A sensitive immunoassay for von Willebrand factor

We have developed an ELISA specific for canine von Willebrand factor antigen (vWF:Ag) that also strongly reacts with the VWF:Ag of humans and many other vertebrates. This assay was designed to avoid the use of immunoreagents of human origin, however, commercially available antibodies to human vWF:Ag may also be used. von Willebrand factor (vWF) was quantitated using a modified double-sandwich ELISA with polyclonal antibodies specific for canine vWF:Ag. The assay was as sensitive for measuring canine vWF:Ag as previously published immuno-radiometric assays and the most sensitive ELISA for human vWF:Ag. Employing commercially available antibodies to human vWF:Ag in the same double-sandwich configuration, the lower limit of detection for human vWF:Ag was 4.8 x 10(-6) units/ml, lower by a factor of ten than previously reported ELISAs. In addition, a wide range of vWF:Ag levels can be determined with just a single plasma dilution. The assay readily distinguishes type III von Willebrand disease from other types of von Willebrand disease having very low levels of vWF. This vWF ELISA can be used to evaluate large numbers of plasma samples simultaneously and is therefore well-suited for large-scale screening programs.     

Von Willebrand factor and platelet adhesiveness

A modification of Salzman's method has been used in an attempt to provide an assay in vitro for the von Willebrand factor. Platelet adhesiveness was increased in von Willebrand's disease by previously coating the beads with normal or haemophilic plasma or cryoprecipitate, whereas von Willebrand plasma had no corrective effect. Antihaemophilic factor (AHF) concentrates were studied in the same way and results compared with experiments in vivo.     

von Willebrand factor release and thrombomodulin and tissue factor expression in Rickettsia conorii-infected endothelial cells.

Mediterranean spotted fever, a tick-borne rickettsiosis caused by Rickettsia conorii, may lead to small-vessel or deep-vein thrombosis. In order to evaluate the role of endothelial cell alteration in this lesion, we infected human endothelial cells derived from umbilical veins with R. conorii. We report the induction of two previously unreported prothrombotic mechanisms in rickettsial disease: (i) a progressive decline in thrombomodulin antigen and (ii) early expression of tissue factor, and, as described for R. rickettsii infection, later release of von Willebrand factor from Weibel-Palade bodies. Thrombomodulin expression in infected endothelial cells, measured by the thrombin-dependent activation of protein C or flow cytometric analysis, decreased steadily between 4 and 24 h after inoculation with rickettsiae. R. conorii infection induced tissue factor expression, measured by clotting assay and flow cytometric analysis, which was detectable 2 h postinoculation, reached its maximum 4 h postinoculation, and progressively decreased thereafter. Infection resulted in a relatively late release of von Willebrand factor antigen into the culture medium. A double-label immunofluorescence assay for the simultaneous evaluation of von Willebrand factor and R. conorii showed that the depletion of cytoplasmic von Willebrand factor stored in Weibel-Palade bodies was due to a direct effect of the intracellular R. conorii. These disturbances of endothelial function observed with R. conorii-infected cells may provide a paradigm for the elucidation of thrombotic pathobiology with Mediterranean spotted fever.     

von Willebrand disease

von Willebrand disease is a common inherited bleeding disorder characterized by excessive mucocutaneous bleeding. Characteristic bleeding symptoms include epistaxis, easy bruising, oral cavity bleeding, menorrhagia, bleeding after dental extraction, surgery, and/or childbirth, and in severe cases, bleeding into joints and soft tissues. There are three subtypes: types 1 and 3 represent quantitative variants and type 2 is a group of four qualitative variants: (1) type 2A—characterized by defective von Willebrand factor-dependent platelet adhesion because of decreased high-molecular-weight von Willebrand factor multimers, (2) type 2B—caused by pathologically increased von Willebrand factor-platelet interactions, (3) type 2M—caused by decreased von Willebrand factor-platelet interactions not based on the loss of high-molecular-weight multimers, and (4) type 2N—characterized by reduced binding of von Willebrand factor to factor VIII. The diagnosis of von Willebrand disease requires specialized assays of von Willebrand factor and/or molecular genetic testing of von Willebrand factor. Severe bleeding episodes can be prevented or controlled with intravenous infusions of virally inactivated plasma-derived clotting factor concentrates containing both von Willebrand factor and factor VIII. Depending on the von Willebrand disease type, mild bleeding episodes usually respond to intravenous or subcutaneous treatment with desmopressin, a vasopressin analog. Other treatments that can reduce symptoms include fibrinolytic inhibitors and hormones for menorrhagia.     

Identification of von Willebrand disease type 2N (Normandy) in Australia: a cross-laboratory investigation using different methods

We report on a cross-laboratory study of type 2N von Willebrand disease (vWD). We tested 101 selected plasma samples for factor VIII and factor VIII binding activity of von Willebrand factor (vWF). Of these plasma samples, 31 were cotested by 2 specialist centers using different detection procedures for vWF-factor VIII binding: there was good agreement between results obtained by chromogenic assay and enzyme-linked immunosorbent assay. In total, 8 patients with type 2N vWD were identified. The 2-stage factor VIII assay detected a deficiency of factor VIII relative to vWF antigen in all 8 patients; the 1-stage factor VIII assay detected a relative deficiency in only 3 patients. Four patients were homozygous for the most common type 2N mutation (R854Q), 3 patients were presumed to be compound heterozygotes, and in 1 patient no type 2N mutations were identified. In this study of patients from 5 specialist centers in Australia, type 2N vWD was found in 5 families. The 2-stage factor VIII assay was more useful as a screening test than the 1-stage assay, and both vWF-factor VIII binding assays were equally effective.     

Von Willebrand factor and von Willebrand's disease: a complex protein and a complex disease

Von Willebrand factor is a complex protein which is important in several ways for normal hemostasis. Von Willebrand's disease results when there is either a quantitative or qualitative disorder of von Willebrand factor. In this review, the structure and function of von Willebrand factor are discussed. Additionally, the current laboratory and clinical classification of von Willebrand's disease and closely related variants are outlined.     

The use of high-dose intravenous gamma-globulin in acquired von Willebrand syndrome

Acquired von Willebrand syndrome has been reported in patients with a variety of primary diseases, many immunologic in nature. Usually, an autoantibody to von Willebrand factor can be identified. These patients often experience severe hemorrhages requiring large doses of cryoprecipitate or factor VIII concentrates, thus exposing them to viral and allergic complications. The success of intravenous gamma-globulin in the treatment of other autoimmune diseases prompted us to treat two patients with acquired von Willebrand syndrome with high-dose intravenous gamma-globulin. Two days after initiation of therapy, von Willebrand factor and factor VIII rose to normal levels in both patients. Patient 1 underwent dental surgery, and patient 2 underwent a splenectomy without increased bleeding and without additional factor coverage or desmopressin acetate therapy. Thus, intravenous gamma-globulin is efficacious for acquired von Willebrand syndrome and obviates the need for replacement therapy with its attendant complications.     

Acquired Von Willebrand syndrome

We report a case of acquired von Willebrand syndrome in association with multiple myeloma, diagnosed in a 54 year-old man suffering from sudden onset of mucocutaneous bleeding. The acquired von Willebrand syndrome is a rare bleeding disorder with laboratory findings similar to those of inherited von Willebrand disease. Diagnosis, etiology and pathophysiology of acquired von Willebrand syndrome are reviewed to establish a differential diagnosis with inherited von Willebrand disease. Identification of the underlying disease responsible for the acquired von Willebrand factor defect and the bleeding diathesis is necessary to choose among the therapeutic options the appropriate treatment.     

The rapid differentiation of type IIb von Willebrand's disease from platelet-type (pseudo-) von Willebrand's disease by the "neutral" monoclonal antibody binding assay.

The differentiation of type IIb von Willebrand's disease from other variants of von Willebrand's disease, especially platelet-type (pseudo-) von Willebrand's disease, poses a significant clinical problem because, although they are similar in the clinical and diagnostic laboratory settings, the therapy of type IIb von Willebrand's disease is different from the therapy of platelet-type von Willebrand's disease. This discrimination has required cumbersome assays using fresh platelet-rich plasma that often yielded equivocal results. Because it was shown by other researchers that type IIb von Willebrand factor binds to normal platelets with increased avidity at low concentrations of ristocetin, it was reasoned that von Willebrand factor from patients with type IIb von Willebrand's disease would also bind to formalin-fixed washed platelets at low concentrations of ristocetin. Using the radiolabeled "neutral" monoclonal antibody AVW1 to label plasma von Willebrand factor, the binding of von Willebrand factor to formalin-fixed washed platelets was studied as a function of ristocetin concentration. These studies demonstrated that the 125I-AVW1 von Willebrand factor from 13 patients with type IIb von Willebrand's disease binds to formalin-fixed washed platelets at significantly lower concentrations of ristocetin than plasma von Willebrand factor from 18 normal individuals, 3 patients with platelet-type von Willebrand's disease and 8 patients with other variant forms of von Willebrand's disease. This radiolabeled "neutral" monoclonal antibody technique provides a rapid, simple method for the differentiation on frozen plasma samples of type IIb von Willebrand's disease from platelet-type and other variants of von Willebrand's disease.     

A new automated screening assay for the diagnosis of von Willebrand disease

A new, automated assay for von Willebrand factor (vWF) activity has recently become commercially available (HemosIL vWF activity assay, Instrumentation Laboratories, Lexington, MA). We prospectively studied 61 specimens from 58 patients undergoing laboratory testing for suspicion of von Willebrand disease with this new method, in comparison with the established ristocetin cofactor method. Assays for factor VIII and vWF antigen were also performed using an established method on an MDA-180 coagulation analyzer (bioMérieux, Durham, NC) and a new method on an ACL TOP coagulation analyzer (Instrumentation Laboratories). Blood types were determined. The results showed no significant difference between the assays for factor VIII (mean, 97% for MDA-180 and ACL TOP; P = .494) or vWF antigen (mean, MDA-180, 109%; ACL TOP, 111%; P = .766). The mean result for the ristocetin cofactor assay was 106% vs 93% with the automated vWF activity (P = .007). The automated activity assay was 100% sensitive and 86% specific for detecting vWF abnormalities and seems to be a suitable screening test. Abnormal results should be followed up with a ristocetin cofactor activity assay for confirmation. Further study is recommended to confirm these conclusions.     

Von Willebrand's disease: combined qualitative and quantitative abnormalities.

Studies of von Willebrand's disease typically show either a quantitative or a qualitative abnormality of the factor VIII/von Willebrand factor protein. In studies of five patients we found a combination of quantitative and qualitative defects. Quantitative abnormalities included decreased levels of procoagulant, antigen and von Willebrand factor activities in the plasma and in the column fractions of cryopercipitate from gel chromatography. Qualitative abnormalities included annormal migration or shape of the crossed antigen-antibody arcs, disproportionate reduction of von Willebrand factor in relation to antigen, altered gel elution patterns of procoagulant, and von Willebrand factor activities the negative carbohydrate stain of the VIII protein on polyacryl-amide-gel electrophoresis and, in two patients, a decreased sialic acid content of factor VIII protein. Our studies indicate that von Willebrand's disease comprises both quantitative and qualitative defects.     

Le syndrome de Willebrand acquis : du diagnostic au traitement

Acquired von Willebrand syndrome is a rare bleeding disorder, which has been related in various diseases including lymphoproliferative disorders or autoimmune diseases. Its diagnosis is an important step before treatment of patients and particularly in case of bleeding. We report four cases from Caen Hemophilia Treatment Center, diagnosed and treated from 1999 to 2008. Mucocutaneous bleeds in every case were the same as in hereditary von Willebrand disease. All patients had no personal or family history of bleeding. Phenotype was identified as type 2 von Willebrand disease with a loss of high molecular weight multimers. Anti-von Willebrand factor inhibitor screening was positive for three patients. The etiological diagnosis was one chronic lymphocytic leukaemia, two monoclonal gammapathies of undetermined significance (MGUS) and one undetermined case. The management of patients need two stages: first infusions of factor von Willebrand/factor VIII concentrates to stop bleeds, then treatment of the underlying disease such as chemotherapy, corticotherapy and treatment with high doses of polyvalents immunoglobulins. In every case, treatment was effective and improved patient's quality of life.     

肺癌患者凝血功能与肺癌分期的关系

目的 探讨肺癌患者凝血功能与肺癌分期的关系.方法 采用ELISA、免疫比浊法检测140例晚期恶性肿瘤患者和30例正常人的凝血、抗凝活性相关的实验室指标,包括血浆纤维蛋白原（ Fb）,抗凝血酶Ⅲ （AT-Ⅲ）,血管性血友病因子 （ vWF）,D-二聚体（ D-D）.结果 晚期恶性肿瘤患者的血浆D-D、Fb、AT-Ⅲ、vWF抗原的水平较正常人显著升高;在肺癌Ⅲ期、Ⅳ期与Ⅰ期相比明显增高（P＜0.05）,差异有统计学意义;Ⅱ期与Ⅰ期相比增高不明显（P〉0.05）,差异没有统计学意义.结论 肺癌患者存在凝血、抗凝、纤溶系统的激活,机体呈现高凝状态,并且高凝状态与肺癌临床分期呈正相关.     

Acquired von Willebrand's disease. Evidence for a quantitative and qualitative factor VIII disorder.

To define further the factor VIII abnormality in acquired von Willebrand's disease, we performed immunoelectrophoresis of factor VIII antigen, as well as quantitative measurements of the antigen, factor VIII procoagulant activity and von Willebrand factor activity on plasma from an affected 57-year-old man who also had a poorly differentiated lymphocytic lymphoma. No evidence for an inhibitor against factor VIII procoagulant activity or von Willebrand factor activity was detected, but immunoelectrophoresis showed none of the less anodic forms of factor VIII antigen. There were concomitant decreases in total antigen (0.19 U per milliliter) and von Willebrand factor levels (0.12 U per milliliter). Factor VIII-procoagulant activity was borderline low (0.45 U per milliliter). Correction of both the abnormal immunoelectrophoresis pattern and the quantitative abnormalities followed radiotherapy of the lymphoma. The factor VIII abnormalities might have resulted from binding or destruction of theless anodic forms of factor VIII antigen by the malignant lymphocytes.     

血管组织中Plaur和Plat诱导血管性血友病因子释放促血栓形成

背景：目前,深静脉血栓形成的分子病因学机制及其形成的核心调控网络仍未完全阐明,对于深静脉血栓的早期诊断预测也无理想的方法。 目的：观察创伤性深静脉血栓形成大鼠静脉内皮细胞中Plaur和Plat的促血栓形成作用。 方法：采用股静脉钳夹联合下肢石膏制动构建大鼠创伤性深静脉血栓模型。依据取材时间及是否有血栓形成分为血栓形成前组、血栓形成组和血栓不形成组,分别于造模后2.5,25 h取大鼠股静脉内皮细胞用于实验。 结果与结论：基因芯片分析及real-time PCR结果均显示创伤后2.5 h,大鼠股静脉Plaur、Plau及血管性血友病因子基因表达上调;血栓形成时,Plaur、Plau及血管性血友病因子基因表达上调更显著。信号通路分析显示Plaur、Plau为血管性血友病因子的上游调控基因,血管性血友病因子为触发血小板黏附、聚集及血栓形成的关键基因。提示Plaur、Plau可通过上调血管性血友病因子表达,引发血小板黏附、聚集,促进大鼠创伤性深静脉血栓形成。     

Measurement of platelet von Willebrand factor is dependent on method of platelet isolation

The authors have compared platelet von Willebrand factor antigen and ristocetin cofactor activity measurements in a normal population, using two different previously published platelet isolation techniques. Preparation of a platelet lysate by platelet washes using Tyrode's albumin solution and inhibitors yielded a threefold higher vWF antigen and a twofold higher ristocetin cofactor activity measurement as compared to platelets isolated by tris buffered saline washes containing 0.1% (weight/volume) Na2EDTA. Contamination by plasma von Willebrand factor to account for the higher values found by the first method was excluded by monitoring the presence of added purified I125 von Willebrand factor to platelet rich plasma. vWF multimer analysis showed the same distribution of multimers in both preparations but suggested a relative decrease in lower molecular weight multimers with the second method. Platelet isolation can result in significant loss of platelet von Willebrand factor, with a preferential loss of lower molecular weight multimers, likely resulting from unintentional platelet release.     

Variations of plasma Willebrand factor and fibrinogen in coronary pathology]

In order to determine a marker of prethrombotic states, reliable and easy to measure, we studied 200 patients under 60 years of age admitted to hospital for a precordial chest pain. Four groups were established: transmural myocardial infarction, acute infarction without Q wave, unstable angina and atypical chest pain. Fibrinogen, von Willebrand factor, cholesterol, and triglyceride levels were measured as well as the white cell and platelet counts. There was a statistically significant correlation between transmural myocardial infarction and increased levels of fibrinogen, von Willebrand factor and white blood cells. Von Willebrand factor was already increased in the acute phase of transmural infarction, reached a maximum on the fifth day and then decreased slowly during the following ten days. This study suggests that plasma von Willebrand factor could be a reliable marker of transmural myocardial infarction in the acute phase or during the two weeks following the thrombotic event.     

Platelet aggregation induced by 1-desamino-8-D-arginine vasopressin (DDAVP) in Type IIB von Willebrand's disease

Type IIB von Willebrand's disease is a distinct form of this disorder, in which there are abnormal factor VIII/von Willebrand factor multimers in plasma (but normal multimers in platelets) and heightened interaction between the von Willebrand factor and platelets in the presence of ristocetin. We have found that infusion of desmopressin acetate (1-desamino-8-D-arginine vasopressin [DDAVP]), an agent used in the treatment of von Willebrand's disease, causes platelet aggregation and thrombocytopenia in patients with Type IIB disease. In vitro, platelets in normal plasma and those obtained from patients with Type IIB disease before DDAVP infusion aggregated upon the addition of platelet-poor plasma from Type IIB patients treated with DDAVP. Platelet aggregation was associated with adsorption of multimers of factor VIII/von Willebrand factor onto the platelets and was inhibited by EDTA. We conclude that in Type IIB von Willebrand's disease, DDAVP releases an abnormal factor with platelet-aggregating properties. DDAVP should not be used to treat patients with Type IIB disease, since the presence of platelet aggregates in the circulation may be harmful.     

Additional factor XII (hageman factor) deficiency in hemophilia a and in von willebrand syndrome

Summary Factor XII plasma levels were investigated with several methods in patients with hemophilia A and B and von Willebrand syndrome. There seem to be some families with hemophilia A or von Willebrand syndrome, who have an additional, congenital, partial lack of factor XII (Hageman factor). The mode of inheritance is independent of the other coagulation disorder. Frequently, the first indication of an additional factor XII deficiency is the disproportionate prolongation of the activated partial thromboplastin time (PTT) as regards the factor VIII level. The average factor XII level in patients with hemophilia A and von Willebrand syndrome is significantly lower than in normal subjects or patients with hemophilia B. It cannot be excluded that the frequently low levels of factor XII in patients with severe hemophilia are acquired and probably due to liver cell damage.Dedicated to Professor F. Hartmann, MD     

Use of intravenous immunoglobulin in patients with acquired von Willebrand syndrome

Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those for congenital von Willebrand disease. Unlike the congenital form, AVWS usually occurs in individuals with no personal or family history of bleeding disorders. According to an international registry, AVWS is mainly associated with lymphomyeloproliferative, immunologic, and cardiovascular disorders, as well as with solid tumors and other miscellaneous conditions; however, the prevalence of AVWS in these underlying disorders is still unknown. von Willebrand factor (VWF) is synthesized normally in most AVWS patients, and the low plasma VWF levels are from its accelerated removal from plasma by five different mechanisms, including autoantibodies. Because of the reduced half-life of endogenous-exogenous plasma VWF, bleeding of AVWS cannot be managed with desmopressin or factor VIII/VWF concentrates. Clinical use of intravenous immunoglobulin (IVIg) in AVWS has been reported since 1988. IVIg is most effective in AVWS with type immunoglobulin (Ig) G monoclonal gammopathies of undetermined significance and in other cases with IgG autoantibodies. IVIg can correct factor VIII and von Willebrand factor complex activities for about 15-20 days, and repeated injections induce remission of AVWS in these patients. Prospective studies are required to evaluate the efficacy and safety of IVIg in AVWS.