Type 2M VWD Research Articles

Management of von Willebrand Disease for Orthognathic Surgery: Evidenced-Based Recommendations and Scoping Review of the Literature

BackgroundThe general management of von Willebrand Disease has evolved over the past several decades. Advances in blood product replacement manufacturing have led to changes in perioperative management of the bleeding diathesis in its various subtypes. Surgical management follows changes in medical management, but unique perioperative considerations exist depending on the type of surgery. Orthognathic surgery is considered a major surgery that can have life-threatening complications in patients with von Willebrand Disease (vWD) and updates in evidenced-based management are presented. ObjectivesThe objectives of this report are to review available literature regarding management of patients with von Willebrand Disease and other bleeding disorder undergoing orthognathic surgery and to provide a case report regarding the management of a patient vWD type 2M undergoing orthognathic surgery. Eligibility CriteriaArticles included for review were studies addressing patients with bleeding disorders undergoing orthognathic surgery. Sources of EvidencePubMed and Ovid were queried using the terms “orthognathic surgery OR jaw deformity” AND “von Willebrand OR bleeding disorders OR hemophilia.” Additional references relevant to the review were obtained from the reference lists of available articles ultimately deemed appropriate for inclusion in the review. MethodsA recent example case of a patient with type 2M vWD undergoing orthognathic surgery and the patient’s perioperative management is presented for consideration. A scoping review of the literature was undertaken for evaluation of available literature using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Review (PRISMA-ScR). ResultsInsufficient evidence exists for significant analysis of perioperative management strategies previously reported compared to the currently utilized management strategy for the case presented. A review of the available literature with reference to updated literature regarding management of patients with vWD undergoing surgical procedures other than orthognathic surgery yields primarily case reports, two randomized controlled trials, and one systematic review. The results indicate that an evolution in the general management of patients with vWD undergoing surgery can be applied to patients undergoing orthognathic surgery and that the addition of hemostatic agents such as tranexamic acid may be considered with a low level of evidence. ConclusionsFor patients undergoing orthognathic surgery, the use of cryoprecipitate in large quantities has been replaced with the use of Humate-P infusion and will likely be replaced with the use of recombinant von Willebrand factor (VWF). The timing of infusion of adjuncts preoperatively for patients with vWD can potentially be extended to the previous day, and the addition of medications to aid in hemostasis such as tranexamic acid may be considered. Prophylactic use of additional VWF adjuncts should be considered in the postoperative setting, and close monitoring is prudent. The use of newer agents may allow for the expansion of craniomaxillofacial surgery procedures that can be safely considered in patients with vWD.

A case report of type 2M Von Willebrand disease with unique mutations

Abstract Von Willebrand disease (vWD) is the most common inherited bleeding disorder. It results from quantitative or qualitative defects of the von Willebrand factor (vWF) protein, and is divided into three types, namely type 1, type 2(2A, 2B, 2M, and 2N), and type 3 vWD. Here we report a case of 55-year-old female who presented with recurrent gastrointestinal bleeding due to angiodysplasia. Her past medical history is significant for vWD (type unknown) with hemoptysis, epistaxis and menorrhagia requiring Humate-P replacement multiple times. She has one sister with mild vWD of unknown type and 4 other siblings with no bleeding history. Coagulation testing shows normal PT and aPTT. Von Willebrand panel shows vWF antigen of 33%, VWF ristocetin cofactor activity of 15%, and Factor VIII of 87%. The ratio (VWF: RCo/VWF: Ag) is low (0.45) and the multimer pattern is normal. This is consistent with type 2M vWD. VWD gene sequencing shows heterozygous variants of c.4241T>G (p.Val1414Gly) and c.5227G>A (p.Val1743Met). Of note, one variant (c.4241T>G/p.Val1414Gly) has been reported in two individuals with type 2A but not in type 2M vWD. This may truly represent different types of vWD caused by same mutation or could be due to variation in testing and/or result interpretation among laboratories. The other variant c.5227G>A(p.Val1743Met) in A3 domain has never been reported with uncertain significance. Further investigation including vWF panel testing and genetic analysis in family members would help characterize those mutations.

Thermodynamic stabilization of von Willebrand factor A1 domain induces protein loss of function.

The von Willebrand disease (vWD) is the most common hereditary bleeding disorder caused by defects of the von Willebrand Factor (vWF), a large extracellular protein in charge of adhering platelets to sites of vascular lesions. vWF performs this essential homeostatic task via specific protein-protein interactions between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIBα). The two naturally occurring vWF A1 domain mutations G1324A and G1324S, near the GPIBα binding site, induce a dramatic decrease in platelet adhesion, resulting in a bleeding disorder classified as type 2M vWD. However, the reason for the drastic phenotypic response induced by these two supposedly minor modifications remains unclear. We addressed this question using a combination of equilibrium-molecular dynamics (MD) and nonequilibrium MD-based free energy simulations. Our data confirms that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. G1324A and G1324S mutations hardly changed the per-residue flexibility of the A1 domain but induced a global conformational change affecting the region near the binding site to GPIBα. Furthermore, we observed two significant changes in the vWF A1 domain upon mutation, the global redistribution of the internal mechanical stress and the increased thermodynamic stability of the A1 domain. These observations are consistent with previously reported mutations increasing the melting temperature. Overall, our results support the idea of thermodynamic conformational restriction of A1-before the binding to GPIBα-as a crucial factor determining the loss-of-function of the G1324A(S) vWD mutants.

The elusive and heterogeneous pattern of type 2M von Willebrand disease: A diagnostic challenge.

To investigate type 2M heterogeneity and to establish the most appropriate diagnostic flowchart. Hemostatic and genetic VWF analyses were performed in 14 type 2M VWD patients carrying the p.G1324S, p.R1374H, p.R1374C, p.A1344_A1350del, or p.F1293L mutations. PFA-100 was always significantly prolonged, and ristocetin-induced platelet aggregation (RIPA) and VWF ristocetin cofactor (VWF:RCo) greatly reduced or absent. Plasma VWF antigen (VWF:Ag) was reduced except in the p.G1324S patient, while platelet VWF:Ag was normal or near normal except in the p.R1374C patients. The ratio of collagen binding (VWF:CB) to VWF:Ag was normal or near normal except in patients carrying the p.R1374H and p.A1344_A1350del mutations, whose large VWF multimers were slightly reduced. Multimer patterns were normal or lacking in large oligomers, or with larger than normal VWF components. Only PFA100, RIPA and VWF:RCo were always abnormal. We thus propose a minimal diagnostic test battery: RIPA (demonstrating the defective VWF-platelet interaction), VWF:Ag (exploring VWF synthesis), and VWF:CB and its ratio (to explore multimer patterns). Other tests would only serve for confirmation, if necessary.

A Short N-Terminal Fragment in Front of the A1 Domain of Von Willebrand Factor Prevents the A1 from Binding the Platelet By Masking a Region Clustered By Type 2B VWD Mutations

Blood glycoprotein von Willebrand Factor (VWF) is constantly circulating in the blood vessel, with its platelet GPIb-IX recognizing unit-the A1 domain-being masked to avoid its adventitious binding and activating platelets. This "self-mask" mechanism, however, remains poorly understood. The impediment in identifying the self-mask ligand is to have the right pair of VWF fragments with opposite binding behavior for comparison. Here we characterize using hydrogen-deuterium exchange mass spec a pair of VWF fragments containing the A1 domain with opposite association to platelets. The results suggest that the short N-terminal region (1238-1260) in front of the A1 domain might involve in protecting the VWF A1 domain by masking a critical region near the binding site for GPIb.We expressed and purified using baby hamster kidney cells two C-terminally His-tagged proteins: both contain the entire A1 domain (1273-1453), with one has more extra N-terminal residues (1238-1472, named lA1) than the other (1261-1472, named sA1). Both proteins seem to be glycosylated and stable without obvious deformation for over one week when stored at 4°C. The analytical ultracentrifugation analysis determined that both proteins are primarily in the monomeric form: the sedimentation coefficient (S) and the derived apparent molecular weight for lA1 are 2.50 S and 36.6 kDa, and those for sA1 are 1.37 S and 27.4 kDa. Interaction of lA1 and sA1 to GPIb, as determined by flow cytometry, is surprisingly opposite: only sA1, but not lA1, efficiently binds washed platelets at the concentration similar to the physiological concentration of vWF in the blood vessel. Increasing lA1 concentration 10 times did not improve its binding. Replacing the platelets with CHO cells that express human GPIb-IX complex exhibited similar result. Implied in these results are that either lA1 adopt a different conformation that disadvantages its binding or the binding site on A1 is masked by the extra amino acids in lA1 compared to sA1. Protein hydrogen-deuterium exchange is a sensitive assay to determine the local conformational change and the existence of the masked region. We acquired hydrogen-deuterium exchange data for both proteins and got nearly 100% sequences coverage from over 150 overlaid peptides for each protein, with on average over 10 redundancy per residue, which allows us to compare the hydrogen-deuterium exchange rate/extent of the A1 domain in lA1 and sA1 in good resolution. The results show that the global hydrogen-deuterium exchange pattern is the same between the two, indicating that the tertiary structures of the two proteins are similar. However, a contiguous region in lA1 is clearly protected from hydrogen-deuterium exchange compared to that in sA1 (indicated by the colorful region in figure 1). The protected region is located at the bottom of the globular A1 domain, including three loop regions (1272-1276; 1304-1310; and 1332-1340) and a part of the a1 helix (1289-1300). Compared to sA1, in this protected region of lA1 the deuterium exchange extents are reduced by 20-60% even after 3-hour incubation. Furthermore, 11 of out of 15 identified "gain-of-function" mutation sites of type 2B VWD are located in this masked region (P1266, H1268, C1272, M1304, R1306, R1308, I1309, S1310, W1313, P1337, R1341); the other 4 are located adjacently (V1314, V1316, L1460, A1461). This good alignment implies that the "gain-of-function" might associate with the detachment of the identified masking ligand in this region.Based on our findings, we propose that this short region in front of the A1 domain may involve in masking the A1 domain from interacting with platelet GPIb-IX. If confirmed, this internal masking ligand might offer new clues on how the mutations on A1 domain affect its role -as "gain-of-function" in type 2B VWD and "loss-of-function" in type 2M VWD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Retrospective Analysis of Bleeding Phenotype and Von Willebrand Factor Exon 28 Polymorphism D1472H at a Single Institution

▪Introduction: The laboratory work-up of von Willebrand Disease includes measurement of Von Willebrand Factor (VWF) antigen (VWF:Ag), VWF activity (ristocetin cofactor activity, VWF:RCo), and factor VIII Activity (FVIII), where VWF:Ag and VWF:RCo below 30-40% in the appropriate clinical setting are consistent with VWD. Accurate diagnosis of type 2 VWD, marked by a qualitative deficiency in the VWF protein, is imperative for appropriate therapeutic interventions. Exon 28 sequencing is frequently pursued to confirm the diagnosis of type 2 VWD. An exon 28 polymorphism, D1472H, results in impaired VWF binding to ristocetin, affecting the clinical assay of VWF activity as measured by ristocetin cofactor activity, but not resulting in impaired VWF function in vivo. This polymorphism is common in the healthy population and does not result in a hemorrhagic phenotype. We reviewed our institution’s experience with exon 28 sequencing with regard to the D1472H polymorphism, laboratory phenotype, and ultimate clinical diagnosis.Methods: Following IRB approval, patients who underwent exon 28 sequencing during evaluation for clinically significant bleeding between the years of 2003-2013 were identified. A retrospective chart review was conducted: patient demographics, VWF:RCo (lowest recorded), VWF:Ag (obtained on same day as the lowest VWF:RCo), and clinical diagnosis were collected. Descriptive statistics were employed to analyze the data.Results: Seventy-three patients underwent exon 28 sequencing during their work up for symptomatic bleeding between 2003-2013. The median age at testing was 7 years (10 months – 56 years), 41% were females. Of the 73 patients tested, D1472H polymorphism results for 65 patients were available for review. Fourteen patients (21.5%) were found to be heterozygous for the D1472H polymorphism. No patients were found to be homozygous. There was no difference between the mean VWF:RCo/VWF:Ag ratio for the D1472H heterozygous group (64% [47-81%]) and the group without the polymorphism (64% [48-80%]). VWF:RCo was lower in the heterozygotes with type 1 VWD (23.5% [14.5%-32.5%]) than in the group without the polymorphism (31% [18.5%-43.5]) (Table 1).In the group heterozygous for the D1472H polymorphism, 5/14 were diagnosed with type 1 VWD (Table 2). Two were diagnosed with type 2M VWD. One patient was diagnosed with type 1 VWD and mild hemophilia, 1 had type 1 VWD and Factor XI deficiency, 1 had type 2B VWD, 1 had chronic thrombocytopenia. Three patients did not have bleeding disorders.Conclusion: At our institution, heterozygosity for the exon 28 D1472H polymorphism was not associated with decreased VWF:RCo or VWF:RCo/VWF:Ag ratios as compared to patients without this polymorphism in all patients tested. For patients diagnosed with type 1 VWD, the VWF:RCo was lower in the group heterozygous for the D1472H mutation. These patients had sub-normal levels of VWF:Ag and VWF:RCo in the setting of clinically significant bleeding. Thus, the provisional diagnosis of VWD was appropriate despite the potential effect of the polymorphism on the VWF:RCo. These results are inconsistent with previously reported studies where presence of the D1472 polymorphism resulted in lower VWF:RCo/VWF:Ag ratios and inaccurate diagnosis of VWD. This study highlights the challenges associated with the laboratory diagnosis of VWD and the importance of continued research into the development of reliable VWD testing that more closely mimics the protein’s in vivoactivity. Abstract 2849. Table 1VWF Assay Results for patients with and without the D1472 polymorphismD1472H+ all comersD1472H - all comersD1472H + no VWDD1472H + type 1 VWDD1472H - no VWDD1472H - type 1 VWDNumber of patients1451371420VWF Ag52%54%90%38%117%47%VWF:RCo37%37%72%23%100%31%VWF:RCo/VWF:Ag0.640.640.80.60.850.65Abstract 2849. Table 2Patients with D1472H + and type 1 VWDPatient No.Age at testing (years)SexVWF:AgVWF:RCoVWF:RCo/VWF:AgDiagnosis118M47%27%0.57type 1 VWD23M23%14%0.61Type 1 VWD339F23%16%0.70type 1 VWD416F25%13%0.52type 1 VWD56M57%31%0.54type 1 VWD656M40%27%0.68type 1 VWD, factor XI deficiency74M53%37%0.70type 1 VWD, mild hem A DisclosuresNo relevant conflicts of interest to declare.

Towards improved diagnosis of von Willebrand disease: Comparative evaluations of several automated von Willebrand factor antigen and activity assays

Introductionvon Willebrand disease (VWD) is reportedly the most common bleeding disorder and arises from deficiency and/or defects of von Willebrand factor (VWF). Laboratory diagnosis and typing has important management implications and requires a wide range of tests, including VWF activity and antigen, and involves differential identification of qualitative vs quantitative defects. MethodsWe have assessed several VWF antigen and activity assays (collagen binding [VWF:CB], ristocetin cofactor [VWF:RCo] and the new Siemens INNOVANCE assay [VWF:Ac], employing latex particles and gain of function recombinant glycoprotein Ib to facilitate VWF binding and agglutination without need for ristocetin) using different instrumentation, including the new Sysmex CS-5100, with a large sample test set (n=600). We included retrospective plus prospective study designs, and also evaluated desmopressin responsiveness plus differential sensitivity to high molecular weight VWF. ResultsVWF:Ag and VWF:RCo results from different methods were respectively largely comparable, although some notable differences were evident, including one high false normal VWF:Ag value (105 U/dL) on a type 3 VWD sample, possibly due to heterophile antibody interference in the latex-based CS-5100 methodology. VWF:Ac was largely comparable to VWF:RCo, but VWF:CB showed discrepant findings to both VWF:RCo and VWF:Ac with some patients, most notably patients with type 2M VWD. Conclusions(a) VWF:Ag on different platforms are largely interchangeable, as are VWF:RCo on different platforms, except for occasional (some potentially important) differences, and manufacturer recommended methods may otherwise require some assay optimization; (b) VWF:RCo and VWF:Ac are largely interchangeable, except for occasional differences that may also relate to assay design (differing optimizations); (c) VWF:CB provides an additional activity to supplement VWF:RCo or VWF:Ac activity assays, and is not interchangeable with either.

Alanine Scanning Mutagenesis of the VWF-A1 Flanking Regions of VWF and the Impact on Ristocetin-induced VWF Interaction with Platelets

Von Willebrand disease is a bleeding disorder with reduced or abnormal function of VWF. While VWF has at least three functions (binding to FVIII, platelet GPIb, and collagen), the function of VWF has been quantified primarily using techniques in which ristocetin promotes the binding of VWF to platelet GPIb. It is recognized that the physiologic agonist for VWF-platelet interactions is shear but ristocetin facilitates a change in VWF structure to induce binding to platelet GPIb. For most individuals, this assessment of VWF function is valid, but over the past several years our group, the Zimmerman Program for the Molecular and Clinical Biology of VWD, has identified “polymorphisms” that do not affect VWF function but interfere with ristocetin binding to VWF in those laboratory tests of VWF function that utilize ristocetin. The two “polymorphisms” are P1467S and D1472H. P1467S is a rare sequence variant not seen in 12,900 alleles in the 1000-genome project. In contrast, the D1472H is a polymorphism seen in 63% of African Americans and in 17% of Caucasians. Since these two sequence variations are physically located in close proximity just C-terminal from the A1 loop, we initiated a systematic study of the adjacent N- and C-terminal sides of the A1 domain by alanine scanning mutagenesis.We concentrated on the sequence between p.C1237 – p.C1272 and p.C1458 – p.K1491 and replaced each amino acid with an alanine. These sequences were then transferred into a full-length expression construct. The individual altered VWF proteins were studied by GPIb-binding in two solid phase assays. The first assay, VWF:RCo, is an ELISA using an antibody-captured recombinant GPIb-alpha in which ristocetin-induced bound VWF is detected using an anti-VWF monoclonal antibody. The second assay, VWF:IbCo, is a similar assay, but the expressed GPIb contained two mutations, D235Y and M239V, that confer spontaneous binding to VWF without the addition of ristocetin as our laboratory has previously published.Where possible all assays were done at similar concentrations of VWF antigen and expressed as a ratio of VWF:IbCo/VWF:RCo compared to VWF:Ag concentration. An increased ratio indicates a reduction in ristocetin-induced binding. A pronounced difference was identified at several amino acids between p.D1459 and p.P1471. Interestingly, the D1472H polymorphism was not abnormal by this approach (D1472A) – thereby indicating that alanine substitution for the aspartic acid does not confer the same conformation that the histidine does in African Americans. Although earlier studies by Mohri and coworkers (JBC 1988) suggest a second binding site, only a minimal signal was identified for the region between p.C1237 –p.P1251. Hot spots were identified with VWF:IbCo/VWF:RCo scores 7- to 30-fold higher than background. To date, natural sequence variations have not yet been identified in these positions. Studies by Scott and coworkers (JBC 1991), suggested that ristocetin binding to proteins was proportional to the presence of X-P-G-X' beta-turns. The region of VWF that we are studying contains one of these sequences and is associated with a major effect on the VWF:IbCo/VWF:RCo signal. Since ristocetin dimerizes at a concentration of 1 mg/ml and the dose used in VWF:RCo is usually 1 mg/ml, these studies support the concept that alterations in ristocetin binding brought about by sequence variations may give the false impression that VWF function is profoundly affected when it is only the in vitro assay that is abnormal.Alanine scanning mutagenesis provides further substantiation of the role of the VWF-A1-loop in the binding of ristocetin to VWF. “Potential” Type 2M VWD subjects with sequence variations in this region may not have VWF functional abnormalities. Therefore, such individuals may require additional studies – particularly if bleeding symptoms are not identified in the presence of significantly abnormal laboratory testing. Disclosures:No relevant conflicts of interest to declare.

A family with type 2M VWD with normal VWF:RCo but reduced VWF:CB and a M1761K mutation in the A3 domain

HaemophiliaVolume 18, Issue 1 p. e33-e33 LETTERS TO THE EDITORS A family with type 2M VWD with normal VWF:RCo but reduced VWF:CB and a M1761K mutation in the A3 domain D. KEELING, D. KEELING Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorJ. BEAVIS, J. BEAVIS Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorR. MARR, R. MARR Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorK. SUKHU, K. SUKHU Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorP. BIGNELL, P. BIGNELL Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this author D. KEELING, D. KEELING Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorJ. BEAVIS, J. BEAVIS Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorR. MARR, R. MARR Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorK. SUKHU, K. SUKHU Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this authorP. BIGNELL, P. BIGNELL Oxford Haemophilia and Thrombosis Centre, Oxford, UKSearch for more papers by this author First published: 17 October 2011 https://doi.org/10.1111/j.1365-2516.2011.02676.xCitations: 18 David Keeling, Oxford Haemophilia and Thrombosis Centre, Churchill Hospital, Oxford, OX3 7LJ, UK. Tel.: +0 18 65 22 5318; fax: +0 18 65 22 5608; e-mail: david.keeling@ndm.ox.ac.uk Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume18, Issue1January 2012Pages e33-e33 RelatedInformation

VWF Binding to Types I, III or VI Collagen In the ZPMCB-VWD with Identification of Subjects with Selective Reduced Plasma VWF:CB

Abstract 235 Von Willebrand Disease (VWD) is a common bleeding disorder caused by quantitative (types 1 and 3) and qualitative (type 2) abnormalities in von Willebrand factor (VWF). Defective VWF binding to collagen (VWF:CB) has been identified in VWD patients, but the type(s) and amount of collagen vary between recommended assays. We measured VWF:CB using separate assays for types I, III, and VI collagen in plasmas from 233 healthy controls and 315 VWD index cases (261 type 1, 37 type 2 and 17 type 3) recruited into the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD). Additional studies included VWF antigen (Ag), VWF ristocetin-cofactor (RCo), VWF multimer, and VWF gene sequence analysis. VWF:CB was tested using ELISA assays with type I human placental collagen (5 ug/ml), type III human placental collagen (1 ug/ml), and by a commercial human placental type VI VWF:CB assay (Technoclone, Austria). Normal ranges were established for VWF:CB and VWF:CB/Ag ratios with the ZPMCB-VWD healthy control population. Three subjects (2 healthy controls and 1 type 1 VWD subject) were identified with reduced VWF:CB and reduced VWF:CB/Ag ratios to type VI collagen, despite normal results with type I and type III collagen. The two control subjects had VWF:CB/Ag ratios of 0.43 and 0.51 for type VI collagen. The patient with type 1 VWD had absent type VI VWF:CB and a VWF:Ag of 54 IU/dL with a history of clinical epistaxis and an EU Bleeding Score of 8 (normal less than or equal to 3). VWF sequencing demonstrated that these three individuals had an A1 loop polymorphism, R1399H, which has been previously reported as a VWF polymorphism. No other individuals in our study had this sequence change. Of particular note was that the VWF:CB/Ag ratios for types I and III collagen were each normal – suggesting a selective abnormality. As anticipated, collagen binding with all collagens was undetectable in the type 3 VWD subjects. Type 2A and 2B VWD subjects demonstrated reduced VWF:CB and VWF:CB/Ag ratios to all three types of collagen. Type 2M and 2N VWD subjects exhibited normal VWF:CB/Ag ratios to all three types of collagen, but one type 2M patient with an I1425F A1 loop mutation had a reduced VWF:CB/Ag ratio of 0.58. In the VWF A1 domain crystal structure, amino acid 1425 is in close proximity to amino acid 1399, suggesting the conformation of that region may be critical to type VI collagen binding to VWF. Since the frequency of the R1399H polymorphism is estimated to be 2% of the population (Sadler and Ginsburg, 1993), defective binding to type VI collagen may be an important contributor to the variability in the bleeding phenotype of VWD. Disclosures: Lentz: Novo Nordisk: Consultancy. Montgomery:GTI Diagnostics, Inc: Consultancy.

A Tiered Management Program To Conserve Replacement Therapy Resources for a Rural Population of Women with Type 2M von Willebrand Disease during Labor and Delivery.

Introduction: Type 2M Von Willebrand Disease (VWD) results from a mutation in the Von Willebrand factor (VWF) gene resulting in decreased or absent binding to platelet glycoprotein Ib. Bleeding episodes in type 2M VWD are variable, but can be severe or life-threatening; effective treatment requires administration of exogenous normal VWF. Pregnant women with type 2M VWD require close monitoring to prevent adverse bleeding events. Following a fatal post-partum hemorrhage in a woman from a large kindred with type 2M VWD, routine prophylactic doses of VWF were administered both pre- and postpartum to women with this disorder. This population is uninsured and utilized product is donated for their care. To determine if the amount of VWF could be safely reduced, a tiered risk stratification and replacement therapy program was developed to manage women with type 2M vWD during labor, delivery and the peuperium. We report the results of this tiered risk stratification in terms of the total product utilized and the rate of bleeding experienced.Methods: Between Jan–Mar 2007, we followed 7 Amish women from a large kindred with type 2M VWD (mutation 4120 C-->T on exon 28). This rural community has limited access to sophisticated hematologic monitoring and is uninsured. Women were risk stratified into 1 of 3 tiers (T) based on their personal or immediate family history of bleeding with childbirth. A nurse provided peripartum monitoring via home visits on postpartum days 1, 3, 5, 10 with a portable hematocrit (Hct) machine. Women in T1 were at lowest risk and given VWF replacement only in the event of excessive bleeding or postpartum Hct <30; T2 (intermediate risk) were given a single prophylactic VWF replacement dose immediately prior to delivery then monitored as T1; T3 received prophylactic VWF dose immediately prior to delivery, at 12 hours postpartum, then monitored by serial Hct per the other tiers. Women requiring Cesarean section were placed into T3 but treated with further VWF replacement due to the surgical intervention.Results: The 7 women fell into the following tiers; T1: 4; T2: 2; T3: 1. Of the 4 women in T1, none experienced excessive bleeding and no VWF therapy was required. In the 2 women in T2, there were no bleeding events requiring additional VWF. Postpartum Hct remained >30 on all evaluations in T1 and 2. One woman was delivered by elective caesarean section for complicated twin pregnancy, and given VWF prior to surgery and once daily for three consecutive postoperative days. She had no bleeding complications, and maintained Hct >30. Compared with the prior method of required pre- and post-partum prophylactic VWF administration regardless of tier or Hct, this pilot protocol safely decreased VWF usage in women assigned to T1 and 2 without adverse bleeding outcome or the need for transfusion.Conclusions: Women with type 2M VWD can be safely managed with a tiered risk stratification system and close postpartum clinical/Hct monitoring that utilizes conservative VWF replacement. For T1 and 2 this lead to a reduction in VWF administration of 83% compared with the prior method of mandatory pre- and postpartum VWF administration. This further translated into a substantial cost savings while still achieving safe hemostatic outcomes. Women at highest risk continue to receive prophylactic VWF pre- and postpartum to prevent serious hemorrhagic events.

Novel Mutations in Types 2 & 3 von Willebrand Disease and Correlation with von Willebrand Factor Multimer Patterns.

Background & Objectives: Diagnosis and classification of VWD are currently based on integration of personal and family bleeding histories and results of protein-based diagnostic tests (VWF:Ag, VWF:RCo, FVIII:C, VWF:CB and VWF multimers) which have performance limitations. Genetic testing is emerging as a complementary diagnostic tool. We have identified mutations and correlated VWF multimer patterns in our patients (pt) with types 2 & 3 VWD, and report novel candidate mutations.Patients & Methods: Unrelated pt with type 3 (n=6) and subtypes 2 (n=22) VWD, from Mayo Comprehensive Hemophilia Center, consented to this IRB approved study. PCR amplification, from genomic DNA, of VWF gene (splice junctions, coding and promoter regions), avoiding pseudogene amplification, followed by ABI® sequencing and analysis (Mutation Surveyor: SoftGenetic®) were performed. Mutations were compared to VWD (ISTH), NCBI NR nucleotide and DV SNP databases. Comparison with available VWF sequences from other vertebrate species was performed. Selected regions not known to contain mutations remain to be sequenced. VWF multimer analysis was performed using a novel in-gel immunostaining and infrared imaging system.Results: Type 2A VWD (n=10), a novel mutation in exon 28 (E28) (V1524G: 2 pt) with characteristic VWF multimer abnormalities (decreased HMWM, increased satellite banding, suggesting enhanced VWF proteolysis). Type 2B VWD (n=6), 2 pt were compound heterozygous for 2 previously reported mutations, the first in E28 (R1306W)/E48 (T2647M) and the second in E28 (V1316M)/E20 (R854Q). Type 2M VWD (n=4):a.an 84 yr female with personal (spontaneous and post-surgical) and family bleeding histories, VWF:RCo 72%, VWF:Ag 118% (RCo:Ag ratio 0.61), normal FVIII and platelets, and aberrant VWF multimer banding pattern without substantive reduction of HMWM, had a novel mutation in E52 (S2775C; conserved in mouse, rat, chicken and dog);b.a 14 yr female with a history of bleeding, VWF:RCo 34%, VWF:Ag 68% (RCo:Ag ratio 0.5) and normal FVIII and platelets, was compound heterozygous for a known mutation (R1399C subtype not classified in VWD database) and two novel mutations: E30 (P1725S) (conserved in mouse, rat, dog, cow and chimp) & E49 (T2666M) (conserved in mouse, dog, rat not in chicken). VWF multimers demonstrated abnormal banding pattern without substantive reduction of HMWM;c.2 unrelated pt and families with ultra-large and smeary multimers, previously classified as Vicenza variant, had R1374C mutations and were reclassified as Type 2M VWD. Type 2N VWD (n=1) previously reported mutation E20 (R854Q) was confirmed.Type 3 VWD, 2 pt had novel mutations, the first in E22 (E950X) and the second was a compound heterozygote for E43 (R2478Q)/E40 (E2322V) mutations. In general, patients with similar mutations had similar multimer patterns.Conclusions: We report novel candidate mutations in types 2 & 3 VWD and demonstrate novel VWF multimer patterns with our in-gel staining system. Multimer patterns correlated well with the underlying genotype. The novel mutations are likely causative, given their occurrence at highly conserved residues, but this needs to be confirmed with expression studies. Integration of genetic testing into the diagnostic workup of VWD will potentially lead to more accurate diagnosis and subtyping of VWD, and may further refine protein based testing and provide additional biological insight into VWD.

Functional Assay of VWF Measured by Its Interaction with Platelet GPIb without the Use of Ristocetin.

The clinical laboratory assessment of von Willebrand factor (VWF) includes measurement of the amount of VWF protein (VWF:Ag) and the functional interaction of VWF with platelets. The universally accepted measure of VWF function utilizes the ability of VWF to bind to platelets (or platelet GPIb) in the presence of ristocetin resulting in VWF-dependent agglutination of platelets measured quantitatively as ristocetin cofactor activity (VWF:RCo) by platelet aggregometry or turbidometry. An international reference standard has been assigned a biologic activity in international units by the WHO and the SSC of the ISTH. VWF:RCo has a recognized wide variability and reproducibility using different assays or when performed in different clinical laboratories. Alternative assays with improved reproducibility have recently been suggested that use recombinant GPIb in an ELISA assay of VWF binding, but this binding still requires ristocetin. Patients have been reported that have reduced interaction with ristocetin such that the VWF:RCo is markedly reduced (<0.12 IU/dL) yet patients have no bleeding symptoms even with major surgical challenge. Furthermore, recent studies done on normals entered into the ZPMCB-VWD have demonstrated a systematic problem with the measurement of VWF:RCo in African Americans in whom there VWF:RCo is significantly reduced compared with their VWF:Ag. Since this difference does not appear to have a clinical phenotype, a functional assay of VWF in the absence of ristocetin might be desirable. Such an assay should be sensitive to the measurement of the more-functional large VWF multimers, correlate with VWF:Ag in patients with reduced VWF function, and remain unaffected by mutations that affect VWF binding of ristocetin but do not have a bleeding phenotype. Since, platelet-type VWD variants of GPIb bind normal plasma VWF in the absence of added ristocetin and preferentially bind large VWF multimers, we explored an assay using combinations of the three GPIb mutations, G233V, D235Y, M239V, either individually or together to measure VWF function in the absence of ristocetin. Initially cell-based assays using mutant GPIB expressed in HEK293T cells were used in flow cytometry assays in which test VWF binding to mutant GPIb was measured using a labeled anti-VWF monoclonal antibody. Results were expressed in VWF:IbCo units. A normal curve was developed using serial dilutions of reference plasma previously standardized against both the ISTH and WHO VWF standards based on the VWF:Ag international standard. Patient samples included 41 normal, 16 type 2M, 5 type 2B, and 5 type 2A VWD patient plasmas. Samples included those with apparent type 2M VWD but without clinical symptoms and African American samples with a reduced VWF:RCo/VWF:Ag (RCo/Ag) ratio. Of the 16 type 2M VWD samples, 7 had markedly reduced VWF:IbCo (consistent with the VWF:RCo assay) and 9 had normal VWF:IbCo. African Americans with the SNPs associated with reduced RCo/Ag ratios had VWF:IbCo assays that correlated with their VWF:Ag in contrast to the abnormal RCo/Ag ratios identified by standard assays. Type 2A patients exhibited reduced VWF:IbCo assays and multimer size seemed to correlate with VWF:IbCo activity. Thus, measurement of VWF function using the VWF:IbCo assay may more directly correlate with VWF function and avoid some of the pitfalls and functional variability of VWF:RCo assays.

A Prospective Comparision of Intranasal (I.N.) and Subcutaneous (S.C.) Desmopressin (DDAVP) Therapy in Type 2M Von Willebrand Disease (VWD).

DDAVP is effective therapy for VWD patients with documented adequate biologic response. Few data evaluating DDAVP exist in the less common type 2M VWD Seventy one members of an Amish kindred diagnosed with type 2 M VWD with abnormal VWF:Rco <35% and C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene have been identified; 11 previously participated in an evaluation of weight-based trial dose of I.N. DDAVP(Stimate® Nasal Spray: 1.5 mg/ml concentration, 2 puffs weight >50 kg); 9/11 were further evaluated for response to S.C. DDAVP (0.3 microgram/kg). These 9 patients included 5 males, 4 females; age range: 21 to 58 years; weight range: 59–101.8 kg. Response utilizing ristocetin cofactor activity (VWF:Rco), factor VIII activity (VIII:C), and VWF antigen (VWF:Ag). Was assessed at 90 minutes post Stimate® administration and at 60 and 120 minutes post S.C. administration; values were also obtained at 4 and 6 hours post S.C. DDAVP to assess pharmacokinetics. Response to DDAVP was defined as VWF:Rco >40%. Five people (55%) responded to Stimate®; three (33%) responded to S.C. DDAVP. There were no major adverse effects. Two males who had responded to I.N. route did not respond to S.C. route; one female who responded to S.C. DDAVP did not respond to Stimate®. Responses fell below 40% at 4 and 6 hours post-dose. Responses to DDAVP are thus observed in a subset of individuals with type 2M VWD. Although all individuals tested exhibit the 4120C>T mutation, the response to DDAVP as measured by VWF: Rco levels varied from 1.7 to 4.9 fold. Therefore response to DDAVP in these patients appears not to be solely based upon the mutation causing the VWD phenotype but may be modified by other environmental and genetic factors. DDAVP responsiveness is an interesting phenotypic trait to be studied. Response may vary based on route of administration; this report highlights the necessity of assessing response to a trial dose by the intended route of administration prior to clinical use. Both I.N. and S.C. DDAVP may be used for minor bleeding episodes in those patients with type 2M VWD with adequate response to a trial dose. Both may be self administered as home based first line hemostatic therapy and are well tolerated.RESPONSE TO I.N. DDAVP(2005)AGE/SEXWEIGHT(KG)PRE VWF:RCo(%)POST VWF:RCo(%)POST/PRE VWF:RCo RATIO58/M94.811423.826/M69.710484.848/M83.214684.929/M79.410282.849/F58.812201.743/F7910353.521/F59.210404.022/F51.810383.827/M76.211524.7RESPONSE TO S.C. DDAVP (2006)AGE/SEXWEIGHT (KG)PRE VWF:RCo(%)PEAK POST VWF:RCo(%)POST/PRE VWF:RCo RATIO58/M101.813423.226/M72.710333.548/M88.114614.429/M82.710313.149/F70.511211.943/F79.510464.621/F5910494.922/F62.710393.927/M7910373.7

High Dose Intranasal Desmopressin (DDAVP) Therapy in Type 2M Von Willebrand Disease (VWD): A Prospective Open Label Trial.

DDAVP is effective treatment for VWD patients with adequate biologic response to administration of a trial dose. Intranasal therapy offers the advantage of ease of administration. Few prospective data evaluating response to intranasal DDAVP exist, especially in less common types such as type 2M VWD. As part of a study of a large Amish kindred of VWD in Northern Indiana, we conducted a prospective open label trial to assess response rate to a single dose of high concentration intranasal DDAVP in 11 affected individuals. Stimate® Nasal Spray (1.5 mg/ml) with weight based dosing was used. Laboratory parameters obtained prior to and 90 minutes after administration of Stimate® to assess for response included ristocetin cofactor activity (VWF:RCo), factor VIII activity(VIII:C), and VWF antigen (VWF:Ag) values. VWF:RCo ≥ 40% at 90 minutes post Stimate® administration was defined as adequate response. Of 212 evaluated members, 54 affected members were identified as defined by abnormally low VWF levels (VWF:RCo levels <35%) and confirmed by presence of C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene. Multimer analysis in a subset of the affected individuals showed a normal pattern. Eleven adult Amish patients (5 males, 6 females), ranging in age from 20 to 56 years, were enrolled. Baseline VWF:RCo values ranged from 10% to 14%. Ten patients reported no active bleeding symptoms at the time of initiation of the trial, while one had ongoing problems with gingival bleeding. All patients weighed >50 kg; hence two intranasal Stimate® spray puffs were administered to each patient. Five of eleven patients had response to intranasal DDAVP as evidenced by ≥ 40% VWF:RCo levels 90 minutes after receiving Stimate® (Response range: 4.0 fold to 4.86 fold rise). The remaining patients did not meet definition of response (Response range: 1.6 fold to 3.8 fold rise). There were no major adverse effects. Nine patients had mild facial flushing alone; one patient had flushing and headache; one patient was asymptomatic. After Stimate® administration, the patient with gingival bleeding reported complete cessation of bleeding sustained for 24 hours. This is the first and largest report to date assessing response to intranasal DDAVP therapy in 2M VWD patients. High concentration intranasal DDAVP may be used for minor bleeding episodes in those patients with adequate response to a trial dose. It is easily self administered as home based first line hemostatic therapy. Intranasal DDAVP is well tolerated with minimal adverse effects. Prospective clinical correlation of efficacy is underway in this population.RESULTS OF STIMATE® TESTINGAGE/SEXBASELINE FVIII:C (%)BASELINE VWF:AG(%)BASELINE VWF:RCO(%)90 MINUTE FVIII:C(%)90 MINUTE VWF:AG(%)90 MINUTE VWF:RCO(%)56/M623011164784225/M5230<101821114847/M5731141961086827/M4620<10114552848/F66291289382025/M351911156935241/F5423<10106563520/F2715<1096524024/F2712<1091492420/F3419<10146163834/F4216<101125824

Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

AbstractThe aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

Type 2M vWD Resulting from a Lysine Deletion within a Four Lysine Residue Repeat in the A1 Loop of von Willebrand Factor

Type 1 von Willebrand disease is characterized by a decreased plasma concentration of functionally normal von Willebrand factor (vWF) whereas type 2M is characterised by an abnormal vWF displaying decreased affinity for platelets. In these two types of patients, the multimeric structure of vWF is normal. We report here the identification, in two unrelated families from the UK and Algeria, of an in-frame 3 bp deletion, at the heterozygous state, resulting in the deletion of a lysine residue within a four lysine repeat at position 642-645 of the mature vWF subunit (del K 1405-1408 in pre-pro vWF). The patients who have a discrepancy between vWF antigen level and vWF ristocetin cofactor activity exhibited decreased ristocetin-induced binding but only a slight decrease in the percentage of high molecular weight (HMW) multimers in plasma. Recombinant vWF harbouring this deletion did not bind to platelet GPIb in the presence of ristocetin or botrocetin although the protein is multimerized. Consequently, this lysine deletion was considered as a type 2M vWD mutation.

Search for mutations in a segment of the exon 28 of the human von Willebrand factor gene: new mutations, R1315C and R1341W, associated with type 2M and 2B variants.

von Willebrand Disease (vWD) is the most frequently inherited bleeding disorder in humans, and is caused by a qualitative and/or quantitative abnormality of the von Willebrand factor (vWF). A large number of defects that cause qualitative variants have been located in the A1 domain of the vWF, which contains sites for interaction with platelet glycoprotein Ib (GPIb). We have developed a new approach to detect mutations based on DdeI digestion and single-strand conformation polymorphism analysis. A segment of 487 nucleotides, extending from intron 27 to codon 1368 of the pre-pro vWF was amplified from genomic DNA. The cleavage with DdeI yields two fragments of appropriate size for this kind of analysis and confirms that the gene, rather than the pseudogene, is being investigated. Six families with type 2B vWD, one type 2M vWD family, and one another type 2A vWD family were studied. After sequencing the fragments with an altered electrophoretic pattern, we found four mutations previously described--R1308C, V1316M, P1337L, and R1306W--in patients with 2B vWD. The last one arose de novo in the patient. In addition, two new candidate mutations were observed: R1315C and R1341W. The first one was associated to type 2M vWD, whereas the one second cosegregated with type 2B vWD. The fact that these new mutations were not found in 100 normal alleles screened further supports their causal relationship with the disease. These mutations, which induce either a gain or a loss of function, further show an important regulatory role of this region in the binding of vWF to GPIb and its implications in causing disease.

Molecular Modeling of Ligand and Mutation Sites of the Type A Domains of Human von Willebrand Factor and Their Relevance to von Willebrand's Disease

Abstractvon Willebrand factor (vWF) is a large multimeric, multidomain glycoprotein found in platelets, endothelial cells and plasma. The A1, A2, and A3 domains in vWF mediate binding to glycoprotein Ib, ristocetin, botrocetin, collagen, sulphatides, and heparin and provide a protease cleavage site. Mutations causing types 2B, 2M, and 2A von Willebrand's disease (vWD) are located in the A1 and A2 domains. Homology modeling was performed to provide a molecular interpretation of vWF function and mutation sites. This was based on our previous alignment of 75 vWF-A sequences, the doubly wound α/β fold seen in recent vWF-A crystal structures from complement receptor type 3 and lymphocyte function-associated antigen-1, and our new alignment of 28 vWF A1 and A2 sequences from different species. The active site in doubly-wound α/β folds forms a crevice that is located at the switch point between the two halves of the central β-sheet, and usually contains two metal-binding Asp residues in the vWF-A superfamily. Although one of these Asp residues is absent from the A1, A2, and A3 domains, this crevice is shown to correspond to the ristocetin binding site in the A1 domain and the protease cleavage site in the A2 domain. The residues R571-K572-R578-R579-K585 are found to be conserved in 28 A1 sequences and are predicted to constitute the heparin binding site in the A1 domain. Inspection of the type 2M vWD mutation sites that are involved in downregulation of glycoprotein Ib (GpIb) binding to vWF shows that these are spatially clustered at the carboxyl-edge of the β-sheet and above it in the A1 domain and may directly perturb GpIb binding. In contrast, the type 2B vWD mutation sites that are involved in upregulation of GpIb binding to vWF are spatially clustered at the amino edge of this β-sheet and below it and are located on the opposite side of the A1 domain from the type 2M mutation sites. The type 2B mutations are located between the heparin and GpIb binding sites. Because heparin binding inhibits the interaction with GpIb, this provides an explanation of vWF upregulation. The type 2A vWD mutation sites in the A2 domain correspond to buried residues that are otherwise 100% conserved across all 28 species, and are likely to be important for the correct folding of the A2 domain and its physiologically important protease site.