A Novel VWD Variant Identified in a Multi-Generational Canadian Ojibway Kindred with a Type 2B VWD Phenotypic Expression

Introduction: Type 2 VWD is caused by variants in the von Willebrand factor (VWF) gene leading to impaired function. Distinction between subtypes of Type 2 has traditionally relied on a panel of assays including VWF antigen (VWF:Ag), VWF ristocetin-cofactor activity (VWF:RCo), VWF multimers, and Factor VIII (FVIII:C). In type 2A, there is either impaired synthesis or accelerated degradation of the more adhesive VWF high molecular weight multimers (HMWMs). In type 2B, enhanced VWF-platelet interactions result in binding and removal of the HMWMs, impairing platelet adhesion. Type 2B VWD can manifest spontaneous platelet agglutination and thrombocytopenia. The inheritance patterns of Type 2A and 2B VWD are generally described to be autosomal dominant and fully penetrant. We identified a multigenerational Canadian First Nations Ojibway kindred with VWD harboring a missense variant in exon 28 (A>T transversion at nucleotide 4898), leading to a single amino acid substitution (p.Asn1633Ile). Some individuals in the kindred are heterozygous, and some homozygous, for the causative variant. Objectives: The objective of this case series are to describe the genetic basis and pattern of inheritance of this variant of VWD, and to characterize the laboratory hemostasis characteristics in the affected individuals. Methods: A retrospective case review was conducted on all affected individuals in the kindred known or suspected to harbour this variant. There were 20 individuals seen at our centre over 32 years who were included in this study. We collected clinical and laboratory data to characterize the bleeding phenotype. Results: Sixteen of the 20 individuals in the study were known or inferred to be heterozygous, and 4 were homozygous. We were able to examine laboratory hemostasis in ten individuals in the study (Table 1). The platelet count, FVIII:C and VWF:Ag levels were within or slightly above reference range for all individuals. In the heterozygous individuals, VWF:RCo was mildly reduced, VWF multimer testing showed loss of HMWMs, and ristocetin-induced platelet aggregation (RIPA) testing normal aggregation to a higher dose (1.25 mg/ml), a pattern consistent with mild Type 2A VWD (Table 1). In homozygous individuals, VWF:RCo was markedly reduced, and while RIPA was minimal with low-dose ristocetin, aggregation to the higher dose was normal despite the very low ristocetin cofactor activity in the plasma. The PFA-100 (collagen-epinephrine) closure times were markedly prolonged in the homozygous subjects and in one heterozygous subject, exceeding 300 seconds. Discussion/Conclusion: This novel VWF variant confers a laboratory phenotype consistent with type 2A VWD in the heterozygotes. Homozygotes have more severely impaired hemostatic function. The conventional hallmark of type 2B VWD (hyperresponsiveness to low-dose ristocetin) was not seen, but the fact that full aggregation was obtained in homozygotes despite ristocetin cofactor activities of only 5-6% in the plasma suggests some degree of hyperaggregability, and hence a phenotype more in keeping with Type 2B than 2A. In the future, it would be interesting to see how this missense variant affects GP1b binding to VWF, as RIPA and VWF:RCo are not entirely comparable assays. Disclosures No relevant conflicts of interest to declare.

The possibility to obtain reagent from platelet concentrates with long storage time for determination of von Willebrand factor ristocetin cofactor activity (vWF:RCo)

Введение. Фактор Виллебранда (ФВ) является важным компонентом системы гемостаза, и отклонение от нормы его содержания или состава вызывает развитие различных типов болезни Виллебранда. Определение ристоцетин-кофакторной активности ФВ (ФВ:РКФА) является особенно значимым при выявлении таких типов болезни Виллебранда как 2А, 2В и 2М, при которых содержание антигена ФВ находится в нормальных пределах, а ФВ:РКФА значительно снижена. Цель исследования: получение реагента для измерения ФВ:РКФА из концентратов тромбоцитов с длительным сроком хранения, обеспечивающего правильность диагностики. Материалы и методы. Функционально полноценные тромбоциты получали из донорских тромбоцитарных концентратов со сроком хранения 7–14 суток. Результаты. При создании реагента для измерений ФВ:РФКА нами был подобран метод отбора функционально полноценных донорских тромбоцитов для получения фиксированных формальдегидом клеток. На основе таких тромбоцитов был разработан реагент, позволяющий измерять ФВ:РКФА с использованием как агрегационного, так и агглютинационного метода. Выявлено соответствие результатов определения ФВ обоими методами с коэффициентом корреляции 0,96. Проведена оценка агрегационного метода измерения ФВ:РКФА по результатам участия в международной программе внешнего контроля качества UK NEQAS for Blood Coagulation (Великобритания). Заключение. Показано, что использование полученного из фиксированных тромбоцитов реагента позволяет правильно измерять ФВ:РКФА. Introduction. Von Willebrand factor (vWF) is an important component of hemostatic system and deviations from the norm of its content or composition are the cause of various types of von Willebrand disease development. Determination of ristocetin-cofactor activity of vWF (vWF:RCo) is particularly important for identifying 2A, 2B and 2M types of von Willebrand disease, in which the content of vWF antigen is within the normal range, and vWF:RCo signifi cantly reduced. Aim: to obtain a reagent for the measurement of vWF:RCo from platelet concentrates with a long shelf life, providing correct diagnostics. Materials and methods. Fully functional platelets were obtained from donor platelet concentrates with a shelf life of 7–14 days. Results. We discovered the method for selection of fully functional donor platelets to produce formaldehyde-fi xed cells. On the basis of these platelets we developed a reagent that allows to measure vWF:RCo using both aggregation and agglutination methods. The appropriateness was found between the results of vWF determination by both methods with a correlation coeffi cient 0.96. The aggregation method of measuring vWF:RCo was evaluated by a result of participation in the international program of external quality control UK NEQAS for Blood Coagulation (United Kingdom). Conclusion. It is shown that the use of a reagent obtained from fi xed platelets allows for the correct measurement of vWF:RCo.

Laboratory Assessment of Anti-Coagulant Properties of a Von Willebrand Factor Targeted Aptamer

Introduction: Many cardiovascular procedures including percutaneous coronary intervention, coronary bypass surgery, and peripheral vascular surgery require antithrombotic therapy during the procedure. Heparin is frequently used, due to low cost, ease of monitoring and reversibility with protamine. Antiplatelet agents such as aspirin and clopidogrel are also frequently used but these agents are not reversible. Alternative reversible antithrombotic strategies that interfere with platelet-mediated thrombosis are needed. We are investigating the use of a reversible agent that is specific for von Willebrand factor (VWF) for use during cardiovascular procedures. We previously developed an antidote-controlled 80 oligonucleotide RNA aptamer Ch-9.14-T10 (T10). (Nimjee, S.M., Lohrmann, Wang, H, et al. Molecular Therapy 2012; 2:391-397). T10 bound to VWF with high affinity and inhibited thrombosis in a murine carotid artery damage model. The aptamer also greatly increased bleeding in mice that were surgically challenged by tail transection and blood loss decreased when complementary antidote oligonucleotides were administered. In addition, T10 inhibited primary hemostasis in the PFA-100 assay and this inhibition was completely reversed by the antidote. This study further characterizes the T10 aptamer, as well as a truncated version of the T10 aptamer, using clinical laboratory assays that would be used to monitor antithrombotic therapies during cardiovascular procedures. Methods: We initiated our study with T10 and then developed a truncated version, T25, a 60 oligonucleotide with which we performed additional investigations. We recruited normal donors for analysis of aptamer and antidotes. All participants had normal platelet counts, VWF activity and antigen levels, and were not taking antiplatelet agents. Laboratory tests included: ristocetin induced platelet agglutination (RIPA), VWF activity, ristocetin cofactor activity, cone and plate(let) analyzer, thromboelastography (TEG) and thromboelastometry (ROTEM). Results: We first determined the effects of the VWF aptamers on RIPA, VWF activity, and ristocetin cofactor activity to confirm functional inhibition of VWF. We found that T10 (500-750 nM) and T25 (250-750nM) completely inhibited RIPA depending on the donor. T25 also inhibited VWF activity with a Ki of 160-320 nM and ristocetin cofactor activity with an IC50 of 3000 nM. Employing a cone and plate(let) analyzer we found that T25 (750 nm) reduced platelet adhesion by 33-49% on a polystyrene surface under arterial flow conditions (1800 s-1, for 2 minutes). The aggregate size was also reduced by 30%. As ACT and TEG are frequently used during cardiovascular procedures, we performed several measures with our VWF aptamer. T10 had no effect on the ACT (730-2000nM), TEG (1600 nM), or ROTEM (750 nM). Standard methods were used for the TEG (Kaolin activation followed by CaCl2) and ROTEM (EXTEM assay) Topf et al. reported a modified ROTEM protocol where they found that incubating citrated whole blood with ristocetin decreased the clot strength as measured by the maximum amplitude (Thromb.Haemost 2011; 105:1091). We reasoned that an aptamer that bound to VWF would inhibit the effect of ristocetin on the maximum amplitude. We found that T25 did inhibit the effect of ristocetin and titration studies showed that 200-400 nM T25 was needed to restore the maximum amplitude to at least 80% of its original value. An example of the ROTEM tracings is shown in figure 1. Conclusion: The VWF-specific aptamer T25 can be detected by several clinical assays, including the RIPA, VWF activity assays, and the cone and plate(let) analyzer. It is not detectable by an ACT or standard TEG or ROTEM, however. We are able to detect the aptamer using a modified ROTEM and are further defining the adaptability of this assay to facilitate monitoring during cardiovascular procedures. Citrated whole blood from was incubated with buffer or ristocetin (0.7 mg/ml) or ristocetin and T25 (300 nM) as indicated. Clotting was initiated with EXTEM (tissue factor pathway activation) and CaCl2. Figure 1. VWF Aptamer T25 Restores the Decrease in Clot Strength by Ristocetin in Citrated Whole Blood from a Normal Donor. Figure 1. VWF Aptamer T25 Restores the Decrease in Clot Strength by Ristocetin in Citrated Whole Blood from a Normal Donor. Disclosures Becker: Regado Biosciences: Membership on an entity's Board of Directors or advisory committees.

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 1.VWF 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 2.Patients 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 Disclosures No relevant conflicts of interest to declare.

Performance of two new automated assays for measuring von Willebrand activity: HemosIL AcuStar and Innovance

SummaryThe ristocetin cofactor activity assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity but remains difficult to perform, and the coefficient of variation of the method is high (about 20–30%). This study evaluated and compared the performance for measuring the VWF activity of two newly commercialised assays [VWF:Ac Innovance (VWF:Ac) and VWF:RCo Acustar (VWF:RCo Acu)] with the reference VWF:RCo aggregation in 123 pathological plasma samples. The correlation and concordance between both new tests (VWF:RCo-Acu and VWF:Ac) and the reference VWF:RCo were good. The results of the VWF activity to VWF antigen ratio were also comparable whatever the method for the classification of VWF deficiency in all patients. Our results showed that both new tests could replace the “gold standard” VWF:RCo in aggregometry with several benefits: they are fully automated, easier and faster to perform, better adapted to emergency situations if necessary.