56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr.

Vorsitz

Prof.

PD Dr.

Assessment of cellular and plasmatic determinants of the APC response to thrombin in factor V Leiden carriers using an endothelial cell-based ex vivo model

Background The thrombophilic factor V Leiden (FVL) mutation shows a highly variable clinical expressivity. We have shown in vivo that asymptomatic FVL carriers respond to extrinsic coagulation activation with higher activated protein C (APC) formation rates than those with a history of venous thromboembolism (VTE). Aim of this study was to investigate potential modulating factors of the APC response in an ex vivo model of the PC pathway using endothelial colony forming cells (ECFCs) and autologous plasma.Methods ECFCs were isolated from FVL carriers with (VTE+) or without (VTE-) previous VTE and healthy controls (n=7 each). After being overlaid with autologous defibrinated plasma, thrombin formation was initiated by addition of tissue factor (1 pmol/L). Thrombin and APC formation were measured over time using oligonucleotide-based enzyme capture assays (OECAs). Additionally, cross-over experiments with FVL and non-FVL cells/plasma were conducted as well as measurements of ECFC-dependent APC formation in HEPES buffer containing thrombin and the zymogen protein C (PC). Thrombomodulin and endothelial protein C receptor (EPCR) expression were quantified in cell-ELISAs. Inactivation kinetics of exogenously added APC were monitored in plasma by OECA.Results Thrombin formation rates did not differ, but APC formation rates in plasma were higher in the FVL VTE- cohort than in the FVL VTE+ cohort (P=0.011) and healthy controls (P=0.003) as indicated by the ratio between the area under the curve (AUC) of APC generation to the thrombin AUC. Cross-over experiments with non-FVL cells and FVL VTE+ plasma yielded increased APC formation in comparison to the autologous approach (P=0.009), indistinguishable from FVL VTE- plasma. The APC AUC of all other combinations and the thrombin AUC did not differ. APC formation in the purified system, APC inactivation kinetics, thrombomodulin and EPCR expression as well as plasma levels coagulation factors and inhibitors did not differ significantly between cohorts.Conclusion Consistent with previous in vivo experiments, the APC response to thrombin formation was impaired in FVL carriers with a history of VTE in comparison to asymptomatic FVL carriers. However, this difference disappeared when FVL cells were replaced by non-FVL cells in cross-over experiments, suggesting that endothelial cell-specific factors contribute to the APC response and thus to thrombotic risk in FVL carriers. Further studies are warranted to identify these factors.Offenlegung Interessenkonflikt: J.O. has received research funding from Bayer, Biotest, CSL Behring, Octapharma, Pfizer, Swedish Orphan Biovitrum, and Takeda; consultancy, speakers bureau, honoraria, scientific advisory board, and travel expenses from Bayer, Biogen Idec, BioMarin, Biotest, Chugai Pharmaceutical Co., Ltd., CSL Behring, Freeline, Grifols, LFB, Novo Nordisk, Octapharma, Pfizer, F. Hoffmann-La Roche Ltd., Sanofi, Spark Therapeutics, Swedish Orphan Biovitrum, and Takeda. The other authors declare no competing financial interests.

Hemostaseology

Freier Vortrag (Reinhold-Eckstein-Investigator-Award)

Bleeding, FVIII activity, and safety 3 years after gene transfer with valoctocogene roxaparvovec: results from GENEr8-1

Background Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) provides endogenous factor VIII (FVIII) production to prevent bleeding in people with severe hemophilia A. The aim is to evaluate outcomes 3 years after receiving valoctocogene roxaparvovec.Methods The open-label, multicenter phase 3 GENEr8-1 trial (NCT03370913) evaluated 6x1013 vg/kg valoctocogene roxaparvovec in 134 adult men with severe hemophilia A (FVIII ≤1 IU/dL) without inhibitors. Bleeds and FVIII use were self-reported after regular prophylaxis ended (scheduled for week [W] 4) through data cutoff. Comparisons to baseline on FVIII prophylaxis were performed in 112 HIV-negative participants enrolling from a non-interventional study (rollover population). FVIII activity per chromogenic assay and quality of life (QOL) per Haemo-QOL-A were assessed in 132 HIV-negative participants (modified intent-to-treat [mITT] population). Safety was assessed in all participants. Results Median follow-up was 162 weeks (N=134); 131 participants completed W156. Over 3 years in 112 rollover participants, mean annualized treated bleeding rate was 0.8 bleeds/year, mean annualized rate of all bleeds was 1.3 bleeds/year, and mean FVIII utilization was 125 IU/kg/year (Table). During Y3, 73.2% of 110 rollover participants had 0 treated bleeds and 61.6% had no bleeds. At W156, mean and median FVIII were 18.8 and 8.4 IU/dL (mITT, N=132; Figure); Overall, 10/132 (7.6%) participants resumed prophylaxis. Mean Haemo-QOL-A Total Score change from baseline to W156 was +6.6 (n=122; P &lt;0.0001). No new safety signals emerged. In Y3, 34/134 participants (25.4%) had alanine aminotransferase (ALT) elevation.Conclusion Valoctocogene roxaparvovec provided robust hemostatic efficacy relative to FVIII prophylaxis for 3 years, with QOL improvement and stable safety.Offenlegung Interessenkonflikt: Johannes Oldenburg received research support from Bayer, Biotest, CSL-Behring, Novo Nordis, Octapharma, Pfizer, Takeda; advisor for Bayer, Biogen Idec, BioMarin, Biotest, Chugai, CSL-Behring, Freeline, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Spark, Swedish Orphan Biovitrum, Takeda; lecturer at Bayer, Biogen Idec, BioMarin, Biotest, Chugai, CSL-Behring, Freeline, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Spark, Swedish Orphan Biovitrum, Takeda; travel support from Bayer, Biogen Idec, Biotest, Chugai, CSL-Behring, Freeline, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Spark, Swedish Orphan Biovitrum, Takeda

Freier Vortrag

Hijacking of the fibrinolytic system by B-cell acute lymphoblastic leukaemia and its therapeutic targeting

Background The fibrinolytic system consists of the proenzyme plasminogen and its active form, plasmin, a serine protease. The activation of plasmin is tightly regulated by activators such as tissue plasminogen activator (tPA) and various inhibitors. A role of this plasminogen activation system in the microenvironment of solid tumours is being unravelled, but a function of this pathway in the bone marrow microenvironment (BMM), where haematological cancers usually originate, has so far been elusive.Methods We employed the retroviral transduction/transplantation model of B-cell acute lymphoblastic leukaemia (B-ALL), mice deficient for annexin A2 (ANXA2), a calcium-binding protein, which is also found in a complex with the plasminogen receptor protein, S100A10, and various in vitro assays including modeling of the extracellular matrix (ECM) to show the role of degradation of the ECM for B-ALL progression. Results ANXA2 was shown to be essential for plasmin activation and remodeling of the ECM in the BMM, thereby shortening survival in B-ALL. Induction of B-ALL in tPA-deficient mice led to fibronectin accumulation in the BMM and significant survival prolongation. Increased ECM density due to ANXA2 deficiency resulted in an entrapment of growth factors such as insulin-like growth factor 1, altering downstream signaling in B-ALL cells. Secretion of interleukin (IL)-6 by B-ALL cells increased hepatic generation of plasminogen/plasmin/tPA. Therapeutically, inhibition of plasmin activation by ε-aminocaproic acid (EACA), an anti-hemorrhagic drug, in murine models of B-ALL led to survival prolongation and fibronectin accumulation in the BMM.Conclusion Our data suggest that B-ALL cells condition their environment by increasing plasminogen/plasmin generation in the liver by IL-6, thereby, influencing the remodeling of ECM proteins in the BMM and, ultimately, B-ALL progression. We propose that inhibition of plasmin-mediated degradation of the ECM by EACA may be beneficial as an adjunct therapy for B-ALL.Offenlegung Interessenkonflikt: We declare no relevant conflicts of interest.

Antibody-induced platelet phosphatidylserine mediates increased prothrombotic potential in heparin-induced thrombocytopenia

Background Heparin-induced thrombocytopenia (HIT) is a severe adverse event caused by antibodies (Abs) reactive to platelet factor 4 (PF4) and heparin. While HIT Abs are well characterized to activate PLTs via PLT Fc-gamma-RIIA, the contribution of different Ab-induced PLT subpopulations to the prothrombotic environment in HIT remains elusive. In this study, we hypothesized that HIT Abs have the potential to induce a procoagulant PLT phenotype that contributes to the prothrombotic condition in HIT. Methods Using flow cytometry (FC), we analyzed whether HIT Abs have the capability to induce changes in the expression level of the PLT surface activation marker P-selectin (CD62p) and the negatively charged procoagulant membrane phospholipid phosphatidylserine (PS). To investigate whether Ab-induced procoagulant PLTs contribute to increased thrombin generation and thrombus formation, calibrated automated thrombogram (CAT) analysis and an ex vivo thrombosis model were utilized, respectively.Results HIT patient Abs induced a procoagulant (CD62p/PS double positive) PLT phenotype with increased thrombin generation potential. Most importantly, spike-in of whole blood samples with HIT Ab-induced procoagulant PLTs prior to perfusion through a collagen coated microfluidic system resulted in a significant increase of thrombus formation in a heparin- as well as PLT Fc-gamma-RIIA dependent manner. Interestingly, HIT Ab-induced thrombus formation was unaffected by inhibiting the interaction of PLTs with neutrophils via CD62p-glycoprotein ligand-1 (PSGL-1) axis. Contrary, increased thrombin and most importantly, HIT Ab-induced thrombus formation were significantly reduced in the presence of the specific PS blocking protein Lactadherin. Conclusion Based on our mechanistic studies we conclude that increased PLT PS rather than CD62p on the surface of HIT Ab-induced procoagulant PLTs might be an essential factor for increased prothrombotic conditions typically observed in HIT. Offenlegung Interessenkonflikt: There a no conflicts of interest to declare.

Treatment of immune thrombocytopenia (ITP) with eltrombopag - Results of the 5th interim analysis of the study RISA

Background Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated thrombocytopenia due to increased platelet destruction and decreased megakaryopoiesis. Eltrombopag (EPAG) is an oral thrombopoietin-receptor agonist, which is proved to be effective and safe in the treatment of ITP. Here we present data from the 5th interim analysis of the non-interventional prospective study RISA.Methods RISA is a prospective multicenter non-interventional study in Germany. It started in December 2015, enrollment stopped in December 2021, and the observational phase will end by December 2023. Dosage of EPAG and treatment corresponded to the Summary of Product Characteristics (SmPC) and routine of treating physicians. In addition to platelet counts and bleeding events, fatigue is also assessed by using the FACIT-Fatigue (FACIT-F) questionnaire. Statistical analysis is solely descriptive. Data cutoff for this 5th interim analysis was 11th Feb 2022Results 302 of the enrolled patients received at least one dose of EPAG and completed at least one post baseline visit. Mean duration of ITP at baseline was 5.4±7.5 years. Mean age was 62.8±17.4 years. Median treatment duration was 14 months. Treatment with EPAG was carried out at a median dosage of 50 mg daily. Median platelet counts increased from baseline 34.0x109/L to 91.5x109/L within one month and remained stable above 90x109/L until the end of the observation period. The number of bleeding events per patient-year decreased, from 1.35 at baseline to 0.59 and 0.16 after one and two years, respectively. In contrast to the improvement in platelet count and incidence of bleeding events, no significant improvement in fatigue score was observed.Conclusion EPAG therapy in ITP effectively increases platelet counts and reduces bleedings. Our preliminary data show that about 82 % of patients responded to EPAG. This seems to be consistent with the results of the open extension study EXTEND [Wong RSM et al. Blood 2017; 130: 2527-36], in which a similarly high overall response rate (86%) was found. In general, treatment with EPAG was well tolerated, with no new safety signals from our data set. However, ITP-related fatigue needs further investigation.Offenlegung Interessenkonflikt: OM discloses honoraria from Amgen, Grifols, Novartis, ArgenX, and SOBI for lectures and andvisory boards; RS has nothing to disclose; TS discloses honoraria from Novartis, Amgen, Alexion, Janssen, AOP, Celgen/BMS, Sobi, ArgenX, Grifols, AstraZeneca; BF has nothing to disclose; MR discloses honoraria from Roche, Celgene, Abbvie, Janssen-Cilag; DK has nothing to disclose; MW has nothing to disclose; CvdH discloses honoraria from BMS, Novartis, AstraZeneca, Ipsen, Pierre Fabre, Böhringer Ingelheim, and Ipsen; SD has nothing to disclose; CW is employee of Novarits Pharma GmbH; MS has nothing to disclose.Invited talks abstract/summary

Identification of prothrombotic variants in patients with Cerebral Vein Thrombosis using Next-Generation Sequencing

Background Prothrombotic hereditary risk factors for cerebral vein thrombosis (CVT) are underinvestigated but of clinical interest to better understand the underlying pathophysiology and stratify patients for the risk of recurrence. Our exploratory study aimed to identify hereditary prothrombotic risk factors in CVT patients using next generation sequencing (NGS).Methods In our outpatient clinic, 183 patients presented in the years 1999-2021 with a history of CVT. An initial screening identified a number of common prothrombic risk factors, including Factor V Leiden (rs6025) and Prothrombin G20210A (rs1799963). All patients without relevant findings (58 individuals) were invited to participate in a subsequent genetic analysis of 55 relevant genes using NGS. The prevalence of the identified variants in the study population was compared with a general European population, accessing the NCBI database of Genotypes and Phenotypes Release 2.Results Three intron variants (ADAMTS13: rs28446901, FN1: rs56380797, rs35343655) were identified to occur with a significantly higher frequency in the CVT patient cohort compared to the general European population. Furthermore, the combined prevalence of at least two of four potentially prothrombic single nucleotide polymorphisms (SNPs) [FGA (rs6050), F13A1 (rs5985), ITGB3 (rs5918), and PROCR (rs867186)] was significantly higher for five out of six possible combinations in the CVT subjects.Conclusion SNPs rs6050 and rs5985 affect fibrin characteristics, rs5918 enhances fibrin binding, and rs867186 reduces the profibrinolytic effect of APC. According to a higher frequency of at least two of these variants in CVT patients, altered blood clot properties combined with increased platelet binding/reduced fibrinolysis might increase the risk for thromboembolic events, such as CVT. Identification of hereditary prothrombic variants makes NGS a valuable tool for supporting standard diagnostics.Offenlegung Interessenkonflikt: No coflicts of interest.

Hemostaseology

Appointment

Vorsitz

Programme