VWF propeptide and ratios between VWF, VWF propeptide, and FVIII in the characterization of type 1 von Willebrand disease.

During posttranslational modifications of von Willebrand factor (VWF), the VWF propeptide (VWFpp) is cleaved. The ratio between VWFpp and VWF antigen (VWF:Ag) and the ratio between factor VIII (FVIII:C) and VWF:Ag may be used to assess synthesis and clearance of VWF. We analyzed the contribution of VWFpp and ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag in the pathophysiological characterization of type 1 von Willebrand disease (VWD) in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD (MCMDM-1VWD) study. The VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were increased among patients compared with unaffected family members and healthy controls. The VWFpp/VWF:Ag ratio was higher in individuals heterozygous for missense mutations than in those heterozygous for null alleles. In contrast, the FVIII:C/VWF:Ag ratio was highest among heterozygotes for VWF null alleles. The ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag indicate that the pathophysiological mechanisms of type 1 VWD include reduced production and accelerated clearance of VWF, but that often a combination of both mechanisms is implicated.

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

Type 3 von Willebrand disease (VWD3) is characterized by unmeasurable von Willebrand factor (VWF) levels in plasma and platelets and severe but variable hemorrhagic symptoms. To identify and characterize the causal mutations, we screened 10 Italian patients with VWD3 by several techniques including Multiplex Ligation-dependent Probe Amplification to identify large insertions and deletions, High Resolution Melting and PCR coupled with Sanger sequencing. Fourteen different mutations scattered throughout the VWF gene were identified, 10 of which were novel. As expected, most of these mutations caused null alleles: five were deletions (del exons 1-3, del exon 17, c.2157delA, c.2269delCT, and c.3940delG), three nonsense (p.Q1526X, p.E1549X, and p.C2448X) and three potential splice-site mutations (c.658-2A>G, c.7729+7C>T, and c.8155+1G>T). Three candidate missense mutations (p.C2184S, p.C2212R, and p.C2325S) were also identified. Missense mutations and the putative splice-site defects were confirmed to be disease related by in vitro expression studies and mRNA analysis. None of these patients have developed alloantibodies against VWF. This study extends our previous finding that most of the mutations that we identified in VWD3 patients arise independently and are scattered throughout the entire VWF gene.

ADAMTS13 and anti-ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission.

BACKGROUND: From 20 to 50% of patients who survive an acute episode of the acquired form of thrombotic thrombocytopenic purpura relapse but clinical and laboratory markers of recurrence are not well established. DESIGN AND METHODS: In 109 patients enrolled in an international registry we evaluated, in the frame of a retrospective cohort study, the predictive role of the metalloprotease ADAMTS13 as measured in plasma during remission. Anti-ADAMTS13 antibodies and von Willebrand factor were also evaluated in a smaller number of the same patients. RESULTS: Median values of ADAMTS13 activity and antigen were significantly lower in patients with recurrent thrombotic thrombocytopenic purpura than in those with no recurrence (activity: 12% vs. 41%; p=0.007; antigen: 36% vs. 58%; p=0.003). A severe deficiency of ADAMTS13 activity (10% or less) was associated with a higher likelihood of recurrence (odds ratio 2.9; 95% confidence interval 1.3 to 6.8; p=0.01). Anti-ADAMTS13 antibodies were also more prevalent in patients with recurrent thrombotic thrombocytopenic purpura (odds ratio 3.1; 95% confidence interval 1.4 to 7.3; p=0.006). The presence during remission of both severe ADAMTS13 deficiency and anti-ADAMTS13 antibodies increased the likelihood of recurrence 3.6 times (95% confidence interval 1.4 to 9.0; p=0.006). The presence of ultralarge von Willebrand factor multimers and of associated diseases or conditions did not increase recurrence. CONCLUSIONS: Survivors of an acute episode of acquired thrombotic thrombocytopenic purpura with severely reduced levels of ADAMTS13 and/or with anti-ADAMTS13 antibodies during remission have an approximately three-fold greater likelihood of developing another episode of thrombotic thrombocytopenic purpura than patients with higher protease activity and no antibody.

Evaluation of a new turbidimetric assay for von Willebrand factor activity useful in the general screening of von Willebrand disease.

We evaluated a new assay (HemosIL VWF Activity on ACL-Futura) in the screening of VWD. Samples from healthy donors and previously diagnosed VWD patients were blindly analyzed by this new activity assay and standard VWF:RCo. Results agreed and both assays showed a similar sensitivity for the screening of VWD.

Von Willebrand factor cleaving protease (ADAMTS-13) in 123 patients with connective tissue diseases (systemic lupus erythematosus and systemic sclerosis).

BACKGROUND AND OBJECTIVES: Autoantibodies inactivating the von Willebrand factor (VWF) cleaving protease, ADAMTS-13, are among the most frequent causes of thrombotic thrombocytopenic purpura (TTP). We evaluated whether or not ADAMTS-13 deficiency and autoantibodies inactivating the protease prevalent in patients with the prototypic autoimmune diseases systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). DESIGN AND METHODS: We measured, in parallel, the protease and VWF antigen (VWF:Ag) in 123 patients, 36 of whom had SLE and 87 of whom had SSc. In 14 patients with either disease who had low plasma protease levels (below 40%) we also looked for anti-ADAMTS-13 inactivating antibodies. RESULTS: ADAMTS-13 levels were significantly lower in SLE (p=0.0013) and in SSc (p=0.0002) than in normal controls. No anti-ADAMTS activity was measurable in patients with low ADAMTS-13 levels. VWF:Ag was high in both SLE and SSc (p=0.001). INTERPRETATION AND CONCLUSIONS: Systemic connective tissue diseases are other conditions besides TTP that are associated in some instances with low but detectable levels of ADAMTS-13. Autoantibodies inactivating protease activity are not the cause of the low plasma levels of ADAMTS-13.

The thrombospondin-1 N700S polymorphism is associated with early myocardial infarction without altering von Willebrand factor multimer size.

The N700S polymorphism of thrombospondin-1 (TSP-1) has been identified as a potential genetic risk factor for myocardial infarction (MI). In a large case-control study of 1425 individuals who survived a myocardial infarction prior to age 45, the N700S polymorphism was a significant risk factor for myocardial infarction in both homozygous (odds ratio [OR] 1.9, 95% confidence interval [CI] 1.1-3.3, P = .01) and heterozygous carriers of the S700 allele (OR 1.4, 95% CI 1.1-3.3, P = .01). TSP-1 has been shown to reduce von Willebrand factor (VWF) multimer size, and the domain responsible for VWF-reducing activity has been localized to the calcium-binding C-terminal sequence. As the N700S polymorphism was previously shown to alter the function of this domain, we investigated whether the altered VWF-reducing activity of TSP-1 underlies the observed prothrombotic phenotype. The TSP1 N700S polymorphism did not influence VWF multimer size in patients homozygous for either allele nor was there a significant reduction of VWF multimer size following incubation with recombinant N700S fragments or platelet-derived TSP-1.

Plasma levels of von Willebrand factor regulate ADAMTS-13, its major cleaving protease.

ADAMTS-13, the metalloprotease that disposes physiologically of the most thrombogenic multimers of von Willebrand factor (VWF), tends to be low in plasma when VWF is high. We evaluated the behaviour of these two proteins in naturally occurring, experimental and clinical situations associated with VWF levels spanning from undetectable to supranormal. ADAMTS-13 was approximately 10% higher (and VWF 35% lower) in 65 healthy individuals of blood group O than in 65 individuals of groups A, B and AB. Thirty-three patients with type 3 von Willebrand disease (VWD) with undetectable plasma VWF had approximately 35% higher levels of ADAMTS-13 than a comparable group of healthy individuals with normal VWF. When VWF was raised to supranormal levels by desmopressin (DDAVP) in 10 healthy volunteers, ADAMTS-13 decreased by approximately 20%, with no change of the protease in three patients with severe VWD who had no post-DDAVP VWF rise. When VWF was raised from very low to normal levels by the infusion of VWF-containing plasma concentrates in four patients with type 3 VWD and one with type 1 VWD plasma, ADAMTS-13 decreased in parallel. These data show that throughout a large spectrum of plasma VWF levels there is a negative association between this protein and the activity of its major cleaving protease.

Plasma levels of the von Willebrand factor-cleaving protease in physiological and pathological conditions in children.

The hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are rare disorders characterized by thrombocytopenia, hemolytic anemia, and ischemic organ failure due to thrombotic occlusions in arterioles. The recent observation that a von Willebrand factor-cleaving protease (VWF-CP) is low in the plasma of patients with TTP but normal in those with HUS has potentially offered a new specific tool for differential diagnosis. In this study, the authors evaluated the plasma levels of the VWF-CP during the neonatal state and healthy childhood and in some pathological pediatric conditions. The protease was measured in 16 healthy newborns, 20 healthy children aged 5-18 years, patients with diabetes mellitus type 1 (n = 7), acute viral hepatitis (n = 10), chronic viral hepatitis (n = 10), transfusion-dependent beta-thalassemia major (n = 10), acute varicella infection (n = 11), the nephrotic syndrome (n = 11), and familial Mediterranean fever (n = 10). Mean protease levels were significantly lower in newborns than in healthy children (50.5 +/- 16.1% vs. 83.3 +/- 16.3%)(p = .0001). In patients with acute viral hepatitis, protease levels were also significantly reduced (40.2 +/- 27% v s. 83.3 +/- 16.3% in healthy children)(p = .0001). Other patient groups had normal protease levels. In conclusion, low protease levels are far from being a specific beacon for TTP. The current paradigm that a single laboratory test may enable physicians to distinguish TTP from HUS seems to be challenged by these and other findings.

von Willebrand factor cleaving protease (ADAMTS-13) and ADAMTS-13 neutralizing autoantibodies in 100 patients with thrombotic thrombocytopenic purpura.

The congenital or acquired deficiency of the von Willebrand factor (VWF) cleaving protease, ADAMTS-13 has been specifically associated with a diagnosis of thrombotic thrombocytopenic purpura (TTP), a microangiopathy characterized by the formation of occlusive platelet thrombi. The mechanisms of TTP were investigated in 100 patients diagnosed on the basis of the presence of at least three of the following: thrombocytopenia, haemolytic anaemia, elevated serum levels of lactate dehydrogenase and neurological symptoms. Plasma levels of ADAMTS-13 were severely reduced (<10% of normal) in 48%, moderately reduced (between 10% and 46%) in 24% and normal (>46%) in 28%. A neutralizing antibody was the cause of the deficiency in 38% of the cases, with a higher prevalence of this mechanism (87%) in the 48 patients with severely reduced ADAMTS-13. Double heterozygosity for a 29 base pair (bp) deletion and a nucleotide insertion and homozygosity for a 6 bp deletion in the ADAMTS13 gene were identified only in two patients born from consanguineous marriages. In conclusion, this study indicated that ADAMTS-13 was normal in nearly one-third of patients with TTP and that ADAMTS-13 deficiency was not associated with the presence of neutralizing antibodies in more than half of the patients.

Patients with localized and disseminated tumors have reduced but measurable levels of ADAMTS-13 (von Willebrand factor cleaving protease).

BACKGROUND AND OBJECTIVES: Patients with disseminated malignancies have been noted to have a deficiency of von Willebrand factor (VWF) cleaving protease, ADAMTS-13. The very low or undetectable plasma levels of this protease are said to be similar to those found in patients with thrombotic thrombocytopenic purpura (TTP). This observation, which challenges the paradigm that severe ADAMTS-13 deficiency is a specific diagnostic marker for TTP, remains so far unconfirmed. DESIGN AND METHODS: We measured the protease and VWF antigen (VWF:Ag) in parallel in 49 Iranian patients with solid tumors, which in 29 cases were localized (stages I and II) and in 20 disseminated (stage IV). Forty-nine healthy individuals matched with cases for sex, age and smoking habits were taken as controls. RESULTS: Patients with disseminated tumors had lower mean plasma levels of ADAMTS-13 than those with localized tumors, but these differences did not reach the level of statistical significance (p=0.059). However, in no patient was the level of ADAMTS-13 below 18% of normal, at variance with previous findings of lower or unmeasurable levels (<15%). The level of ADAMTS-13 was significantly lower in patients with localized tumors than in controls ( p = 0.0003 ), but higher than in patients with disseminated disease (p=0.0001 vs controls). INTERPRETATION AND CONCLUSIONS: Malignancy, whether localized or disseminated, is another condition associated with low ADAMTS-13 levels not accompanied by signs and symptoms of TTP and other thrombotic microangiopathies.

Molecular defects in type 3 von Willebrand disease: updated results from 40 multiethnic patients.

Type 3 von Willebrand disease (VWD) is characterized by unmeasurable von Willebrand factor (VWF) levels in plasma and platelets and severe hemorrhagic symptoms. We have characterized at the molecular level a group of 40 patients (12 Italians, 14 Iranians, and 14 Indians) to evaluate genetic heterogeneity among these populations. Some of these patients have been previously investigated by us (mutations shown in italics); they are included in this study to provide a more comprehensive pattern of gene defects in type 3 VWD. Patients' DNA were first tested for more frequently reported mutations, then screened by single-strand conformation polymorphism and direct sequence analysis. Fifty gene defects were identified, of which 45 are novel. As expected most of these defects caused null alleles, 17 being nonsense mutations (Q218X, W222X, R365X, R373X, Y610X, W642X, E644X, Q706X, Q1311X, S1338X, Q1346X, Y1542X, R1659X, E1981X, E2129X, R2434X, and Q2544X), 12 small deletions (191delG, 276delT, 788del24, 2016del4, 2157delA, 2269delCT, 2435delC, 4092delAC, 6182delT, 7294delGT, 7683delT, and 8241del9), 4 small insertions (4414insC, 7130insC, 7137insT, and 7674insC), 8 possible splice site mutations (1110(-1)G-->A, 1946(-4)C-->T, 3108(+5)G-->A, 3379(+1)G-->A, 5053(+1)G-->A, 5170(+10)C-->T, 6977(-1)G-->C, and 7729(+7)C-->T), 8 candidate missense mutations (D47H, S85P, D141N, D141Y, C275S, C1071F, C2174G, and C2804Y), and 1 large gene deletion (exons 23-52). Only 2 of these patients have developed alloantibodies against VWF. This study extend our previous finding that mutations responsible for type 3 VWD are scattered throughout the entire VWF gene and that there is no founder effect in these three populations studied.

An association of candidate gene haplotypes and bleeding severity in von Willebrand disease (VWD) type 1 pedigrees.

von Willebrand disease (VWD) type 1 is difficult to diagnose because of bleeding variability and low heritability of von Willebrand factor (VWF) levels. We compared a bleeding severity score and bleeding times to candidate gene haplotypes within pedigrees of 14 index cases, using a covariance components model for multivariate traits (Mendel: QTL Association). These pedigrees included 13 affected and 40 unaffected relatives, as defined by plasma ristocetin cofactor (VWF:RCo) levels. The bleeding severity score was derived from a detailed history. Donors were genotyped using a primer extension method, and 9 candidate genes were selected for analysis. VWF:RCo levels had the strongest influence on bleeding severity score and bleeding time. ITGA2 haplotype 2 (807C) and ITGA2B haplotype 1 (Ile(843)) were each associated with increased bleeding severity scores (P < .01 and P < .01, respectively). GP6 haplotype b (Pro(219)) was also associated with increased scores (P = .03) after adjustment for donor age. No association was observed with 6 other candidate genes, GP1BA, ITGB3, VWF, FGB, IL6, or TXA2R. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (-1793G; P = .02). These results establish that genetic differences in the adhesion receptor subunits alpha(2), alpha(IIb,) and GPVI can influence the phenotype of VWD type 1.

von Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome.

Whether measurement of ADAMTS13 activity may enable physicians to distinguish thrombotic thrombocytopenic purpura (TTP) from hemolytic uremic syndrome (HUS) is still a controversial issue. Our aim was to clarify whether patients with normal or deficient ADAMTS13 activity could be distinguished in terms of disease manifestations and multimeric patterns of plasma von Willebrand factor (VWF). ADAMTS13 activity, VWF antigen, and multimeric pattern were evaluated in patients with recurrent and familial TTP (n = 20) and HUS (n = 29). Results of the collagen-binding assay of ADAMTS13 activity were confirmed in selected samples by testing the capacity of plasma to cleave recombinant VWF A1-A2-A3. Most patients with TTP had complete or partial deficiency of ADAMTS13 activity during the acute phase, and in some the defect persisted at remission. However, complete ADAMTS13 deficiency was also found in 5 of 9 patients with HUS during the acute phase and in 5 patients during remission. HUS patients with ADAMTS13 deficiency could not be distinguished clinically from those with normal ADAMTS13. In a subgroup of patients with TTP or HUS, the ADAMTS13 defect was inherited, as documented by half-normal levels of ADAMTS13 in their asymptomatic parents, consistent with the heterozygous carrier state. In patients with TTP and HUS there was indirect evidence of increased VWF fragmentation, and this occurred also in patients with ADAMTS13 deficiency. In conclusion, deficient ADAMTS13 activity does not distinguish TTP from HUS, at least in the recurrent and familial forms, and it is not the only determinant of VWF abnormalities in these conditions.