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Summary Background In individuals with borderline von Willebrand factor (VWF) plasma levels, second-level tests are required to confirm or exclude von Willebrand disease (VWD). These tests are time-consuming and expensive. Objective To assess which parameters can predict VWD diagnosis in individuals with borderline VWF levels (30–60 IU dL−1). Methods Nine hundred and fifty individuals with bleeding episodes or abnormal coagulation test results were investigated with first-level tests (blood count, prothrombin time, activated partial thromboplastin time, blood clotting factor VIII, VWF ristocetin cofactor activity [VWF:RCo], and VWF antigen), and 93 (62 females and 31 males; median age, 28 years; interquartile range 15–44) had borderline VWF:RCo levels. All underwent second-level investigations to confirm or exclude VWD. A multivariable logistic regression model was fitted with sex, age, bleeding score, family history, VWF:RCo and ABO blood group as predictors, and used to predict VWD diagnosis. Results Forty-five of the 93 individuals (48%) had VWD (84% type 1). A negative linear relationship between VWF:RCo levels and risk of VWD diagnosis was present, and was particularly evident with blood group non-O [adjusted odds ratio 7.00 (95% confidence interval [CI] 1.48–33.11) for every 5 IU dL−1 decrease in VWF:RCo]. The other variable clearly associated with VWD diagnosis was female sex (adjusted odds ratio 5.76 [95% CI 1.47–22.53]). The area under the receiver operating characteristic curve of the full logistic model was 0.89 (95% CI 0.82–0.95). Conclusions In individuals with borderline VWF, the two strongest predictors of VWD diagnosis are low VWF:RCo levels (particularly in those with blood group non-O) and female sex. This predictive model has a promising discriminative ability to identify patients with borderline VWF levels who are likely to have VWD.

Summary Introduction We characterized four unrelated patients with von Willebrand disease type 2A/IIE, sharing the same von Willebrand factor (VWF) in-frame deletion (p.[P1127_G1180delinsR];[=]) resulting from exon 26 skipping (Δ26). Objectives To identify the VWF mutations and how they caused the mRNA splicing alteration, to evaluate the deletion by in vitro expression studies, and to assess whether or not the heterogeneity of the patients’ phenotype might be related to a different degree of expression of the deleted subunit in patient plasma VWF. Methods Sequence analysis was performed with patient genomic DNA and platelet mRNA. Semiquantitative RT-PCR was also carried out to compare the expression of the wild-type (WT) and Δ26 alleles in the four patients. In silico analysis was performed with prediction splicing programs. Expression studies were performed to evaluate mutant recombinant VWF (rVWF) (Δ26 and Δ26/WT) as compared with WT rVWF. Results Three patients shared the synonymous single-nucleotide substitution (SSS) c.[3390C>T];[=], whereas the novel mutation c.[3380-2A>G];[=] was present in the fourth patient. Semiquantitative RT-PCR of platelet mRNA revealed a different ratio of the WT and Δ26 alleles in the patients, consistent with the different VWF:FVIIIB values present in patient plasma. Expression studies confirmed reduced VWF–FVIII binding of rVWF-Δ26/WT. Conclusions SSS can induce alternative splicing, and those like c.3390C>T, which impact on the poorly conserved splicing regulatory elements, are difficult to predict, so that their role can be evaluated only by mRNA analysis. Moreover, these mutations seem to have different effects on the efficiency of alternative splicing, producing heterogeneous VWF variants among the four patients.

Background Octapharma PPGmbH has recently modified its manufacturing process for solvent/detergent-treated plasma to incorporate a prion reduction step, in which a 3 log reduction has been demonstrated. The current study was undertaken to assess the impact of this procedure on haemostatic variables in the new product OctaplasLG in comparison with standard Octaplas. Methods Production batches of standard Octaplas (n = 4) and OctaplasLG (n = 16) were assessed for levels of coagulation factors, physiological protease inhibitors, markers of activation and procoagulant microparticles. Global haemostasis was assessed by a thrombin generation test (TGT) and rotational thromboelastometry (ROTEM). Results Mean levels of factors: II, V, VII, IX, X, XI, XII and XIII, VWF:Ag, antithrombin, protein C and free protein S were all > 75 u/dl. ADAMTS-13 activity levels were normal. Factor VIII and VWF:RCo were > 55 u/dl. TGT and ROTEM were similar in both preparations, and microparticles were present at negligible levels. Two units of OctaplasLG had slightly elevated levels of Prothrombin Fragments 1 + 2, but D-Dimer and thrombin-antithrombin complexes were normal in all batches. Conclusion These studies indicate that the affinity chromatography procedure used in OctaplasLG does not appear to adversely affect the proven haemostatic quality of Octaplas, while offering a selective reduction in the concentration of pathological prion proteins.

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.

von Willebrand disease (VWD) is the most common inherited bleeding disorder. In this perspective article, Drs. Federici and Canciani review how correlating the clinical, laboratory and genetic features of von Willebrand disease has led to improved understanding of its pathophysiology. They show how this has rationalized classification, which in turn can be used to improve treatment of these patients. See related paper on page 679.

Type 2B von Willebrand disease (VWD2B) is caused by an abnormal von Willebrand factor (VWF) with increased affinity for the platelet receptor glycoprotein Ib-α (GPIb-α) that may result in moderate to severe thrombocytopenia. We evaluated the prevalence and clinical and molecular predictors of thrombocytopenia in a cohort of 67 VWD2B patients from 38 unrelated families characterized by VWF mutations. Platelet count, mean platelet volume, and morphologic evaluations of blood smear were obtained at baseline and during physiologic (pregnancy) or pathologic (infections, surgeries) stress conditions. Thrombocytopenia was found in 20 patients (30%) at baseline and in 38 (57%) after stress conditions, whereas platelet counts were always normal in 16 patients (24%) from 5 families carrying the P1266L/Q or R1308L mutations. VWF in its GPIb-α–binding conformation (VWF–GPIb-α/BC) was higher than normal in all except the 16 cases without thrombocytopenia (values up to 6-fold higher than controls). The risk of bleeding was higher in patients with thrombocytopenia (adjusted hazard ratio = 4.57; 95% confidence interval, 1.17-17.90) and in those with the highest tertile of bleeding severity score (5.66; 95% confidence interval, 1.03-31.07). Prediction of possible thrombocytopenia in VWD2B by measuring VWF–GPIb-α/BC is important because a low platelet count is an independent risk factor for bleeding.

Summary. We analyzed the association of bleeding severity with candidate gene haplotypes within pedigrees of 11 index cases of von Willebrand disease (VWD) type 2 (two type 2A, three type 2B and six type 2M), using the QTL Association model (MENDEL 5.5). In addition to the 11 index cases, these pedigrees included 47 affected and 49 unaffected relatives, as defined by VWF mutations and/or phenotype. A bleeding severity score was derived from a detailed history and adjusted for age. Donors were genotyped using a primer extension method, and eight candidate genes were selected for analysis. VWF antigen (or ristocetin cofactor activity) levels had the strongest influence on bleeding severity score. After Bonferroni correction for multiple testing, only ITGA2 promoter haplotype -52T was associated with an increased bleeding severity score (P

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 ( 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.

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 (Ile843) were each associated with increased bleeding severity scores (P < .01 and P < .01, respectively). GP6 haplotype b (Pro219) 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 α2, αIIb, and GPVI can influence the phenotype of VWD type 1.

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.

To cite this article: Castaman G, Baronciani L, Canciani MT, Federici AB. Different bleeding risk in type 2A and 2M von Willebrand disease: a 2-year prospective study in 107 patients: a reply to a rebuttal. J Thromb Haemost 2012; 10: 1458–60.See also Favaloro EJ, Bonar R, Marsden K. Different bleeding risk in type 2A and 2M von Willebrand disease: a 2-year prospective study in 107 patients: a rebuttal. This issue, pp 1455–8.

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.

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.

Patients with monoclonal gammopathies of uncertain significance (MGUS) may develop an acquired bleeding disorder similar to congenital von Willebrand disease, called acquired von Willebrand syndrome (AvWS). In these patients, measures to improve hemostasis are required to prevent or treat bleeding episodes. We diagnosed 10 patients with MGUS and AvWS: 8 had IgGκ (3) or λ (5) MGUS and 2 IgM-κ MGUS. Three therapeutic approaches were compared in them: (1) desmopressin (DDAVP), (2) factor VIII/von Willebrand factor (FVIII/vWF) concentrate, and (3) high-dose (1 g/kg/d for 2 days) intravenous Ig (IVIg). In patients with IgG-MGUS, DDAVP and FVIII/vWF concentrate increased factor VIII and von Willebrand factor in plasma, but only transiently. IVIg determined a more sustained improvement of the laboratory abnormalities and prevented bleeding during surgery (short-term therapy). In addition to the standard 2-day infusion protocol, a long-term IVIg therapy was performed in 2 patients with IgG-MGUS: repeated (every 21 days) single infusions of IVIg did improve laboratory abnormalities and stopped chronic gastrointestinal bleeding. On the other hand, IVIg failed to correct laboratories abnormalities in patients with IgM-MGUS. These comparative data obtained in a relative large and homogeneous group of patients with AvWS and MGUS confirm that DDAVP and FVIII/vWF concentrates improve the bleeding time (BT) and FVIII/vWF measurements only transiently, whereas IVIg provides a sustained treatment of AvWS associated with IgG-MGUS, but not with IgM-MGUS. © 1998 by The American Society of Hematology.

In normal platelet alpha-granules von Willebrand factor (VWF) is stored with multimerin and factor V in an eccentric electron-lucent zone. Because the platelet stores of VWF are deficient in 'platelet low' type 1 and type 3 von Willebrand disease (VWD), we investigated their electron-lucent zone proteins. The patients with VWD had partial to complete deficiencies of plasma and platelet VWF but normal alpha-granular multimerin and factor V, and normal alpha-granular fibrinogen, thrombospondin-1, fibronectin, osteonectin and P-selectin. In type 3 VWD platelets, alpha-granular electron-lucent zones lacking VWF-associated tubules were identified and multimerin was found in its normal alpha-granular location. These findings indicate that the formation of the electron-lucent zone and the sorting of multimerin to this region occur independent of VWE The isolated abnormalities in VWF suggests a VWF gene mutation is the cause of 'platelet low' type 1 VWD.

SummaryType 2B von Willebrand disease (vWD) is typically characterized by enhanced ristocetin-induced platelet aggregation (RIPA) caused by increased von Willebrand factor (vWF) affinity for platelets. Furthermore, absence of larger vWF multimers in plasma is characteristic of the originally described type IIB patients, now considered a subgroup of type 2B. We describe here three affected members of a family presenting with prolonged bleeding time, thrombocytopenia, markedly enhanced RIPA and spontaneous platelet aggregation, but normal plasma vWF antigen and ristocetin cofactor activity. Larger plasma vWF multimers, albeit decreased, were present in relatively greater proportion than in other type IIB patients. Genetic studies performed in two of these patients resulted in the identification of a previously unreported A→G transition at nucleotide 4175 in the sequence of the pre-pro-vWF cDNA, corresponding to the substitution Ile546→Val in the mature vWF subunit. This mutation appears to be responsible for an unusual type 2B phenotype, with greater enhancement of the vWF platelet interaction than in typical cases but partial preservation of the larger vWF multimers in plasma.

The ristocetin cofactor assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity in the diagnosis of von Willebrand's Disease (VWD). However, the assay suffers from poor reproducibility and sensitivity at low levels of VWF and is labour intensive. We have undertaken an evaluation of a new immunoturbidimetric VWF activity (VWF:Ac) assay (INNOVANCE(®) VWF Ac. Siemens Healthcare Diagnostics, Marburg, Germany) relative to an established platelet-based VWF:RCo method. Samples from 50 healthy normal subjects, 80 patients with VWD and 50 samples that exhibited 'HIL' (i.e. Haemolysis, Icterus or Lipaemia) were studied. VWF:Ac, VWF:RCo and VWF:Ag were performed on a CS-analyser (Sysmex UK Ltd, Milton Keynes, UK), all reagents were from Siemens Healthcare Diagnostics. The VWF:Ac assay, gave low intra- and inter-assay imprecision (over a 31-day period, n = 200 replicate readings) using commercial normal (Mean 96.2 IU dL(-1), CV < 3.0%) and pathological (Mean 36.1 IU dL(-1), CV < 3.5%) control plasmas. The normal and clinical samples exhibited good correlation between VWF:RCo (range 3-753 IU dL(-1)) and VWF:Ac (rs = 0.97, P < 0.0001), with a mean bias of 5.6 IU dL(-1). Ratios of VWF:Ac and VWF:RCo to VWF:Ag in the VWD samples were comparable, although VWF:Ac had a superior lower level of detection to that of VWF:RCo (3% and 5% respectively). A subset (n = 97) of VWD and HIL samples were analysed for VWF:Ac at two different dilutions to assess the effect on relative potency, no significant difference was observed (P = 0.111). The INNOVANCE(®) VWF Ac assay was shown to be reliable and precise.

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. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.

Summary. Background: Binding of von Willebrand factor (VWF) multimers of ultra-large size to platelets is considered the triggering mechanism of microvascular thrombosis in thrombotic thrombocytopenic purpura (TTP). Objective: To assess the potential of VWF-related measurements as markers of disease activity and severity in TTP. Methods: VWF antigen (VWF:Ag), platelet glycoprotein-Ib-α binding-conformation (GPIb-α/BC) and multimeric pattern were investigated in 74 patients with acquired TTP during acute disease, remission or both and 73 healthy controls. In patients with both acute and remission samples available, VWF ristocetin co-factor activity (VWF:RCo) and collagen binding (VWF:CB) were also measured. The relationships of study measurements with the presence of acute disease and remission and with markers of disease severity were assessed. Results: VWF:Ag and VWF-GPIb-α/BC were higher in TTP patients than controls (P

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is due to quantitative and/or qualitative defects of von Willebrand factor (VWF). Despite the improved knowledge of the disease, detailed data on VWD types requiring specific treatments have not been reported thus far. To determine the number and types of VWD requiring therapy with desmopressin (DDAVP) and/or VWF/FVIII concentrates in Italy, a national registry on VWD (RENAWI) was organized. Only 16 of 48 centers included VWD in the RENAWI with diagnoses performed locally. Patients with uncertain results were retested by two expert laboratories using multimeric analysis and mutations of the VWF gene. A total of 1234 of 1529 (81%) cases satisfied the inclusion criteria and could be classified as VWD1 (63%), VWD2A (7%), VWD2B (6%), VWD2M (18%), VWD2N (1%), and VWD3 (5%). VWD types were also confirmed by DNA analyses and occur in young adults (83%), mainly in women (58%). Mucosal bleedings (32 to 57%) are more frequent than hematomas (13%) or hemarthrosis (6%). Most patients were exposed to an infusion trial with desmopressin (DDAVP) and found responsive with the following rates: VWD1 (69%), VWD2A (26%), VWD2M (29%), and VWD2N (71%). However, DDAVP was not always used to manage bleeding in all responsive patients and VWF/FVIII concentrates were given instead of or together with DDAVP in VWD1 (30%), VWD2A (84%), VWD2B (62%), VWD2M (63%), VWD2N (30%), and VWD3 (91%). Data of the RENAWI showed that correct VWD identification and classification might be difficult in many Italian centers. Therefore, evidence-based studies should be organized only in well-characterized patients tested by laboratories that are expert in the clinical, laboratory, and molecular markers of VWD.

Summary. Background: Over the last 4 years ADAMTS-13 measurement underwent dramatic progress with newer and simpler methods. Aims: Blind evaluation of newer methods for their performance characteristics. Design: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS-13-deficient plasma (arbitrarily set at 0%) into one normal-pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested ‘blind’ 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. Results: There were eight functional and three antigen assays. Linearity of observed-vs.-expected ADAMTS-13 levels assessed as r2 ranged from 0.931 to 0.998. Between-run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10–15% for five methods and up to 20% for the remaining three. F-values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS-13 levels (the higher the F-value, the better the capacity) ranged from 3965 to 137. Between-method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS-13 offer the best performance characteristics. Conclusions: New assays for ADAMTS-13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.

Summary Missense mutations are not considered a common cause of type 3 von Willebrand’s disease (VWD), the most severe defect of von Willebrand factor (VWF) characterized by undetectable levels of this protein in plasma and platelets. Nevertheless, several missense mutations have been identified in these patients. In this study, we report the cases of two Italian patients with type 3 VWD, both compound heterozygotes for different missense mutations and null alleles, p.D141Y/c.2016_2019del and p.C275S/p.W222X. We performed in vitro expression studies of the candidate missense mutations, both located in the D1 domain of VWF propeptide, to confirm their link with the disease and to understand the mechanisms of type 3 VWD responsible in these patients. Mutant and wild-type (WT) expression vectors were used for transient transfection and co-transfection studies in COS-7 cells. Single construct transfections of both missense mutations showed a strongly reduced but detectable secretion of recombinant (r)VWFs (approximately 15% of WT), with essentially only dimers being visualized on multimeric analysis. As expected, expression of a single construct of either mutation with the WT, showed mildly reduced secretion (approximately 40% of WT) and a full set of multimers. These expression studies indicate that the two amino acids D141 and C275 are key residues in the tertiary structure of the VWF propeptide. Their replacement with a tyrosine and a serine, respectively, might compromise propeptide folding, affecting both its intracellular survival and its capacity to mediate multimerization. Co-expression of hybrid rVWFs confirmed the recessive inheritance pattern of these missense mutations.

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.

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta- thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.

Summary. Background: In a patient previously diagnosed with type 2A von Willebrand disease (VWD) [absence of high and intermediate molecular weight von Willebrand factor (VWF) multimers and markedly reduced ristocetin-induced platelet aggregation (RIPA)], an infusion test of desmopressin was followed by mild thrombocytopenia. This led to further laboratory investigations of his affected brother and of family members, who showed different phenotypic patterns compatible with type 1, 2A, 2B and an uncertain classification of VWD. The two brothers were compound heterozygotes (C275R/P1337L), whereas the others members of the family were heterozygous for C275R (a novel mutation in the D1 domain) or P1337L (a type 2B mutation in the A1 domain). Objective and methods: To evaluate the role of the combined effect of the two mutations in the two brothers, C275R and P1337L recombinant (r) VWFs were transiently expressed in COS-7 cells. Results: Recombinant VWF levels secreted in cell media were similar for wild-type (WT), P1337L and hybrid P1337L/WT rVWFs, reduced for hybrids C275R/P1337L and C275R/WT rVWFs, and strongly reduced for C275R rVWF. All rVWFs had a full set of multimers except C275R rVWF, which had only dimers. P1337L rVWF and C275R/P1337L rVWF showed the highest degree of binding to glycoprotein (GP) Ibα and the lowest to collagen, followed by P1337L/WT rVWF (with an intermediate level of binding to both ligands), and by WT rVWF with the lowest level of binding to GPIbα and the highest to collagen. Conclusion: These results suggest that the two compound heterozygous patients have a circulating VWF mainly mutated in the A1 domain (P1337L). This peculiar type 2B VWF variant showed a remarkably high affinity for the GPIbα platelet receptor, leading to the loss of high and intermediate molecular weight multimers and hence to decreased RIPA, as in type 2A VWD.

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.

A novel mutation, R1308L (3923G > T) was present in the heterozygous state in five members of a family with type 2B von Willebrand disease (VWD) characterized by a full set of von Willebrand factor (VWF) multimers in plasma and by the absence of thrombocytopenia before and after desmopressin (DDAVP). The defect (R1308L) was located at the same amino acid position of one of the most common mutations associated with type 2B VWD (R1308C), which is characterized by the loss of high molecular weight VWF multimers (HMWM) in plasma and the occurrence of thrombocytopenia. To understand the mechanisms of this defect, the novel (R1308L) and 'common' (R1308C) mutations were expressed in COS-7 cells, either alone or, to mimic the patients' heterozygous state, together with wild-type VWF. R1308L recombinant VWF (rVWF) had a higher affinity for the platelet glycoprotein Ibalpha (GPIbalpha) receptor than wild-type rVWF, R1308C rVWF showing an even higher affinity. A novel finding was that both mutant rVWFs showed a similarly reduced binding to collagen type I and type III in comparison with wild-type rVWF. The latter finding suggests a more important role than recognized so far for the VWF A1 domain in VWF binding to collagen, which may contribute to the in vivo hemostatic defect associated with type 2B VWD.

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.

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).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.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).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.

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.

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.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.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).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.

Congenital or immunomediated deficiencies of the metalloprotease that cleaves physiologically von Willebrand factor (vWF) reduce or abolish the degradation of ultralarge vWF multimers that cause the formation of intravascular platelet thrombi in patients with thrombotic thrombocytopenic purpura (TTP). There is little knowledge on the behavior of the protease in other physiological and pathologic conditions. Such knowledge is important to evaluate the specificity of low protease plasma levels in the diagnosis of TTP. Using an enzyme immunoassay, the protease was measured in 177 control subjects of different ages, in 26 full-term newborns, and in 69 women during normal pregnancy. Because TTP is often associated with multiorgan involvement and acute phase reactions, clinical models of these pathologic conditions were also investigated, including decompensated liver cirrhosis (n = 42), chronic uremia (n = 63), acute inflammatory states (n = 15), and the preoperative and postoperative states (n = 24). Protease levels were lower in healthy persons older than 65 than in younger persons. They were low in newborns but became normal within 6 months, and they were lower in the last 2 trimesters of pregnancy than in the first. Protease levels were also low in patients with cirrhosis, uremia, and acute inflammation, and they fell in the postoperative period. There was an inverse relation between low protease and high plasma levels of vWF antigen and collagen-binding activity. In conclusion, low plasma levels of the vWF cleaving protease are not a specific beacon of TTP because the protease is also low in several physiological and pathologic conditions.

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.

Ristocetin Cofactor and Collagen Binding Activities Normalized to Antigen Levels for a Rapid Diagnosis of Type 2 von Willebrand Disease Single Center Comparison of Four Different Assays

Background: In von Willebrand disease type 2B (VWD2B) the abnormal VWF spontaneously binds to circulating platelets, usually resulting into loss of high molecular weight multimers (HMWM) and thrombocytopenia. In these patients VWF:RCo/Ag ratio is indicative of reduced HMWM and roughly correlated with the severity of VWD2B defect. Physiological concentrations of Chloride ions (Cl−) inhibit the hydrolysis of VWF by ADAMTS-13. Cl− acts as an allosteric effector and its specific binding to VWF A1 domain results into a reduced availability of A2 to be proteolised by ADAMTS-13. Type 2B VWF R1306W showed a reduced affinity to Cl− (>KdCl−) and an increased (>kcat/Km) susceptibility to proteolysis by ADAMTS13 (De Cristofaro, R. et al Molecular mapping of the chloride-binding site in VWF. J Biol Chem2007; 281: 30400–11). Aim: To determine if other VWD2B mutations affect Cl– binding by changing the proteolysis of mutant VWF by ADAMTS-13. Methods: Five distinct mutant recombinant (r)A1-A2-A3 proteins (Table), have been prepared along with the WT. Determination of KdCl− of each rA1- A2-A3 was achieved by intrinsic protein fluorescence, performed as a function of both Cl− concentrations and temperature. Study on the kinetic rates of hydrolysis of each rA1- A2-A3 by ADAMTS-13 was evaluated using HPLC methods. Results and Conclusions: The results (Table) confirmed that affinity of Cl− (KdCl−) was decreased in all mutant rA1-A2-A3s compared to the WT at different extent, with an inverse correlation to the catalytic specificity (kcat/Km) of ADAMTS-13 interaction. Higher VWF:RCo/Ag ratios were found associated with lower values of kcat/Km in our studied patients. This finding strongly support that the reduced affinity of type 2B VWF to Cl− might contribute to the depletion of HMWM. Table (patients) WT --- R1341Q (4) R1341W (3) H1268D (1) V1316M (4) R1306W (15) KdCl− (mM) 156 191 359 481 526 540 kcat/Km (M−1s−1) 7.25x104 8.8x104 1.2x105 1.39x105 1.6x105 1.95x105 VWF:RCo/Ag ratio (range) 0.7 – 1.2 0.8 (0.77–0.84) 0.7 (0.6–0,8) 0.5 0.3 (0.2–0.7) 0.5 (0.25–1.0)

Background: Type 2B von Willebrand disease (VWD) is due to a unique gain-of-function variant of von Willebrand factor (VWF) that spontaneously interacts with circulating platelets resulting in loss of VWF high molecular weight multimers (HMWM) in plasma and reduced platelet counts, in most cases. Diagnosis of 2B is based on increased ristocetin-induced platelet aggregation (RIPA) in patient’s platelet rich plasma (PRP). Type 2B “Malmo or New York” is caused by the mutation P1266L that is associated with increased RIPA, but with normal multimeric distribution in plasma and no thrombocytopenia. Aims: To determine the incidence and the molecular defects of this rare VWF variant among a large population of VWD patients regularly followed at two Northern Italian Hemophilia Centers. Methods: Criteria for VWD type 2B were those recommended by the ISTH-SSC-SC on VWF. RIPA and multimeric analysis were performed according to standardized methods. Bleeding severity score (BSS) was calculated in all patients enrolled in this study. Sequence analysis of the portion of exon 28 encoding for the A1 domain was performed to identify nucleotide substitutions in all cases where RIPA was Results: 74 patients from 16 different families satisfied the criteria for VWD type 2B. Seven patients from 3 unrelated families clearly showed normal multimeric distribution in plasma and no thrombocytopenia also after stimuli. Clinical and laboratory parameters of the seven affected members are reported as mean values (ranges): BSS=2.5 (2–4); RIPA=0.56 (0.5–0.7) mg/ml; platelet count=255 x 10^9/L (172–353); VWF:RCo=46 U/dL (21–66); VWF:Ag=53 U/dL (30–67); FVIII=61 U/dL (54–76). Patients from family A showed five distinct substitutions (3686T>G, 3692A>C, 3735G>A, 3789G>A, 3797C>T), patient B1 two (3789G>A, 3797C>T) and patient from family C three (3692A>C, 3789G>A, 3797C>A). Two of these were silent, whereas the other caused the following amino acid changes: three in family A (V1229G, N1231T, P1266L), one in B1 (P1266L) and two in family C (N1231T, P1266Q). Mutations V1229G and N1231T have already been reported, whereas mutation P1266Q is novel. The substitutions found in families A and B clearly correspond to the pseudogene sequence, suggesting gene conversion into VWF gene. In family C, the nucleotide mutation causing P1266Q substitution is not consistent with this mechanism: moreover the patients do not show the substitution 3735G>A found in the pseudogene. Conclusion: We confirm that the substitution of proline 1266 with leucine appears to be the main cause of type 2B “Malmo/New York” phenotype while its replacements with a glutamine apparently results in lower VWF activities in family C. Based on these data, gene conversion appears to be more frequent than expected and capable to generate different set of mutations.

The introduction of plasma exchange therapy in early 1970s significantly reduced the rate of mortality in patients affected by thrombotic thrombocytopenic purpura (TTP), a disease characterized by thrombocytopenia and microangiopathic hemolytic anemia. A similar improvement was never achieved in the prevention of the disease recurrence. Still, 20–50% of patients, who survived the fatal disease, experience a relapse one month or even years after the acute episode of TTP. There is no pathognomic marker or laboratory test that can be used for the surveillance of TTP during remission and predict which patients will relapse. We have retrospectively analyzed for the first time at remission the role of ADAMTS13, anti-ADAMTS13 autoantibodies and von Willebrand Factor (VWF) in 109 patients who survived the acute episode of TTP. ADAMTS13 activity and ADAMTS13 antigen levels were measured as described by Gerritsen et al (TH 1999) and Feys HB et al. (JTH 2006), respectively. The total anti-ADAMTS13 autoantibodies (with and without neutralizing activity) were measured by western blot analysis and the presence of neutralizing anti-ADAMTS13 autoantibodies was checked according to Gerritsen et al (TH 1999). VWF antigen was measured using an ELISA assay and VWF multimers analysis was carried out using low-resolution SDS-agarose gel electrophoresis and exposing gels to human anti-VWF antibodies labeled with I125 for autoradiography (Ruggeri & Zimmerman, Blood 1981). All variables have been statistically analyzed in 2 subgroups of patients with or without TTP recurrence, in order to understand the role of each variable as a potential predictor marker for recurrence. Univariate and multivariate analysis were carried out to evaluate adjusted and unadjusted odds ratios (Ors) with 95% confidence intervals (CI) as a measure of the relative risk of relapse associated with the risk factors under investigation. Our data showed that the median value of ADAMTS13 activity and antigen levels at remission were significantly lower in patients with recurrent TTP than in patients with no relapse (ADAMTS13 activity: 12% vs. 41%; p=0.007; ADAMTS13 antigen: 36% vs 58%; p=0.003). Furthermore, the prevalence of patients with severe ADAMTS13 deficiency (≤10%) was significantly higher in the group of patients who relapsed (OR=2.9 CI95% 1.3–6.8, p=0.01). The prevalence of anti-ADAMTS13 autoantibodies (with or without neutralizing activity) resulted to be significantly higher in patients with recurrent TTP (OR= 3.1 CI 95% 1.4–7.3, p=0.006). A higher VWF antigen levels or the presence of ultralarge VWF (ULVWF) multimers at remission did not increase the risk of recurrence (p=0.4 for VWF:Ag and p=0.7 for ULVWF multimers). In conclusion, our data showed that the association of severe ADAMTS13 deficiency and the presence of anti-ADAMTS13 autoantibodies is a negative prognostic marker at remission and increases the relative risk of TTP recurrence by 3.6 times (OR=3.6 CI95% 1.4–9). Therefore our results would suggest that our efforts should go in the direction of maintenance therapy which aims at reducing or abolishing the presence of antibodies during remission and increasing the level of ADAMTS13 in plasma in order to prevent the recurrence of TTP.

Background and Objectives. Patients with severe forms of von Willebrand’s disease (VWD) may have frequent episodes of mucocutaneous bleeding and also of hemarthrosis or hematomas. They are usually unresponsive to desmopressin (DDAVP) and must be treated with high- or intermediate-purity VWF/FVIII concentrates (VWFc). Due to the different VWF/FVIII composition of the VWFc, timing and dosage of infusions are still not completely standardized. Aim of this study was to evaluate the efficacy and safety of repeated infusions of VWFc given on demand, for bleeds and surgery, and on secondary long-term prophylaxis to our cohort of severe VWD patients. Design and Methods. This is a cohort study on 473 VWD patients regularly followed up at our Center for more than two years. Inclusion criteria: patients proven to be unresponsive to DDAVP and treated with VWFc with > 25 exposure days (ED)/year (> 50,000 FVIII U/year) during the previous 2 years. All patients were characterized by a bleeding severity score derived from a detailed history of 11 symptoms. Since VWFc available in Italy are still labeled in FVIII IU, patients were given 60 or 40 FVIII IU/Kg of high- (Alphanate, Fanhdi) or intermediate-purity (Haemate-P) in case of on demand or long term prophylaxis, respectively. Efficacy of VWFc used on demand versus prophylaxis (every other day or twice a week) regimens was based on resolution/reduction of bleeds and rated as excellent/good versus partial/poor clinical responses. Safety was measured by monitoring side effects and pre-post infusion FVIII levels during the first two weeks of treatment. Results. 32/473 (7%) patients only met the inclusion criteria. They were severe (bleeding scores >15; VWF:RCo baseline levels 95%, >85%, >90% surgeries, bleeds, prophylaxis, respectively. Prophylaxis could stop bleeding in 8 patients and largely reduced hospitalization for PRBC transfusions in the remaining 3. As far as safety, FVIII levels were always

Background: Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder caused by abnormal von Willebrand factor (VWF) that displays increased affinity to the platelet glicoprotein 1b alpha (GpIba) and is due to a group of mutations clustered within VWF A1 domain. Such an enhanced 2BVWF-GpIba binding usually result in loss of large VWF multimers and moderate-mild thrombocytopenia. A llama-derived antibody fragment (AuVWFa11) recognizing the GpIba-binding conformation has been recently developed (Blood 2005;106:3035). Aims and design of the study: to further explore the usefulness of AuVWFa11 in type 2B diagnosis, we have prospectively tested AuVWFa11 in our cohort of 16 patients previously characterized by platelet count, VWF multimers and mutations. Methods: Data of platelet count with mean platelet volume (MPV) and morphologic evaluation of the blood smear to search for giant platelets or aggregates were associated with the history of physiologic or pathologic stress conditions such as pregnancy, infections, surgery or use of DDAVP. All patients were diagnosed by ristocetin induced platelet agglutination (RIPA) in the Platelet Rich Plasma (PRP), ristocetin cofactor activity (VWF:RCo) with VWF antigen (VWF:Ag), multimeric structure of VWF. Mutations within VWF A1 domain were searched for and confirmed by sequencing exon 28. AuVWFa11 was tested in 40 normal individuals (expressed as % of active VWF in normal pool plasma =0.70±0.13) and in type 2B. Results: Data (mean ± SD) of the AuVWFa11 tested in the 16 patients with type 2B VWD are correlated with the main phenotypic data and genotype (Table1). Platelet count 3 correlate with loss of large VWF multimers and mild-moderate thrombocytopenia. Conclusions: The AVWF11a can show activated VWF in most type 2B VWD patients, especially when 2B VWF mutants induce significant loss of large multimers and thrombocytopenia. Therefore AuVWF11a can be a useful additional tool in the diagnosis of type 2B VWD. | Mutation (n) | RIPA (mg/ml) | VWF.Ag (U/dL) | Plat Count (×10^9/L) | MPV (micron^3) | Loss of HMW Mult | AuVWFa11 (ratioNPP) | |:------------ | ------------ | ------------- | -------------------- | -------------- | ---------------- | ------------------- | | R1306W (5) | 0.65 | 40±9 | 165±39 | 10.3±2.3 | YES | 3.7±1.5 | | R1308C (3) | 0.72 | 53±16 | 163±61 | 11.5±1.9 | YES | 3.3±2.3 | | R1308L (3) | 0.50 | 48±13 | 341±104 | 8.1±3.1 | NO | 0.5±0.2 | | I1309V (1) | 0.40 | 115 | 222 | 11.8 | PARTIAL | 2.1 | | V1316M (2) | 0.50 | 32±7 | 119±30 | 9.2±2.4 | YES | 4.4±0.1 | | P1337L (1) | 0.50 | 48 | 222 | 9.5 | PARTIAL | 1.3 | | R1341Q (1) | 0.67 | 43 | 422 | 9.9 | PARTIAL | 2.9 | Table 1

An uncommon missense variant of thrombospondin-1 representing an asparagine (Asn-700) to serine substitution (Ser-700) has been identified as a potential genetic risk factor for myocardial infarction (MI). Thrombospondin-1 (TSP-1) is a glycoprotein stored in human platelet alpha granules and has been shown to regulate von Willebrand factor (VWF) multimer size. The TSP-1 domain responsible for VWF-reducing activity was localized to the calcium-binding C-terminal sequence at position Cys 974. Considering the proximity of the N700S TSP-1 polymorphism and the demonstration that the Ser-700 polymorphism alters the calcium binding affinity of this domain, we investigated whether altered VWF-reducing activity of Ser-700 polymorphism underlies the observed prothrombotic phenotype. Plasma samples were analyzed from a cohort of patients (N=31) who survived an acute myocardial infarction at age 45 or younger compared with age-matched controls (N=19). The relative percentage of high molecular weight VWF multimers did not differ significantly between individuals homozygous for either Ser-700 or Asn-700 (p=.20). Similarly, the relative percentage of high molecular weight multimers was 31.2% for the Ser-700 homozygous MI patients (N=21) versus 31.1% (p=.95) in the Asn-700 healthy controls (N=10). These findings were confirmed using the collagen binding assay to assess VWF activity relative to antigen concentrations. The mean ratio of CBA/Ag was .98 in the Ser-700 patients versus 1.0 in the Asn-700 control group (p=.22). Mean plasma thrombospondin-1 concentrations did not differ between the two groups. The VWF reducing capacity of TSP-1 was also assessed in vitro using recombinant TSP-1 N700S constructs containing the last EGF-like repeat, all type-3 repeats (calcium-binding domains), and C-terminal sequence. Incubation of Ser-700 peptide, Asn-700 peptide, or platelet purified full-length TSP-1 with ultra-large VWF (using either TTP plasma or recombinant ultra-large VWF) did not result in any significant reduction of VWF multimer size under static conditions. In conclusion, the association between coronary artery thrombosis and the TSP-1 Ser-700 polymorphism is not mediated through altered VWF reducing activity.

The inherited deficiency of the von Willebrand factor-cleaving protease, ADAMTS13, is usually associated with severe forms of thrombotic thrombocytopenic purpura (TTP). Among the reported 53 mutations on ADAMTS13 gene, none has been described on TSP1-6 repeat domain. We investigated an Iranian man with history of chronic recurrent TTP and very low plasma levels of ADAMTS13 activity (2.3%). He had his first TTP episode at the age of 21 years and 6 subsequent episodes occurred without precipitating events. Genetic analysis revealed a homozygous deletion of nucleotides 2930–2935 (GTGCCC), in exon 23 of ADAMTS13 gene, leading to the replacement of Cys977 residue by a Trp and the deletion of Ala978 and Arg979, in the TSP1-6 repeat domain. The patient’s brother, homozygous for the same mutation, has no clinical manifestation of TTP so far, despite the same degree of ADAMTS13 deficiency. The patient’s parents resulted to be heterozygous. To explore the mechanism of ADAMTS13 deficiency, wild type (ADAMT13WT) and mutant (ADAMTS13del6bp) expression vectors were transiently transfected in HEK293 and COS-7 cells. The enzymatic activity of the expressed rADAMTS13 proteins was evaluated by measuring the extent of VWF multimer degradation, using a quantitative immunoblotting assay. rADAMT13WT was able to degrade multimers completely (100% of activity), whereas rADAMTS13del6bp had enzymatic activity reduced to ~10% of WT. Both WT and mutant proteases present in conditioned media and cell lysates, were analyzed by Western blot analysis, using anti-V5 monoclonal antibody against the C-terminal tag of rADAMTS13. Blots showed a dense band of ~190 KDa in the medium, corresponding to the secreted rADAMT13WT protein. Cells transfected with ADAMTS13del6bp showed a fainter band roughly estimated to be 5% of the WT obtained by densiometric analysis. Therefore, pulse-chase labelling experiments were performed to evaluate the presence of a secretion pathway alteration. After 60 min pulse with [35S] methionine the maximum level of rADAMT13WT in conditioned media was found at 24 hours. rADAMTS13del6bp was minimally present after 3 hours only, no band being detected after 7 hours of chase. Differential immunofluorescence studies were performed using an anti-V5 monoclonal antibody against ADAMTS13 proteins and monoclonal antibodies recognizing markers of Cis-Golgi and ER. The merging studies in WT and mutant transfected cells showed that rADAMTS13WT was mostly localized in the perinuclear area, suggesting a primary localization in the Golgi apparatus, whereas rADAMTS13del6bp showed less intense staining diffusely throughout the cytoplasm, only a minimal amount of the mutant protein being localized in the Cis-Golgi and ER. The structural consequences of the deletion mutation in the TSP1-6 domain may cause a loss of the known antiparallel, three-stranded fold characterizing the architecture of the TSP-like domains. In these domains, each strand is capped by disulfide bonds on each end. This study suggests that the residue Cys977 could be involved in the formation of disulphide bonds responsible for a correct folding of one of the TSP1-like domains of ADAMTS13. Therefore the deletion could lead to uncorrected folding which reflects an intracellular degradation and secretion defect of the mutant protease without any intracellular accumulation. These results reflect the severe deficiency of ADAMTS13 in the patient’s plasma.

Background and Objectives. Patients with severe forms of von Willebrand’s disease (VWD) may have frequent episodes of mucocutaneous bleeding and also of hemarthrosis or hematomas. However, little retrospective or prospective data on secondary long term prophylaxis in VWD are available. Aim of this study was to evaluate the efficacy and safety of fixed regimens of prophylaxis with factor VIII/VWF concentrates in our cohort of VWD patients with recurrent joint and gastrointestinal (GI) bleeds. Design and Methods. This is a cohort study on 452 VWD patients regularly followed up at our Center for at least three years. 89/452 cases (20%) were treated with FVIII/VWF concentrates during the last two years because of one or more bleedings and 11/89 (12%) were included in a long term prophylaxis program because of frequent recurrence of bleeds at the same sites. All patients were characterized by a bleeding severity score derived from a detailed history of 11 symptoms. Since concentrates available in Italy are still labelled in FVIII IU, patients were given 40 FVIII IU/Kg of high- (Alphanate, Fanhdi) or intermediate-purity (Haemate-P) concentrates, two times a week (joint bleeds) or every other day (GI bleeds) to maintain FVIII/VWF levels higher than baseline during prophylaxis. Effectiveness of prophylaxis was based on resolution/reduction of bleeding as well as on numbers of transfused packed red blood cells (PRBC) and days of hospitalization. Safety was measured by monitoring side effects and FVIII levels before and after every injection during the first three weeks of prophylaxis. Results. All the 11 patients were severe, as shown by high bleeding scores (> 20) and VWF:RCo baseline levels

We analyzed the association of bleeding severity with eight candidate gene haplotypes within pedigrees of twelve index cases of VWD type 2 (six type 2M, three type 2A and three type 2B), using a covariance components model for multivariate traits (Mendel 5.5: QTL Association). In addition to the 12 index cases, these pedigrees included 58 affected and 55 unaffected relatives, as characterized by plasma Ristocetin cofactor (VWF:RCo), levels, VWF antigen (VWF:Ag) levels, ristocetin induced platelet agglutination (RIPA), VWF multimeric analysis and specific mutations of VWF. A bleeding severity score was derived from a detailed questionnaire. The severity of bleeding episodes was ranked from 0 to 5 in each of eleven bleeding categories: epistaxis, cutaneous symptomatic bleeding, bleeding from minor wounds, oral cavity bleeding, gastrointestinal bleeding, bleeding associated with tooth extraction, surgery, postpartum hematoma, muscle hematoma, hemarthrosis, and menorrhagia. Donors were genotyped using specific fluorescent-labeled oligonucleotides in a melting curve analysis (LightTyper, Roche, Inc.). VWF:RCo levels, followed closely by VWF:Ag levels, had the strongest influence on bleeding severity score. ITGA2 promoter haplotype -52T and ITGA2B haplotype 1 (Ile843) were each associated with increased bleeding severity scores (p=0.039 and p = 0.024, respectively), and the association of both remained significant when bleeding severity score was adjusted for age (p=0.012 and p = 0.010, respectively). These associations persisted even if menorrhagia, the only gender-associated bleeding category, was excluded from the analysis. This is the first report of a statistically significant association of ITGA2 haplotype -52T with risk for bleeding in vivo, a finding that is consistent with the significant attenuation of ITGA2 transcription by this promoter haplotype in in vitro studies. No statistically significant association was observed with the major haplotypes of six other candidate genes, GP1BA, ITGB3, GP6, VWF, FGB, and IL6. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (−1793G) (p=0.04), although the impact of this VWF haplotype 1 association was not of itself sufficiently strong to directly influence bleeding severity score. These results establish that genetic differences in the expression or activity of integrin receptor subunits alpha-2 and alpha-IIb can influence the bleeding phenotype of VWD type 2. Together with the results of our previous studies, these findings demonstrate that platelet receptor gene haplotypes can influence bleeding tendency not only in VWD type 1 but also in VWD type 2. Interestingly, the influence of GP6 haplotype b (Pro219) on bleeding seen in VWD type 1 was not evident in this study of VWD type 2 pedigrees. Further studies on additional VWD type 1 and type 2 pedigrees, particularly pedigrees with diverse racial and/or ethnic backgrounds, will enable us to distinguish the variety and extent of genetic differences that can influence bleeding tendency in this disease.