Type 2A (IIH) von Willebrand disease is due to mutations that affect von Willebrand factor multimerization

Abstract

Summary. Background: Type IIH von Willebrand disease was reported 20 years ago as a novel variant characterized by the loss of the largest multimers in plasma and platelets and absence of the typical triplet structure. Objectives and methods: The propositus and his daughter have been reinvestigated and characterized at the molecular level. The identified mutations were expressed in COS‐7 cells to evaluate the mechanism of this variant. Results and Discussion: The propositus had normal von Willebrand factor (VWF):ristocetin cofactor activity (RCo) and high VWF antigen (VWF:Ag) values, with a low VWF:RCo/VWF:Ag ratio (0.51). No abnormalities were found in his daughter, except for the reduced triplet structure in plasma VWF and diminished ultralarge VWF (ULVWF) multimers in platelets. Three mutations were identified in the propositus: 604C>T (R202W), 4748G>A (R1583Q), and 2546G>A (C849Y). The amounts of secreted recombinant VWF (rVWF) were apparently increased for R202W (130%), R202W‐R1583Q (131%), and R202W‐R1583Q/WT (121%), reduced for C849Y (72%) and C849Y/WT (83%), and normal for R1583Q (107%) and R202W‐R1583Q/C849Y (102%). In cell lysates, higher values were found in association with the C849Y mutation. A normal multimeric pattern was found in R1583Q rVWF, mainly dimers in R202W rVWF, and intermediate molecular weight multimers in C849Y rVWF. Hybrid R202W‐R1583Q/WT and C849Y/WT rVWFs had a nearly normal multimeric pattern, whereas in hybrid R202W‐R1583Q/C849Y rVWF there was a loss of large/intermediate multimers. Conclusions: The propositus phenotype seems to be due to mutations R202W and C849Y, both affecting the VWF multimerization process and, for C849Y rVWF, intracellular survival. The absent triplet multimeric structure in the propositus and its reduction in his daughter appears to be related to the lack of ULVWF multimers, which mainly contribute to the formation of satellite bands.

Abnormal VWF modifies megakaryocytopoiesis: studies of platelets and megakaryocyte cultures from patients with von Willebrand disease type 2B

“…Recombinant mutant R1306W-VWF (VWF2B) was obtained using stable cell lines as described. 17 Fibrinogen (FG) was purified from human plasma as reported. 18 Human fibronectin (FN) was obtained from Sigma-Aldrich.…”

Molecular characterization, recombinant protein expression, and mRNA analysis of type 3 von Willebrand disease: Studies of an Italian cohort of 10 patients

“…HRM analysis was then carried out for 25 exons (number 4,6,8,9,11,12,16,17,19,20,21,23,25,30,34,35,36,38,39,40,41,44,46,48,51) in all patients. This is a mutation scanning technique that monitors the progressive change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured with marginal increases in temperature [18].…”

“…(c.6634T>C), and pSV-VWFH-C2325S (c.6973T>A) were generated by site-directed mutagenesis (QuikChange 1 II XL Site-Directed Mutagenesis kit; Stratagene, Cedar Creek, TX), using as template the expression vector pSV-VWFH [11] and specifically designed oligonucleotides [20].…”

“…COS-7 cells were grown at full confluence into a six-well cell culture plate and were transfected with the appropriated amount of expression vector [wild-type (WT), mutant and both to generate mutant/WT recombinant VWF hybrids] using jetPEI TM transfection reagent (PolyPlus-transfection, Euroclone, Pero, Italy) as previously reported [20]. After 72 h of incubation, conditioned media and cell lysates were harvested and collected [11], recombinant VWF (rVWF) antigen levels were quantified and were expressed as mean values ± standard deviation.…”

Identification and Functional Analysis of a Novel von Willebrand Factor Mutation in a Family with Type 2A von Willebrand Disease

“…von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by mutations in the VWF gene [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], which result in quantitative and/or qualitative defects in VWF [2], [19]. VWD is classified into three types: types 1 and 3 refer to different degrees of quantitative VWF deficiencies, whereas type 2 refers to all qualitative VWF deficiencies.…”