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Bleeding Disorders 17 th Congress of the European Hematology Association Bleeding disorders 0458 PLASMA CONCENTRATION OF PROTEIN Z AND PROTEIN Z-DEPEND - ENT PROTEASE INHIBITOR IN PATIENTS WITH HAEMOPHILIA A 0457 L Bolkun 1, M Galar 2, D Lemancewicz 2, K Mazgajska-Barczyk 2, E Cichocka 2, J Kloczko 2, J Piszcz 2 NINE NOVEL ADAMTS13 MUTATIONS IN CONGENITAL PREGNANCY 1University Hospital, Bialystok, Poland ASSOCIATED ADULT ONSET TTP 2Haematology Department, Bialystok, Poland K Langley , E Heelas, M Underwood, J Iseppi, I Mackie, S Machin, M Scully UCL, UK, United Kingdom Background. The potential role of alterations in protein Z concentrations in the pathogenesis of coagulation has been investigated in several studies which, ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I however, yielded conflicting results. Protein Z deficiency may induce bleeding domains 13) deficiency is associated with thrombotic thrombocytopoenia pupu - as well as prothrombotic tendencies and it might occur as an inherited disor - ra (TTP), a rare, life threatening thrombotic microangiopathic anaemia. TTP der. Aims. The purpose of the present study was to explore the concentration may be acquired, usually due to the presence of autoantibodies, or congeni - of protein Z and protein Z-dependent protease inhibitor in patients with tal. The congenital form can present in childhood, adolescence or adulthood, haemophilia A. Additionally; it examined the correlation between PZ/ZPI plas - but in adults usually occurs secondary to haemostatic challenge. The differ - ma concentration and bleeding rate and joint bleeding per year. Materials and ences in age of onset are likely to result from the individual repertoire of muta - Methods. The study was based on fifty male patients: 25 with severe, 15 with tions in the ADAMTS13 gene, located on Chromosome 9.DNA sequencing was moderate and 10 with a mild form of haemophilia A. Median age at the time of performed on nine patients with pregnancy associated, adult onset TTP, two of samples taking was 29 (range 19-55 years). None of these patients had under - whom were sisters. Eight of the patients were Caucasian and one of Asian ori - gone prophylaxis of bleeding with FVIII (factor VIII) before the study. All stud - gin. All the patients presented with a markedly reduced ADAMTS13 activity of ied subjects received FVIII on demand, doses depending on the kind of bleed - < 5% (FRETs NR 60-123%) and negative for anti-ADAMTS13 IgG autoantibody. ing. Haemophilia A patients with confirmed hepatitis B or C and with inhibitor Using population data from the 1000 Genomes Project and Hapmap we con - of factor VIII had been excluded from the study. The control group consisted firmed the presence of 3 single nucleotide polymorphisms (SNPs), 10 synony - of 90 healthy male blood donors at median age of 30 (range 19-52). The year - mous SNPs, 5 missense mutations, 4 frame shift mutations and 4 intronic ly bleeding rate and joint bleeding rate were calculated on the basis of data from mutations.Six novel exonic mutations and 3 novel intronic mutations were iden - medical records and home-treatment reports from 2005 to 2011.Quantitative tified. These included two exonic missense mutations: R497C and R1219Q. assessment of protein Z and ZPI in the plasma was performed using commer - Four frame shift mutations were found: p.A111QfsX18, p.N667TfsX31, cial test (Asserachrom Protein Z Elisa Kits, Diagnostica Stago, France) with p.M486VfsX47 and p.I1339SfsX21, one of which occurred in both of the two expected mean value (±SD), 1.56±0.61 μg/mL) and Enzyme-linked Immunosor - siblings. The intronic mutations detected were c.L987+11, c.L1786+90 and bent Assay Kit for Protein Z Dependent Protease Inhibitor (ZPI) (USCN Life Sci - c.Q1245+81. Some SNPs had a higher prevalence in our patients than antic - ence Inc, China). Quantitative determination of FIX in the plasma was done ipated from normal population frequencies, for example R7W is present in 7% using Stago® Deficient IX (Stago,France), with expected normal range of fac - and A1033T is present in 3% of the general Caucasian population, but were tor IX between 60-150%. Active form of FVIII was tested by means of standard found in 7 of the 8 Caucasian patients studied. Exons 4,6,13,15 and 24, exclu - methods. Results. In haemophilia A patients mean plasma concentrations of sively in our cohort, demonstrated several differences from wild type, where - PZ and ZPI were significantly higher than in healthy individuals: PZ (1.87±0.68 as exons 2,3,7,9,10,14,20,22,25,27 showed no variation from wild type in any μg/mL vs 1.49±0.54 μg/mL) and ZPI (5.02±1.11 μg/mL vs 4.22±0.55 μg/mL), of the patients investigated. Notably, seven of the nine patients had both the with p=0.02 and p=0.03, respectively. In the subgroup with severe haemophil - R7W and A1033T SNPs in conjunction with the R1060W mutation. The patients’ ia A, an in-depth analysis revealed a tendency to modulating effect of the PZ full mutational profile (including synonymous mutations) was examined in the (r=-0.53; p=0.072) and a statistically significant one in the case of ZPI (rho=- context of their clinical features. One patient with a moderate clinical profile; only 0.79, p=0.002) on the bleeding rate. It simultaneously disclosed a statistically one heterozygous missense mutation (R1060W) and relatively few exonic dif - significant correlation between the number of bleeds to the joints (20.18±14.1), ferences from wild type (R7W, A900V, A1033T, V970V) however had three PZ (r=-0.72; p=0.04) and ZPI (rho=-0.88, p=0.001). With reference to this par - intronic mutations. It is not yet understood how these may interact to influence ticular group of patients, the study also showed some other statistically mean - the clinical picture, or how their position on the allele may contribute. In con - ingful correspondences: between PZ and ZPI (rho=0.65, p=0.02), PZ and FIX clusions in conjunction with further novel mutations in our cohort, we have also (r=-0.61, p=0.04), as well as ZPI and FVIII (rho=0.78, p=0.002).No such link in documented a higher frequency of SNPs and intronic mutations in association patients with mild and moderate haemophilia A was established. Conclusions. with synonymous SNPS, further work will focus on how these new findings Despite the fact that FVIII deficiency is undoubtedly the main mechanism of result in changes in expression and function of ADAMTS13 in relation to TTP. bleeding in haemophilia A patients, the activity of PZ/PZI complex may play some modulating role in the matter. Further research is required to fully eluci - date the haemostatic role of ZP/ZPI complexes in haemophilia A patients. 0459 WITHDRAWN 186 | haematologica | 2012; 97(s1) Amsterdam, the Netherlands, June 14 – 17, 2012 0460 0461 LOCAL EXPERIENCE IN REVERSAL DABIGATRAN USING ACTIVATED A NOVEL FLOW CYTOMETRY-BASED PLATELET AGGREGATION ASSAY PROTHROMBIN COMPLEX CONCENTRATES (FEIBA) THAT DISCRIMINATES BETWEEN GLANZMANN THROMBASTENIA AND KC Cheung , S Rodgers, S Mcrae LAD-III SYNDROME. IMVS, Adelaide, Australia L Gutiérrez 1, I De Cuyper 1, M Meinders 1, E Van de Vijver 1, A Gerrits 1, D De Kor - te 1, L Porcelijn 2, M De Haas 2, K Seeger 3, A Verhoeven 4, T Kuijpers 5, T van den Background. Dabigatran etexilate (Pradaxa, Boehringer Inhgelheim) is a pro- Berg 1 drug of dabigatran, a reversible direct inhibitor of thrombin that exhibits its effect 1Sanquin Research and Landsteiner Laboratory, Amsterdam, Netherlands by binding directly to the active site of thrombin. The major advantage of dabi - 2Sanquin Diagnostics, Amsterdam, Netherlands gatran over warfarin is that it produces a predictable pharmaco-dynamic effect 3Otto-Heubner-Center for Pediatric and Adolescent Medicine, Charité-Univer - which renders routine laboratory monitoring unnecessary. Several studies sitätsm, Berlin, Germany demonstrated that the efficacy of dabigatran is at least comparable to that of 4AMC, Amsterdam, Netherlands current therapies in preventing stroke related to atrial fibrillation and venous 5Emma Children’s Hospital, AMC, Amsterdam, Netherlands thrombosis following hip and knee replacement surgeries.Like other anticoag - ulants, Dabigatran is associated with a risk of major bleeding with an expect - Background. The main function of platelets is to maintain normal hemostasis. ed rate of 1-2% per annum receiving therapeutic anticoagulation. The optimal Several platelet function tests have been developed to date to be used in the management of a major bleeding complication occurring in patients receiving clinic and in experimental animal models. In particular, platelet aggregation is Dabigatran remains uncertain, and current recommendations regarding the use routinely measured in an aggregometer, which requires normal platelet counts of reversal agents are based on a combination of pre-clinical data, anecdotal and significant blood sample volumes, making difficult analyses in thrombocy - case reports and the absence of effective alternatives. Aims. To determine the topenic patients or infants. For the same reason, analysis of platelet aggrega - usefulness of the CAT in determining the effect of dabigatran at therapeutic con - tion in small rodents can be tedious. Patients with Glanzmann thrombasthenia centrations, and to measure the effect of activated prothrombin complex con - or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) pres - centrate (FEIBA®, Baxter AG, Vienna, Austria) in reversing the haemostatic ent with increased bleeding tendency because of the lack or dysfunction of the defect induced by dabigatran. Methods. The thrombin generation assay was fibrinogen receptor GPIIb/IIIa (integrin αIIb β3), respectively. Although the bleed - performed in platelet poor plasma (PPP) using the Calibrated Automated ing disorder is more severe in LAD-III patients, classic aggregometry or perfu - Thrombogram (CAT) (Thrombinoscope).
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