVIII:C, VWF:Ag and VWF:GPIb‐binding activity) and using a cut‐off of <0.70 for type 2 VWD revealed sensitivity and specificity of, respectively, 92% and 72.4% for VWF:RCo/VWF:Ag, 84% and 89.7% for VWF:GPIbR/VWF:Ag, and 92% and 85.1% for VWF:GPIbM/VWF:Ag, whereas a lowered cut‐off of < 0.60 resulted in reduced sensitivity with increased specificity for all assays.

Conclusion

VWD classification based on FVIII:C, VWF:Ag and VWF:GPIb‐binding activity revealed an overall problem with normal VWF:GPIb‐binding activity/VWF:Ag within type 2, especially type 2A/IIE. Although all assays were practically identical, BC‐VWF:RCo had higher %CV compared with both new assays but comparable lower limit of quantification (LLOQ) ~4 IU dL^?1. No clear improved distinction between type 1 and 2 VWD with new assays was seen. BC‐VWF:RCo and HemosIL^® are ristocetin dependent, whereas INNOVANCE^® does not rely upon ristocetin and is not influenced by VWF polymorphisms increasing VWF:GPIb‐binding activity levels. INNOVANCE^® seems to be the best choice as a first‐line VWF:GPIb‐binding activity assay, providing the best balance between sensitivity and specificity for type 2 VWD.

     

Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD)

Summary.  Background:  Type 1 von Willebrand disease (VWD) is a congenital bleeding disorder characterized by a partial quantitative deficiency of plasma von Willebrand factor (VWF) in the absence of structural and/or functional VWF defects. Accurate assessment of the quantity and quality of plasma VWF is difficult but is a prerequisite for correct classification. Objective:  To evaluate the proportion of misclassification of patients historically diagnosed with type 1 VWD using detailed analysis of the VWF multimer structure. Patients and methods:  Previously diagnosed type 1 VWD families and healthy controls were recruited by 12 expert centers in nine European countries. Phenotypic characterization comprised plasma VWF parameters and multimer analysis using low- and intermediate-resolution gels combined with an optimized visualization system. VWF genotyping was performed in all index cases (ICs). Results:  Abnormal multimers were present in 57 out of 150 ICs; however, only 29 out of these 57 (51%) had VWF ristocetin cofactor to antigen ratio below 0.7. In most cases multimer abnormalities were subtle, and only two cases had a significant loss of the largest multimers. Conclusions:  Of the cases previously diagnosed as type 1 VWD, 38% showed abnormal multimers. Depending on the classification criteria used, 22 out of these 57 cases (15% of the total cohort) may be reclassified as type 2, emphasizing the requirement for multimer analysis compared with a mere ratio of VWF functional parameters and VWF:Ag. This is further supported by the finding that even slightly aberrant multimers are highly predictive for the presence of VWF mutations.     

Laboratory diagnosis of von Willebrand disease

Von Willebrand disease is the most-common inherited bleeding disorder, including both quantitative (types 1 and 3) and qualitative (type 2) defects of von Willebrand factor. Among patients with suspected von Willebrand disease, the laboratory diagnosis requires three levels of testing: screening tests, specific assays for von Willebrand factor to establish the diagnosis, and discriminating tests to allow accurate characterization of the numerous types and subtypes of the disease. Because of their poor sensitivity, normal screening tests do not exclude the diagnosis. In most cases, specific measurements of von Willebrand factor antigen, von Willebrand factor ristocetin cofactor activity, and factor VIII levels in plasma allow differentiation of quantitative (proportionately decreased levels) and qualitative (discrepant levels) deficiencies of von Willebrand factor. Among the latter, a decreased von Willebrand factor ristocetin cofactor activity/von Willebrand factor antigen ratio is in favor of the three subtypes (2A, 2M, and 2B) defined by an abnormal interaction between von Willebrand factor and platelet glycoprotein Ib, whereas a decreased factor VIII/von Willebrand factor antigen ratio suggests subtype 2N, defined by a defective binding of von Willebrand factor to factor VIII. Several discriminating tests are available to definitively characterize each subtype. Moreover, for all variants, the link between phenotype and genotype is established using DNA analysis. In all cases, the precise characterization of type and subtype of von Willebrand disease remains essential for the choice of optimal therapeutic monitoring of each patient. Presented at the Joint Meeting of the World Health Organization and the International Society for Thrombosis and Hemostasis “Impact, Prevention and Control of von Willebrand’s disease” London, October 12–14, 1998     

Reduced von Willebrand factor survival in von Willebrand disease: pathophysiologic and clinical relevance

Summary.  Von Willebrand disease (VWD) is characterized by a wide heterogeneity of clinical and laboratory phenotypes. The complexity of the phenotype is further increased by a highly variable removal rate of von Willebrand factor (VWF) released by desmopressin, which is independent of post-infusion peak level. After the initial demonstration that a reduced VWF survival is present in patients with R1205H mutation (VWD Vicenza), several other mutations, mostly occurring in the VWF D3 domain, have been shown to be associated with accelerated removal of released VWF. Normal subjects with O blood group show reduced survival after desmopressin, underlining the role of different VWF glycosylation present in ABO blood group. Recent evidence suggests that liver and spleen macrophages are responsible for VWF clearance through uptake and endocellular degradation, but it is still not known why some VWF mutants are more prone to increased clearance.