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Abstract: Four variants have been continuously subjected to debate and received different von Willebrand disease (VWD) classifications: p.R1315L, p.R1315C, p.R1374H, and p.R1374C. We chose to comprehensively investigate these variants with full set of VWD tests, protein-modeling predictions and applying structural biology. Patients with p.R1315L, p.R1315C, p.R1374H, and p.R1374C were included. A group with type 2A and 2M was included to better understand similarities and differences. Patients were investigated for phenotypic assays and underlying disease mechanisms. We applied deep protein modeling predictions and structural biology to elucidate the causative effects of variants. Forty-three patients with these variants and 70 with 2A (n = 35) or 2M (n = 35) were studied. Patients with p.R1315L, p.R1374H, or p.R1374C showed a common phenotype between 2M and 2A using von Willebrand factor (VWF):GPIbR/VWF:Ag and VWF:CB/VWF:Ag ratios and VWF multimeric profile, whereas p.R1315C represented a type 2M phenotype. There was an overall reduced VWF synthesis or secretion in 2M and cases with p.R1315L, p.R1374H, and p.R1374C, but not in 2A. Reduced VWF survival was observed in most 2A (77%), 2M (80%), and all 40 cases with p.R1315L, p.R1374H, and p.R1374C. These were the only variants that fall at the interface between the A1-A2 domains. p.R1315L/C mutants induce more compactness and internal mobility, whereas p.R1374H/C display a more extended overall geometry. We propose a new classification of type 2M/2A for p.R1315L, p.R1374H, and p.R1374C because they share a common phenotype with 2M and 2A. Our structural analysis shows the unique location of these variants on the A1-A2 domains and their distinctive effect on VWF., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background:  Low von Willebrand factor (VWF) refers to subjects with plasma levels of 30 to 50 IU/dL. The mechanism of low VWF is poorly understood. We chose to determine the clinical presentation, laboratory phenotype, and underlying mechanisms of low VWF., Material and Methods:  We included 250 patients characterized with low VWF. The International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT) was used to assess clinical symptoms. To determine the underlying mechanisms of low VWF, we used as markers the VWF propeptide (VWFpp) assay and FVIII:C/VWF:Ag ratio for VWF synthesis and the VWFpp/VWF:Ag ratio for VWF clearance. Results were compared with those of 120 healthy controls. Cases with abnormal screening tests were further evaluated for coagulation factor levels and platelet disorders., Results:  The median age of the cohort was 35 years (range 3-85), 21% were children ( n  = 53), 34% were adult males ( n  = 85), and 45% ( n  = 112) were adult females. According to the ISTH-BAT, abnormal bleeding was found in 35% of children, 47% of males, and 49% of females. No association was found between VWF activity levels and ISTH-BAT. Patients showed an overall decreased VWF synthesis/secretion and an enhanced VWF clearance was identified in 33% of them. In 89 patients (36%), there were other hemostasis-related defects, but there was no difference in the ISTH-BAT between the two groups., Conclusion:  Our findings indicate that reduced VWF synthesis/secretion and enhanced VWF clearance are major mechanisms of low VWF levels. Patients with low VWF have significant bleeding manifestations. While other hemostasis defects occurred together with low VWF, this combination did not exacerbate clinical symptoms., Competing Interests: F.P. reports participation at educational meetings and the advisory board of Sanofi, Sobi, Takeda, Roche, and Biomarin. The other authors state that they have no conflict of interest., (Thieme. All rights reserved.)

Background: Enhanced von Willebrand factor (VWF) clearance from plasma is associated with von Willebrand disease (VWD). However, the genetic background of this disease mechanism is not well defined., Objective: To determine VWF variants that are associated with reduced VWF survival., Methods: Two hundred fifty-four patients with VWD (type 1 = 50 and type 2 = 204) were investigated, and the results were compared with 120 healthy controls. The patients were comprehensively characterized for phenotypic and genetic features. The ratio of VWF propeptide (VWFpp)/VWF antigen (VWFpp ratio) was used to establish in each patient the VWF clearance state., Results: Out of 92 variants associated with type 1 (7 were novel) and type 2 VWD, 19 had a VWFpp ratio ranging from 1.7 to 2.2, 24 had a VWFpp ratio between 2.3 and 2.9, and 24 variants had a ratio of ≥3. The VWFpp median ratio in healthy controls was 0.98 (0.55-1.6) so that a cut-off value of >1.6 was considered an indicator of accelerated VWF clearance from plasma. An enhanced VWF clearance was observed in 34% of type 1 cases, 100% of type 1 Vicenza cases, 81% of 2A cases, 77% of 2B cases, 88% of 2M cases, and 36% of 2N cases., Conclusions: An accelerated VWF clearance was found in most patients with type 2A, 2B, and 2M VWD, with a lower proportion of type 1 and 2N. Sixty-seven different variants alone or in combination with other variants were associated with an increased VWFpp ratio. The variants with the highest VWFpp ratio were mostly located in the D3-A1 VWF domains., Competing Interests: Declaration of competing interests F.P. has participated in educational meetings and the advisory board of Sanofi, Sobi, Takeda, Roche, and Biomarin. O.S. received congress support from Kedrion. All other authors report no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The spreading of opium use poses new health related concerns. In some areas of Asia its use is believed to protect from cardiovascular disorders, such as coronary artery disease (CAD). However, whether opium use has an association with CAD is unclear. We aimed to investigate the association between non-medical opium use and CAD. We set up a case-control analysis, i.e., the Milano-Iran (MIran) study by enrolling consecutive young patients who underwent a coronary angiography at the Tehran Heart Center, between 2004 and 2011. Incident cases with CAD were contrasted with controls for opium use. Relative risks were calculated in terms of odds ratios (ORs) by logistic regression models adjusted for age, sex, cigarette smoking, body mass index, hypertension, hyperlipidaemia, and diabetes. Interaction analyses were performed between opium and major cardiovascular risk factors. 1011 patients with CAD (mean age 43.6 years) and 2002 controls (mean age 54.3 years) were included in the study. Habitual opium users had a 3.8-fold increased risk of CAD (95%CI 2.4-6.2) compared with non-users. The association was strongest for men, with a fully adjusted OR of 5.5 (95%CI 3.0-9.9). No interaction was observed for the combination of opium addiction and hypertension, or diabetes, but an excess in risk was found in opium users with hyperlipidaemia (OR 16.8, 95%CI 8.9-31.7, expected OR 12.2), suggesting supra-additive interaction. In conclusion, despite common beliefs, we showed that non-medical opium use is associated with an increased risk of CAD, even when other cardiovascular risk factors are taken into account., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Maino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Type 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions., Objective: The objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS., Methods: An indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay., Results: In total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14)., Conclusions: Anti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used., Competing Interests: Declaration of competing interest statement J.E. received research support from CSL Behring outside the scope of this project and fees for educational activities from Roche and Celgene, which fees go to the institution. J.G. in the last 3 years received fees for lectures or consultancy from LFB outside the scope of this work. F.L. received unrestricted research grants from CSL Behring, Takeda, SOBI, and uniQure. He is a consultant for CSL Behring, Takeda, BioMarin, and uniQure, of which the fees go to the University. He has been a DSMB member of a study sponsored by Roche. P.M.M. is member of the scientific board for the Bayer Awards. Speaker fee from Bayer and Kedrion, Roche for lectures at educational symposia. C.S., in the last 3 years, has received honoraria for consulting or speaker bureau from Bayer, CSL Behring, Takeda, Novonordisk, Sobi, Novartis, Pfizer, Amgen, and Roche. A.T. received research support or honoraria for lectures or consultancy from CSL Behring, Octapharma, and Takeda outside of this work. F.P. reported participation at advisory board of Sanofi, Sobi, Takeda, Roche, and BioMarin. A.B.F. reported advisory board participation of CSL Behring, Grifols, Takeda, Octapharma, LFB, and Kedrion. J.O. has received research funding from Bayer, Biotest, CSL Behring, Octapharma, Pfizer, Swedish Orphan Biovitrum, and Takeda; consultancy, speakers bureau, honoraria, scientific advisory board, and travel expenses from Bayer, Biogen Idec, BioMarin, Biotest, Chugai Pharmaceutical Co., Ltd., CSL Behring, Freeline, Grifols, Novo Nordisk, Octapharma, Pfizer, F. Hoffmann-La Roche Ltd., Sanofi, Spark Therapeutics, Swedish Orphan Biovitrum, and Takeda. GC reported advisory board participation/speaker fee of CSL Behring, Grifols, Takeda, LFB, and Kedrion. The remaining authors declare no competing financial interests., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Several assays are now available to evaluate platelet-dependent von Willebrand factor (VWF) activity., Objective: To report the results obtained using 4 different assays in patients with von Willebrand disease (VWD) carrying variants mainly in the A1 domain, which is critical for VWF binding to glycoprotein Ib (GPIb) and ristocetin., Methods: We evaluated 4 different assays, 2 gain-of-function mutant GPIb binding (VWF:GPIbM) and 2 ristocetin cofactor (VWF:RCo) assays, in 76 patients with type 2 VWD. Patients and healthy controls were tested using VWF:GPIbM enzyme-linked immunosorbent assay (ELISA), VWF:GPIbM automated, VWF:RCo aggregometric, and VWF:RCo automated assays., Results: There was a good correlation (Pearson's r>0.82) and agreement (Bland-Altman plots assessment) between the 4 assays, although several outliers existed among the type 2B without high-molecular-weight multimers (HMWM). The VWF activity/VWF:antigen ratios, calculated for each assay, were used to establish the percentage of a correct diagnosis of type 2 (ratio<0.60) in these patients: VWF:RCo aggregometric, 2A(100%), 2M(78%), 2M/2A(100%), 2B(68%); VWF:RCo automated, 2A(88%), 2M(89%), 2M/2A(100%), 2B(63%); VWF:GPIbM ELISA, 2A(96%), 2M(67%), 2M/2A(67%), 2B(0%); VWF:GPIbM automated, 2A(73%), 2M(44%), 2M/2A(75%), 2B(84%). In type 2B patients with HMWM, all assays gave a ratio ≥0.60., Conclusion: The VWF:GPIbM-automated assay is the most effective to diagnose as type 2 the 2B variants, whereas the VWF:RCo assays are the most effective in detecting 2M and 2M/2A variants. The VWF:GPIbM ELISA greatly overestimates the activity of the type 2B patients lacking HMWM. In this study, the use of a VWF activity/VWF:antigen ratio cut-off of 0.70 halved the number of misdiagnosed patients., (© 2023 The Authors.)

Background: Factor V Leiden (FVL) and factor II c.∗97G>A (rs1799963) are genetic risk factors for venous thromboembolism. Their contribution to coronary artery disease (CAD) is less clear., Objectives: This study aimed to investigate the association between FVL, rs1799963, and premature CAD in Iranians., Methods: We performed a genetic case-control study of 944 cases and 1081 controls from the premature CAD Milano-Iran study, including patients aged 18-55 (female) and 18-45 years (male) who underwent coronary angiography at the Tehran Heart Centre (Iran) in 2004-2011. Cases had luminal stenosis ≥50% in at least 1 main coronary artery or branch. Controls were age- and sex-matched with no CAD history. FVL and rs1799963 were genotyped using TaqMan SNP genotyping assays. Association was tested by logistic regression adjusted for matching factors and ethnicity. Effect modification by sex and cardiovascular risk factors (metabolic [obesity, hypertension, hyperlipidemia, and diabetes], and smoking) was assessed., Results: The risk of premature CAD was increased by 50% in FVL carriers (adjusted odds ratio [adjOR] 1.54 [95% CI, 0.95-2.48]) and slightly reduced in rs1799963 carriers (adjOR 0.71 [95% CI, 0.40-1.27]). These effects were more pronounced in women than men (FVL, adjOR 1.66 vs 1.25; rs1799963, adjOR 0.60 vs 1.07). The risk of premature CAD was substantially increased in carriers of FVL with at least 1 metabolic risk factor compared with noncarriers without metabolic risk factors (adjOR 25.14 [95% CI, 12.51-50.52])., Conclusion: FVL but not FII rs1799963 was associated with an increased risk of CAD in young Iranians. This risk increased considerably when combined with metabolic cardiovascular risk factors., (© 2023 The Authors.)

von Willebrand disease (VWD) type 2 is caused by qualitative abnormalities of von Willebrand factor (VWF). This study aimed to determine the genotypic and phenotypic characterizations of a large VWD type 2 cohort from Milan. We included 321 patients (54% female) within 148 unrelated families from 1995 to 2021. Patients were fully characterized using laboratory phenotypic tests, and the genotypic diagnosis was confirmed by target genetic analysis using Sanger sequencing. Patients were diagnosed with type 2A (n = 98; 48 families), 2B (n = 85; 38 families), 2M (n = 112; 50 families), or 2N (n = 26; 12 families). Eighty-two unique VWF variants, including 8 novel variants, were found. The potential pathogenic effect of novel variants was assessed by in silico analysis. Most patients were heterozygous for a single variant (n = 259; 81%), whereas 37 cases (11%) had 2 variants (4 homozygous, 9 in trans, and 24 in cis). Twenty-five patients (8%) had ≥3 variants, mainly as a result of gene conversions. Among the 82 distinct variants identified, 5 different types, including missense (n = 64), gene conversion (n = 10), synonymous (n = 1), deletion (n = 4), and splice (n = 3), were observed. The results from this large cohort showed that VWD type 2 is invariably due to variants that do not prevent the synthesis of the protein, and a vast majority of patients (88%) had missense variants. Given the complexity of type 2 diagnosis and the necessity of performing several phenotypic tests, genetic analysis for patients suspected of having type 2 is beneficial to establish the correct diagnosis., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Type 3 von Willebrand disease (VWD) is a severe bleeding disorder caused by the virtually complete absence of von Willebrand factor (VWF). Pathophysiological mechanisms of VWD like defective synthesis, secretion, and clearance of VWF have previously been evaluated using ratios of VWF propeptide (VWFpp) over VWF antigen (VWF:Ag) and factor (F)VIII coagulant activity (FVIII:C) over VWF:Ag., Objective: To investigate whether the VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios may also be applied to understand the pathophysiological mechanism underlying type 3 VWD and whether VWFpp is associated with bleeding severity., Methods: European and Iranian type 3 patients were enrolled in the 3WINTERS-IPS study. Plasma samples and buffy coats were collected and a bleeding assessment tool was administered at enrolment. VWF:Ag, VWFpp, FVIII:C, and genetic analyses were performed centrally, to confirm patients' diagnoses. VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were compared among different variant classes using the Mann-Whitney test. Median differences with 95% confidence intervals (CI) were estimated using the Hodges-Lehmann method. VWFpp association with bleeding symptoms was assessed using Spearman's rank correlation., Results: Homozygosity/compound heterozygosity for missense variants showed higher VWFpp level and VWFpp/VWF:Ag ratio than homozygosity/compound heterozygosity for null variants ([VWFpp median difference, 1.4 IU/dl; 95% CI, 0.2-2.7; P = .016]; [VWFpp/VWF:Ag median difference, 1.4; 95% CI, 0-4.2; P = .054])., Fviii: C/VWF:Ag ratio was similarly increased in both. VWFpp level did not correlate with the bleeding symptoms (r = .024; P = .778)., Conclusions: An increased VWFpp/VWF:Ag ratio is indicative of missense variants, whereas FVIII:C/VWF:Ag ratio does not discriminate missense from null alleles. The VWFpp level was not associated with the severity of bleeding phenotype., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: von Willebrand factor propeptide (VWFpp) plays an important role in VWF multimerization and storage. VWFpp mutations have been previously associated with types 1, 3 and 2A/IIC von Willebrand disease (VWD)., Aims: To characterize the novel p.Thr274Pro variant identified in two unrelated type 1 VWD patients., Methods: Phenotype tests were performed to evaluate patients' plasma and platelets following the current ISTH-SSC guidelines. Molecular analysis was performed using next-generation sequencing. The pcDNA3.1-VWF-WT and mutant pcDNA3.1-VWF-Thr274Pro expression vectors were transiently transfected into HEK293 cells to evaluate recombinant (r)VWF constitutive and regulated secretion. For the latter, the transfected cells were stimulated with phorbol-12-myristate-13-acetate. Immunofluorescence staining was performed to assess the localization of WT-rVWF and Thr274Pro-rVWF in endoplasmic reticulum, lysosomes, cis-/trans-Golgi and pseudo-Weibel Palade bodies., Results: Biochemical characterization of patients' plasma samples indicated a type 1 VWD diagnosis. Both patients were heterozygous for the p.Thr274Pro variant. Hybrid Thr274Pro/WT-rVWF showed a secretion reduction of 36±4% according to patients' plasma VWF:Ag levels, whereas Thr274Pro-rVWF secretion was strongly impaired (21±2%). The amount of rVWF in cell lysates was nearly normal for both Thr274P (62±17%) and Thr274Pro/WT-rVWF (72±23%). The regulated secretion was impaired for Thr274Pro/WT-rVWF, whereas Thr274Pro-rVWF was not released at all. Immunofluorescence staining revealed no particular differences between WT and Thr274Pro-rVWF, although Thr274Pro-rVWF showed less pseudo-Weibel Palade bodies with a rounder shape than WT-rVWF., Conclusions: The novel p.Thr274Pro mutation has a dominant effect and it is responsible of patients' type 1 VWD phenotype through a combined mechanism of reduced synthesis, impaired secretion and multimerization., (© 2022 The Authors. Haemophilia published by John Wiley & Sons Ltd.)

Background: We previously described the association between rare ADAMTS13 single nucleotide variants (SNVs) and deep vein thrombosis (DVT). Moreover, DVT patients with at least one rare ADAMTS13 SNV had a lower ADAMTS13 activity than non-carriers., Aims: To confirm ADAMTS13 variants association with DVT and reduced plasma ADAMTS13 activity levels in a larger population. To investigate the role of VWF and F8 variants., Methods: ADAMTS13, VWF and F8 were sequenced using next-generation sequencing in 594 Italian DVT patients and 571 controls. Genetic association testing was performed using logistic regression and gene-based tests. The association between rare ADAMTS13 variants and the respective plasmatic activity, available for 365 cases and 292 controls, was determined using linear regression. All analyses were age-, sex- adjusted., Results: We identified 48 low-frequency/common and 272 rare variants. Nine low-frequency/common variants had a P<0.05, but a false discovery rate between 0.06 and 0.24. Of them, 7 were found in ADAMTS13 (rs28641026, rs28503257, rs685523, rs3124768, rs3118667, rs739469, rs3124767; all protective) and 2 in VWF (rs1800382 [risk], rs7962217 [protective]). Rare ADAMTS13 variants were significantly associated with DVT using the burden, variable threshold (VT) and UNIQ (P<0.05), but not with C-ALPHA, SKAT and SKAT-O tests. Rare VWF and F8 variants were not associated with DVT. Carriers of rare ADAMTS13 variants had lower ADAMTS13 activity than non-carriers (ß -6.2, 95%CI -11,-1.5). This association was stronger for DVT patients than controls (ß -7.5, 95%CI -13.5,-1.5 vs. ß -2.9, 95%CI -10.4,4.5)., Conclusions: ADAMTS13 and VWF low-frequency/common variants mainly showed a protective effect, although their association with DVT was not confirmed. DVT patients carrying a rare ADAMTS13 variants had slightly reduced ADAMTS13 activity levels, but a higher DVT risk. Rare VWF and FVIII variants were not associated with DVT suggesting that other mechanisms are responsible for the high VWF and FVIII levels measured in DVT patients., Competing Interests: F. P. reports participation at educational meetings and advisory board of Sanofi, Sobi, Takeda and Roche. I. M. received honoraria for participating as a speaker at educational meetings organized by Instrumentation Laboratory and Sanofi-Genzyme. The other authors state that they have no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials

Background: Severe coronavirus disease 2019 (COVID-19) is characterized by an increased risk of thromboembolic events, with evidence of microthrombosis in the lungs of deceased patients., Objectives: To investigate the mechanism of microthrombosis in COVID-19 progression., Patients/methods: We assessed von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin-cofactor (VWF:RCo), VWF multimers, VWF propeptide (VWFpp), and ADAMTS13 activity in a cross-sectional study of 50 patients stratified according to their admission to three different intensity of care units: low (requiring high-flow nasal cannula oxygenation, n = 14), intermediate (requiring continuous positive airway pressure devices, n = 17), and high (requiring mechanical ventilation, n = 19)., Results: Median VWF:Ag, VWF:RCo, and VWFpp levels were markedly elevated in COVID-19 patients and increased with intensity of care, with VWF:Ag being 268, 386, and 476 IU/dL; VWF:RCo 216, 334, and 388 IU/dL; and VWFpp 156, 172, and 192 IU/dL in patients at low, intermediate, and high intensity of care, respectively. Conversely, the high-to-low molecular-weight VWF multimers ratios progressively decreased with increasing intensity of care, as well as median ADAMTS13 activity levels, which ranged from 82 IU/dL for patients at low intensity of care to 62 and 55 IU/dL for those at intermediate and high intensity of care., Conclusions: We found a significant alteration of the VWF-ADAMTS13 axis in COVID-19 patients, with an elevated VWF:Ag to ADAMTS13 activity ratio that was strongly associated with disease severity. Such an imbalance enhances the hypercoagulable state of COVID-19 patients and their risk of microthrombosis., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: Deep vein thrombosis (DVT) is a common multi-factorial disease with a partially understood aetiology. Although the roles of high factor (F)VIII and von Willebrand factor (VWF) levels are recognized, that of ADAMTS13 is still unclear., Aim: To assess the association between ADAMTS13 activity levels, VWF antigen (VWF:Ag) and FVIII coagulant activity (FVIII:C) levels and DVT., Materials and Methods: 365 Italian DVT patients and 292 age- and sex-matched controls were considered. Plasma ADAMTS13 activity was measured using FRETS-VWF73 assay. VWF:Ag and FVIII:C were measured using immunoassay and one-stage clotting assay (ACL TOP analyzer), respectively. Quartile analyses were performed to evaluate the individual association between ADAMTS13 activity, VWF:Ag, FVIII:C and DVT. The combined effect of high VWF levels (> 4th quartile) and low ADAMTS13 levels (< 1st quartile) was evaluated using binary variables. All models were age- and sex-adjusted. Estimated risks were reported as Odds ratio (OR) with 95% confidence intervals (CI)., Results: ADAMTS13 activity was lower in DVT patients (94% vs. 98% of controls). Patients with an ADAMTS13 activity <1st quartile (86%) showed a 1.6-fold increased risk of DVT (95%CI, 1.05-2.55). The combination of low ADAMTS13 activity and high VWF:Ag levels was associated with a 15-fold increased risk (95%CI, 7.80-33.80). VWF:Ag and FVIII:C were associated to DVT with a dose-response relationship., Conclusions: ADAMTS13 activity < 86% was associated with a moderate risk of DVT. The co-presence of low ADAMTS13 activity and high VWF levels resulted in a strong synergistic effect on DVT risk. The association of VWF:Ag and FVIII:C with DVT was confirmed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: von Willebrand disease (VWD) diagnosis starts with first level tests: factor VIII coagulant activity, VWF antigen (VWF:Ag) and platelet-dependent VWF activity (VWF:RCo, VWF:Ab, VWF:GPIbR or VWF:GPIbM). The VWF collagen binding (VWF:CB) assay measures the binding capacity of von Willebrand factor (VWF) to collagen., Aim: To assess, in previously diagnosed VWD patients, the performance of a fully automated chemiluminescent test panel including VWF:Ag, VWF:GPIbR and VWF:CB assays., Methods: The patients, historically evaluated using in-house VWF:Ag and VWF:CB assays and an automated latex enhanced immunoassay VWF:GPIbR method, were re-evaluated using the VWF test panel HemosIL AcuStar., Results: The VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag obtained by means of AcuStar showed an overall good concordance with the corresponding data obtained at the time of the historical diagnosis. When discrepancies occurred, these were generally due to the lower VWF:CB/VWF:Ag obtained with AcuStar as compared with that obtained with the historical methods and this affected particularly the diagnosis of VWD type 2M. Together, the AcuStar VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag were able to distinguish type 1 from types 2A, 2B and 2M, whereas no distinction was possible between type 2A and 2B., Conclusion: The AcuStar panel offers a good performance in the differential diagnosis between VWD type 1 and 2A/2B patients. A high rate of coincidence with historical diagnosis was obtained for VWD types 3, 2A/2B and 1. Even though in some cases more tests (eg, RIPA/multimeric analysis) are needed to complete an accurate VWD classification, the AcuStar panel is considered a sensitive, rapid and reliable tool to diagnose VWD patients., (© 2020 John Wiley & Sons Ltd.)

Essentials ISTH Bleeding Assessment Tool (ISTH-BAT) is used to assist the diagnosis of bleeding disorders. We examined whether the ISTH-BAT is capable of predicting the risk of future bleeding. 136 subjects were administered the ISTH-BAT and followed for up to four years. The ISTH-BAT score failed to predict the risk of future bleeding., Summary: Background The ISTH Bleeding Assessment Tool (ISTH-BAT) is a diagnostic tool used in subjects with suspected inherited bleeding disorders. Aim To evaluate whether the ISTH-BAT, applied at first work-up in a tertiary-care center, predicts the risk of subsequent bleeding events. Methods This was an observational cohort study including all consecutive subjects, of either sex and any age, referred between 2011 and 2015 because of a suspected bleeding disorder. The analysis was restricted to those with an ISTH-BAT score of ≥ 3. Incidence rates (IRs) of major bleeding (MB) and clinically relevant non-major bleeding (CRNMB) events were calculated as the number of events over accrued person-years. The main analysis was performed with Cox regression analysis, assessing an ISTH-BAT score of ≤ 5 versus a score of > 5, as well as the score as a continuous variable, and various covariates (sex, age, and presence/absence of a final diagnosis). Results One hundred and thirty-six subjects had a median ISTH-BAT score of 4 (range 3-18). Eleven subjects (8.1%) had a bleeding event during follow-up (one MB event; 10 CRNMB events). The overall IR of bleeding events per 100 person-years was 3.7 (95% confidence interval [CI] 1.8-6.6). No difference was observed between subjects with an ISTH-BAT score of ≤ 5 and those with a score of > 5 (hazard ratio [HR] 1.2, 95% CI 0.3-4.6). The results were similar when the ISTH-BAT score was considered as a continuous variable (HR 1.1, 95% CI 0.9-1.4). The IR of bleeding was increased in individuals with a diagnosis of a hemostatic defect (IR of 7.5 per 100 person-years; HR 3.0, 95% CI 0.8-11.8). Conclusions The ISTH-BAT does not identify patients at increased risk of future bleeding events., (© 2017 International Society on Thrombosis and Haemostasis.)

Introduction: We characterized five patients affected with von Willebrand disease (VWD) carrying the p.Arg1379Cys mutation. One was diagnosed as VWD type 1 and four as type 2M. The 2M patients also have the variant p.Ala1377Val in cis with p.Arg1379Cys., Aim: To evaluate the role of p.Ala1377Val and p.Arg1379Cys von Willebrand factor (VWF) variants to explain patients' phenotype., Methods: Conventional phenotype tests were used to evaluate patients' plasma and platelets. Direct sequence analysis of exon 28 was carried out. The allele frequency of p.Ala1377Val was evaluated using online database. pcDNA3.1-VWF-WT and mutant (A1377V, R1379C and A1377V-R1379C) expression vectors were transiently transfected in HEK293 cells. The capacity of WT and mutant recombinant (r)VWF (along with patients' plasma VWF) to bind glycoprotein Ibα (GpIbα) were evaluated, using two ELISA assays. One with a wild-type (WT) recombinant (r)GpIbα at increasing ristocetin concentrations (from 0 to 1.50 mg mL -1 ) and the other with a gain-of-function mutant rGpIbα (VWF:GPIbM)., Results: The substitution c.4130C>T (p.Ala1377Val) was reported as rare variant in online databases. At 0.25 mg mL -1 of ristocetin, WT, A1377V and R1379C showed 6, 7.5 and 12-fold increased binding to rGpIbα, respectively. A1377V-R1379C rVWF showed no increased binding to rGpIbα at the same ristocetin concentration and reached the highest binding, of only 3-fold increased, at 1.50 mg mL -1 of ristocetin. The VWF:GPIbM showed strongly reduced values for the A1377V-R1379C rVWF and the 2M patients' plasma., Conclusion: Our study showed that the presence of both p.Ala1377Val and p.Arg1379Cys mutations (synergistic effect) abolishes the binding of rVWF to rGpIbα, explaining patients' 2M phenotype., (© 2016 John Wiley & Sons Ltd.)

Background: Deep vein thrombosis (DVT) genetic predisposition is partially known., Objectives: This study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies., Methods: Wild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity of recombinant ADAMTS13 were measured by ELISA and FRETS-VWF73 assays, respectively. The replication studies were performed in an Italian case-control study (Milan study; 298/298 patients/controls) using a next-generation sequencing approach and in a Dutch case-control study (MEGA study; 4306/4887 patients/controls) by TaqMan assays., Results: In vitro results showed reduced ADAMTS13 activity for three SNVs (p.Val154Ile [15%; 95% confidence interval [CI] 14-16], p.Asp187His [19%; 95%[CI] 17-21], p.Arg421Cys [24%; 95%[CI] 22-26]) similar to reduced plasma ADAMTS13 levels of patients carriers for these SNVs. Therefore these three SNVs were interrogated for risk association. The first replication study identified 3 heterozygous carriers (2 cases, 1 control) of p.Arg421Cys (odds ratio [OR] 2, 95%[CI] 0.18-22.25). The second replication study identified 2 heterozygous carriers (1 case, 1 control) of p.Asp187His ([OR] 1.14, 95%[CI] 0.07-18.15) and 10 heterozygous carriers (4 cases, 6 controls) of p.Arg421Cys ([OR] 0.76, 95%[CI] 0.21-2.68)., Conclusions: Three SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced ex vivo and in vitro ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT., Competing Interests: F. Peyvandi has received honoraria for participating as a speaker at satellite symposia and educational meetings organized by Bayer, Biotest, CSL Behring, Grifols, Novo Nordisk, and Sobi. She is recipient of research grant funding from Alexion, Biotest, Kedrion Biopharma, and Novo Nordisk received by Fondazione Luigi Villa, and she has received consulting fees from Kedrion Biopharma, LFB and Octapharma. She is a member of the Ablynx scientific advisory board. The other authors do not have any conflict of interests to disclose. The above statement does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials.

Congenital thrombotic thrombocytopenic purpura (TTP) is a rare form of thrombotic microangiopathy, inherited with autosomal recessive mode as a dysfunction or severe deficiency of ADAMTS-13 (A Disintegrin And Metalloprotease with ThromboSpondin 1 repeats Nr. 13), caused by mutations in the ADAMTS-13 gene. About 100 mutations of the ADAMTS-13 gene were identified so far, although only a few characterised by in vitro expression studies. A new Asp to Gly homozygous mutation at position 173 of ADAMTS-13 sequence was identified in a family of Romanian origin, with some members affected by clinical signs of TTP. In two male sons, this mutation caused a severe (< 3%) deficiency of ADAMTS-13 activity and antigen level, associated with periodic thrombocytopenia, haemolytic anaemia and mild mental confusion. Both parents, who are cousins, showed the same mutation in heterozygous form. Expression studies of the mutant ADAMTS-13, performed in HEK293 cells, showed a severe decrease of the enzyme's activity and secretion, although the protease was detected inside the cells. Molecular dynamics found that in the D173G mutant the interface area between the metalloprotease domain and the disintegrin-like domain significantly decreases during the simulations, while the proline-rich 20 residues linker region (LR, 285-304) between them undergoes extensive conformational changes. Inter-domain contacts are also significantly less conserved in the mutant compared to the wild-type. Both a decrease of the inter-domain contacts along with a substantial conformational rearrangement of LR interfere with the proper maturation and folding of the mutant ADAMTS-13, thus impairing its secretion.

Background: Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin-induced platelet agglutination (RIPA) test. The major limitation of RIPA is the requirement of fresh blood sample., Objectives: In this study, we evaluated the VWF gain-of-function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH-SSC guidelines., Patients/methods: Seventy-six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays., Results: The mean (minimum-maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84-6.11) than in healthy controls (1.05, 0.87-1.34), type 1 (0.85, 0.51-1.15), 2A (1.20, 0.36-2.82), and 2M (1.07, 0.91-1.38) (P < 0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio., Conclusions: Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients., (© 2015 International Society on Thrombosis and Haemostasis.)

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., (© 2014 International Society on Thrombosis and Haemostasis.)

Background: Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self-association and binding to the platelet receptor glycoprotein (GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα., Objectives: To assess the mechanism responsible for the enhanced interaction of the p.R1306W VWF mutant with the platelet receptor., Methods: The interaction of GPIbα with wild-type (WT) and p.R1306W VWF multimers and A1-A2-A3 constructs was investigated with surface plasmon resonance spectroscopy. Analysis of the static VWF conformation in solution was performed with dynamic light scattering spectroscopy. The shear stress-induced self-association of VWF multimers was investigated with atomic force microscopy (AFM) over a 0-60 dyn cm(-2) range., Results: WT VWF did not interact with GPIbα under static conditions, whereas the mutant at ~ 2 μg mL(-1) already bound to the receptor. By contrast, the WT and p.R1306W-A1-A2-A3 constructs showed comparable affinities for GPIbα (Kd  ~ 20 nm). The hydrodynamic diameter of resting R1306W VWF multimers was significantly greater than that of the wild type (210 ± 60 nm vs. 87 ± 22 nm). At shear forces of < 14 dyn cm(-2) , the p.R1306W multimers rapidly changed conformation, entering a regime of self-aggregation, which, in contrast, was induced for WT VWF by shear forces of > 30 dyn cm(-2) . Mechanical stretching AFM experiments showed that p.R1306W multimers needed less energy per length unit (~ 10 pN) to be stretched than the WT protein., Conclusions: The increased affinity of p.R1306W VWF for GPIbα arises mostly from higher sensitivity to shear stress, which facilitates exposure of GPIbα binding sites., (© 2013 International Society on Thrombosis and Haemostasis.)

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&#95;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., (© 2013 International Society on Thrombosis and Haemostasis.)

Autoimmune thyroid disease (AITD) has been reported in patients with multiple sclerosis (MS) receiving interferon-beta (IFN-β), but not in those receiving Glatiramer acetate (GA). CXCL10 is a chemokine playing a pathogenetic role in AITD and MS. Our aim was to evaluate the effects on CXCL10 secretion of IFN-β and GA, alone and in combination with TNF-α, in primary cultures of thyrocytes (PCT). Significant and dose-dependent secretions of CXCL10 were induced by IFN-β but not GA. TNF-α synergistically increased IFN-β induced CXCL10 secretion. These results may provide an explanation for the occurrence of AITD during IFN-β, but not during GA, treatment for MS., (Copyright © 2011 Elsevier B.V. All rights reserved.)