ALBERT

Description: Key Points D1472H sequence variation is associated with a decreased VWF:RCo/VWF:Ag ratio in type 1 VWD subjects. D1472H sequence variation is not associated with an increase in bleeding as measured by bleeding score in type 1 VWD subjects.

Description: 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.

Description: Type 1 VWD is the most common form of VWD and is characterized by quantitative deficiency of VWF. Mechanisms causing type 1 VWD include decreased VWF synthesis due to promoter polymorphisms, decreased VWF secretion with intracellular retention/degradation, and increased clearance of VWF from plasma (type 1C VWD). VWF and its propeptide (VWFpp) are released into plasma in equimolar amounts but have very different half-lives (8 – 12 hours and 2-3 hours, respectively). The assay of VWFpp can be used to assess VWF synthesis, secretion, and clearance. Reduced VWFpp level indicates reduced VWF synthesis or secretion. An increased VWFpp/VWF:Ag ratio is expected when VWF is cleared rapidly from plasma (type 1C VWD), while decreased VWF secretion/intracellular retention results in a normal ratio. We enrolled 502 type 1 VWD index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of the confirmed type 1 VWD cases, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL, and 12 met the criteria for “Type 1 – Severe” with VWF:Ag of 1 – 5 IU/dL and VWFpp/VWF:Ag 〈 3. Type 1C VWD comprised 22% of all type 1 VWD cases. The type 1C cohort included several individuals previously diagnosed as type 2A (11), type 2M (2), and “Unclassified” (3). The most significant reclassification involved 7 cases previously diagnosed as type 3 VWD – these patient had detectable VWF:Ag (2 – 6 IU/dL) and VWFpp (14 – 66 IU/dL), and elevated VWFpp/VWF:Ag (4.2 – 33.0). Although plasma VWF:Ag is very low in these patients, they might be expected to have normal platelet stores of VWF:Ag, unlike type 3 VWD patients. Type 3 VWD patients in our study had undetectable (

Description: Warfarin remains the primary agent used in long-term antithrombotic treatment and prophylaxis. The major adverse effect associated with its use is bleeding. When intracerbral or other life threatening hemorrhage occurs in patients on warfarin, or urgent surgical intervention is indicated, timely reversal may favorably impact outcomes. Warfarin effect is commonly reversed with fresh frozen plasma (FFP) and vitamin K. The efficacy of FFP in this setting has been questioned, and fluid overload is a common complication. In addition, thawing and administering significant volumes of FFP may delay correction. We instituted a policy for rapid delivery and administration of Prothrombin Complex Concentrate (PCC) in the setting of the need for urgent reversal of warfarin. Patients were triaged by emergency services to determine the need for urgent warfarin reversal. Hematology telephone consultation was obtained, and patients received 25–50 units/Kg of PCC, (Proplex-T, Baxter). PT/INR was recorded before and immediately (within 30 minutes) after dosing, and 24 hours post dosing. Urgent surgical interventions were performed immediately after PCC. We report data from retrospective chart review of 2 years experience with this protocol. 58 patients were treated, with a mean age of 71y (range 26–92). Indications for Warfarin: atrial fibrillation (38), DVT (5), pacer or defibrillator (3), prosthetic aortic valve (4), cardiomyopathy (1), A-V fistula clot (1), undetermined from chart review (6). Concomitant bleeding risks were identified in 15 patients, including renal failure (4), thrombocytopenia (4), anti-platelet therapy (3) and liver disease (4). 29 Patients received FFP in the course of their care as directed by their primary care service. 12 patients presented with extreme (〉10) elevations of their INR. INR results are reported below: INR pre-PCC (Mean) INR post-pcc (Mean) INR 24 hr post-PCC (Mean) All Patients (N=58) 11.7 1.4 1.5 Pre INR 〈 10 (N=46) 5.4 1.3 1.5 Pre INR ≥ 10 (n=12) 35.3 1.8 1.7 No FFP Used (N=29) 12.2 1.2 1.2 16 Patients underwent invasive or surgical procedures immediately after PCC. Hemostasis was judged to be normal in all cases. Patients were monitored throughout their hospital stay for thrombotic complications. 2 patients had non-Q myocardial infarction after their therapy. Both had catastrophic presentations and histories of cardiac revascularization. One patient had a line-associated thrombosis 17 days after PCC and one had possible extension of chronic calf thrombophlebitis by doppler evaluation 3 days after PCC. These events were felt to be more likely attributable to underlying disease than PCC. No patients experienced fluid overload related to PCC use. Patients with intracerebral hemorrhage (N=22) had a mortality rate of 45%, which compares favorably with the recently published figure of 52%. Responses to PCC were similar in patients not receiving plasma as part of their care (N=29). 30 patients had a higher INR at 24 hours when compared to the INR post infusion, however the change was small (Mean INR Δ=0.3). Recent protocol refinements include routine use of IV Vitamin K, No FFP use and Q6 hour PT/INR monitoring for possible additional PCC dosing in the first 24 hours. Immediate reversal and the absence of volume complications make the use of PCC superior to FFP for the reversal of warfarin effect in the urgent setting, and wider use of this intervention is appropriate.

Description: Abstract 2208 Diagnosis of von Willebrand disease (VWD) currently relies on two assays of von Willebrand factor (VWF), the VWF antigen ELISA (VWF:Ag) and the VWF ristocetin cofactor activity assay (VWF:RCo). The latter exploits the capacity of ristocetin to induce VWF – platelet interactions as a measure of VWF function. Ristocetin, however, is a non-physiologic agonist as shear stress is the physiologic stimulus inducing VWF to bind platelets in vivo. Recently we have reported that the VWF:RCo/VWF:Ag ratio is decreased in individuals with an A1 domain polymorphism, D1472H. The lack of bleeding in subjects with this polymorphism suggests D1472H does not create a physiologic defect in VWF – platelet interactions. D1472H is directly adjacent to a known ristocetin-binding area in the VWF A1 region (Leu 1457 – Pro 1471), supporting the hypothesis that D1472H affects the ability of ristocetin to bind VWF. Similarly, a heterozygous sequence change leading to P1467S (located in the same ristocetin binding domain) resulted in an undetectable VWF:RCo but no bleeding symptoms were noted in affected subjects. To further investigate the cause of this observation, we developed a method to study the binding of ristocetin to VWF directly. Maleic anhydride microtiter plates were used to capture ristocetin via its amine groups. A VWF source, either plasma or recombinant VWF (rVWF), was then added, wells washed, and VWF binding detected using anti-VWF antibodies. Both plasma and rVWF bound to the captured ristocetin similarly with ristocetin plating concentrations ranging from 0.01 to 1 mg/ml. Specificity of ristocetin dependent VWF binding was confirmed, as preincubation of ristocetin with rVWF decreased binding of rVWF to immobilized solid-phase ristocetin. No detectable binding was present for a full length rVWF construct with the entire A1 loop deleted (del 1242–1478) or a construct missing part of the A1 loop (del 1392–1402). VWF binding to ristocetin was inhibited by both monoclonal and polyclonal antibodies directed against the VWF A1 loop. VWF A1 loop constructs with the A1 domain polymorphisms D1472H and P1467S showed decreased binding to ristocetin when compared to a wild-type A1 loop construct. Wild-type A1 loop binding to ristocetin in our assay was 0.98 while 1472H A1 loop binding was reduced at 0.77 (p

Description: Type 2A von Willebrand disease (VWD) is characterized by the absence of large von Willebrand factor (VWF) multimers and decreased platelet binding function. These variants are classified as either group 1 (2A-1) with impaired assembly and secretion of VWF multimers, or group 2 (2A-2) with increased susceptibility to proteolysis by ADAMTS13. However, laboratory parameters in individual patients may not discriminate between 2A-1 and 2A-2; this must be established through expression studies. Type 2A VWD patients recruited through the TS Zimmerman Program For The Molecular And Clinical Biology Of VWD (ZPMCB VWD) were phenotypically identified based upon laboratory parameters. By sequencing genomic DNA we identified 13 potentially causative sequence variations in the VWF D2, D3, A1, and A2 domains of type 2A VWD patients. Expression studies were performed to examine the effect of these mutations on VWF processing and proteolysis, and VWF regulated storage. The VWF variants L1503R, S1506L, and V1607D demonstrated severely impaired secretion and lacked mid- and high-molecular weight multimers, consistent with a group 1 classification (2A – 1). Some variants including I1568N, G1579R, and G1631D were normally multimerized and secreted, but had an increased susceptibility to cleavage by ADAMTS13 cleavage, indicative of group 2 classification (2A – 2). Five variants involving cysteine residues (C1190S, C1099P, C1272R/S/Y) had multimer abnormalities, a modest reduction in secretion, and variably enhanced ADAMTS-13 cleavage. These variants did not easily fit the criteria for either 2A subgroup. We also identified sequence variations (M740I and I1380V) that had no effect on VWF secretion, structure, or proteolysis that are likely innocuous polymorphisms but are present in patients with a type 2A phenotype. VWF is stored for regulated release in endothelial cell Weibel-Palade bodies and in platelet alpha-granules. The effect of mutations on VWF regulated storage has been documented for few VWF variants. The 2A variants were expressed in HEK293 cells, immunostained, and examined by confocal microscopy. Several variants including C1272S/Y, L1503R, S1506L, and V1607D did not form storage granules. A loss or reduction in endothelial cell Weibel-Palade bodies may explain the diminished desmopressin response observed in many type 2A VWD patients. In sum, our data indicate that 2A-2 variants are associated with VWF A2 domain mutations and normal regulated storage, while 2A-1 mutations are not restricted to a particular domain and result in loss of regulated VWF storage. Mutations involving cysteines may not affect VWF secretion, but are likely to cause abnormalities in multimer structure. In summary, type 2A VWD appears to result from at least three intersecting mechanisms: intracellular retention, defective multimerization, or increased plasma proteolysis.

Description: Atypical Hemolytic Uremic Syndrome (aHUS) is a rare disease of hemolysis, thrombocytopenia, and organ dysfunction (predominantly renal or CNS) that is often attributed to mutations in the alternate pathway of the complement system. To aid in the evaluation of patients with aHUS, a 15-gene next generation sequencing (NGS) panel was developed. Included in the panel are several genes within the highly homologous region of complement activation (RCA). Sixteen exon pairs across five genes in this region (CFH, CFHR1, CFHR3, CFHR4, and CFHR5) have greater than 95% sequence identity. This leads to difficulties in aligning corresponding NGS reads to the appropriate exons. Accordingly, reference databases for normal populations, as generated by whole genome or whole exome sequencing, are lacking in variant frequency data for these and many other highly-homologous genes, leaving an increased dependence on predictive tools in attempt to classify the pathogenicity of variants. A detail-oriented approach, including allele-specific PCR and Sanger sequencing of longer amplicons to confirm appropriate alignment of reads, allowed for interrogation of these difficult regions for which polymorphism frequency data is sparse. An example of a challenging NGS alignment is reads attributed to CFHR3 exon 5 or CFHR4 exon 9. The reference sequences for these exons differ by one base pair, which correspond to CFHR3 c.721 C and CFHR4 c.1415 T. Combining allele-specific PCR and Sanger sequencing uncovered a variant in CFHR3 (c.721C〉T) which corresponds to the reference nucleotide for CFHR4. Allele-specific PCR involved designing primers in the introns of these genes which results in sequencing a region 770 bases long, or more than three times the length of a typical NGS read. Due to the length of the sequence identity, the NGS read corresponding to this variant always aligned bioinformatically to the incorrect gene (CFHR4) as the reference allele. The variant was analyzed with several algorithms including SIFT, Polyphen2, Condel, and Mutation Taster which predicted it was benign, but the accuracy of these tools is uncertain. This variant was not previously reported in either aHUS patients or the normal population; however, over a 16 month period, allele-specific PCR and Sanger sequencing for this variant of unknown significance was performed on all patient samples and was detected in 14% of the samples tested. In order to better categorize the pathogenicity of the variant, it was necessary to determine whether the subset of patient DNA tested was enriched for the variant because it was associated with aHUS, or whether the variant was present at such a high frequency in the normal population. A high-throughput melt-curve assay was developed. CFHR3 exon 5 and a portion of the adjacent introns was amplified by allele specific PCR and melting curve analysis was performed to determine the genotype using FRET probe technology on the LC480 instrument. DNA extracted from normal blood donor samples, including 96 African Americans, 74 Caucasians, and 112 Hispanics were screened. Overall, 31% of the population was heterozygous for the variant and 16% of the population was homozygous for the variant. The allele was most common in the African American population where 25% of the population was homozygous for the variant and least common in the Caucasian population where 68% of the population was wild-type. This data provides evidence that the variant is benign and not associated with an increased risk of aHUS. Focused large-gene panels, such as this one for aHUS, highlight the ability to meet challenges associated with NGS technology in regions of high sequence identity. We presented our approach for resolution of sequencing information to allow for appropriate classification of variant pathogenicity, resulting in decreased reporting of clinically insignificant results. Disclosures Friedman: Novo Nordisk: Consultancy; Alexion: Speakers Bureau; Instrumentation Laboratories: Consultancy; CSL Behring: Consultancy, Honoraria.

Description: Mild platelet function defects (MPFD) are a variable group of common inherited disorders characterized by mucocutaneous bleeding and excessive bleeding with trauma, surgery and procedures. The diagnosis is often based on subjective interpretations of expensive platelet function tests that require a substantial volume of blood. Furthermore, the diagnosis of MPFD can significantly limit participation in contact sports and physically demanding careers. To explore these issues, a retrospective chart review of patients from the Comprehensive Center for Bleeding Disorders of the BloodCenter of Wisconsin with a diagnosis of MPFD, and who had at least one follow-up evaluation was undertaken. The majority of patients had been evaluated by a two-step process; initially screened with CBC, PT, PTT, TT and a von Willebrand factor (VWF) panel. If these tests were normal, platelet function was assessed with light transmission aggregometry and chemiluminesence detection of ATP release in platelet rich plasma by the agonists TRAP, arachidonic acid (0.5 nM), ADP (10 uM), collagen (2.5 ug/mL) and high and low dose ristocetin. One hundred forty-eight patients diagnosed with a primary MPFD between 1982-2014 and with at least one recorded follow-up were identified. Bleeding scores were assigned by a system incorporating principles developed for the ISTH bleeding assessment tool. Patients ranged in age from 2 months to 57 years. For assessment of age on platelet function, patients were grouped as follows: Group A 16 years (N=26). At diagnosis, median bleeding score was 3 (range 0-12). Bleeding scores increased with age (p

Description: While von Willebrand disease (VWD) is the most common inherited bleeding disorder, most patients have quantitative defects in von Willebrand factor (VWF). The qualitative variants, collectively termed type 2 VWD, are less common, but also in general more severe than type 1 VWD. However, despite a common laboratory phenotype of decreased VWF:RCo/VWF:Ag ratio for types 2A, 2B, and 2M VWD, the clinical phenotype is highly variable. We examined index cases and affected family members enrolled in the Zimmerman Program with a phenotypic diagnosis of type 2 VWD. All subjects had factor VIII (FVIII), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and multimer distribution analyzed in a central laboratory. For calculation of mean VWF:RCo values, a level of 5 was assigned to subjects with VWF:RCo below the laboratory lower limit of detection of 10 IU/dL. A platelet binding assay was also performed using a gain of function GPIb containing 2 mutations that enable spontaneous binding to VWF in the absence of ristocetin (VWF:GPIbM). Full length VWF gene sequencing was performed for all index cases. Targeted sequencing was performed for family members to ascertain the presence or absence of sequence variations found in the index case. Bleeding symptoms were quantified using the ISTH bleeding assessment tool and reported as bleeding scores (BS). Mean FVIII, VWF:Ag, VWF:RCo, and BS are listed in the table below for each type 2 variant. For type 2A VWD, 113 subjects have been enrolled to date. All had an abnormal multimer distribution with loss of high molecular weight multimers. 6 type 2A subjects had a VWF:RCo/VWF:Ag ratio of ≤0.7. The lowest VWF:RCo levels were seen in the type 2A cohort with 60%