[Generalized seizure during placement of an axillary plexus block]. / Generalisierter Krampfanfall während der Anlage einer axillären Plexusblockade.

A young patient experienced a generalized seizure during the placement of an axillary plexus block. The mechanisms, essentially the presumed intravascular administration, which led to the local anesthetic toxicity as the cause of this event, are discussed. This case is an example of how visualization of the anatomy by ultrasound can give a false impression when certain details are not respected. It is assumed that the main mechanism in this case was venous compression by the ultrasound transducer.

Ex vivo human placental transfer study on recombinant Von Willebrand factor (rVWF).

Deficiency or mutation of von Willebrand factor (VWF) leads to a coagulation disorder (von Willebrand disease; VWD) which requires a lifelong therapy. For avoiding maternal complications treatment may be necessary also in pregnancy, but placental transfer to the fetus might impact its coagulation system and evoke undesired side effects. As VWF is a very large molecule it may be assumed that it does not pass the placental barrier. To prove this hypothesis the materno-fetal transfer of recombinant VWF (rVWF) has been analyzed ex vivo in a total of 21 valid dual side placenta perfusions. Three groups of five placentas each have been perfused with physiological and up to ten-fold increased concentrations of rVWF for 2 h. Six placentas have been used for control perfusions. A series of different control parameters has been assessed for documentation of intactness and functionality of the placenta and the perfusion system. In not a single analysis, independent of time and concentration, rVWF was detected in the fetal circuit. In the maternal circuit VWF concentration decreased slightly during perfusion. These results demonstrate that recombinant VWF does not pass the human placenta.

An international collaborative study to compare different von Willebrand factor glycoprotein Ib binding activity assays: the COMPASS-VWF study.

Essentials New VWF activity assays are increasingly used but information on their comparability is limited. This is an ISTH SSC-organized study (expert labs, 5 countries) to compare all available assays. VWF activity by six assays correlated well with each other. The new assays show improved characteristics - minor differences are noted. SUMMARY: Background Several new assays have become available to measure von Willebrand factor (VWF) activity. The new assays appear to have improved performance characteristics compared with the old reference standard, ristocetin cofactor activity (VWF:RCo), but information is limited about how they compare with VWF:RCo and each other. Methods The von Willebrand factor Subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) designed a collaborative study involving expert laboratories from several countries to compare available tests with each other and with VWF:RCo. Eight laboratories from five countries were provided with blinded samples from normal healthy individuals and well-characterized clinical cases. Laboratories measured VWF activity using all tests available to them; data from six laboratories, not affected by thawing during transportation, are included in this study. Results All tests correlated well with VWF:RCo activity (r-values ranged from 0.963 to 0.989). Slightly steeper regression lines for VWF:Ab and VWF:GPIbM were clinically insignificant. The new assays showed improved performance characteristics. Of the commercially available assays, the VWF:GPIbR using the AcuStar system was the most sensitive and could reliably detect VWF activity below 1 IU dL-1 . The lower limit of the measuring interval for the VWF:GPIbM and the VWF:GPIbR assays was in the 3-4 and 3-6 IU dL-1 range, respectively. Inter-laboratory variation was also improved for most new assays. Conclusion All VWF activity assays correlated well with each other and the VWF:RCo assay. The slight differences in characteristics found in the COMPASS-VWF study will assist the VWF community in interpreting and comparing activity results.

Acquired von Willebrand syndrome in congenital heart disease surgery: results from an observational case-series.

Essentials Bleeding complications during congenital heart disease surgery in neonatal age are very common. We report the perioperative incidence of acquired von Willebrand syndrome (aVWS) in 12 infants. aVWS was detected in 8 out of 12 neonates and infants intraoperatively after cardiopulmonary bypass. Ten patients received von Willebrand factor concentrate intraoperatively and tolerated it well. SUMMARY: Background Cardiac surgery of the newborn and infant with complex congenital heart disease (CHD) is associated with a high rate of intraoperative bleeding complications. CHD-related anatomic features such as valve stenoses or patent arterial ducts can lead to enhanced shear stress in the blood stream and thus cause acquired von Willebrand syndrome (aVWS). Objective To evaluate the intraoperative incidence and impact of aVWS after cardiopulmonary bypass (CPB) in neonates and infants with complex CHD. Patients/Methods We conducted a survey of patients aged < 12 months undergoing complex cardiac surgery in our tertiary referral center. Twelve patients, whose blood samples were analyzed for aVWS before CPB and immediately after discontinuation of CPB on a routine basis, were eligible for the analysis. von Willebrand factor antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), collagen binding activity (VWF:CB), VWF:multimers and factor VIII activity (FVIII:C) were determined. Results aVWS was diagnosed by VWF multimer analysis in 10 out of 12 patients (83%) prior to surgery and intraoperatively at the end of CPB in 8 out of 12 patients (66%). Ten patients received VWF/FVIII concentrate intraoperatively as individual treatment attempts during uncontrolled bleeding. They tolerated it well without intraoperative thrombotic events. One patient suffered a transient postoperative cerebral sinuous vein thrombosis. Conclusions aVWS is of underestimated incidence in complex CHD surgery. These data may offer a new approach to reduce the risk of severe bleedings and to achieve hemostasis during high-risk pediatric cardiac surgery by tailoring the substitution with von Willebrand factor concentrate.

Identification and characterization of the elusive mutation causing the historical von Willebrand Disease type IIC Miami.

UNLABELLED: Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation. SUMMARY: Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo.

Clopidogrel Resistance in Neurovascular Stenting: Correlations between Light Transmission Aggregometry, VerifyNow, and the Multiplate.

BACKGROUND AND PURPOSE: Clopidogrel resistance is blamed for thromboembolic complications in neurovascular stent placement. Platelet-function assays are weakly standardized. The aim of this study was to correlate the results of 3 different platelet-inhibition measurements (from light transmission aggregometry, the VerifyNow P2Y12 test, and the Multiplate analyzer) and their relation to periprocedural thromboembolic complications in elective neurovascular stent placement. MATERIALS AND METHODS: Clopidogrel resistance was determined on the day of the intervention according to predefined platelet reactivity cutoff values. All 3 tests were performed in 103 consecutive neurovascular stent-placement procedures in 97 patients (extracranial, n = 77; intracranial, n = 26). RESULTS: The clopidogrel resistance rates were 47.6% (light transmission aggregometry), 50.5% (VerifyNow), and 35.9% (Multiplate). In 67% of the patients, clopidogrel resistance was present according to at least one method. The correlations of qualitative results that classified a patient as responsive or resistant to clopidogrel were 67.9% for light transmission aggregometry with VerifyNow, 77.7% for light transmission aggregometry with the Multiplate, and 66% for VerifyNow with the Multiplate. Periprocedural thromboembolic complications (n = 9) occurred more frequently in patients who were determined by all 3 methods to be clopidogrel resistant. The difference was most pronounced with light transmission aggregometry (complication rates, 14.4% [clopidogrel-resistant patients] vs 3.7% [clopidogrel-responsive patients]). Sensitivity and specificity rates of clopidogrel resistance in relation to embolic complications were, respectively, 78% and 55% for light transmission aggregometry, 67% and 51% for VerifyNow, and 44% and 67% for the Multiplate. CONCLUSIONS: Clopidogrel resistance is a frequent finding in patients who undergo neurovascular stent placement. The correlations among the different testing methods are only modest and differ considerably. Light transmission aggregometry results seem to correlate with thromboembolic complications more accurately than with VerifyNow and Multiplate point-of-care methods.

Interactions of von Willebrand factor and ADAMTS13 in von Willebrand disease and thrombotic thrombocytopenic purpura.

The function of von Willebrand factor (VWF), a huge multimeric protein and a key factor in platelet dependent primary haemostasis, is regulated by its specific protease ADAMTS13. The ADAMTS13 dependent degradation of VWF to its proteolytic fragments can be visualized as a characteristic so-called triplet structure of individual VWF oligomers by multimer analysis. Lack of VWF high molecular weight multimers (VWF-HMWM) or their pathologically enhanced degradation underlies a particular type of von Willebrand disease, VWD type 2A with a significant bleeding tendency, and may also be observed in acquired von Willebrand syndrome due to cardiovascular disease. In these conditions multimer analysis is an obligatory and powerful tool for diagnosis of VWD. The opposite condition, the persistence of ultralarge VWF (UL-VWF) multimers may cause the microangiopathic life-threatening disorder thrombotic thrombocytopenic purpura (TTP). During the course of active TTP, UL-VWF is consumed in the hyaline thrombi formed in the microvasculature which will ultimately result in the loss of UL-VWF and VWF-HMWM. Therefore, VWF multimer analysis is not a valid tool to diagnose TTP in the active phase of disease but may be helpful for the diagnosis of TTP patients in remission.

Germline de novo mutations and linkage markers vs. DNA sequencing for carrier detection in von Willebrand disease.

Linkage analysis in autosomal inherited von Willebrand disease (VWD) is important to diagnose the carriers and reduce the burden of severe type VWD. The study was designed to identify the carriers and estimate the frequency of variable number of tandem repeats (VNTR) instability in VWD families. Carrier detection was performed in eight recessive type 3 VWD (VWD3) families using VNTRs VWF1 and VWF2, RsaI (789Thr/Ala) linkage markers, multimer analysis and DNA sequencing. Moreover, five dominant VWD families were studied through DNA sequencing and multimer analysis. Frequency of VWF VNTR instability was investigated in 20 VWD families. In VWD3 families, a total of 22 (81.5%) carriers were identified using VWF1 and VWF2 markers. However, only 13(48.1%) carriers were identified through RsaI markers. Mutation screening revealed 22(81.5%) carriers in VWD3 and 4 (33.3%) carriers in VWD2 families. In comparison to DNA sequencing, the accuracy of VWF1 and VWF2 markers in VWD3 was 85.7% while RsaI could identify 68.2% carriers accurately. Mutations p.R1205H and p.C1272R were identified as de novo in families. Multimer analysis confirmed the identified carriers in VWD2 families. Three VWD families were found to be carrying VNTR instability for VWF1 and VWF2 locus. VNTRs could be an effective linkage markers for carrier detection in VWD3 families. However, in the event of germline de novo mutations and VNTR instability, it may confound risk of misdiagnosis of carriers. Multimer analysis could be an alternative way of carrier detection in dominant type 2A and type 2B VWD families.

von Willebrand disease type 2A phenotypes IIC, IID and IIE: A day in the life of shear-stressed mutant von Willebrand factor.

The bleeding disorder von Willebrand disease (VWD) is caused by mutations of von Willebrand factor (VWF), a multimeric glycoprotein essential for platelet-dependent primary haemostasis. VWD type 2A-associated mutations each disrupt VWF biosynthesis and function at different stages, depending on the VWF domain altered by the mutation. These effects cause considerable heterogeneity in phenotypes and symptoms. To characterise the molecular mechanisms underlying the specific VWF deficiencies in VWD 2A/IIC, IID and IIE, we investigated VWF variants with patient-derived mutations either in the VWF pro-peptide or in domains D3 or CK. Additionally to static assays and molecular dynamics (MD) simulations we used microfluidic approaches to perform a detailed investigation of the shear-dependent function of VWD 2A mutants. For each group, we found distinct characteristics in their intracellular localisation visualising specific defects in biosynthesis which are correlated to respective multimer patterns. Using microfluidic assays we further determined shear flow-dependent characteristics in polymer-platelet-aggregate formation, platelet binding and string formation for all mutants. The phenotypes observed under flow conditions were not related to the mutated VWF domain. By MD simulations we further investigated how VWD 2A/IID mutations might alter the ability of VWF to form carboxy-terminal dimers. In conclusion, our study offers a comprehensive picture of shear-dependent and shear-independent dysfunction of VWD type 2A mutants. Furthermore, our microfluidic assay might open new possibilities for diagnosis of new VWD phenotypes and treatment choice for VWD patients with shear-dependent VWF dysfunctions that are currently not detectable by static tests.

Deep intronic 'mutations' cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA.

BACKGROUND: In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and non-genetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. METHODS: We determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LR-PCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. RESULTS: All VWF tests were normal. The LR-PCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. CONCLUSION: By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

Pregnancy in Upshaw-Schulman syndrome.

The Upshaw Schulman syndrome (MIM #274150) is a hereditary deficiency of the von Willebrand factor cleaving protease (ADAMTS13) due to homozygous or compound heterozygous mutations in the ADAMTS13 gene. Patients are prone to bouts of thrombotic thrombocytopenic purpura. However, disease manifestation needs a second trigger event. Pregnancy is a known risk factor for TTP. Patients with USS may manifest during pregnancy and the postpartum period or relapse with a TTP bout. Before plasma therapy mortality for both the mother and the fetus was high, but even nowadays when plasma is delivered, therapy is challenging, still bearing a high risk for miscarriage or long term sequelae for the mother. In this report on pregnancies in three mothers with USS, plasma therapy was increased in frequency and amount given with regard to platelet count or ADAMTS13 activity, thus leading to a successful outcome.

Third Åland islands conference on von Willebrand disease, 26-28 September 2012: meeting report.

The first meeting of international specialists in the field of von Willebrand disease (VWD) was held in the Åland islands in 1998 where Erik von Willebrand had first observed a bleeding disorder in some members of a family from Föglö and a summary of the meeting was published in 1999. The second meeting was held in 2010 and a report of the meeting was published in 2012. Topics covered included progress in understanding of VWD over the last 50 years; multimers; classification of VWD; pharmacokinetics and laboratory assays; genetics; treating the paediatric patient; prophylaxis; geriatrics; gene therapy and treatment guidelines. This third meeting held over 3 days covered the structure and function of von Willebrand factor (VWF); type 1 VWD, the most common form of the disease; a lifespan of pharmacokinetics in VWD; detecting inhibitors in VWD patients; and special challenges in understanding and treating the female VWD patient.

Increased amounts of von Willebrand factor are bound to microparticles after infusion of desmopressin.

Effects of desmopressin (DDAVP) in platelet disorders and primary haemostasis cannot be attributed solely to the increase in FVIII/VWF (von Willebrand factor), as VWF/FVIII concentrates have no effect in these circumstances. Microparticles (MP) can support haemostasis by expression of phospholipids, tissue factor and VWF on their surface. We hypothesized that significant amounts of VWF are bound to MP after DDAVP administration and that consequently depletion of MP should influence VWF:Ag and VWF:RCo plasma levels. Platelet-poor plasma was either obtained well from healthy controls or before and after DDAVP administration from patients with von Willebrand's disease (type 1 or possible type 1) or patients with other bleeding disorders as controls. Concentrations of MP and VWF parameters were determined before and after MP depletion by different methods (magnetic bead selection, plasma microfiltration, ultracentrifugation). Platelet MP and VWF-bearing MP were significantly increased after DDAVP. MP depletion by magnetic bead selection led to a significant reduction in VWF:Ag (-18.0%) and VWF:RCo (-27.7%) plasma levels without changes in VWF multimer composition. As results were similar for DDAVP control subjects, the amount of VWF bound to circulating microparticles was significantly higher after DDAVP administration compared with healthy controls (reduction -11.7%). DDAVP leads to a release of microparticles and increases the amount of VWF bound to microparticles which might explain the clinical efficacy of DDAVP in platelet disorders.

von Willebrand's disease: a report from a meeting in the Åland islands.

von Willebrand's disease (VWD) is probably the most common bleeding disorder, with some studies indicating that up to 1% of the population may have the condition. Over recent years interest in VWD has fallen compared to that of haemophilia, partly the result of focus on blood-borne diseases such as HIV and hepatitis. Now the time has come to revisit VWD, and in view of this some 60 international physicians with clinical and scientific interest in VWD met over 4 days in 2010 in the Åland islands to discuss state-of-the-art issues in the disease. The Åland islands are where Erik von Willebrand had first observed a bleeding disorder in a number of members of a family from Föglö, and 2010 was also the 140th anniversary of his birth. This report summarizes the main papers presented at the symposium; topics ranged from genetics and biochemistry through to classification of VWD, pharmacokinetics and laboratory assays used in the diagnosis of the disease, inhibitors, treatment guidelines in different age groups including the elderly who often have comorbid conditions that present challenges, and prophylaxis. Other topics included managing surgeries in patients with VWD and the role of FVIII in VWF replacement, a controversial subject.

Prospective evaluation of a pediatric bleeding questionnaire and the ISTH bleeding assessment tool in children and parents in routine clinical practice.

BACKGROUND: Diagnosing mild bleeding disorders (BDs) in children is difficult. Bleeding scores (BSs) have been proposed for obtaining standardized quantitative histories. OBJECTIVES: To compare the Canadian pediatric bleeding questionnaire (PBQ) with the new ISTH bleeding assessment tool (ISTH BAT) for the determination of BS in a routine pediatric outpatient setting. METHODS: One hundred children with a suspected BD were enrolled in this cross-sectional study. Bleeding scores were calculated for all children and their natural parents. For all children, extensive laboratory investigations were performed. RESULTS: Based on laboratory tests, 56 children were diagnosed as having no BD, 11 were diagnosed with possible VWD, 12 with VWD 1, 11 with VWD 2, five with possible platelet defects, and five with mild factor deficiencies. Both questionnaires were able to discriminate between no BD and VWD (P = 0.0001), but the area under the receiver characteristics curve to detect any mild BD was only 0.76. Despite the inherited nature of the BD, a family score did not increase the ability to discriminate between no BD and VWD (P = 0.2052). There was no significant difference between the two tools used (P = 0.3253) or simple qualitative criteria, such as yes/no questions regarding bleeding (P = 0.3477). CONCLUSIONS: The two tools translated into German did not differ substantially. Both were able to discriminate between no BD and a possible BD with acceptable accuracy. A BS of < 2 makes a BD unlikely. Simple qualitative criteria were similar; however, to allow comparison of studies and follow-up in patients over time, we recommend the ISTH BAT.

Reduced von Willebrand factor secretion is associated with loss of Weibel-Palade body formation.

BACKGROUND: von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. OBJECTIVES: We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel-Palade body (WPB)-like granules. METHODS: Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild-type VWF. Expression was also examined in HEK293 cells that form WPB-like granules when transfected with wild-type VWF. RESULTS: Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB-like granules, while R2663C was capable of WPB-like granules. Heterozygous expression of VWF variants had a negative impact on wild-type VWF with a reduction in elongated WPB-like granules in co-transfected cells. CONCLUSIONS: Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB-like granules.

[Von Willebrand factor and ADAMTS13 balancing primary haemostasis]. / Regulation der primären Hämostase durch von-Willebrand-Faktor und ADAMTS13.

Von Willebrand factor (VWF) is an adhesive, multi-functional huge multimerized protein with multiple domains harboring binding sites for collagen, platelet glycoprotein receptors and coagulation factor VIII (FVIII). The functional domains enable VWF to bind to the injured vessel wall, to recruit platelets to the site of injury by adhesion and aggregation and to bind and protect FVIII, an important cofactor of the coagulation cascade. VWF function in primary haemostasis is located in particular in the arterial and micro-circulation. This environment is exposed to high shear forces with hydrodynamic shear rates ranging over several orders of magnitude from 10⁻¹ to 105 s-1 and requires particular mechanisms to enable platelet adhesion and aggregation under these variable conditions. The respective VWF function is strictly correlating with its multimer size. Lack or reduction of large VWF multimers is seen in patients with von Willebrand disease (VWD) type 2A which correlates with reduction of both VWF:platelet GPIb-binding and VWF:collagen binding and a bleeding phenotype. To prevent unlimited platelet adhesion and aggregation which is the cause of the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), VWF function is regulated by its specific protease ADAMTS13. Whereas a particular susceptibility of VWF to ADAMTS13 proteolysis is the cause of a frequent VWD type 2A phenotype, lack or dysfunction of ADAMTS13, either acquired by ADAMTS13 antibodies or by inherited ADAMTS13 deficiency (Upshaw-Schulman Syndrome), causes TTP. Therefore VWD and TTP represent the opposite manifestations of VWF related disorders, tightly linked to each other.