Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD)

Summary.  Background:  Type 1 von Willebrand disease (VWD) is a congenital bleeding disorder characterized by a partial quantitative deficiency of plasma von Willebrand factor (VWF) in the absence of structural and/or functional VWF defects. Accurate assessment of the quantity and quality of plasma VWF is difficult but is a prerequisite for correct classification. Objective:  To evaluate the proportion of misclassification of patients historically diagnosed with type 1 VWD using detailed analysis of the VWF multimer structure. Patients and methods:  Previously diagnosed type 1 VWD families and healthy controls were recruited by 12 expert centers in nine European countries. Phenotypic characterization comprised plasma VWF parameters and multimer analysis using low- and intermediate-resolution gels combined with an optimized visualization system. VWF genotyping was performed in all index cases (ICs). Results:  Abnormal multimers were present in 57 out of 150 ICs; however, only 29 out of these 57 (51%) had VWF ristocetin cofactor to antigen ratio below 0.7. In most cases multimer abnormalities were subtle, and only two cases had a significant loss of the largest multimers. Conclusions:  Of the cases previously diagnosed as type 1 VWD, 38% showed abnormal multimers. Depending on the classification criteria used, 22 out of these 57 cases (15% of the total cohort) may be reclassified as type 2, emphasizing the requirement for multimer analysis compared with a mere ratio of VWF functional parameters and VWF:Ag. This is further supported by the finding that even slightly aberrant multimers are highly predictive for the presence of VWF mutations.     

ADAMTS-13, von Willebrand factor and related parameters in severe sepsis and septic shock

BACKGROUND: Insufficient control of von Willebrand factor (VWF) multimer size as a result of severely deficient ADAMTS-13 activity results in thrombotic thrombocytopenic purpura associated with microvascluar thrombosis and platelet consumption, features not seldom seen in severe sepsis and septic shock. METHODS: ADAMTS-13 activity and VWF parameters of 40 patients with severe sepsis or septic shock were compared with those of 40 healthy controls of the same age and gender and correlated with clinical findings and sepsis outcome. RESULTS: ADAMTS-13 activity was significantly lower in patients than in healthy controls [median 60% (range 27-160%) vs. 110% (range 63-200%); P < 0.001]. VWF parameters behaved reciprocally and both VWF ristocetin cofactor activity (RCo) and VWF antigen (VWF:Ag) were significantly (P < 0.001) higher in patients compared with controls. Neither ADAMTS-13 activity nor VWF parameters correlated with disease severity, organ dysfunction or outcome. However, a contribution of acute endothelial dysfunction to renal impairment in sepsis is suggested by the significantly higher VWF propeptide and soluble thrombomodulin levels in patients with increased creatinine values as well as by their strong positive correlations (creatinine and VWF propeptide r(s) = 0.484, P < 0.001; creatinine and soluble thrombomodulin r(s) = 0.596, P < 0.001). CONCLUSIONS: VWF parameters are reciprocally correlated with ADAMTS-13 activity in severe sepsis and septic shock but have no prognostic value regarding outcome.     

von Willebrand factor: two sides of a coin

Everyone experiences minor bleeding and clotting, and many illnesses feature extremes of hemorrhage or thrombosis. Recent advances have illuminated the ways in which von Willebrand factor (VWF) contributes to both kinds of hemostatic emergency, whether mundane or life threatening, often through disturbances in VWF synthesis or catabolism. von Willebrand factor multimer assembly depends on the ability of the propeptide to promote disulfide bond formation in the Golgi, possibly by acting as a pH-sensitive oxidoreductase. Once secreted into the blood, multimers are subject to competing processes of clearance and of proteolysis by ADAMTS-13. Defects in the secretion or intravascular clearance of VWF can cause exceptionally severe forms of von Willebrand disease (VWD) type 1. Defects in the assembly of VWF multimers, or exaggerated proteolytic degradation by ADAMTS-13, can cause VWD type 2A and contribute to VWD type 2B. Conversely, defects in the feedback proteolysis of VWF by ADAMTS-13 can cause thrombotic thrombocytopenic purpura (TTP). The pathophysiologic importance of VWF is not limited to the dramatic phenotypes of VWD and TTP. In fact, VWF level also correlates with thrombosis risk and inversely with bleeding risk within the apparently healthy population. More research is needed to understand how VWF function is regulated, and to enable physicians to use this knowledge for the benefit of their patients.     

von Willebrand factor is a major determinant of ADAMTS-13 decrease during mouse sepsis induced by cecum ligation and puncture

Summary.  Background:  During sepsis, von Willebrand factor (VWF) is abundantly secreted; the main mechanism regulating its size involves specific proteolysis by the metalloprotease ADAMTS-13. Objectives:  To determine whether ADAMTS-13 consumption due to its binding to, and/or cleavage, of VWF contributes to its decrease during sepsis and whether abrogating or enhancing ADAMTS-13 activity influences sepsis outcome. Methods:  ADAMTS-13 activity was evaluated in a model of sepsis induced by cecum ligature and puncture (CLP) in wild-type and Vwf ^−/− mice. Sepsis outcome was studied in those mice and in Adamts-13 ^−/− mice. Finally, survival was studied in wild-type mice injected hydrodynamically with the human ADAMTS-13 gene. Results:  In wild-type mice, CLP-induced sepsis elicited a significant ADAMTS-13 decrease, and a strong negative correlation existed between VWF and ADAMTS-13. In Vwf ^−/− mice, CLP also induced severe sepsis, but ADAMTS-13 was not significantly diminished. Notably, Vwf ^−/− mice lived significantly longer than wild-type mice. In contrast, Adamts-13 ^−/− mice and wild-type mice were comparable with regard to thrombocytopenia, VWF concentrations, absence of thrombi, and survival. Hydrodynamic hADAMTS-13 gene transfer with the pLIVE expression vector resulted in high and stable ADAMTS13 activity in CLP mice; however, no impact on survival was observed. Conclusions : VWF secretion is a major determinant of ADAMTS-13 decrease in the CLP model, and plays an important role in sepsis-induced mortality, but the complete absence of its regulating protease, ADAMTS-13, had no detectable impact in this sepsis model. Furthermore, increasing ADAMTS-13 activity had no impact on survival.     

Thrombotic thrombocytopenic purpura

This overview summarizes the history of thrombotic thrombocytopenic purpura (TTP) from its initial recognition in 1924 as a most often fatal disease to the discovery in 1997 of ADAMTS-13 deficiency as a major risk factor for acute disease manifestation. The cloning of the metalloprotease, ADAMTS-13, an essential regulator of the extremely adhesive unusually large von Willebrand factor (VWF) multimers secreted by endothelial cells, as well as ADAMTS-13 structure and function are reviewed. The complex, initially devised assays for ADAMTS-13 activity and the possible limitations of static in vitro assays are described. A new, simple assay using a recombinant 73-amino acid VWF peptide as substrate will hopefully be useful. Hereditary TTP caused by homozygous or double heterozygous ADAMTS-13 mutations and the nature of the mutations so far identified are discussed. Recognition of this condition by clinicians is of utmost importance, because it can be easily treated and--if untreated--frequently results in death. Acquired TTP is often but not always associated with severe, autoantibody-mediated ADAMTS-13 deficiency. The pathogenesis of cases without severe deficiency of the VWF-cleaving protease remains unknown, affected patients cannot be distinguished clinically from those with severely decreased ADAMTS-13 activity. Survivors of acute TTP, especially those with autoantibody-induced ADAMTS-13 deficiency, are at a high risk for relapse, as are patients with hereditary TTP. Patients with thrombotic microangiopathies (TMA) associated with hematopoietic stem cell transplantation, neo-plasia and several drugs, usually have normal or only moderately reduced ADAMTS-13 activity, with the exception of ticlopidine-induced TMA. Diarrhea-positive-hemolytic uremic syndrome (D+ HUS), mainly occurring in children is due to enterohemorrhagic Escherichia coli infection, and cases with atypical, D- HUS may be associated with factor H abnormalities. Treatment of acquired idiopathic TTP involves plasma exchange with fresh frozen plasma (FFP), and probably immunosuppression with corticosteroids is indicated. We believe that, at present, patients without severe acquired ADAMTS-13 deficiency should be treated with plasma exchange as well, until better strategies become available. Constitutional TTP can be treated by simple FFP infusion that rapidly reverses acute disease and--given prophylactically every 2-3 weeks--prevents relapses. There remains a large research agenda to improve diagnosis of TMA, gain further insight into the pathophysiology of the various TMA and to improve and possibly tailor the management of affected patients.     

The active conformation of von Willebrand factor in patients with thrombotic thrombocytopenic purpura in remission

Summary.  Background:  Functional deficiency of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients is associated with circulating ultralarge von Willebrand factor (VWF) molecules that display spontaneous platelet-binding capacities. Upon remission, however, ADAMTS13 activity does not always return to baseline. Objective:  To study ADAMTS13 and VWF-related features in TTP patients in remission. Methods:  ADAMTS13 activity, anti-ADAMTS13 antibodies, VWF antigen, ultralarge VWF and levels of VWF that circulate in a glycoprotein Ibα-binding conformation were determined in plasma samples of 22 acquired TTP patients in remission between 1 month and 6 years after achieving remission. The composition of active multimers was investigated with a novel immunoprecipitation assay based on monoclonal antibody AU/VWF-a12, which specifically recognizes the active conformation of VWF. Results:  ADAMTS13 activity was undetectable in 23% of the patients, even years after they had achieved remission, and lack of ADAMTS13 activity was associated with increased active VWF levels and the presence of ultralarge VWF multimers. Active VWF levels and ultralarge VWF were also associated with blood groups. Results from immunoprecipitation experiments revealed the full range of multimers to be present. Conclusion : ADAMTS13 deficiency and the concurrent presence of ultralarge VWF and increased active VWF levels can be detected in TTP patients for years after they have achieved remission. Immunoprecipitation results suggest that the active conformation of VWF may be present in the lower molecular weight multimers, but future studies are necessary to confirm our findings.     

Use of a mouse model to elucidate the phenotypic effects of the von Willebrand factor cleavage mutants,Y1605A/M1606A and R1597W

Background:  von Willebrand Factor (VWF) is tightly regulated by the metalloproteinase ADAMTS13, which cleaves VWF to reduce VWF multimer size and binding affinity for collagen and platelets. Objective:  This study examines two VWF mutations, R1597W (enhanced cleavage) and Y1605A‐M1606A (decreased cleavage), to determine their impact on VWF, in addition to ADAMTS13‐mediated cleavage. Methods:  In vitro mouse ADAMTS13 digestions were performed on recombinant proteins. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA, following which VWF antigen, multimer profile and VWF propeptide levels were determined. A ferric chloride injury model of thrombosis was also evaluated. Results:  In vitro ADAMTS13 digestion of full‐length mouse VWF required > 97‐fold higher ADAMTS13 levels for Y1605A/M1606A, and 68% lower ADAMTS13 levels for R1597W compared with wild type. In vivo, R1597W had reduced VWF:Ag and both mutations exhibited increased VWF propeptide/VWF:Ag ratios. R1597W multimers show a lower molecular weight profile compared with wild type and Y1605A/M1606A mice. When co‐injected with Adamts13 cDNA, Y1605A/M1606A multimers were larger compared with wild type, and R1597W showed only a single multimer band and decreased clearance via VWFpp/VWF:Ag ratio. R1597W was associated with reduced thrombus formation but normal platelet accumulation in a ferric chloride injury model while Y1605A/M1606A had a loss of occlusive thrombi but increased platelet accumulation compared with wild type. Conclusions:  This study demonstrates that mutations that alter ADAMTS13 cleavage also can affect VWF clearance, VWF antigen level, multimer structure and thrombotic potential in the VWF knockout hydrodynamic injection model.     

Evidence that high von Willebrand factor and low ADAMTS-13 levels independently increase the risk of a non-fatal heart attack

Summary.  Background:  A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) may influence von Willebrand factor (VWF) levels and consequently the risk of myocardial infarction (MI). Moreover, ADAMTS-13 influences hemostatic plug formation in mouse models. We therefore studied their associations in the Glasgow MI Study (GLAMIS). Methods and results:  We measured ADAMTS-13 and VWF antigen levels by ELISAs in stored plasma from a case–control study of 466 MI cases and 484 age- and sex-matched controls from the same north Glasgow population. There was no correlation between ADAMTS-13 and VWF levels in cases or controls. ADAMTS-13 levels correlated positively with serum cholesterol and triglycerides and body mass index, and negatively with high-density lipoprotein-cholesterol. VWF levels correlated with age, fibrinogen and C-reactive protein. In multivariable analyses including risk factors, VWF correlated positively with risk of MI, and ADAMTS-13 correlated negatively with risk of MI. These associations were independent of each other. The association of ADAMTS-13 with risk of MI was observed only in multivariable analysis. Conclusions:  VWF and ADAMTS-13 levels were not associated in this study, and showed associations with MI risk in opposite directions but of similar strength. The association of ADAMTS-13 with MI is influenced by lipid levels, and consequently requires further investigation.     

Prevalence of the ADAMTS-13 missense mutation R1060W in late onset adult thrombotic thrombocytopenic purpura

Summary.  Background:  Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor-cleaving protease (ADAMTS-13) activity. ADAMTS-13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. Objectives:  We sought to identify common ADAMTS-13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. Patients/Methods/Results:  We detected a missense mutation (C3178T) in exon 24 of ADAMTS-13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls ( n  = 100). Three of the patients had pregnancy-associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1-7 domain of ADAMTS-13. In vitro expression of mutant and wild-type ADAMTS-13 demonstrated that R1060W caused severe intracellular retention of ADAMTS-13 (<5% secretion) without affecting its metalloprotease activity. One homozygous and five heterozygous patients were identified. No other causative mutations were discovered, yet all six patients had ADAMTS-13 activity levels <5% at presentation (normal: 66–126%). Antibodies/inhibitors to ADAMTS-13 were detected in three/five heterozygous patients, and all six patients had subnormal antigen levels. Six asymptomatic first-degree relatives, including those of two probands with antibodies, were also heterozygous for C3178T; all but one had subnormal ADAMTS-13 activity. Conclusion:  The high prevalence of R1060W ADAMTS-13 in adult onset TTP, together with its absence in childhood congenital TTP cases reported elsewhere, suggests it may be a factor in the development of late onset TTP.     

Diagnostic workup of patients with acquired von Willebrand syndrome: a retrospective single-centre cohort study

Summary.  Background:  Diagnosis of acquired von Willebrand syndrome (AVWS) remains challenging. Diagnostic algorithms suggest the use of factor VIII (FVIII:C), von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and collagen-binding capacity (VWF:CB), but the sensitivity of these and other laboratory tests for the diagnosis of AVWS is unknown. Objectives:  To analyze the capacity of laboratory tests, including point-of-care testing (POCT), for the identification of patients with AVWS. Patients/methods:  Thirty-five consecutive patients were enrolled with AVWS diagnosed because of a history of recent onset of bleeding, a negative family history of von Willebrand disease, and abnormal plasma VWF multimers. Results:  According to our inclusion criteria, all patients had bleeding symptoms, and the VWF high molecular weight multimers were either decreased or absent. Regarding POCT, PFA-100 was inconclusive, due to anemia or thrombocytopenia, in 29%; the sensitivity was 80% in the remaining patients. The sensitivity of VWF:Ag (23%), VWF:RCo/Ag ratio < 0.7 (26%), VWF:CB/Ag ratio < 0.7 (46%), anti-VWF antibodies (15%) and VWF propeptide/Ag ratio (22%) was too low to rule out the disease. A combination of VWF:Ag < 50 IU dL⁻¹, VWF:RCo/Ag ratio < 0.7 and VWF:CB/Ag ratio < 0.8 yielded a sensitivity of 86%. Patients diagnosed only because of abnormal VWF multimers showed similar clinical characteristics as other patients. Conclusions:  Early diagnosis of AVWS is difficult, due to lack of sensitivity of the tests used. A substantial number of patients present with normal or increased test results, emphasizing the importance of multimer analysis in all patients with suspected AVWS.     

Altered megakaryocytopoiesis in von Willebrand type 2B disease

Summary.  Type 2B von Willebrand disease (VWD2B) is caused by gain-of-function amino acid substitutions in the von Willebrand factor (VWF) A1 domain. These allow facilitated binding of mutated VWF to platelet GPIbα with prolonged lifetimes of VWF bonds and enhanced ADAMTS-13 cleavage of large VWF multimers. A bleeding rather than prothrombotic syndrome is due to: (i) decreased large VWF multimers in plasma; (ii) limited thrombus formation; and (iii) thrombocytopenia affecting some but not all patients. Accumulating evidence points to an altered megakaryocytopoiesis in VWD2B with the production of enlarged or giant platelets showing an abnormal ultrastructure and, in a cohort of patients, the presence of circulating platelet agglutinates. In fact, evidence from in vitro cultures and marrow aspirates suggests that the upregulated VWF function can lead to abnormal VWF trafficking in megakaryocytes, a modified platelet production with interacting proplatelets, and the presence or even release of platelet agglutinates in the bone marrow.     

Pathogen reduction of fresh plasma using riboflavin and ultraviolet light: effects on plasma coagulation proteins

BACKGROUND: Treatment with riboflavin and ultraviolet (UV) light reduces the pathogens present in blood components. This study assessed changes to the coagulation proteins that had occurred during this treatment of fresh plasma units before freezing.
STUDY DESIGN AND METHODS: Twenty fresh plasma units (230 ± 30 mL) were treated by the Mirasol process (CaridianBCT Biotechnologies) and frozen within 8 hours of donation. Plasma units were combined with 35 mL of a 500 µmol/L riboflavin solution in an illumination bag to achieve a final concentration of approximately 60 µmol/L riboflavin. The bag was placed in the Mirasol illuminator and exposed to UV light (6.24 J/mL). Samples were frozen before and after treatment.
RESULTS: Recoveries observed were 67.7 ± 3.9% Factor (F)XI, 68.5 ± 3.3% FVIII:C, 78.8 ± 4.5% fibrinogen, 78.9 ± 4.1% FV, 79.0 ± 4.2% FVII, 79.0 ± 8.6% F IX, 79.7 ± 2.6% FX, and 85.0 ± 3.7% FII. Von Willebrand factor (VWF) antigen, VWF:ristocetin cofactor, and ADAMTS13 recoveries were 87.0 ± 7.1, 85.5 ± 6.6, and 73.3 ± 15.2%, respectively, while that of protein C was 83.6 ± 2.6%. A loss of high-molecular-weight VWF multimers was observed in most units. Recoveries for protein S, antithrombin, and plasmin inhibitor were greater than 90%. The mean FVIII:C concentration, after treatment, was 0.76 ± 0.17 IU/mL.
CONCLUSIONS: As with other pathogen reduction technologies, the Mirasol process resulted in some loss of coagulation factor activity. For most Mirasol-treated units and for most of the tested factors this is unlikely to have clinical impact, but trials are required to demonstrate this.     

Decreased ADAMTS-13 (A disintegrin-like and metalloprotease with thrombospondin type 1 repeats) is associated with a poor prognosis in sepsis-induced organ failure

OBJECTIVE: The inability to regulate the inflammatory response initiated upon infection leads to severe sepsis, characterized by widespread microvascular injury and thrombosis, organ ischemia, and dysfunction. A disintegrin-like and metalloprotease with thrombospondin type 1 repeats (ADAMTS)-13 regulates primary hemostasis by proteolyzing von Willebrand factor (VWF). Decreased ADAMTS-13 has been reported in disseminated intravascular coagulation due to severe sepsis. The present study investigates whether the sepsis-related dysregulation of endothelial activation leads to specific changes of ADAMTS-13. DESIGN: Case-control study. SETTING: Adult intensive care unit in a university hospital. PATIENTS/SUBJECTS: Three groups were studied: a case group of 30 patients with severe sepsis, a control group of 29 patients with comparable organ failure unrelated to sepsis, and 30 age- and gender-matched healthy subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Significantly lower ADAMTS-13 activity was observed in patients with severe sepsis (43.2%; interquartile range, 32.7, 67.0) than in patients with organ failure unrelated to sepsis (67.8%; 57.4, 87.9; p < .05) and healthy subjects (105.6%; 87.2, 125.6; p < .001). Accordingly, ADAMTS-13 antigen was more decreased in patients with severe sepsis than in patients with organ failure unrelated to sepsis and healthy subjects. VWF antigen was higher in patients with severe sepsis than in patients with organ failure unrelated to sepsis and healthy subjects. We found strong negative correlations in severe sepsis but not in organ failure unrelated to sepsis, between ADAMTS-13 activity and 1) VWF antigen; 2) thrombomodulin; 3) interleukin-6; 4) Acute Physiology and Chronic Health Evaluation II score; 5) shock; 6) acute renal injury. Moreover, patients above the median of ADAMTS-13 activity presented a higher survival compared with those below the median in the patients with severe sepsis but not in the patients with organ failure unrelated to sepsis. In contrast, there was no significant association between VWF antigen and survival in either the severe sepsis group or the group with organ failure unrelated to sepsis. CONCLUSIONS: We observed low ADAMTS-13 activity and antigen in severe sepsis and in other conditions associated with organ dysfunction. ADAMTS-13 levels were significantly associated with differences in morbidity, mortality, and variables of inflammation and endothelial dysregulation only in severe sepsis patients. This suggests that ADAMTS-13 deficiency may have a pathophysiological relevance specific to severe sepsis.     

A new L1446P mutation is responsible for impaired von Willebrand factor synthesis, structure, and function

We report on a new mutation (4337T-->C) in exon 28 of the von Willebrand factor (VWF) gene, resulting in a substitution of L with P at residue 1446 (L1446P) of pre-pro-VWF. The defect is transmitted as a dominant trait and induces a reduced VWF synthesis, an abnormal VWF multimer pattern and a deficient VWF-platelet glycoprotein Ib interaction. The proband had low plasma and platelet VWF antigen levels, a reduced VWF collagen-binding capacity, and a disproportionately low VWF ristocetin cofactor activity, associated with the absence of ristocetin-induced platelet aggregation. Multimer analysis showed that the smaller multimers were slightly low, whereas the larger ones were significantly reduced or absent, with a clear cutoff between the two patterns. Similar hemostatic findings were observed in the proband's sister and nephew. Desmopressin administration restored VWF levels to near normal, but this was not so for VWF ristocetin cofactor activity or ristocetin-induced platelet aggregation. VWF multimers improved after desmopressin, moreover, with the larger forms restored and the smaller ones still relatively more represented. Recombinant P1446 VWF synthesis was reduced at heterozygous level, and its multimer pattern was similar to that observed in plasma VWF. These findings confirm the role of L1446P mutation in determining the von Willebrand disease (VWD) phenotype observed in our patients. Given the lack of large and intermediate VWF multimers, and the fact that the VWF-platelet interaction defect appears to be partially independent of multimer pattern, the VWD associated with L1446P mutation may belong to the type 2A/2M VWD variant.     

Platelet-derived VWF-cleaving metalloprotease ADAMTS-13

The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra-large (UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin-stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation-dependent protease. A recent study shows that platelets contain the VWF-cleaving metalloprotease ADAMTS-13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell-derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS-13 antibody from the plasma of a patient with acquired TTP. ADAMTS-13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS-13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor-activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS-13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells.     

Type 2M von Willebrand disease variant characterized by abnormal von willebrand factor multimerization

We describe a von Willebrand disease (VWD) variant characterized by low plasma and platelet von Willebrand factor (VWF), impaired ristocetin-induced VWF binding to platelet glycoprotein Ib (GPIb), and abnormal VWF multimer pattern not associated with the absence of large forms. A C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene was found; this mutation introduces a cysteine at the codon for Arg 611 of mature VWF. In addition to the decreased factor VIII (FVIII) and VWF levels, ristocetin-induced platelet aggregation (RIPA) was almost absent, and VWF ristocetin cofactor activity (VWF:RCo) was significantly more decreased than VWF antigen. The patients (mother and son) also showed a defect in VWF collagen-binding activity. Plasma VWF multimers were decreased, with no limit in the size of large forms, and the normal discontinuous multimer organization was replaced by a diffuse smear, especially detectable in the large forms. This picture was emphasized by 1-deamino-8-D -arginine vasopressin (DDAVP) infusion, so that the abnormal VWF multimers appeared to have a molecular weight higher than those present in, or released by, human umbilical vein endothelial cells. DDAVP also increased FVIII and VWF levels but did not normalize the GPIb-dependent VWF functions expressed as RIPA and VWF:RCo. We include this variant in type 2M VWD, focusing on the abnormality in GPIb-dependent VWF function. We advance that this defect depends on the mutation in the GPIb binding domain of VWF rather than the abnormal VWF multimer pattern.     

Preclinical evaluation of a semi‐automated and rapid commercial electrophoresis assay for von Willebrand factor multimers

Background

The von Willebrand factor (VWF) multimer test is required to correctly subtype qualitative type 2 von Willebrand disease (VWD). The current VWF multimer assays are difficult, nonstandardized, and time‐consuming. The purpose of this study was to evaluate the clinical utility of the commercial VWF multimer kit by Sebia (Lisses, France), an electrophoresis technique yielding same‐day results.

Methods

Ten healthy volunteer plasma samples, in‐house reference plasma (IRP) and commercial normal plasma (CNP) samples, 10 plasma samples from patients with a known VWD type, 1 hemophilia A plasma sample, and 7 external quality assurance (EQA) samples were analyzed using the commercial VWF multimer kit. Additional coagulation testing included measurements of VWF antigen (VWF:Ag), VWF activity (VWF:Ac), and FVIII activity (FVIII:C).

Results

The CNP results revealed a relative loss of the highest molecular weight multimers; therefore, IRP was preferred as the reference sample. The interpretations of 10 patients with a known VWD type could be successfully reproduced and agreed with previous VWF multimer results. In all EQA surveys, the multimer results and final VWD diagnosis agreed with expert opinion.

Conclusions

The VWF multimer assay by Sebia is easy to perform and can be successfully implemented in any clinical laboratory for second‐stage evaluation of VWD. The resolution power of multimer distribution is adequate to correctly classify VWD types 1, 2A, 2B, and 3.

     

Measurement of von Willebrand factor-cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods

Summary.  A severely deficient ADAMTS-13 activity (<5%) is a key laboratory finding confirming the diagnosis of thrombotic thrombocytopenic purpura (TTP), whereas a mildly or moderately decreased activity is found in various other conditions. Laboratory tests for ADAMTS-13 activity must reliably identify a severe deficiency and detect inhibitory antibodies against ADAMTS-13. We carried out a multicenter comparison of different assays for ADAMTS-13 activity in plasma, including the quantitative immunoblotting of degraded von Willebrand factor (VWF) substrate, the residual collagen binding activity and ristocetin cofactor activity of degraded VWF, and an immunoradiometric assay. The main goal was to investigate whether all assays concordantly identified severe ADAMTS-13 deficiency and detected inhibitory antibodies. ADAMTS-13 activity was determined by five laboratories in 30 plasma samples of patients with hereditary and acquired TTP and other conditions. ADAMTS-13 activity values of the samples ranged from <3% to > 100%. Concerning the identification of a severe ADAMTS-13 deficiency, good interassay and interlaboratory agreement was observed with only one false-negative and two false-positive results by two laboratories using a collagen binding assay. For samples with normal or mildly to moderately reduced ADAMTS-13 activity, results were less concordant. There was good agreement for the detection of strong inhibitors. We conclude that all assays investigated are useful as a screening test in suspected TTP. Further assay improvement is needed, however.     

An influence of ABO blood group on the rate of proteolysis of von Willebrand factor by ADAMTS13

Summary.  The susceptibility of von Willebrand factor (VWF) of blood group O, A, B and AB to proteolysis by the ADAMTS13 metalloprotease was investigated. Multimeric analysis indicated that the rate of VWF proteolysis differed between blood groups and was greater for group O VWF than for non-O VWF in the rank order O ≥ B > A ≥ AB. Measurement of the collagen binding activity of VWF of each blood group following proteolysis for a fixed time interval corroborated the results obtained on multimer analysis: the loss of collagen binding activity was greater for VWF of group O compared with non-O VWF, in the rank order O ≥ B > A ≥ AB. Ristocetin was found to increase the rate of VWF proteolysis approximately two-fold; the differential between blood groups was retained in the presence of ristocetin.     

The effect of exercise on von Willebrand factor and ADAMTS-13 in individuals with type 1 and type 2B von Willebrand disease.

BACKGROUND: The effect of exercise on von Willebrand factor (VWF) and ADAMTS-13 levels in individuals with von Willebrand disease (VWD) has never been reported. OBJECTIVES: The aim was to quantify the effect of a standardized exercise protocol on individuals with type 1 and type 2B VWD. PATIENTS/METHODS: Thirty individuals from three groups (10 controls, 11 with type 1 VWD and 9 with type 2B VWD) completed the Standard Bruce Protocol Treadmill Test. A bleeding questionnaire was administered and blood tests were performed pre- and immediately postexercise. The groups were well matched for age, gender and body mass index (BMI). RESULTS: There was a correlation in all groups between the metabolic equivalents (METS) achieved and the degree of change of VWF and FVIII:C levels (P < 0.002, Pearson's correlation). There was a significant postexercise increase in VWF:Ag, VWF:RCo, FVIII:C and activated VWF levels in both the control group and in the type 2B VWD group, but not in the type 1 VWD group. Specific to the type 2B VWD group was an increase in the percentage of high molecular weight multimers (P = 0.022), a decrease in the mean platelet count compared with the other groups (P < 0.001) and an increase in the ADAMTS-13 level (P = 0.001). CONCLUSIONS: There are significant differences in the effects of exercise on individuals with type 1 and type 2B VWD compared with controls. Further clinical studies are necessary to evaluate exercise as a therapeutic option in VWD.