ALBERT

Description: Abstract 4657 Introduction: Pregnancy is a hypercoagulable state, and thromboembolism is the leading cause of antepartum and postpartum maternal mortality. Women with thrombophilic mutations (factor V leiden, prothrombin, and MTHFR) and inherited bleeding disorders, such as deficiency of factor XIII and fibrinogen, have been shown to be at increased risk of pregnancy loss. However, the risk of miscarriage in women with other inherited bleeding disorders has been discussed controversially. Due to the lack of data, it cannot be determined if the risk of miscarriage is increased in women with von Willebrand disease (vWD). The aim of our study was to clarify the association between inherited bleeding disorders and pregnancy loss. Patients and Methods: Subjects Concerning this investigation we included 91 female patients with two [n=46] or more [n=45] miscarriages occurring prior to 28 weeks of gestation and/or stillbirth without apparent reason. The median age of the examined group at the time of first fetal loss was 29 years, ranging from 17 to 41 years. Methods At first we compiled a detailed clinical history of bleedings of all patients. Subsequently, we performed various tests to gather information regarding coagulation abnormalities and thrombophilic defects. Therefore a molecular and functional assessment of the following data was performed: Coagulation factors, vWF:Ag, vWF:RCo, phospholipid antibodies, hyperhomocysteinaemia (HHCY), protein S (PS), protein C (PC), antithrombin (AT) and FV-Leiden mutation (G1691A), FII mutation (G20210A) and MTHFR C677T. Results: In our investigated population consisting of 91 women we registered 299 pregnancies of which 240 resulted in fetal loss, 232 prior to week 28 of pregnancy and 8 stillbirths. Seven out of 91 patients (8%) were carriers of inherited coagulation disorders; vWD: n=2 (2%), FVII deficiency: n=3 (3%), thrombocytopathy: n=2 (2%). In our study collective there was no increased rate of patients with vWD. None of the patients showed a FXIII- or fibrinogen deficiency. However, 17 patients (19%) have a bleeding diathesis. In 55 patients (60%) we could detect the following thrombophilic defects: FV-Leiden (G1691A): n=10, MTHFR C677T: n=42, PS: n=1, PC: n=1, APS: n=1. Conclusion: The incidence of vWD patients in our miscarriage collective is the same as the overall incidence of vWD patients in the general population. Therefore vWD is not associated with an increased risk of fetal loss. Disclosures: No relevant conflicts of interest to declare.

Description: Background The present study was performed to assess the association of prothrombotic risk factors and underlying conditions, e.g. smoking and obesity, with unexplained recurrent miscarriage (uRM) in white women. Methods and Results From 1998 to 2003, 133 Caucasian women aged 18–42 years (median 28 years) suffering from uRM were consecutively enrolled. In patients and 133 age-matched healthy controls prothrombotic risk factors (factor V (FV) G1691A, factor II (FII) G20210A, MTHFR T677T, 4G/5G plasminogen activator inhibitor (PAI)-1, lipoprotein (Lp) (a), protein C (PC), protein S (PS), antithrombin (AT), antiphospholipid/anticardiolipin (APA/ACA) antibodies) as well as associated environmental conditions were investigated. 70 (52.6%) of the patients had at least one prothrombotic risk factor compared with 26 control women (19.5%; p 30 mg/dL, increased APA/ACA and BMI 〉 25 kg/m2 in combination with a prothrombotic risk factor were found to be significantly associated with uRM. Associations of the heterozygous FII variant, MTHFR or 4G/4G PAI-1 genotypes, deficiency states of PC, PS and AT, as well as the combination of smoking with at least one prothrombotic risk factor did not reach statistical significance. In multivariate analysis, increased Lp(a) (odds ratio (OR): 4.7/95% confidence interval (CI): 2.0–10.7), the FV mutation (OR:3.8/CI:1.4–10.7), and increased APA/ACA (OR: 4.5/CI: 1.1–17,7) had independent associations with uRM. Conclusion In Caucasian women uRM is associated with the presence of elevated Lp(a) or further prothrombotic risk factors.

Description: Introduction Acquired hemophilia A (AHA) is an autoimmune disease caused by the development of inhibitory autoantibodies against factor VIII (FVIII) resulting in severe hemorrhages. Associated pathologies, such as autoimmune disease, malignancy and pregnancy are observed in approximately 50% of patients. Aim To elucidate the relationship between an underlying disease, the bleeding tendency and patients immunological profile the characteristics of anti-FVIII autoantibodies in AHA patients with (n=6) and without an underlying disease (n=9) were determined. Patients and Methods The median age of this cohort (n=15) was 71 years with two-thirds older than 70 years. Treatment parameters were analysed and patients were classified according to there bleeding tendency into a mild, moderate and severe phenotype. FVIII domain specificity of anti-FVIII autoantibodies was analysed in ELISA by binding to (i) FVIII fragments (heavy (HC) and light chain (LC), A2 and C2 domain) and (ii) single human domain hybrid human/porcine FVIII proteins. The amount of FVIII-specific IgGs was measured by ELISA and their relative contribution to the total amount of anti-FVIII IgG was calculated from standard curves for FVIII-specific IgG1, IgG2, IgG3, and IgG4. Results All but one patient were treated with bypassing agents including activated FVII, activated prothrombin complex concentrate or porcine FVIII. All patients received immunosuppressive treatments. 14/15 achieved initial complete remission with 6 patients experiencing another episode of inhibitors. Characteristics of anti-FVIII autoantibodies in AHA patients with or without an underlying disease were similar: FVIII-specific autoantibodies targeted primarily the FVIII LC with a dominance of epitopes located C2 domain compared to the C1 domain. FVIII-specific antibodies belonged to the subclasses IgG1, IgG2, and IgG4. The individual IgG subclass levels did not correlate with the total amount of anti-FVIII antibodies or inhibitory anti-FVIII antibodies in Bethesda units. IgG1 and 2 vs IgG4 levels did not correlate with bleeding tendency. Patients with a mild bleeding phenotype only recognized the C2 domain, whereas other patients had antibodies against C2 and or other domains. Although lower levels of FVIII activity (FVIII:C) were observed in disease-associated AHA patients with a median FVIII:C of 0.5% (range, 0-6%) compared to 1.5% (range, 0-10%) in idiopathic AHA patients, this difference was not statistically significant. FVIII:C levels and FVIII inhibitor titers at clinical presentation did not correlate to the severity of bleeds: the median FVIII:C level in patients who had strong bleeds was 0.5% (range, 0-10%), moderate bleeds 3.6% (range, 0-6%), and mild bleeds 1.2% (range, 1-6%). The FVIII inhibitor titer at presentation was similar in patients who had mild, moderate and severe bleeding tendency with a median of 35 BU/mL (range, 29-55 BU/mL), 49.5 BU/mL (range, 9-156 BU/mL), and 17.3 BU/mL (range, 2.2-614 BU/mL), respectively. Conclusion The presented data challenges the view from other small cohorts that differential immunological profiles exist between disease-associated and idiopathic AHA patients. Data on the influence of epitopes and IgG subclasses on outcome in AHA patients remains conflicting and needs further study. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Antithrombotic therapy with Rivaroxaban [RIVA] is increasingly being administered for secondary TE prophylaxis in adults. The objective of the present study was to evaluate efficacy and safety of standard RIVA administered as routine medication in an outpatient cohort of pts with TE. Furthermore, on an explorative basis we investigated the influence of RIVA on coagulation factors and biomarkers, and the impact of RIVA monitoring during routine administration. Methods: In 140 consecutively admitted whiteoutpts (15-82 yrs; male 56%) with TE and standard RIVA medication (2x 15 mg followed by 20 mg absolute) recruited between January 2013 and January 2014, a comprehensive monitoring of RIVA through (24h) and peak levels (2h, 4h; Xa-based chromogenic substrate S-2732; Haemochrom Diagnostica) alongwith anti-factor-Xa-activities [Xa; Xa-based assay, Haemochrom Diagnostica], selected coagulation factors and biomarkers (factors II, V, VIII, von-Willebrand-Ristocetin-cofactor [RICO], antithrombin [AT], protein C [PC], D-Dimer, prothrombin fragment F1+2 [F1+2], dRVVT-ratio) was performed during routine follow-up. Efficacy endpoints were defined as any TE or thrombus progression during treatment, safety endpoints were defined as significant bleeding requiring any medical intervention, such as dose reduction, withdrawal of RIVA or death related to therapy. Blood samples were taken during routine follow-up visits in the study centers on a monthly (RIVA start) to 3-months (maintenance) interval. Apart from descriptive analysis non-parametric statistics was performed. In addition, chi-square or Fisher’s exact test was applied. Results: During the study period of 15 months in 140 pts 210 follow-up visits including analyses of 420 individual blood samples were performed. Median pt age was 49yrs, with no difference between males and females. Median (min-max) body weight [bw] per kg was 85 (50-151). Median (min-max) daily RIVA dose per kgbw was 0.2 mg (0.09-0.51).Due to a significant lower bw the median daily RIVA dose of 0.24 mg (0.1-0.51) in females was significantly higher compared to males with 0.20 mg (0.09-0.4; p

Description: Acquired hemophilia A (AHA) is a rare autoimmune disease caused by development of inhibitory anti-factor VIII (fVIII) antibodies (also called inhibitors) resulting in severe hemorrhages. In addition, inhibitor development is the most serious complication of today's replacement therapy in patients with hereditary X-linked hemophilia A (HA) disorder. Earlier studies showed that antibodies in AHA and HA inhibitor plasmas are both primarily directed to the A2 and C2 domains suggesting that these two domains are the predominant immunogenic fVIII regions (Fulcher et al, 1987; Prescott et al, 1997; Lollar, 2004). However, the C1 domain also makes a major contribution to the humoral anti-fVIII immune response in hemophilic mice (Healey et al, 2007), which motivated us to analyze the frequency and epitope specificity of anti-C1 antibodies in AHA and HA inhibitor patient plasma. The frequency of domain-specific antibodies were studied by antibody binding to human A2, C1 and C2 domains presented as (i) single human domain (SHD) human/porcine hybrid fVIII and (ii) HSA-fusion proteins. While similar frequencies of A2- and C2-specific antibodies were observed for both applied mapping strategies the use of isolated C1 domain resulted in much higher detection level of anti-C1 antibodies compared to the use of the human C1 domain human/porcine hybrid fVIII protein. As homologue-scanning mutagenesis relies on differences among human and porcine sequences these results suggest the presence of a large number of cross-reactive anti-C1 antibodies binding to species-conserved epitopes. Overall, anti-C1 antibodies were detected in 90 of 115 (78%) AHA and 36 of 63 (57%) HA inhibitor patients. Two well-characterized monoclonal C1 inhibitors, human LE2E9 (Jacquemin et al, 2000) and murine MAb 2A9 (ASH 2014 poster, Batsuli et al) were used for indirect epitope mapping of anti-C1 antibodies in AHA patients by competition binding studies. Our results for AHA patients with non-crossreactive anti-C1 antibodies only (n=11) show that antibody binding to human C1 domain human/porcine hybrid fVIII (HP53) protein was completely blocked in the presence of MAb 2A9. In contrast, antibody binding to the isolated C1 domain was only partially reduced in the presence of MAb 2A9 for a selected number of (high responding) AHA patients (n=10) suggesting the presence of a second population of crossreactive anti-C1 antibodies that exclusively bind to conserved amino acid residues. Competition binding to native and denatured fVIII and HP53 proteins revealed that MAb 2A9 and LE2E9 bind mutually exclusive to a conformational C1 epitope involving amino acid residues that are not conserved between humans and pigs. Consequently, essential binding residues were identified for both C1 inhibitors via the use of HP53 variants, in which surface exposed non-conserved amino acid residues on the human C1 domain were substituted for porcine residues. The results of this mutational analysis showed that despite their competitive binding different amino acid residues are essential for binding of MAb 2A9 and LE2E9. These findings are in agreement with the different specific inhibitory activities of the two C1 inhibitors (97 BU/mg vs 10000 BU/mg). Finally, HSA-C1 point mutants were used to directly map essential epitope residues of anti-C1 antibodies in AHA and HA inhibitor patient plasma. Our study demonstrates that a large number of AHA and HA inhibitor patients (126 of 178; 71%) have anti-C1 antibodies that comprise at least two different populations, crossreactive and non-crossreactive to porcine fVIII. Therefore, in addition to the A2 and C2 domains, the C1 domain seems to significantly contribute to the immune response to fVIII in these patients. As recent data point toward a functional role of the fVIII C1 domain for membrane-, fX-, and von Willebrand factor-binding (Lü et al, 2011) the clinical relevance of anti-C1 antibodies should be analyzed in further studies. Disclosures Tiede: Leo Pharma: Consultancy, Honoraria; Novo Nordisk: Consultancy, Honoraria, Research Funding; Coachrom: Research Funding; SOBI: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Octapharma: Other: Investigator, Speakers Bureau; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Investigator, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Königs:Bayer: Research Funding, Speakers Bureau; Biotest: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Sobi: Consultancy; CSL Behring: Research Funding, Speakers Bureau; Intersero: Research Funding; NovoNordisk: Speakers Bureau.

Description: Background: Venous thromboembolism [TE] is a multifactorial disease and antithrombotic therapy with Rivaroxaban [RIVA] is increasingly being administered for TE treatment in adults. Aim: The objective of the present study was to evaluate efficacy and safety of standard RIVA administered as routine medication in an outpatient cohort of patients with TE. Methods: In 212 consecutively admitted outpatients (14-

Description: Key Points This study is the first to assess prognostic factors in patients with AHA treated according to a uniform immunosuppressive regimen. Residual factor VIII activity and inhibitor concentration at baseline are potentially useful predictors of remission.

Description: Key Points The fVIII C1 domain contributes significantly to the immune response against fVIII in acquired and congenital hemophilia inhibitor patients. B-cell epitopes identified for monoclonal murine and human C1 inhibitors are recognized by antibodies present in patients with hemophilia.

Description: Abstract 2273 Introduction Reversal of pharmacologic anticoagulation is an issue that arises when an anticoagulated patient has a major bleeding or when an emergency surgery needs immediate correction of coagulation. However, the new oral anticoagulants (NOAC) Rivaroxaban (anti-FXa) and Dabigatran (anti-FIIa) lack specific antidotes and only limited data is available regarding the antidotal effect of non-specific haemostatic agents. Therefore, in this ex vivo study reversal of anticoagulant activity after the administration of either 20mg Rivaroxaban or 150mg Dabigatran was tested in vitro using two different PCC (Beriplex®, Cofact®), aPCC (FEIBA®; factor eight inhibitor bypassing activity) or rFVIIa (NovoSeven®) at various concentrations. Patients and Method Subjects 10 healthy caucasian subjects (female: n=5; male: n=5) were first randomized to receive either 20 mg Rivaroxaban or 150 mg Dabigatran in one oral dose. The patients had to abide a 7-day wash-out period before administration of the respective second drug. Methods Citrated venous blood was taken right before (T0) and 2 hours after administration (T2) of either Dabigatran or Rivaroxaban. The potential of 4 commercially available haemostatic agents to reverse the anticoagulant effect of the NOACs was evaluated. • Beriplex: 0.25; 0.5 (corresp. to 25 U/kg); 1; 2 U/mL • Cofact: 0.25; 0.5 (corresp. to 25 U/kg); 1; 2 U/mL • FEIBA:0.25; 0.5; 1 (corresp. to 80 U/kg); 2 U/mL • NovoSeven: 1.25; 2.5; 5; 10 μg/mL (a dose of 90–100 μg/kg corresponds to a plasma level of about 2 μg/mL) Thrombin generation using Calibration Automated Thrombinography (CAT) was the primarily applied assay, along with the parameters aPTT, PT, thrombin time, Rivaroxaban- and Dabigatran levels and Ecarin time. Parameters of interest concerning the thrombin generation assay (TGA) were the endogenous thrombin potential (ETP), Peak (maximum reaction velocity) and Lag time (LT; length of the latent phase). Thrombin generation in platelet poor plasma was initiated by adding 1 pM tissue factor and 4 μM phospholipids. Results 2 hours after administration (T2), Rivaroxaban showed remarkable inhibitory effects on the investigated TGA parameters ETP, Peak and Lag time, with a more pronounced inhibitory effect on the Peak. In contrast, Dabigatran at T2 only showed a slight effect on ETP and no effect on the Peak whereas LT was significantly prolonged (fourfold). Rivaroxaban-induced inhibition of ETP and Peak were reversed by FEIBA in a concentration dependent manner with an over-correction for the two highest concentrations (1 and 2 U/mL). Cofact and rFVIIa restored ETP dose-dependently and both reached baseline T0 at their highest concentrations. Compared to FEIBA, rFVIIa and Cofact only had a slight but dose-dependent effect on the Peak. Interestingly, the other PCC, Beriplex, did not show any reversal effects on ETP and Peak. Regarding Rivaroxaban-prolonged LT all concentrations of rFVIIa and FEIBA were responsible for a significant LT-reduction close to baseline, whereas both PCCs did not correct prolonged LT. Regarding Dabigatran, all doses of rFVIIa, Cofact and FEIBA reduced the LT, with a more pronounced and dose-dependent effect of Cofact and FEIBA. For Beriplex only a slight reduction in LT was observed. Conclusion FEIBA and rFVIIa showed significant reversal of anticoagulant activity already at low therapeutic concentrations for both anticoagulants in TGA. Surprisingly, regarding both investigated PCCs, only Cofact showed an antidotal effect on TGA parameters, especially in higher therapeutic concentrations (1 and 2 U/mL). Considering that the main difference between the two PCCs is that Beriplex contains small amounts of heparin and Cofact does not, it is conceivable that this may explain the observed differences in TGA. Further clinical validation is needed. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4445 In contrast to congenital von Willebrand Syndrome therapy of acute bleeding in acquired von Willebrand Syndrome (AWS) with von Willebrand factor containing concentrates (F VIII/vWF) is often ineffective. In patients with IgG monoclonal antibodies therapy with intravenous immunoglobulins (IVIG) is reported to be effective whereas patients with IgM antibodies seem not to respond to IVIG. Over the last years we diagnosed and treated several patients with AWS either prior to surgery or due to acute bleeding. About half of the patients responded adequately to IVIG as reported in the literature. One patient with no response to IVIG 4 days after the last dose received DDAVP with a significant increase in von Willebrand factor-antigen and ristocetin cofactor and normalized half life. Dental surgery could be performed without any bleeding complications under daily infusions of DDAVP. Another patient with partial response to IVIG and contraindications to DDAVP received F VIII/vWF prior to dental extraction. In contrast to former recoveries with a shortened half life of the infused concentrate (about 2 hours), half life of vWF was significantly prolonged following prior IVIG treatment. Another patient responded sufficiently to single therapy with F VIII/vWF. A further patient with suspected diagnosis of moderate hemophilia A had received DDAVP for severe epistaxis with good response and sufficient half lifes. He was transferred to our centre and we diagnosed AVW with IgM antibodies. We performed another recovery and half life with DDAVP with adequate response. According to our experience we propose the following therapeutic algorithm: A recovery with DDAVP should be performed first line if treatment is not contraindicated. In case of insufficient half lifes and/or intended major surgery, a recovery and half life with F VIII/vWF concentrate should follow. In case of significantly reduced half life of wWF an attempt with IVIG is necessary. If response to IVIG is not adequate further treatment with DDAVP or F VIII/vWF is indicated. As treatment with IVIG is not predictable in all patients with IgG antibodies and is very costly a therapeutic attempt with DDAVP in the first line and F VIII/vWF second line is worthwhile. In major surgery requiring prolonged replacement therapy, IVIG treatment, if effective, is less costly than treatment with FVIII/vWF. Disclosures: No relevant conflicts of interest to declare.