Acquired von Willebrand Syndrome: A Comprehensive Review and a Nordic Perspective.

Acquired von Willebrand Syndrome in Cardiac Patients

Introduction Bleeding is the most frequent adverse event in patients with continuous flow mechanical circulatory support (CF-MCS) and has been linked to the occurrence of acquired von Willebrand syndrome (aVWS). MCS devices cause an increased shear-induced proteolysis of von Willebrand factor (VWF) by ADAMTS13, leading to aVWS. Hence, specifically blocking ADAMTS13 might be an efficient way to rescue the loss of HMW VWF multimers in CF-MCS patients. Purpose To investigate if blocking ADAMTS13, using an in-house developed inhibitory anti-ADAMTS13 monoclonal antibody (mAb), prevents the loss of high molecular weight (HMW) VWF multimers in in vitro CF-MCS systems and to determine the efficacy of this therapy in a CF-MCS calf model. Methods Human blood was perfused through in vitro CF-MCS systems (Heartmate II and Impella CP, axial flow heart pumps) in the presence of the inhibitory or control mAb (20 μg/mL). Bovine blood was perfused through an in vitro Impella 5.5 system with the inhibitory mAb (20 μg/mL) or PBS. Next, Impella 5.5 pumps were implanted in calves. One dose of the inhibitory mAb (600 μg/kg) or PBS was injected eight days after Impella implantation. Plasma samples were analysed for VWF multimers, VWF antigen (VWF:Ag) and VWF collagen binding activity (VWF:CB). Results A time-dependent decrease in HMW VWF multimers was observed in both in vitro CF-MCS systems in the presence of the control mAb, leading to a 70% reduction of HMW VWF multimers, 180 minutes (min) after blood perfusion (p=0.01 for HM II and p=0.0003 for Impella). This was also reflected by a severely decreased VWF:CB/VWF:Ag ratio (0.59±0.11 and 0.52±0.10 at 180 min versus 1.00±0.06 and 1.07±0.09 before perfusion, for the HM II (p=0.03) and Impella (p=0.001) respectively). Interestingly, blocking ADAMTS13 using the inhibitory mAb prevented the loss of HMW VWF multimers in both systems (p=0.50 for the HM II and p=0.06 for the Impella, 180 min after the start of perfusion). The preservation of HMW VWF multimers was also reflected by normal VWF:CB/VWF:Ag ratios (0.92±0.16 and 0.97±0.11 at 180 min versus 0.93±0.09 and 1.19±0.12 before perfusion for the HM II (p=0.75) and Impella (p=0.06) respectively). Blocking bovine ADAMTS13 using the inhibitory mAb could prevent the loss of HMW VWF multimers in the in vitro Impella 5.5 system, showing that the calf is a good preclinical animal model to study the in vivo effect of this novel therapy. Impella implantation in the calves led to a decrease in HMW VWF multimers (Figure 1A and B). Hence, this animal model represents the VWF laboratory features of MCS-induced aVWS. Moreover, the loss of HMW VWF multimers after pump implantation could be rescued after injection of the inhibitory mAb (Figure 1A and B). Conclusion Blocking ADAMTS13 rescues MCS-induced VWF proteolysis in calves. Hence, inhibiting ADAMTS13 function could become a promising therapeutic strategy to rescue aVWS-induced bleeding in MCS patients. Figure 1. Impella calf model Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Fund for Scientific Research Flanders

In 1926, Erik von Willebrand, a Finnish internist andacademic, evaluated and described a 5-year-old girlwith extreme bleeding and bruising due to what hadbeen designated the A˚landic haemorrhagic diseaseby inhabitants of this archipelago island in the Gulfof Bothnia. Hjordis, the propositus, was the ninth of12 children born to a pedigree in which four femalesiblings had already died before the age of 4 withuncontrolled haemorrhage and in which 23 of 66family members, predominantly females, had expe-rienced significant bleeding and bruising complica-tions [1]. In fact, Hjordis herself eventually died atthe age of 13 during her fourth menstrual cycle.Professor von Willebrand mistakenly concluded thatthis bleeding diathesis was an unusual form ofhaemophilia and decided to label the new diseaseas pseudo-haemophilia to differentiate it from thesex-linked recessive haemophilia A. Little did vonWillebrand realize that this disease, eventually tobecomeeponymous[vonWillebranddisease(VWD)],would become the most commonly diagnosed con-genital bleeding disorder, with a prevalence rangingbetween 1 per 10 000 individuals to 1.3% [2]. Type1 VWD is the most common subtype, representingup to 75–80% of all cases while the subtype 3 VWDoccurs in approximately 1 per million population inthe United States and Europe [2]. Determination ofthe exact prevalence of VWD is hindered somewhatby the heterogeneity of clinical and diagnosticlaboratory features. Even Professor von Willebrandappreciated the challenges of diagnosing VWD as hecollaborated with Professor Rudolf Ju¨gens at theBerlin University to examine patients blood sampleswith a newly invented kapilla¨rtrombometer appa-ratus [3]. Although they were technically mistakenwhen they attributed the bleeding manifestations ofVWD to a platelet defect, their observations wereremarkably prescient since it was not until early1970s that the existence of a specific von Willebrandfactor (VWF) glycoprotein separate from factor VIIIwas finally appreciated and demonstrated to supportplatelet adhesion to the subendothelial matrix ofdamaged blood vessels.Over the last 81 years since von Willebranddescribed the bleeding disorder, there have beennumerous attempts and approaches to refine thediagnosis of the disease so that appropriate andefficacious treatment can be delivered. The clinicalphenotype of VWD includes, in part, information onwhether bleeding is spontaneous or related to surgi-cal or physical trauma; family history and inheri-tance pattern; menstrual history in women; age ofonset of bleeding (to distinguish between acquired vs.inherited coagulation disorders); and sites of bleed-ing(mucocutaneousvs.visceral,intra-articular,intra-muscular, or soft tissue locations). Subsequentlaboratory testing is necessary to exclude or confirmthe diagnosis and to further classify the subtype ofVWD. This article will focus on selected vagariesassociated with the ability to utilize clinical pheno-typic information gathered through the history andphysical examination to predict the presence ofVWD. The clinical diagnosis of VWD must be based,however, on more than physician suspicion andintuition. Thus, the need for confirmatory testing inthe laboratory. The frailties of conventional labora-tory testing for diagnosing VWD and the use of anin vitro surrogate of the bleeding time will also be

Acquired von Willebrand syndromes: clinical features, aetiology, pathophysiology, classification and management

Recessive type 3 von Willebrand disease (VWD) is caused by homozygosity or double heterozygosity for two non-sense mutations (null alleles). Type 3 VWD is easy to diagnose by the combination of a strongly prolonged bleeding time (BT), absence of ristocetine-induced platelet aggregation (RIPA), absence of von Willebrand factor (VWF) protein, and prolonged activated partial thromboplastin time (aPTT) due to factor VIII:coagulant (FVIII:C) deficiency. VWD type 3 is associated with a pronounced tendency to mucocutaneous and musculoskeletal bleedings since early childhood. Carriers of one null allele are usually asymptomatic at VWF levels of 50% of normal. Recessive severe type 1 VWD is caused by homozygosity or double heterozygosity for a missense mutation. Recessive type 1 VWD differs from type 3 VWD by the presence of detectable von Willebrand factor: antigen VWF:Ag and FVIII:C levels between 0.09 and 0.40 U/mL. Patients with recessive type 1 VWD show an abnormal VWF multimeric pattern in plasma and/or platelets consistent with severe type 2 VWD. Carriers of a missense mutation may have mild bleeding and mild VWF deficiency and can be diagnosed by a double VWF peak on cross immunoelectrophoresis (CIE). There will be cases of mild and moderate recessive type 1 VWD due to double heterozygosity of two missense mutations, or with the combination of one missense mutation with a non-sense or bloodgroup O. Mild deficiency of VWF in the range of 0.20 to 0.60 U/mL, with normal ratios of von Willebrand factor: ristocetine cofactor/antigen VWF:RCo/Ag and VWF:collagen binding/antigen (VWF:CB/Ag), normal VWF multimers, and a completely normal response to desmopressin acetate (DDAVP) with VWF level rising from below to above 1.00 U/mL are very likely cases of so-called pseudo-VWF deficiency in individuals with normal VWF protein and gene. Autosomal dominant type 1 VWD variants are in fact type 2 variants caused by a heterozygous missense mutation in the VWF gene that produces a mutant VWF protein that has a dominant effect on normal VWF protein produced by the normal VWF allele with regard to the synthesis, processing, storage, secretion, and/or proteolysis of VWF in endothelial cells. A DDAVP challenge test clearly differentiates between dominant type 1 VWD phenotype and dominant type 2 M VWD.

Acquired Von Willebrand syndrome (AVWS) can contribute to bleeding complications in patients with ventricular assist devices (VADs). AVWS results from shear stress, which causes unfolding of the high-molecular-weight (HMW) multimers of Von Willebrand factor (VWF) with subsequent cleavage. Loss of the HMW multimers of VWF is the leading finding in AVWS. In consequence, binding of VWF to collagen and to platelets is impaired. The onset of AVWS after VAD implantation is not yet determined. We examined VAD patients for presence of an AVWS in the early, intermediate, and late phase after VAD implantation. Patients with a biventricular Thoratec-PVAD(®) (BVAD, n = 6) or a left-ventricular HeartMateII(®) (HMII, n = 11) were analyzed prior to VAD implantation and after 1, 3, 14, 30, and 60 days. Diagnosis of AVWS based on VWF:ristocetin cofactor activity/VWF:VWF antigen (VWF:RCo/VWF:Ag), collagen-binding capacity:VWF antigen (VWF:CB/VWF:Ag), and multimeric analysis. In addition, we analyzed the number of bleeding episodes, which required surgical intervention. No patient had an AVWS prior to VAD implantation. An AVWS was identified already in the very early postoperative period, that is, in almost all patients on the first day and in all patients on the third day. The AVWS was also detected in the majority of patients in the further course. Nine of all 17 patients suffered bleeding complications and required a total of 25 interventions due to hemorrhages. Forty percent of re-interventions were carried out within the first 10 days after implantation; five of these were necessary within the first 24h. The AVWS is present already in the early postoperative phase after VAD implantation. Therefore, reduced shear stress has to be an important feature of newly developed assist devices in the future.

Dear Sir, It has been reported that approximately 30% of the genetic variations that influence von Willebrand Factor (VWF) levels in plasma are due to the ABO blood group of the individuals. The effect of ABO blood type on VWF expression has, therefore, been the subject of many studies over the years. Plasma VWF levels were reported to be significantly lower in group O individuals than in non-O individuals, which correlates with the increased risk of bleeding of the former. Explanations for the reduced levels of VWF in group O individuals range from the effect of ABO blood group on the rate of synthesis/secretion of VWF, to an effect on the survival of the protein and its clearance from plasma. The VWF cleaving protease ADAMTS-13, (the 13th member of the ADAMTS family of metalloproteases characterized by the combination of a disintegrin-like and metalloprotease with thrombospondins type 1 motif), was found to dispose of VWF physiologically by cleaving the peptide bond between tyrosine and methionine in the central A2 domain of VWF. The gene for ADAMTS-13 is located on chromosome 9q approximately 140,000 nucleotides from the ABO locus; this close proximity may also play a role in the 30% genetic variation that ABO exerts on VWF levels1. Maintaining a balance between VWF and ADAMTS-13 is crucial for blood haemostasis. While many pathological conditions are associated with an imbalance between these two proteins, several physiological factors have also been found to play a role in affecting the expression of these proteins. In this study we aimed to determine the effects of age, gender and ABO phenotype on the activity and antigenic levels of ADAMTS-13 in healthy males and females of Arab ethnicity. A hypothesis that the lower levels of VWF in group O individuals are mirrored by higher levels of ADAMTS-13 was also tested in this study. After obtaining consent, venous blood was collected into vacuum collection tubes containing sodium citrate (3.8 %, w/v)-(Becton, Dickinson and Company, New Jersey, USA), from 200 apparently healthy subjects (100 males and 100 females). All subjects were non-smokers and were undergoing a routine check-up at the time of blood collection. Standard, commercially available, enzyme-linked immunosorbent assay (ELISA) kits were used to determine levels of the studied protein according to the manufacturer’s description (Technoclone, Vienna, Austria). In order to measure VWF antigen levels, we used a sandwich ELISA with co-incubation of VWF and a secondary conjugated antibody (anti-VWF-POX) in a single step. The ADAMTS-13 antigen assay involved adding first ADAMTS-13 and then, after a washing step, a conjugate working solution containing anti- ADAMTS-13 POX. For the ADAMTS-13 activity assay, a recombinant VWF fragment was immobilised onto an ELISA plate, which encodes the A2 domain and the ADAMTS-13 cleavage site at Tyr1605-Met1606 and is tagged with S-transferase (GST)-histidine (GST-VWF73-His). After adding plasma, the residual, cleaved VWF fragment is measured by using a second monoclonal antibody [horseradish peroxidase (HRP)-conjugated monoclonal anti-N10] that recognises only the cleaved VWF fragment. The chromogenic substrate tetramethylbenzidine (TMB) was used to detect the reaction in all the assays. Since race was not a factor in this study, as all subjects were of Arab ethnicity, we focused on the effects of age, gender and ABO blood group on VWF and ADAMTS-13 levels. A non-parametric Spearman’s correlation analysis was performed to investigate the effects of age on the investigated proteins. As previously reported2, higher levels of VWF were found with older age (r=0.269, p<0.001). Given the size of the cohort, it is difficult to determine the degree to which the level changes with increasing age. Our analysis also showed that ADAMTS-13 activity decreased with age (r= −0.257, p<0.001), while ADAMTS-13 antigen levels were not affected by increasing age. It is not clear why there is this discrepancy, but the absolute difference between the two proteins appears to be small and is not likely to be of any physiological or clinical relevance (Figure 1). Whether the higher VWF antigen levels in older individuals is a consequence of lower activity of ADAMTS-13 is subject for further analysis. Figure 1 VWF antigen levels increase with age (r = 0.269), while ADAMTS-13 activity levels decrease (r= −0.257) (p 0.05). In order to determine whether gender had an influence on the findings, we compared VWF and ADAMTS-13 levels in males and females, regardless of blood group type. After controlling for age, females had significantly lower levels of VWF (p<0.001) compared to those in males (Table I). We also found that females had higher levels of ADAMTS-13 antigen (p<0.001); the combination of the results for VWF and ADAMTS-13 antigen levels could indicate that females are more prone to bleeding, but further investigation is recommended in a larger cohort to support or refute this hypothesis. Table I Comparison of ADAMTS-13 and vWF levels between males and females and between O blood group and non-O blood group subjects. Results are expressed as median (range). Eighty-one subjects (40.5 %) in our population had the O blood group and their median age was 32 years (range, 18–70 years), while 119 (59.5 %) were non O-blood group and had a median age of 33 years (range, 15–76). There was not a statistical difference in the age between the two group (p>0.05). While subjects with O blood group had significantly lower VWF antigen levels than those with non-O blood groups (p=0.003), there were no differences in ADAMTS-13 antigen and activity levels between the two groups (Table I). ADAMTS-13 levels continued to be not different when individual groups were compared (using the Kruskal-Wallis test), but VWF was significantly different (p=0.001) with levels increasing in the following order: O<A<B<AB (results not shown). After incorporating gender into the ABO blood group analysis, we found that only group O females had significantly lower VWF levels than non-O females [45% (16–275) vs 59% (18–181), p 0.05]. The lack of a difference may be related to ethnicity. It is well documented that ethnicity plays an important role in determining VWF levels3. To our knowledge, no studies on VWF and ABO blood group have previously been conducted in subjects of Arab ethnicity; hence this finding may be unique to our population although a study on a larger population is recommended as the small sample size represent a limitation to the current study. ADAMTS-13 antigen and activity levels were not different between the two groups in either gender (p>0.05). These findings suggest that whatever is causing lower levels of VWF is not related to quantitative changes in ADAMTS-13 antigen and/or activity but may be more related to structural difference in VWF protein in subjects of different blood groups. Although it is still not clear how ABO group can influence the proteolysis of VWF, it has been suggested that in group O individuals, the A2 domain (the site of VWF proteolysis by ADAMTS-13) adopts a conformation more permissive for ADAMTS-13 cleavage. A and B antigens were found to protect against VWF proteolysis, while VWF purified from group O blood has been shown to be cleaved faster by ADAMTS-13 protease2. ABO(H) sugars were also reported to affect the susceptibility of VWF to ADAMTS13 cleavage. O’Donnell et al. reported that a reduction in the number of terminal sugars on N-linked glycan increases the susceptibility of VWF to ADAMTS-13 proteolysis4. A study published in 2010 reported that the degree of sialylation modulated by ABO blood group (rather than ABO group itself) is the reason for altered proteolysis of VWF by ADAMTS-135. Here we have presented the first report on the effect of age, gender and ABO on the expression of VWF and ADAMTS-13 in healthy Arabs. VWF levels increased with age, while ADAMTS-13 activity decreased; however, despite being statistically significant, the correlation between age and these two proteins was weak. We confirmed that the levels of VWF antigen are lower in individuals with O blood group, but only in female subjects, who also had higher ADAMTS-13 antigen levels. ADAMTS-13 antigen and activity levels were not affected by ABO blood group. A more detailed analysis in a larger group of subjects is recommended.

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Are intravenous immunoglobulins really inappropriate in acquired von Willebrand syndrome?

Von Willebrand factor propeptide (VWFpp) is a fragment of a new synthesized VWF molecule that plays an important part in the biosynthesis of this protein. After completion of multimerisation of von Willebrand factor (VWF) dimers in the Golgi apparatus, as result of furin proteolysis VWFpp is truncated/disconnected from the mature VWF molecule to form a non-covalent complex with VWF. The VWFpp-VWF complex is stored in endothelial and platelet storage granules and released into the bloodstream where it dissociates into VWFpp and VWF. Plasma VWFpp level and ratio of plasma VWF propeptide (VWFpp) to VWF antigen (VWF:Ag) i.e VWFpp /VWF:Ag ratio are important biomarkers of VWF synthesis/release/clearance. These biomarkers have distinct therapeutic utility as they help to identify patients with von Willebrand disease (VWD) in whom DDAVP (desmopressin 1-desamino-8-D-arginine vasopressin) is ineffective due to rapid VWF clearance. They also have significant diagnostic value because they help to differentiate between congenital VWD subtypes as well as between congenital VWD and acquired von Willebrand Syndrome (AVWS). The purpose of the study was to determine the VWFpp level and the VWFpp/VWF: Ag ratio in patients with VWD and AVWS and to assess the significance of these biomarkers for diagnosis and management of congenital and acquired von Willebrand disease. Our study involved 120 VWD patients; 21 with AVWS and 111 healthy controls. Study results confirm that VWFpp level and the VWFpp/VWF: Ag ratio have significant value fordiscrimination between severe type 1 VWD and type 3 VWD as well as between acquired and congenital VWD which has serious implications for therapy. The biomarkers are also useful for identification of patients in whom DDAVP treatment may prove ineffective. In type 1 VWD (< 30 IU/ml) the VWFpp/VWF: Ag ratio was elevated in 57% of patients while in the group of patients with threshold VWF values (‘Low VWF’, 30–50 IU/dL) the ratio was within normal, which may be suggestive of other underlying causes of VWF deficiency. Assays in patients with non-neutralizing anti-VWF antibodies (AVWS) have shown that the VWFpp/VWF: Ag ratio can have significant impact on monitoring therapy and assessment of remission in patients with anti VWF antibodies.

Background: Hemodynamic alterations in Fontan patients (FP) are associated with hemostatic dysbalance and Fontan-associated liver disease. Studies of other hepatopathologies indicate an interplay between cholestasis, tissue factor (TF), and von Willebrand factor (VWF). Hence, we hypothesized a relationship between the accumulation of bile acids (BA) and these hemostatic factors in FP. Methods: We included 34 FP (Phenprocoumon n = 15, acetylsalicylic acid (ASA) n = 16). BA were assessed by mass spectrometry. TF activity and VWF antigen (VWF:Ag) were determined by chromogenic assays. VWF collagen-binding activity (VWF:CB) was assessed via ELISA. Results: Cholestasis was observed in 6/34 FP (total BA ≥ 10 µM). BA levels and TF activity did not correlate (p = 0.724). Cholestatic FP had lower platelet counts (p = 0.013) from which 5/6 FP were not treated with ASA. VWF:Ag levels were increased in 9/34 FP and significantly lower in FP receiving ASA (p = 0.044). Acquired von Willebrand syndrome (AVWS) was observed in 10/34-FP, with a higher incidence in cholestatic FP (4/6) (p = 0.048). Conclusions: Cholestasis is unexpectedly infrequent in FP and seems to be less frequent under ASA therapy. Therefore, ASA may reduce the risk of advanced liver fibrosis. FP should be screened for AVWS to avoid bleeding events, especially in cholestatic states.

Aortic-valve stenosis can be complicated by bleeding that is associated with acquired type 2A von Willebrand syndrome. However, the prevalence and cause of the hemostatic abnormality in aortic stenosis are unknown. We enrolled 50 consecutive patients with aortic stenosis, who completed a standardized screening questionnaire to detect a history of bleeding. Forty-two patients with severe aortic stenosis underwent valve replacement. Platelet function under conditions of high shear stress, von Willebrand factor collagen-binding activity and antigen levels, and the multimeric structure of von Willebrand factor were assessed at base line and one day, seven days, and six months postoperatively. Skin or mucosal bleeding occurred in 21 percent of the patients with severe aortic stenosis. Platelet-function abnormalities under conditions of high shear stress, decreased von Willebrand factor collagen-binding activity and the loss of the largest multimers, or a combination of these was present in 67 to 92 percent of patients with severe aortic stenosis and correlated significantly with the severity of valve stenosis. Primary hemostatic abnormalities were completely corrected on the first day after surgery but tended to recur at six months, especially when there was a mismatch between patient and prosthesis (with an effective orifice area of less than 0.8 cm2 per square meter of body-surface area). Type 2A von Willebrand syndrome is common in patients with severe aortic stenosis. Von Willebrand factor abnormalities are directly related to the severity of aortic stenosis and are improved by valve replacement in the absence of mismatch between patient and prosthesis.

The bleeding tendency in patients with low von Willebrand factor and type 1 phenotype is greater in the presence of impaired collagen‐induced platelet aggregation

Acquired Von Willebrand Syndrome In Mitral Valve Leak

Lenalidomide as a novel treatment for refractory acquired von Willebrand syndrome associated with monoclonal gammopathy