血管性血友病诊治进展

血管性血友病(von Willebrand disease,VWD)是临床上最常见的一种遗传性出血性疾病,其发病机制是患者的血管性血友病因子(von Willebrand factor,VWF)基因突变,导致血浆VWF数量减少或质量异常.国外报道VWD发病率为0.1/万至1/万[1].     

血管性血友病

血管性血友病(von Willebrand disease，vWD)是临床上一种常见的遗传性出血性疾病，其发病机理是患者的血管性血友病因子(von Willebrand factor，vWF)基因突变，导致血浆vWF数量减少或质量异常。vWD相当常见，但轻症患者可无出血表现，故很难准确统计其发病率。国外报道一般在千分之一至五千分之一。     

血管性血友病和血管性血友病因子的研究进展

血管性血友病（von Willebrand disease，vWD）是最常见的遗传性出血性疾病之一。根据多中心研究的结果估计，有症状的vWD患者达113／10^6或更高，而具有vWD危险的人数高达1480／10^6～3580／10^6。1926年芬兰医师von Willebrand首次发现该病，并认识到其是常染色体的遗传性出血性疾病。通过对vWD的病理机制的研究，已明确该病是由于血浆中一种多聚糖蛋白，即血管性血友病因子（vWF）缺陷所致，且这种缺陷是由于vWF基因突变所导致。     

von Willebrand因子抑制剂ARCl779可减少颈动脉内膜切除术后的脑栓子信号数量

抑制von Willebrand因子（von Willebrand factor，vWF）为卒中和心肌缺血的预防提供了一种新的方法，但至今尚未证实其对临床相关终点的疗效。ARCl779是一种通过结合至vWF的A1域并借此阻断其与糖蛋白相互作用而抑制vWF的促血栓形成功能的适配体。1期研究显示，它能够抑制血小板聚集，而且出血风险低于传统抗血小板药物。英国伦敦圣乔治大学临床神经科学系的Markus等进行了一项随机双盲对照试验，旨在探讨ARC1779对颈动脉内膜切除术（carotid endarterectomy，CEA）后即刻脑栓子数量的影响。     

vWF及ADAMTS13在肝脏疾病中的意义

<正>微循环障碍参与了多种疾病的进展,例如脓毒症、多器官衰竭等。血管性假性血友病因子(von Willebrand factor,vWF)可使血小板粘附聚集于血管内皮细胞,导致血小板血栓形成;血管性血友病因子特异性裂解酶(a disintegrin-like and metalloprotease with thrombo-spondin type I repeats 13,     

von Willebrand因子与蛛网膜下腔出血后脑血管痉挛

脑血管痉挛是蛛网膜下腔出血后的并发症之一,也是蛛网膜下腔出血患者残疾和死亡的重要原因,其发病机制至今尚不明确,目前有关血管内皮细胞功能的研究较多.von Willebrand因子(vWF)是反映血管内皮细胞功能的特异标志物,文章就vWF在脑血管痉挛中的作用做了综述.     

糖尿病血管并发症与von Willebrand因子

von Willebrand因子(vWF)参与调节血小板粘附、聚集功能,而血小板这些功能与糖尿病血管并发症有密切关系。本文讨论了vWF合成与代谢、结构与功能、糖尿病病人中的vWF变化以及vWF在糖尿病血管并发症发生、发展中的作用。继续这方面的研究可能有助于探索糖尿病血管并发症的发病机理及新的防治途径。     

低水平von Willebrand因子与脑内出血有关

高血压与颅内出血的联系已非常明确,但相比之下,凝血因素如von Willebrand因子(vWF)对颅内出血危险性有什么影响还不清楚。据最近的Stroke报道,高血压和vWF因子是颅内出血的独立危险因素,它们对颅内出血危险性有协同作用,提示存在两种截然不同的致病途径。     

vWF与多器官功能不全综合征

多器官功能不全综合征（MODS）是目前医学界研究的热点和难点之一，其发病机制至今未能阐明。近年来有关内皮细胞（EC）损伤和MODS的报道较多，von Willebrand因子（vWF)既是反映EC损伤的标志物又参与机体的凝血功能，与MODS关系较为密切。本文拟就vWF与血管内皮细胞损伤、凝血功能障碍及MODS的关系作一综述。     

ADAMTS13与血栓性微血管病

正ADAMTS13是一种含I型血小板结合蛋白基序的解聚蛋白样金属蛋白酶家族第13位成员(a disintegrin-like and metalloprotease with thrombospondin-1 motifs,13th member of the family,ADAMTS13)[1],此前称为血管性血友病因子裂解酶(von Willebrand factor-cleaving protease,vWF-CP)。虽然血栓性血     

Measurement of von Willebrand factor as the marker of endothelial dysfunction in vascular diseases

BACKGROUND:

von Willebrand factor is a blood glycoprotein that is required for normal hemostasis. Its level can be increased by endothelial cell damage.

HYPOTHESIS:

von Willebrand factor is a suitable marker of endothelial dysfunction.

METHODS:

von Willebrand factor activity was determined by ELISA in patients with acute coronary syndromes, acute stroke and chronic vascular diseases, and was compared with the values of healthy controls.

RESULTS:

von Willebrand factor activity of patients in each group was significantly higher (P<0.001) than that of the control group. The values of patients with acute coronary syndrome and acute stroke were significantly higher (P<0.05 and P<0.01, respectively) than those of patients with chronic vascular diseases. von Willebrand factor activity was significantly higher in patients with acute coronary syndrome and acute stroke (P<0.05 and P<0.01, respectively) on the sixth day than on admission.

CONCLUSIONS:

By measuring von Willebrand factor activity, a considerable, significant difference could be found between healthy people and chronic and acute vascular patients. The routine measurement of von Willebrand factor activity in vascular patients as an index of endothelial dysfunction may have clinical importance, because detection of this marker can be a noninvasive way of assisting diagnosis and indicating disease progression.     

An apparently silent nucleotide substitution (c.7056C>T) in the von Willebrand factor gene is responsible for type 1 von Willebrand disease

Background

Nucleotide variations not changing protein sequences are considered silent mutations; accumulating data suggest that they can, however, be important in human diseases.

Design and Methods

We report an altered splicing process induced by a silent substitution (c.7056C>T) in the von Willebrand factor gene in a case of type 1 von Willebrand disease originally classified as lacking von Willebrand factor mutations.

Results

The c.7056C>T synonymous substitution introduces a new donor splice site within exon 41, leading to messenger RNA lacking nucleotides 7055-7081 (c.7055_7081del). The encoded von Willebrand factor protein is predicted to lack amino acids 2352-2360 in the B2 domain. The patient’s von Willebrand disease phenotype was characterized by reduced plasma and platelet von Willebrand factor, which was normal in function and multimer structure. In vitro expression studies demonstrated that co-transfection of equimolar c.7055_7081del and wild-type von Willebrand factor (mimicking the patient’s heterozygous state) induced a 50% lower von Willebrand factor secretion than the wild type, while almost no von Willebrand factor secretion was seen with the mutated von Willebrand factor alone. The secreted von Willebrand factor was structurally and functionally normal, suggesting that the c.7056C>T substitution behaves like a loss-of-function allele.

Conclusions

This is the first report of a synonymous von Willebrand factor substitution being responsible for von Willebrand disease. Our findings suggest the need to reconsider the role of von Willebrand factor polymorphisms in von Willebrand disease.     

Biogenesis of Weibel-Palade bodies in von Willebrand's disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Background

Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand’s disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies.

Design and Methods

The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate.

Results

p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser.

Conclusions

We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.     

An Arg545----Cys545 substitution mutation of the von Willebrand factor in type IIB von Willebrand's disease.

Type IIB is a special variant of von Willebrand's disease, characterized by an abnormal von Willebrand factor which shows an increased interaction with platelets. This interaction sometimes causes platelet aggregation and thrombocytopenia in vivo. It involves the glycoprotein-Ib (GPIb) receptor on platelets and corresponding GPIb-binding sites in the von Willebrand factor. We here demonstrate a C----T mutation at codon 1308 of the von Willebrand factor gene in 2 related patients with IIB von Willebrand's disease. The transition gives rise to a substitution of arginine by cysteine at position 545 of the mature von Willebrand factor subunit. This position is close to the GPIb- as well as the collagen- and heparin-binding domains of the von Willebrand factor. The mutation may change the conformation of the molecule in this region and activate the GPIb-binding domain, which is normally not exposed in the von Willebrand factor of circulating blood.     

The molecular biology of von Willebrand disease

von Willebrand disease is the most common inherited bleeding disorder in humans, with the general population prevalence estimated to be as high as 1% in some studies. This condition exhibits extensive heterogeneity with over 20 distinct subtypes distinguished based on subtle clinical and laboratory differences in presentation. Recent research laboratory advances have shed considerable new light on the molecular basis of this disorder. Specific mutations within the von Willebrand factor gene have been identified in many of the qualitative variants of von Willebrand disease, providing important new insight into the structure and function of this central clotting protein. However, the complex genetic factors determining the clinical severity of type 1 von Willebrand disease, the most common variant, still remain largely unknown and are the subject of current investigation.     

Pregnancy and delivery in women with von Willebrand��s disease and different von Willebrand factor mutations

Background

Pregnancy in von Willebrand’s disease may carry a significant risk of bleeding. Information on changes in factor VIII and von Willebrand factor and pregnancy outcome in relation to von Willebrand factor gene mutations are very scanty.

Design and Methods

We examined biological response to desmopressin, changes in factor VIII and von Willebrand factor and pregnancy outcome in a cohort of 23 women with von Willebrand’s disease characterized at molecular level and prospectively followed during 2000–2007.

Results

Thirty-one pregnancies occurred during the study period. Remarkably, similar changes of factor VIII and von Willebrand factor were observed after desmopressin and during pregnancy in nine women with R854Q, R1374H, V1665E, V1822G and C2362F mutations. Women with von Willebrand’s disease and R1205H and C1130F mutations (17 pregnancies in 12 women) had only a slight increase of factor VIII and von Willebrand factor during pregnancy while their response to desmopressin was marked but short-lived. For these women, two to three desmopressin administrations within the first 48 hours were sufficient to successfully manage vaginal delivery. Two women with recessive von Willebrand’s disease due to compound heterozygosity for different gene mutations had a spontaneous, major increase in factor VIII while von Willebrand factor remained severely reduced. Desmopressin increased factor VIII and was clinically useful in the first case, while a factor VIII/von Willebrand factor concentrate was required in the second patient not responsive to the compound. Factor VIII/von Willebrand factor concentrate was also required for two women with type 2 A von Willebrand’s disease with V1665E mutations who had no von Willebrand factor activity change during pregnancy. In one of them, delayed bleeding occurred 15 days later requiring treatment with Factor VIII/von Willebrand factor concentrate. No miscarriages or stillbirths occurred.

Conclusions

Close follow-up and detailed guidelines for the management of parturition have produced a very low rate of immediate and late bleeding complications in this setting. Desmopressin was effective and safe in preventing significant bleeding at delivery in most of these patients.     

DDAVP in acquired von Willebrand syndrome associated with multiple myeloma

The response to a single intravenous infusion of 1-deamino-8-D-arginine vasopressin (DDAVP, desmopressin) was studied in two patients with acquired von Willebrand syndrome associated with IgG-kappa myeloma. Following infusion of DDAVP (0.3-0.4 micrograms/kg), prolonged bleeding time was normalized; plasma ristocetin cofactor activity, von Willebrand factor antigen, and factor VIII activity were remarkably increased; and high-molecular-weight forms of von Willebrand factor were demonstrated by crossed immunoelectrophoresis in both patients. Excellent hemostasis was achieved following administration of DDAVP in one patient when it was used for the treatment of gum bleeding and for the prophylaxis of bleeding during and after dental extractions. These observations suggest that DDAVP is an effective alternative to blood products for at least some patients with acquired von Willebrand syndrome in addition to patients with inherited von Willebrand disease, hemophilia A, and uremia.     

High-throughput molecular diagnosis of von Willebrand disease by next generation sequencing methods

Genetic analysis of von Willebrand disease by von Willebrand factor gene sequencing has not yet become routine practice. Nevertheless, the prospects for molecular diagnosis have changed dramatically in recent years with the unveiling of next-generation sequencing platforms. With the goal of applying this technology to von Willebrand disease, we designed a strategy for von Willebrand factor gene enrichment and multiplexing based on short polymerase chain reactions. Forty patients were simultaneously analyzed enabling the identification of 43 mutations, including 36 substitutions, 2 intronic splice site mutations, 2 indels, and 3 deletions. By pooling patient genomic DNA before polymerase chain reaction enrichment, indexing samples with barcode tags, and re-sequencing on the next-generation sequencing instrument, at least 350 patients and relatives per run can be simultaneously analyzed in a fast, inexpensive manner. This is one of the first reports in which this technology has been shown to be feasible for large-scale mutation screening by single gene re-sequencing.     

Optimizing therapy with factor VIII/von Willebrand factor concentrates in von Willebrand disease

In von Willebrand disease, the main goals of treatment are to correct the dual defect of haemostasis caused by a reduced or abnormal von Willebrand factor (vWF), i.e. the prolonged bleeding time (BT) and the deficiency of factor VIII coagulant activity (FVIII:C). The synthetic vasopressin analogue, desmopressin (DDAVP), has reduced the need for transfusions in most of the mild forms of von Willebrand disease but DDAVP is ineffective in type 3 and in other severe cases of types 1 and 2 von Willebrand disease. For many years cryoprecipitate has been the mainstay of replacement therapy but, after the introduction of virucidal methods, concentrates containing FVIII/vWF have been considered much safer than cryoprecipitate and proposed in von Willebrand disease management. FVIII/vWF concentrates have been produced and tested by many authors but there is only one report describing four virus-inactivated FVIII/vWF concentrates evaluated in a cross-over randomized trial. According to these in vitro and pharmacokinetic data, the following information can be derived: (a) no FVIII/vWF concentrate had an intact multimeric structure similar to that of normal plasma or of cryoprecipitate; (b) all FVIII/vWF concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed in the concentrate devoid of FVIII:C; (c) no FVIII/vWF concentrate consistently normalized the BT in a sustained fashion. On the other hand, clinical haemostasis can be achieved in the management of bleeding episodes and of surgery for most of von Willebrand disease cases regardless of whether the BT is corrected; in the few rare cases with mucosal bleeding not controlled by FVIII/vWF concentrates, infusion of DDAVP or platelet concentrates can be administered in addition.     

In vitro and in vivo characterization of a high-purity, solvent/detergent-treated factor VIII concentrate: evidence for its therapeutic efficacy in von Willebrand's disease

A factor VIII (FVIII) concentrate, virus-inactivated by the solvent/detergent procedure, was studied in vitro. In contrast with most high-purity, virus-inactivated FVIII concentrates, it contains not only high levels of von Willebrand factor (vWF) antigen and ristocetin cofactor activity but also high molecular weight forms of von Willebrand factor. Furthermore, it is able to promote platelet adhesion on collagen in a perfusion system. In vivo studies performed in patients with different types of von Willebrand's disease provided evidence that this concentrate corrects Duke's bleeding time and prevents or stops haemorrhages. Thus, the particular advantages of this FVIII/vWF preparation are safety, low content of contamination proteins, and efficacy in von Willebrand's disease.