ALBERT

Description: Key Points CLEC4M plays a role in the clearance of VWF. CLEC4M polymorphisms contribute to the genetic variability of VWF plasma levels.

Description: Pregnancy and delivery represent a major hemostatic challenge in von Willebrand disease (VWD). In addition to maintaining adequate hemostasis, von Willebrand factor (VWF) is an important regulator of angiogenesis, with angiodysplasia contributing to morbidity in patients with VWD. Placental development is complex, with vascular development important for fetal growth and viability. It has been hypothesized that antepartum bleeding or abnormal placental angiogenesis may lead to pregnancy loss in women with VWD. Large studies have reported an increased risk of postpartum bleeding in patients with VWD, however, little information is available for rates of pregnancy loss. We conducted a cross-sectional study to determine the rate of pregnancy loss in women with VWD, when compared to a control population. We invited all women with VWD followed by the Southern Alberta Rare Blood and Bleeding Disorders Comprehensive Care Program to participate in an online or paper questionnaire, along with a control group of women without VWD identified through a low-risk obstetrical clinic in Calgary, Alberta. We included patients who were 18 years of age or older with a diagnosis of VWD, defined as VWF antigen and activity levels less than 50 percent or a historical diagnosis, in combination with a bleeding score of 4 or more (condensed MCMDM-1 bleeding questionnaire). Patients were excluded if they had no past pregnancies, no mailing address, or had an alternative bleeding disorder. Our 20-item questionnaire was reviewed and pre-tested by hematologists, obstetricians and laypeople. With patient consent, data was supplemented from clinical and hospital records to verify and expand on the information collected in the questionnaire. The primary outcome was the incidence of spontaneous abortion (fetal loss 24 hour) bleeding post-delivery in women with VWD. In VWD women with and without pregnancy loss, there was no difference in mean bleeding score (9.7 vs. 10.9). There was also no difference in cases of pre-eclampsia, placental abruption or gestational hypertension in VWD women with and without pregnancy loss. Two VWD patients with previous pregnancy loss developed gastrointestinal angiodysplasia later in life. There was no significant increase in the rate of pregnancy loss in women with VWD, however, a larger cohort study is needed to confirm the rate of pregnancy loss before further conclusions can be made. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 16 Type 1 von Willebrand's Disease (VWD) can result from decreased synthesis or accelerated clearance of von Willebrand Factor (VWF), resulting in partial quantitative deficiency. Approximately 35% of individuals with Type 1 VWD do not have a putative mutation in their VWF gene, suggesting that genes other than VWF may contribute to the pathophysiology of this disease. Recently, the CHARGE GWAS meta-analysis identified single nucleotide polymorphisms in the gene encoding the C-type lectin domain family 4 member M (CLEC4M) as being significantly associated with plasma VWF levels in normal individuals. CLEC4M is a pathogen recognition receptor with a polymorphic extracellular neck region consisting of a variable number of tandem repeats (VNTR) (3 – 9 repeats). We hypothesize that CLEC4M binds to and clears VWF from the circulation, and that different CLEC4M VNTR alleles may contribute to differences in plasma levels of VWF in normal subjects and patients with Type 1 VWD. Previously, genotyping of 555 subjects (196 cases with type 1 VWD, and 362 family members) for the CLEC4M VNTR number showed that the most frequently documented alleles were VNTR 5 (29%), 6 (15%), and 7 (53%). Family-based association analysis on kindreds with type I VWD has demonstrated a significant excess transmission of VNTR 6 to the type I VWD phenotype (p=0.005) and an association of this VNTR allele with VWF:RCo (p=0.037). In the present studies, we have complemented this genetic association data with experiments to directly evaluate the ability of CLEC4M to bind and internalize VWF. Further, we characterized the ability of different CLEC4M VNTR alleles to facilitate VWF clearance. Binding of VWF to CLEC4M was assessed with a modified ELISA using a recombinant CLEC4M-Fc chimera. CLEC4M-Fc bound to Humate P (plasma-derived VWF-FVIII) in a dose-dependent manner. CLEC4M-Fc also bound to recombinant human VWF, and factor VIII-free plasma-derived VWF. CLEC4M-Fc demonstrated a 70% increase in binding to de-O-glycosylated Humate P (p=0.041), and a 75% decrease in binding to de-N-glycosylated Humate P (p=0.046) relative to controls. Additionally, pre-incubation of CLEC4M-Fc with the polysaccharide mannan attenuated binding to all VWF preparations by approximately 50%. Binding and internalization of VWF by HEK 293 cells stably expressing CLEC4M (VNTR allele 7) was assessed with immunofluorescence and ELISA. Binding of VWF co-localized with CLEC4M expression on HEK 293 cells. CLEC4M and VWF co-localized with early endosomal antigen-1, suggesting that CLEC4M participates in receptor-mediated endocytosis of VWF. CLEC4M-expressing cells bound and internalized VWF in a dose- and time-dependent manner relative to controls. Preincubation of CLEC4M expressing cells with mannan inhibited VWF binding and internalization by 50% (p=0.0088). The contribution of CLEC4M genetic variability to VWF binding and internalization was measured using HEK 293 cells expressing CLEC4M with 4, 6, 7, and 9 tandem repeats. Decreased binding and internalization of VWF was observed with cells expressing CLEC4M 4 and 9 tandem repeat constructs as compared to CLEC4M with 7 tandem repeats (CLEC4M 4 – 60% reduction, p 〈 0.001; CLEC4M 9 – 45% reduction, p=0.006). Cells expressing the CLEC4M VNTR combination 4 and 9, had a 55% decrease in binding and internalization of VWF relative to cells expressing CLEC4M with 7 VNTRs (p 〈 0.001). These VNTR associated differences in VWF binding/internalization were not accounted for by variances in the CLEC4M expression levels in the transfected HEK 293 cells. These studies demonstrate that the C-type lectin CLEC4M binds to and internalizes VWF through an N-glycan-dependent mechanism. Additionally, it provides further evidence that polymorphisms in the CLEC4M gene contribute to quantitative VWF deficiency. Disclosures: Montgomery: Gen-Probe/GTI Diagnostics: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

Description: There is little safety data available on the use of epidural analgesia during pregnancy in women with von Willebrand disease (VWD), a common disorder comprising up to 0.1% of the population. Despite physiological increases in von Willebrand factor antigen and activity levels to normal or near levels during third trimester in Type 1 VWD patients, epidural analgesia use is varied and often withheld. We conducted a cross-sectional questionnaire in order to further characterize the local practice and provide additional safety data on epidural use in the VWD population. We invited all women with VWD followed by the Southern Alberta Rare Blood and Bleeding Disorders Comprehensive Care Program to participate in an online or paper questionnaire. The questionnaire was part of a larger study identifying pregnancy complications in VWD, with additional questions collected on the diagnosis and severity of VWD, epidural use and associated outcomes, as well as the perceived contraindications of epidural analgesia. All questions were pre-tested by hematologists, obstetricians and laypeople. With patient consent, supplemental data was collected from clinic and hospital records. We included patients who were 18 years of age or older with a diagnosis of VWD, defined as von Willebrand factor (VWF) antigen and activity levels less than 50 percent or a historical diagnosis, in combination with a bleeding score of 4 or more (condensed MCMDM-1 bleeding questionnaire). Patients were excluded if they had no past pregnancies, no mailing address, or had an alternative bleeding disorder. Confidence intervals for proportions were calculated using the Wilson’s score method. Of the 98 women with VWD who met the study inclusion criteria, 31 (32%) completed the questionnaire. There were 25 (81%) diagnosed with Type 1, 5 (16%) with Type 2, and 1 (3%) with Type 3 VWD. The mean bleeding score was 10.3 (SD 3.6). There were a total of 83 pregnancies (mean 2.7, range 1-6), with 60 deliveries assessed because of 20 pregnancy losses, 2 elective terminations, and 1 patient currently pregnancy. Of the 60 deliveries, the rate of epidural use was 28.3% (95% confidence interval 18.5-40.8, n=17). All epidural analgesia use was reported in women with Type 1 VWD. Of the 43 pregnancies without an epidural, the stated reasons were as follows: 10 because of concern for bleeding (23.3%), 25 because of personal preference (58.1%), 7 ‘other’ (16.3%), and 1 participant without a response (2.3%). In the 10 pregnancies where an epidural was not used due to bleeding concerns, 6 patients had Type 1 VWD and 1 patient had Type 2N VWD. The outcome of epidural use in the VWD population was reassuring, 2 women reported their epidural analgesia to be ineffective, and 4 women reported neurological symptoms such as numbness, tingling or weakness. All neurological symptoms were temporary with no persistent deficits or documented complication of bleeding. Due to the retrospective nature of this study, VWF antigen and activity levels at the time of delivery were not available, limiting the conclusions that can be made on the safety of epidural use based on VWF levels. It is our local practice to proceed with epidural analgesia if a patient’s VWF antigen and activity levels measured in third trimester are greater than 50%. For VWF levels less than 50%, epidural analgesia would only be considered with appropriate DDAVP or factor concentrate replacement. In summary, our local rate of epidural use was close to 30% in the VWD population, which was primarily composed of Type 1 VWD patients. Outcome data is reassuring, but a larger cohort study is needed to further quantify rare risks such as neuraxial bleeding. Disclosures No relevant conflicts of interest to declare.

Description: Key PointsThe robustness of the VWF:collagen-binding assay is confirmed in a comprehensive evaluation of VWD collagen-binding defects. Collagen binding by VWF, GPVI, and α2β1 have major albeit overlapping functions in primary hemostasis.

Description: Introduction Hemophilia A and B are the most common severe bleeding disorders. Improvements in carrier detection and prenatal diagnosis of hemophilia have practical implications for family planning as well as peri- and antenatal pregnancy management. Access to counselling and testing is heterogeneous. We evaluated the awareness and usage of prenatal genetic counselling, sex determination and procedures to determine fetal hemophilia status over time in carriers of hemophilia at our hemophilia treatment centre (HTC). Methods Carriers of hemophilia A or B were recruited through our HTC. Research was approved by our institutional ethics board approval. An online link was emailed to participants using Research Electronic Data Capture (REDCap) Clinical Research Unit software. Survey questions explored demographics, family members affected and severity, pregnancy history, awareness and utilization of carrier testing, genetic counselling, prenatal diagnostic technologies and terminations. Responses were analyzed by 2 reviewers for basic statistics and to identify themes in a descriptive qualitative design. Results & Discussion 85 hemophilia carriers were identified; 3 were lost to follow up and 34 had missing or incorrect contact information. 48 survey invitations were distributed with 24 responses received (50% response rate). One response was excluded as the participant was not a carrier (spontaneous mutation in her son). 20 complete and 3 incomplete responses were analyzed. Median age was 44 years (28-73), with sons most commonly affected family member (14/23, 61%). 3 participants reported no pregnancies, 2 did not respond and 1 did not list the years of her pregnancies. The majority received genetic counselling from HTC personnel. Table 1. Awareness and utilization of carrier testing, counselling, pregnancy, prenatal technologies and termination. * Denominator represents number of responses **Chorionic villae and/or amniotic fluid testing Responses were analyzed by year of pregnancy: prior to 2000 (n=8) or after 2000 (n=9). No pregnancy dates were given in 6 responses. 100% of participants with pregnancy after 2000 had carrier testing. The majority also received genetic counselling (n=8). Members of this group had access to and utilized prenatal diagnostic technologies; pre-implantation genetic diagnosis (n=1), early prenatal diagnosis (n=2) or invasive testing (n=4). Rationale given included: more time to prepare emotionally (n=3), optimize delivery management (n=2), and as a consideration to terminate pregnancy (n=1). Overall, there was increased but heterogeneous knowledge of available technologies potentially reflecting limited understanding during or ineffectively timed counselling, or limited knowledge by carriers who had completed their families when these technologies became available. Identified categories and themes from text responses include: · Impact of family experience with hemophilia: o Themes: severely affected family members impact family planning and attitudes about hemophilia, prenatal diagnosis does not prompt termination due to improvements in hemophilia care · Impact on medical care: o Themes: early identification may prepare for delivery, concern about overtreatment of carriers during delivery · Impact on psychosocial well-being: o Themes: concern about others' opinions, guilt for having more than one affected child · Need for education: o Themes: importance of collaboration between HTC and health care providers, HTC as the primary source for hemophilia related information Study challenges included limited responses precluding statistical analysis and the ability to submit incomplete responses due to sensitive subject matter. Conclusion Genetic technologies have shown significant advances and now allow early detection of those with hemophilia, including carriers. However, this study shows that experiences and knowledge of these technologies are still widely varied. As a result, hemophilia carriers may benefit from standardized educational materials which may compliment counselling provided by HTCs and may be useful for multigenerational knowledge dissemination. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Epidural and spinal hematoma are rare but potentially devastating complications of neuraxial anesthesia. Individuals with pre-existing bleeding disorders and tendencies are likely at higher risk of these feared complications, however there are currently no comprehensive recommendations to direct the use of neuraxial anesthesia in these patients. Objective Our objectives were to create a set of consensus recommendations to advise clinicians on appropriate treatment thresholds prior to neuraxial anesthesia for patients with a variety of common and uncommon bleeding disorders and tendencies, and to propose safe hemostatic thresholds for administration and removal of neuraxial anesthetics in these individuals. Materials and Methods A steering committee comprised of 14 hematologists, anesthesiologists and methodologists created and refined a set of Delphi statements regarding the use of neuraxial anesthesia in the following pre-existing bleeding disorders and tendencies: von Willebrand disease, immune thrombocytopenia, gestational thrombocytopenia, platelet function disorders, thrombocytopenia associated with hypertensive disorders of pregnancy, coagulation factor deficiencies and fibrinogen disorders. Statements were developed using data obtained from a previously conducted scoping review of existing literature supplemented with the expert opinion of the steering committee in areas where there are minimal published data. Statements include varying hemostatic laboratory parameters in the context of high or low bleeding risk as measured by either a validated bleeding assessment tool (BAT) or pre-defined criteria for excessive bleeding. We plan to recruit approximately 30 international panelists from the fields of hematology and anesthesiology to participate in a modified (i.e. electronic) Delphi technique. Three Delphi rounds will be conducted, during which we will ask panelists to rate their agreement with each Delphi statement on a 5-point Likert scale. Results will be analyzed for statistical consensus, defined as a Cronbach's alpha value of greater than or equal to 0.70. Statements that achieve consensus will be included in the final recommendations. Results Results of the modified Delphi study are pending. Conclusions Administration of neuraxial anesthesia in patients with pre-existing bleeding disorders and tendencies continues to present a significant management challenge for clinicians. We anticipate that the resultant recommendations will become a helpful tool for the future management of neuraxial anesthesia in these patients. Disclosures Martin: Borden Ladner Gervais LLP:Consultancy.Carvalho:Gauss Surgical:Consultancy.Kuter:Dova:Consultancy, Honoraria;Daiichi Sankyo:Consultancy, Honoraria;Actelion (Syntimmune):Consultancy, Honoraria, Other: Travel Expenses, Research Funding;Principia Biopharma:Consultancy, Honoraria, Other, Research Funding;Protalix Biotherapeutics:Consultancy;Shionogi:Consultancy;Protalex:Consultancy, Honoraria, Research Funding;Kezar Life Sciences, Inc:Other, Research Funding;CRICO:Consultancy, Honoraria;Genzyme:Consultancy, Honoraria;Principia:Consultancy, Research Funding;Shire:Consultancy, Honoraria;Shionogi:Consultancy, Honoraria;Sanofi (Genzyme):Consultancy, Honoraria;Incyte:Consultancy, Honoraria;Immunovant:Consultancy, Honoraria;Kyowa-Kirin:Consultancy, Honoraria;Merck Sharp Dohme:Consultancy, Honoraria;Momenta:Consultancy, Honoraria;Novartis:Consultancy, Honoraria;Pfizer:Consultancy, Honoraria;Zafgen:Consultancy, Honoraria;Up-To-Date:Consultancy, Honoraria, Patents & Royalties;UCB:Consultancy, Honoraria;Platelet Disorder Support Association:Consultancy, Honoraria;Argenx:Consultancy, Honoraria, Other: Travel Expenses, Research Funding;Bristol-Myers Squibb:Consultancy, Honoraria, Other: Travel Expenses, Research Funding;Caremark:Consultancy, Honoraria;Immunovant:Other: Travel Expenses, Research Funding;Alnylam:Consultancy, Honoraria, Other: Travel Expenses, Research Funding;Agios:Consultancy, Honoraria, Other: Travel Expenses, Research Funding;Amgen:Consultancy, Honoraria, Other: Travel Expenses, Research Funding;Takeda (Bioverativ):Consultancy, Honoraria, Other, Research Funding;Rigel:Consultancy, Honoraria, Other, Research Funding;Protalex:Consultancy, Honoraria, Other, Research Funding.Lavin:Tremeau Pharmaceuticals:Membership on an entity's Board of Directors or advisory committees;Siemens Healthineers:Other: Speaker's fees;Takeda:Research Funding;Shire:Research Funding.Sholzberg:Takeda:Honoraria, Other: Scientific Advisory Board, Research Funding;Novartis:Honoraria, Other: Scientific Advisory Board;NovoNordisk:Honoraria, Other: Scientific Advisory Board;Octapharma:Honoraria, Other: Scientific Advisory Board, Research Funding;Amgen:Honoraria, Other: Scientific Advisory Board, Research Funding.