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。     

血管性血友病的诊断与治疗

血管性血友病 (ｖｏｎＷｉｌｌｅｂｒａｎｄｄｉｓｅａｓｅ ,ｖＷＤ)是临床上一种常见的遗传性出血性疾病 ,其发病机制是患者的ｖｏｎＷｉｌｌｅ ｂｒａｎｄ因子 (ｖｏｎＷｉｌｌｅｂｒａｎｄｆａｃｔｏｒ ,ｖＷＦ)基因突变 ,导致血浆ｖＷＦ数量减少或质量异常。ｖＷＦ在体内由血管内皮细胞与巨核细胞合成。ｖＷＦ在止血中的主要作用有两个方面 :①与血小板膜糖蛋白 (ＧＰ)Ⅰｂ Ⅸ复合物及内皮下胶原结合 ,介导血小板在血管损伤部位的粘附 ;②作为因子Ⅷ的载体 ,具有稳定因子Ⅷ的作用。此外 ,ｖＷＦ也能结合ＧＰⅡｂ Ⅲａ ,参与了血小板的聚…     

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。     

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。     

血浆血管性假血友病因子对肝纤维化的诊断价值

目的 通过检测各组肝病患者血浆的血管性假血友病因子（VWF）水平与血清Ⅲ型前胶原蛋白（PⅢ）、透明质酸（HA）、层粘连蛋白（LN）之间的比较来进一步探讨血VWF对肝纤维化的诊断价值。方法 用VWF Laurell免疫火箭电泳法,PⅢ、HA、LN均用放射免疫法。结果 发现VWF在各肝病组均比对照组明显升高（P＜0.01）;其血浓度肝硬变＞慢性活动性肝炎＞急性肝炎。VWF与PⅢ在慢性活动性肝炎组中两者虽直线相关（P＜0.05）。结论 VWF血浓度可反映肝损伤及肝纤维化的程度,并随着急性肝炎、慢性活动肝炎及肝硬变的发展呈进行性升高。     

解读血管性血友病的诊断指南

血管性血友病(von Willebrand disease,vWD)是一种常染色体(显性或隐性)遗传的出血性疾病,是由血管性血友病因子(von Willebrand factor,vWF)量的缺乏和(或)质的异常引起。vWF是一种血浆蛋白,在血管损伤部位介导血小板黏附,其与凝血因子Ⅷ(FⅧ)结合可使血循环中FⅧ稳定而不易被降解;vWF缺陷可损害血小板黏附、聚集功能,并可降低血循环中FⅧ水平,从而引起临床出血表现。近年,也见获     

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。     

三个遗传性血管性血友病家系表型诊断和基因型研究

目的 对3个遗传性血管性血友病(vWD)家系进行表型诊断和基因型分析,探讨其分子发病机制.方法 分别采用出血时间(BT)、活化部分凝血活酶时间(APTT)、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活性(vWF∶A)测定,vWF胶原结合试验(vWF∶CB),vWF与FⅧ结合试验(vWF∶FⅧ∶B)和多聚体分析对3例vWD先证者和家系成员进行表型诊断.提取3例先证者外周血基因组DNA,用PCR法扩增vWF基因的52个外显子及侧翼序列,通过直接测序分析vWF基因变异.结果 3例左证者APTT均延长,BT除先证者3外均正常,血浆RIPA、vWF∶RCo、vWF∶Ag、vWF∶A和vWF∶CB均有不同程度的减低.多聚体分析结果显示先证者3中、高分子质量多聚体缺失,而先证者l、2基本正常.对3例先证者进行基因测序,共发现3个杂合突变,分别为vWF基因的G144067A(R2287Q)、G110374A(R1374H)、C110770T(S1506L)以及28号外显子G110667A(D1472H)杂合多态性.结论 R2287Q、R1374H和S1506L这3种杂合错义突变及D1472H杂合多态性是分别导致3例先证者发生遗传性vWD的分子发病机制.其中R2287Q是国际首次报道的新突变.

Abstract：

Objective To analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism. Methods Bleeding time (BT),activated partial thromboplastin time ( APTT), ristocetin-induced platelet aggregation ( RIPA ), von Willebrand factor timer 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. Results APTT were prolonged in all three probands, while BT were normal exferent 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. Conclusion 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.     

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)。     

Performance and clinical utility of a commercial von Willebrand factor collagen binding assay for laboratory diagnosis of von Willebrand disease

BACKGROUND: Von Willebrand disease (VWD) diagnosis and classification usually require a combination of nonspecific and VW-factor (VWF)-specific assays. We evaluated the analytical performance of a commercially available collagen-binding assay (CBA) and its usefulness in conjunction with other assays for laboratory diagnosis of VWD. METHODS: We used a commercial CBA ELISA (Life Technologies) to evaluate 3085 plasma samples. We used standard procedures to perform other assays, including factor VIII activity (FVIII:C), VWF antigen (VWF:Ag), ristocetin cofactor activity, VWF collagen binding capacity (VWF:CB), and VWF multimeric analysis. RESULTS: CBA intra- and interassay CVs were <6% and <13%, respectively. Reference intervals were 45%-198% for VWF:CB and 0.75-1.32 for the VWF:CB/Ag ratio. Of 3085 samples tested, 235 (8%) had results commonly associated with VWD. Multimer analysis and phenotypic data in 156 samples identified VWD types as: 91 (58%) type 1, 62 (40%) type 2, and 3 (2%) type 3. Of the 91 type 1 samples, proportional decreases in functional activity were seen in 75 samples (82%) according to CBA and in 63 samples (69%) according to the ristocetin cofactor assay. Of the type 2 samples, 10 were further identified as probable type 2A, 26 as probable type 2B, 12 as probable type 2M, and 14 could not be subtyped. VWF:CBA/Ag ratios <0.5 occurred in 83% of VWD type 2A and 2B samples, indicating characteristic functional discordance. Mean (SD) VWF:CB values were significantly higher in individuals without group O blood [113 (45)] than in those with group O blood [83 (32)] (t-test, P = 0.007). CONCLUSIONS: The commercial CBA assay produces reliable results and is useful for laboratory diagnosis of VWD.     

The molecular basis of von Willebrand disease

von Willebrand disease (VWD) is a clinically heterogeneous bleeding disorder that reflects a wide array of defects. Quantitative subtypes of the disorder, including types 1 and 3 VWD, result in bleeding due to reduced levels of circulating von Willebrand factor (VWF) protein. Qualitative subtypes, defined as type 2 VWD, act through altered VWF function. A range of molecular defects are responsible for many of these subtypes, including missense, nonsense, splicing, insertion, and deletion mutations, resulting in either dominant or recessive inheritance. While many mutations correspond to selected variants, the basis for variation in expression and the imperfect correlations between genotype and phenotype remain to be understood.     

Acquired von Willebrand disease

Acquired von Willebrand disease (AvWD) is a relatively rare acquired bleeding disorder that usually occurs in elderly patients, in whom its recognition may be delayed. Patients usually present predominantly with mucocutaneous bleeding, with no previous history of bleeding abnormalities and no clinically meaningful family history. Various underlying diseases have been associated with AvWD, most commonly hematoproliferative disorders, including monoclonal gammopathies, lymphoproliferative disorders, and myeloproliferative disorders. The pathogenesis of AvWD remains incompletely understood but includes autoantibodies directed against the von Willebrand factor (vWF), leading to a more rapid clearance from the circulation or interference with its function, adsorption of vWF by tumor cells, and nonimmunologic mechanisms of destruction. Laboratory evaluation usually reveals a pattern of prolonged bleeding time and decreased levels of vWF antigen, ristocetin cofactor activity, and factor VIII coagulant activity consistent with a diagnosis of vWD. Acquired vWD is distinguished from the congenital form by age at presentation, absence of a personal and family history of bleeding disorders, and, often, presence of a hematoproliferative or autoimmune disorder. The severity of the bleeding varies considerably among patients. Therapeutic options include desmopressin and certain factor VIII concentrates that also contain vWF. Successful treatment of the associated illness can reverse the clinical and laboratory manifestations. Intravenous immunoglobulins have also shown some efficacy in the management of AvWD, especially cases associated with monoclonal gammopathies. Awareness of AvWD is essential for diagnosis and appropriate management.     

Classification of von Willebrand disease

Von Willebrand disease (VWD) is known for its marked heterogeneity which was already recognized by von Willebrand in 1926. The basis of phenotypic differentiation are quantitative and qualitative or functional differences between the different types and subtypes of VWD. One of the most important tools in the classification of VWD is multimer analysis that visualizes many of the structural abnormalities of mutant VWF. The introduction of multimer analysis was followed by the identification of an increasing number of different VWD phenotypes that were first reviewed in 1987 by Ruggeri and Zimmerman, thus forming a first classification of the disease. However, the detection of additional phenotypes required a revision of the nomenclature at a time point when only a few types of VWD had already been analyzed on the molecular level. Consequently, the molecular data only played a minor role in the revised classification published by Sadler in 1994. The advent of molecular techniques provided the opportunity for genotype/phenotype studies which recently helped not only to elucidate or confirm important functions of VWF and its steps of post-translational processing but also many disease causing defects. The reproducible correlation between certain phenotypes and particular mutations can now be used for a molecular approach towards a soundly based classification of VWD, equally useful for the clinician and for research requirements.     

Women with von Willebrand disease

The clinical presentation of VWD shows sex-related differences of symptoms. In women the most typical symptoms are menorrhagia, bleeding during and after delivery or abortion and bleeding in connection with caesarean section or gynaecological surgery. Menorrhagia is one of the most common symptoms presented to gynaecologists. In 7-20% of menorrhagia the underlying cause is VWD, in our cohort of 185 women with menorrhagia the prevalence of VWD was even 32%. On the other hand in women with VWD menorrhagia with onset at the menarche can be found in 60-93%, influencing substantially their morbidity and quality of life. During pregnancy women with mild VWD experience a decrease of bleeding tendency due to an increase of endogenous VWF. But as the VWF concentration drops rapidly after delivery, the post-partum period is often associated with significant bleeding complications. In severe forms of VWD the bleeding risk is high during delivery and postpartum period. Laboratory monitoring and therapeutical measures should be continued for 8-10 days after delivery. During menopause the clinical situation improves for most of the women with mild or moderate VWD.     

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