Characterization Of Von Willebrand Factor Research Articles

Lowering the increased intracellular pH of human-induced pluripotent stem cell-derived endothelial cells induces formation of mature Weibel-Palade bodies.

Differentiation of human‐induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial‐like cells (hiPSC‐derived endothelial cells [hiPSC‐ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC‐ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel‐Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC‐ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC‐ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC‐ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC‐ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC‐EC. Mechanistically, pro‐VWF dimers require the lower pH environment of the trans‐Golgi network for maturation and tubulation. These data show that while hiPSC‐ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function.

Diagnosis and therapeutic management in a patient with type 2B-like acquired von Willebrand syndrome

Acquired von Willebrand syndrome (AVWS) usually mimics von Willebrand disease (VWD) type 1 or 2A. However, in rare cases, the characteristics of other VWD types can predominate in AVWS that might require careful consideration of differential treatment options. The diagnosis and the treatment of a case of type 2B-like AVWS are discussed. Diagnosis of AVWS was ascertained by determining ristocetin cofactor activity, ristocetin-induced platelet aggregation, von Willebrand factor antigen, collagen binding and characterization of von Willebrand factor (VWF) multimers. Inhibitor presence was sought through mixing experiments, the Bethesda method, and calculation of the in-vivo recovery and plasma half-life of VWF after administration of factor VIII/VWF concentrate. Mutations in the A1 domain of VWF were ruled out by sequencing of exon 28 of the VWF gene. A 34-year-old male patient, putatively diagnosed with type 2B VWD, and undergoing laparoscopic cholecystectomy, did not respond adequately to perioperative hemostatic treatment with desmopressin and high doses of factor VIII/VWF concentrate, requiring the administration of recombinant activated factor VII. Further diagnostic workup revealed AVWS mimicking type 2B VWD, most likely owing to an autoantibody developed in the course of underlying monoclonal gammopathy of undetermined significance. The presence of AVWS should be considered before a diagnosis of type 2B VWD is made, especially in patients with a history atypical for inherited disease.

Unraveling the mystery of von Willebrand factor.

Almost 35 years ago Jan and I discovered that factor VIII (FVIII) and von Willebrand factor (VWF) were intrinsically related. This feature led to the revelation of VWF as an essential hemostatic entity, deficient in von Willebrand's disease (VWD). We started in the late 1960s, working as PhD students independently of each other, Jan in 1968 on the characterization of FVIII at the ‘Haemophilia Clinic’ [1] and later the Paediatric Clinic of the University of Amsterdam, and I in September 1969 on the characterization of VWF at the Laboratory of Thrombosis and Haemostasis of the University Hospital in Utrecht, a laboratory headed by Dr Jan Sixma consisting of a small research lab and a diagnostic lab.

Characterization of von Willebrand factor in cultured human endothelial cells from patients with different types of von Willebrand disease

Characterization of von Willebrand factor in cultured human endothelial cells from patients with different types of von Willebrand disease

Abnormality of the N-Terminal Portion of von Willebrand Factor in Type IIA and IIC von Willebrand Disease

We have established a new analytical method which allows the characterization of von Willebrand factor (vWF) degradation fragments in minute amounts (10 microliters) of plasma, without the need for immunopurification of vWF. Plasma vWF was hydrolysed by S aureus V-8 protease (V-8 protease) and the cleaved fragments separated by SDS-agarose gel electrophoresis followed by staining with 125I-labeled polyclonal or monoclonal antibodies against vWF and autoradiography. Quantification of the amount of each product was estimated by counting the incorporated radioactivity following excision. V-8 protease limitedly hydrolysed vWF in normal as well as type I von Willebrand disease (vWD) plasma and produced two distinct fragments with identical electrophoretic and antigenic characteristics to those produced from purified vWF, i.e. a C-terminal SpII and a series of N-terminal SpIII fragments (SpIIIa, b and c). The method was applied to further characterize the molecular abnormalities of vWF in eighteen patients with type II vWD. In seven individuals with type IIA and five patients with type IIC, SpIII appeared significantly modified as compared to normal. In type IIA, there was a marked decrease or absence of SpIIIa and an increase of SpIIIb and c. In type IIC, SpIIIb was lacking. In three patients with type IIB and in three patients with type IID, there was no significant modification of SpIII. In all cases, SpII was apparently not modified. In conclusion, the molecular abnormality of vWF in type IIA and IIC vWD appears to reside in SpIII, the N-terminal portion of the vWF-subunit (residues 1 to 1,365).