Two novel ADAMTS13 gene mutations in thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS)

Two novel ADAMTS13 gene mutations in thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS).BackgroundThrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS) are now considered to be variants of one single syndrome called thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS). Key features are thrombocytopenia, hemolytic anemia, and subsequently impaired function of different organs, especially the kidneys and the central nervous system (CNS). One possible reason is the deficiency of von Willebrand factor-cleaving protease (vWF-CP) resulting in persistence of uncleaved, ultralarge von Willebrand factor multimers (ULvWFM).MethodsWe report a patient who was initially diagnosed with Evans syndrome (hemolytic anemia and autoimmune thrombocytopenia) as infant. At 10 years of age he developed HUS-like disease with gastrointestinal tract infection, hemolytic anemia, thrombocytopenia,and acute renal failure. However, enteropathogenic Escherichia coli–like or Shiga-like toxins were not detected.ResultsFurther investigations revealed severe deficiency (<3%; normal >40%) of vWF-CP activity caused by compound heterozygosity of two novel ADAMTS13 gene mutations (1170 G>C [W390C] and 3735 G>A [W1245X]. vWF-CP autoantibodies were not detected. Periodic (every 2 weeks) treatment with fresh frozen plasma (FFP) maintained both platelet level and kidney function within normal range and prevented new episodes of TTP/HUS.ConclusionEnteropathogenic E. coli– and Shiga-like toxin-negative patients who present with hemolytic or thrombocytopenic episodes and HUS like symptoms should be tested for vWF-CP deficiency and other noninfectious reasons for TTP/HUS since plasma substitution possibly provides an efficient therapeutic option for this subgroup of patients

Assessing thrombogenesis and treatment response in congenital thrombotic thrombocytopenic purpura.

Despite clinical remission and normal platelet counts, congenital TTP (cTTP) is associated with non-overt symptoms. Prophylactic ADAMTS13 replacement therapy such as plasma infusion (PI) prevents acute episodes and improves symptomatology. There is no current method to investigate disease severity or monitor the impact of treatment. We utilize a dynamic high shear flow assay to further understand disease pathophysiology and determine the impact of cTTP on symptomatology and therapy, despite normal platelet counts. Whole blood, under high shear, was run over collagen-coated channels, causing platelet adhesion to von Willebrand factor (VWF) multimers. The resulting surface coverage by platelet-VWF thrombus was assessed. The normal range was 6-39% in 50 controls. Twenty-two cTTP patients with normal platelet counts were evaluated. Median pre-treatment surface coverage was 89%, and PI reduced coverage to a median of 44% (p = 0.0005). Patients taking antiplatelets had further reduced coverage when combined with PI and improved non-overt symptoms such as headache, lethargy, and abdominal pain in 100% of patients compared to 74% with PI alone (p = 0.046). We use a dynamic assay to report increased in vitro platelet adhesion and aggregation and additionally demonstrate significantly decreased thrombi following PI, with levels in the normal range levels achieved in patients taking additional antiplatelet therapy

Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p. Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p. Cys2771Arg and p. Cys2773Arg, we found the majority of monomers (87 +/- 5% and 73 +/- 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 +/- 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p. Cys2771Arg, p. Cys2773Arg, and p. Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini;a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis

Exponential Size Distribution of von Willebrand Factor

AbstractVon Willebrand Factor (VWF) is a multimeric protein crucial for hemostasis. Under shear flow, it acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Here, we quantify for the first time, to our knowledge, the size distribution of recombinant VWF and VWF-eGFP using a multilateral approach that involves quantitative gel analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy. We find an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization process during VWF biosynthesis. The distribution is solely described by the extent of polymerization, which was found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution is monitored using fluorescence correlation spectroscopy and compared to a computer simulation of a random cleavage process relating ADAMTS13 concentration to the degree of VWF breakdown. Quantitative assessment of VWF size distribution in terms of an exponential might prove to be useful both as a valuable biophysical characterization and as a possible disease indicator for clinical applications

Distinct Mechanisms of IgM Antibody-Mediated Acquired von Willebrand Syndrome and Successful Treatment with Recombinant von Willebrand Factor in One Patient

Acquired von Willebrand Syndrome (AVWS) is a rare coagulation disorder which can be associated with IgM paraproteinaemia. Recently, recombinant von Willebrand factor (rVWF) has become available for the treatment of bleedings in patients with inherited von Willebrand disease, but experience in patients with AVWS is limited. We report on 2 patients with AVWS with underlying IgM paraproteinaemia with distinct underlying pathomechanisms. In 1 patient, the paraprotein built unspecific complexes with von Willebrand factor (VWF). In the other patient, we were able to detect an IgM antibody against VWF. Bleeding in this patient was successfully treated with rVWF. To our knowledge, this is the first report about the successful use of rVWF in a patient with AVWS with the detection of a VWF-specific antibody

Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort

Several cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however, these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17–18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships

Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion