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Canales: Sandoz: Honoraria; iQone: Honoraria; Janssen: Speakers Bureau; Janssen: Honoraria; Roche: Speakers Bureau; Karyopharm: Honoraria; Sandoz: Speakers Bureau; Novartis: Honoraria; Takeda: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Janssen: Speakers Bureau; Roche: Speakers Bureau; Sandoz: Speakers Bureau; Takeda: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Gilead: Honoraria. Butta:NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Pfizer: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Grifols: Research Funding. Alvarez Román:NovoNordisk,: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; Bayer: Consultancy; Grifols: Research Funding. Jiménez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria.

NETosis is a process suffered by neutrophils that consists in the loss of their function and the release of their nuclear material as large web-like structure called neutrophil extracelular traps (NETs). Many authors demonstrated that NETs participate in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), because the release of autoantigens amplifies inflammatory responses, perpetuating the exacerbation of autoimmunity. On the other hand, NETs may play a prominent role in thrombosis because they serve as a negative charge scaffold to trap platelets and coagulation factors, promoting blood clot formation.

Introduction:Platelet surface glycoproteins (GPs) are highly glycosylated and are key elements for platelet function since most of them constitute receptors for adhesion ligands. However, exact role of their glycan composition is not clear. Under normal conditions, platelets contain sialic acid in the carbohydrate side chains of their GPs, and it has been described that alterations in the degree of their sialinization can affect the clearance of platelets. This mechanism has been proposed as involved in etiopathogenesis of immune thrombocytopaenia (ITP), mainly in those patients who do not respond to treatments. Thus, after the loss of sialic acid, there would be a greater exposure of galactose and of N-acetyl-glucosamine residues on the surface of circulating platelets to hepatic Ashwell-Morell receptors, which could induce their phagocytosis and platelet clearance. On the other hand, procoagulant platelets, defined as the platelet subpopulation that binds functional prothrombinase, exposed on their surface increased levels of P-selectin and GPIb, two glycan rich GPs. So, it is tempting to speculate that changes in glycan residues on platelet surface may induce changes in their function.

Fernandez-Bello: Novartis:Speakers Bureau;Stago:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Current Employment, Research Funding, Speakers Bureau;Roche:Speakers Bureau;SOBI,:Research Funding;Pfizer:Speakers Bureau.García Barcenilla:Pfizer,:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Bayer:Speakers Bureau;Novartis:Speakers Bureau;NovoNordisk:Research Funding, Speakers Bureau.Alvarez Román:Roche:Speakers Bureau;Novartis:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Pfizer,:Research Funding, Speakers Bureau;Bayer:Consultancy;SOBI,:Consultancy, Research Funding, Speakers Bureau;Grifols:Research Funding;NovoNordisk,:Research Funding, Speakers Bureau.Martín:NovoNordisk:Speakers Bureau;SOBI:Research Funding;Pfizer:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Novartis:Speakers Bureau.Rivas Pollmar:Novartis:Speakers Bureau;Roche:Speakers Bureau;Pfizer:Speakers Bureau.Canales:Roche:Honoraria;Celgene:Honoraria;Roche:Honoraria;Janssen:Honoraria;Novartis:Honoraria;Sandoz:Speakers Bureau;Sandoz:Honoraria;Janssen:Speakers Bureau;iQone:Honoraria;Sandoz:Honoraria;Gilead:Honoraria;Takeda:Speakers Bureau;Novartis:Honoraria;Karyopharm:Honoraria;Janssen:Honoraria;Takeda:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau;Sandoz:Speakers Bureau;Roche:Speakers Bureau;Karyopharm:Honoraria.Butta:Grifols:Research Funding;Novartis:Speakers Bureau;ROCHE:Research Funding, Speakers Bureau;Pfizer:Speakers Bureau;SOBI:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Speakers Bureau.Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer:Honoraria;F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer:Consultancy;Grifols, Novo Nordisk, Takeda, Sobi, Pfizer:Research Funding.

Fernandez-Bello: Pfizer:Speakers Bureau;Novartis:Speakers Bureau;Stago:Speakers Bureau;Roche:Speakers Bureau;NovoNordisk:Current Employment, Research Funding, Speakers Bureau;Takeda:Research Funding, Speakers Bureau;SOBI,:Research Funding.de la Corte:Pfizer:Research Funding, Speakers Bureau;NovoNordisk:Research Funding, Speakers Bureau;Takeda:Speakers Bureau;Roche:Research Funding, Speakers Bureau;Sobi:Research Funding, Speakers Bureau;Bayer:Speakers Bureau.Alvarez Román:Novartis:Speakers Bureau;Bayer:Consultancy;Grifols:Research Funding;Pfizer,:Research Funding, Speakers Bureau;Roche:Speakers Bureau;NovoNordisk,:Research Funding, Speakers Bureau;Takeda:Research Funding, Speakers Bureau;SOBI,:Consultancy, Research Funding, Speakers Bureau.Martín:SOBI:Research Funding;NovoNordisk:Speakers Bureau;Novartis:Speakers Bureau;Roche:Speakers Bureau;Pfizer:Research Funding, Speakers Bureau.Rivas Pollmar:Pfizer:Speakers Bureau;Roche:Speakers Bureau;Novartis:Speakers Bureau.García Barcenilla:Novartis:Speakers Bureau;Bayer:Speakers Bureau;Roche:Speakers Bureau;Pfizer,:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Research Funding, Speakers Bureau.Canales:Janssen:Honoraria;Sandoz:Speakers Bureau;Takeda:Speakers Bureau;Karyopharm:Honoraria;Novartis:Honoraria;Celgene:Honoraria;Roche:Honoraria;Gilead:Honoraria;Sandoz:Honoraria;iQone:Honoraria;Janssen:Speakers Bureau;Janssen:Honoraria;Roche:Speakers Bureau;Karyopharm:Honoraria;Sandoz:Speakers Bureau;Novartis:Honoraria;Takeda:Speakers Bureau;Roche:Honoraria;Sandoz:Honoraria;Janssen:Speakers Bureau;Roche:Speakers Bureau.Butta:Takeda:Research Funding, Speakers Bureau;SOBI:Speakers Bureau;Pfizer:Speakers Bureau;ROCHE:Research Funding, Speakers Bureau;Novartis:Speakers Bureau;Grifols:Research Funding;NovoNordisk:Speakers Bureau.Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer:Consultancy;Grifols, Novo Nordisk, Takeda, Sobi, Pfizer:Research Funding;F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer:Honoraria.

Fernandez-Bello: Stago: Speakers Bureau; Pfizer: Speakers Bureau; SOBI,: Research Funding; Roche: Speakers Bureau; Novartis: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Current Employment, Research Funding, Speakers Bureau. Justo Sanz:Takeda: Current Employment. Alvarez Román:Bayer: Consultancy; Grifols: Research Funding; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau. García Barcenilla:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Speakers Bureau; NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Bayer: Speakers Bureau. Canales:Sandoz: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Karyopharm: Honoraria; Roche: Speakers Bureau; Takeda: Speakers Bureau; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Janssen: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Gilead: Honoraria; Janssen: Speakers Bureau; Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding. Butta:Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Grifols: Research Funding; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau.

Introduction:

Background:Joint damage is the most frequent and most debilitating comorbidity of haemophilia and can be prevented by adequate prophylactic treatment. Nevertheless, many causes and not only plasma levels of the factor affect joint damage in severe haemophilia (SH) patients. One to be considered is variability on fibrin polymerization capacity since it might influence the bleeding tendency and, consequently, would affect the joint condition in SH patients. To our knowledge, there are not enough studies about that.

Introduction: Trauma-induced coagulopathy (TIC) is a multifactorial condition secondary to severe trauma. In TIC, early fibrinogen (FI) replacement and low dose of recombinant activated factor VII (rFVIIa) may positively impact outcome. Factor XIII (FXIII), on the other hand, may stimulate in vitro clot formation and clot stability. We hypothesized that combination of FI, rFVIIa and FXIII might normalize clot formation more effectively than the isolated use of each concentrate in a model of TIC.

Butta: Grifols: Research Funding; Novartis: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; SOBI: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Speakers Bureau. Alvarez Román:Grifols: Research Funding; Bayer: Consultancy; Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau. Martín:SOBI: Research Funding; Pfizer: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau. Rivas Pollmar:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Justo Sanz:Takeda: Current Employment. García Barcenilla:NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Pfizer,: Speakers Bureau; Roche: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Speakers Bureau. Canales:Celgene: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria; Roche: Honoraria; Gilead: Honoraria; Sandoz: Honoraria; iQone: Honoraria; Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Sandoz: Honoraria; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding.

Hematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment.

Introduction:Hemophilia A (HA) patients under emicizumab prophylaxis treatment may require the concomitant use of procoagulant factors for breakthrough bleedings or immune tolerance induction. Thromboembolic events have been described with the concomitant use of emicizumab and activated prothrombin complex concentrate (aPCC), but not with recombinant activated factor VII (rFVIIa). Previous studies showed that the in vitrocombination of emicizumab and plasma-derived Factor VIII/Von Willebrand Factor (pdFVIII/VWF) had a non-additive effect on thrombin generation (TG)(Bravo M-I, et al J Thromb Haemost. 2020;18:1934-39). The aim of this study was to evaluate the TG resulting from ex vivocombination of plasma samples from HA patients treated with emicizumab, with a pdFVIII/VWF concentrate (Fanhdi ®, Grifols).

Introduction: Platelet glycoproteins are key contributors to platelet function but their glycans structure is unclear. Alterations in glycan composition have been reported to impact platelet clearance under physiological conditions and in the disease mechanism of immune thrombocytopenia (ITP). Therefore, this study sought to characterize glycan structures in human platelets from healthy control individuals and ITP patients using mass spectrometry (MS)-based glycomics approach, andto compare their glycomic profiles to facilitate understanding of glycan alterations in ITP.

Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count <100×10 9/L with or without bleeding manifestations, and diagnosed by exclusion of other causes of thrombocytopenia.

Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; Bayer: Speakers Bureau; SOBI: Speakers Bureau. Canales: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Eusa Pharma: Consultancy, Honoraria; Incyte: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Jiménez-Yuste: Grifols: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; SOBI: Speakers Bureau; Bayer: Speakers Bureau. Canales: Takeda: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Incyte: Consultancy; Sanofi: Consultancy; Novartis: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; Eusa Pharma: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria. Butta: CSL-Behring: Research Funding; Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novo-Nordisk: Speakers Bureau. Jiménez-Yuste: Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding.

Butta: Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; CSL-Behring: Research Funding; Novo-Nordisk: Speakers Bureau. Canales: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; Incyte: Consultancy; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; Eusa Pharma: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Jiménez-Yuste: Pfizer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Alvarez Román: Octapharma: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

Bravo: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Raventós: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Pérez: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Butta: CSL-Behring: Research Funding; Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novo-Nordisk: Speakers Bureau. Jiménez-Yuste: Bayer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Costa: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®. Willis: Grifols: Current Employment, Other: Grifols is a manufacturer of the pdFVIII/VWF concentrate, Fanhdi®.

Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Introduction:

No relevant conflicts of interest to declare.

Stenmo: Novo Nordisk: Employment. Fernandez-Bello:Novo Nordisk: Research Funding. Ezban:Novo Nordisk: Employment. Butta:Novo Nordisk: Research Funding. Jiménez-Yuste:novo Nordisk: Consultancy.

Introduction: The risk of bleeding in patients with thrombocytopenia is increased with platelet counts less than 20 or 30 x 109/L. Nevertheless, some patients with thrombocytopenia of peripheral and central origin (immune thrombocytopenia [ITP] and myelodysplastic syndromes [MDS] respectively) have fewer bleeding symptoms than expected. This fact suggests there may be compensatory mechanisms for the thrombocytopenia, such as the presence of microparticles (MPs).

Immune thrombocytopenia (ITP) is an example of an autoimmune disease in which B-lymphocytes produce autoantibodies against platelets. Antibody-mediated platelet destruction and suboptimal platelet production leads to a decrease in platelet count. ITP patients with thrombocytopaenia have increased plasma levels of a proliferation-inducing ligand (APRIL), a factor that can promote B-cell maturation and survival.Two new compounds that bind to the thrombopoietin receptor (TPO-R) and activate the megakaryopoiesis have been recently approved for the treatment of chronic ITP as second-line treatment.It has been recently reported an improved regulatory T-cell activity in patients with chronic ITP treated with TPO-R agonists (TPO-RA) (Bao et al, 2010). So we aimed to evaluate the effect of TPO-RA treatment on APRIL plasma levels in ITP patients before (ITP-1) and after responding (ITP-2) to the treatment.This was an observational and prospective study. Thirteen patients with chronic ITP in whom treatment with a TPO-RA was indicated, and thirty-three healthy controls were included. ITP patients were studied at two times: at inclusion (ITP-1), when platelet count was less than 30x109/L for patients without concomitant medication or less than 65x109/L for patients receiving corticosteroids or intravenous immunoglobulin; and after a response to TPO-RA therapy was elicited (ITP-2). The response to TPO-RA was defined as a platelet count >30x109/L in patients without additional treatment or >65x109/L for those with concomitant treatments.EDTA-anticoagulated whole blood was centrifuged at 1,500 g for 15 min at 23°C to obtain platelet poor plasma which was then centrifuged at 10,000 g for 15 min at room temperature. Supernatant plasma was stored at –70°C until analysis. Plasma TPO and APRIL concentrations were determined using a commercially available enzyme-linked immunosorbent assay (ELISA, Duoset-R&D, Minneapolis, Mn, USA). Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain).Comparisons of quantitative variables were made with ANOVA and Dunn test. Results were expressed as mean±SD. Correlations were calculated with Spearman test. Values of p≤0.05 were considered statistically significant.Platelet count in the ITP-1 group ((23±17)x109/L) increased after responding to TPO-RA to values similar to controls (controls: (233±77)x109/L and ITP-2: (140±36)x109/L).TPO plasma level was higher in ITP-1 patients (30.05±26.81 pg/ml, p<0.005) than in healthy controls (7.36±11.74 pg/ml) but not significantly different when compared with the values of the ITP-2 group (26.81±17.62 pg/ml).ITP-1 patients showed significantly higher APRIL plasma levels (37.60+28.73 ng/ml, p<0.0001) than controls (2.20±3.11 ng/ml) and ITP-2 patients (4.92+4.68 ng/ml), indicating that TPO-RA treatment caused a diminution in APRIL plasma levels.When looking into the relationship between APRIL plasma levels and platelet count, a significant correlation was only found in the ITP-1 group (r=-0.5919, p<0.05). This supports the potential role of APRIL in the reduction of platelet counts in ITP patients.ITP patients with thrombocytopaenia that responded to TPO-RA treatment increased their platelet count reducing plasma APRIL levels and without changing the moderately high levels of plasma TPO. Reductions in APRIL levels caused by TPO-RA treatment could be an additional mechanism that contributes to an increased platelet count in ITP patients treated with these agents.Bao W, Bussel JB, Heck S, et al. Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents. Blood. 2010 Nov 25;116(22):4639-4645No relevant conflicts of interest to declare.

Immune thrombocytopaenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopaenia caused by autoantibodies against platelet proteins. Bleeding risk in patients with ITP is increased with platelet counts less than 20 or 30 x 109/L. However, patients with ITP often have few bleeding symptoms despite very low platelet counts suggesting the existence of compensatory mechanisms. Moreover, an increased risk for thrombosis in patients with ITP has been described (Nørgaard M, 2012). It has been recently reported that increased production of platelet- and red cells-derived microparticles (MP) might be one of the causes of increased thrombotic risk in ITP patients (Sewify, 2013).The aim of this study was to evaluate the microparticle-associated and plasma procoagulant activities in ITP patients with thrombocytopaenia.Sixty-eight patients with chronic ITP and platelet count less than 50 x 109/L and twenty-two healthy controls were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 1,500 g for 15 min at 23°C. Platelet-poor plasma obtained was additionally centrifuged twice at 23°C (15 min at 1,500 g, and 2 min at 13,000 g) and aliquots were stored at -70ºC until analysis.Phosphatidylserine-MP (Ph-MP) and tissue factor-MP (TF-MP) dependent procoagulant activities were determined with the ZYMUPHEN kits (Hyphen BioMed, Neuville sur Oise, France) following the manufacturer’s instructions.Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) test as described by Hemker et al (2000) at a final concentration of 1 pM tissue factor and 4 μM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak.To test resistance to protein C, CAT experiments were performed without and with the addition of thrombomodulin (TM) (PPP and PPP with thrombomodulin reagents, Thrombinoscope BV, Maastricht, The Netherlands). Results were expressed as the ratio [(ETP with TM)/(ETP without ETP)]x100.Results were expressed as mean±SD. Comparisons of quantitative variables were made with Mann-Whitney test and correlations with Spearman test. Values of p≤0.05 were considered statistically significant.Ph-MP associated procoagulant capacity in ITP patients was higher than in controls (p<0.05) whereas MP-TF associated procoagulant activity was practically negligible in both groups. Plasma procoagulant activity was higher in ITP patients than in controls (ETP: 1604±616 nM x min in ITP patients and 1302±416, p=0.012 in controls; Peak: 328±123 nM in ITP patients and 203±74 nM in controls, p<0.001). We tested whether the higher procoagulant activity of plasma from ITP patients was due to a resistance to protein C. We observed that the mean Ratio value in ITP patients was slightly higher than the mean Ratio of controls (60±18 and 50±13 respectively, p=0.034). Despite this significant difference in the Ratio, no correlation was found between this value and the CAT parameters.ITP patients with thrombocytopaenia had a higher Ph-MP associated and plasma procoagulant activity than controls. The fact that the increased MP-procoagulant activity was not accompanied by a higher TF-MP associated procoagulant activity brings further support to the previous observation that MPs in ITP patients are from platelets and red cells, as both cells express very low levels of TF (Sewify, 2013). Regarding the increased plasma procoagulant capacity observed in ITP patients, our results suggest that resistance to protein C does not seem to be the main mechanism involved. References • Nørgaard M. Thromb Res. 2012;130 Suppl 1:S74-75.• Sewify EM, et al. Thromb Res. 2013;131:e59-63.Hemker HC, et al. Thromb Haemost 2000;83:589-9.No relevant conflicts of interest to declare.

Immune thrombocytopaenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopaenia caused by autoantibodies against platelet proteins. Bleeding risk in patients with ITP is increased with platelet counts less than 20 or 30 x 109/L. However, patients with ITP often have few bleeding symptoms despite very low platelet counts suggesting the existence of compensatory mechanisms. Moreover, an increased risk for thrombosis in patients with ITP has been described (Nørgaard M, 2012). It has been recently reported that increased production of platelet- and red cells-derived microparticles (MP) might be one of the causes of increased thrombotic risk in ITP patients (Sewify, 2013).

Immune thrombocytopenia (ITP) is an example of an autoimmune disease in which B-lymphocytes produce autoantibodies against platelets. Antibody-mediated platelet destruction and suboptimal platelet production leads to a decrease in platelet count. ITP patients with thrombocytopaenia have increased plasma levels of a proliferation-inducing ligand (APRIL), a factor that can promote B-cell maturation and survival.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders with clonal bone marrow anomalies characterized by ineffective hematopoiesis, morphologic and functional hematopoietic cells abnormalities, and an increased risk of transformation to acute myeloid leukemia. Previous work from our lab has shown that MDS patients had an impairment in platelet activation, more externalization of phosphatidylserine (PS) indicating an increased platelet apoptosis and, in some cases, thrombocytopenia. However, these patients do not bleed at the frequency that would be expected taking into account these facts.The aim of this work was to identify the existence of some compensatory mechanisms that could be counteracting the expected bleeding tendency in these patients.Since apoptosis is accompanied by membrane blebbing and production of PS-exposing procoagulant microparticles (MP) shed from the plasma membrane, we assessed the thrombogenic capacity of MP and evaluate their correlation with exposure of PS. We also studied the ability of plasma to generate thrombin after stimulus.Seventy-five patients with MDS, 36% female, mean age 77 years (range: 41 to 93 years) and sixty-eight healthy controls, 29% female, mean age 52 years (range 20 to 76 years) were included. Whole blood was collected in citrated tubes and centrifuged at 1,500 g for 15 min at 23°C to obtain platelet poor plasma (PPP). PPP was subjected to 2 additional centrifugations at room temperature (first: 15 min at 1,500 g and second: 2 min at 13,000 g) following SSC-ISTH recommendations to obtain platelet free plasma (PFP). MP procoagulant activity was determined in PFP with the ZYMUPHEN MP-Activity kit (HYPHEN BioMed, Neuville sur Oise, France).Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) as described by Hemker at final concentration of 1pM tissue factor and 4 microM phospholipids. Four CAT parameters were recorded: endogenous thrombin potential (ETP, total amount of thrombin generated over time), lag time (time to the beginning of the explosive burst of thrombin generation), time to peak (TTP, time to reach the maximum thrombin concentration) and peak height (maximum thrombin concentration obtained). Apoptosis was determined by assessing the level of PS externalization by the binding of FITC-Annexin V to platelets by flow cytometry. Thrombocytopenia was defined as platelet count less than 50,000 platelets/microliter.Platelet count in MDS patients varied from 4,000 to 478,000 platelets/microliter. We observed an increased MP-associated procoagulant activity in MDS patients with thrombocytopenia compared to controls (p<0.05). This would be related to the increased apoptosis observed in platelets from MDS patients with thrombocytopenia (Spearman test r = 0.655, p <0.01). CAT evaluation of plasma procoagulant activity did not show differences between patients and controls in any of the measured parameters.Our results showed an increased procoagulant activity associated to plasma MPs in MDS with thrombocytopenia that might contribute to the minor risk of bleeding observed in these patients. However, further studies are necessary to assess the clinical significance of this finding.No relevant conflicts of interest to declare.

ITP management has changed since thrombopoietin receptor agonists (TRAs) were approved for its treatment. Current TRAs are recommended for adults at risk of bleeding who relapse after splenectomy, as well as for those where splenectomy is contraindicated and where, at least, one other therapy has failed.The goal of this paper is to present the results of the treatment and follow-up of 21 patients treated with Eltrombopag in our Centre during the last year.We present 21 patients diagnosed with chronic ITP according to the American Society of Hematology guidelines. 7 of them were males and 14 females. The median age was 70 years (range from 26 to 81 years). Previous splenectomy was undergone in 3 patients. 10 patients had previously received two or more therapies for ITP. 11 patients received TRAs as second line treatment.On average, platelet counts at the beginning of the treatment with Eltrombopag were 23.000/μL. All patients started with the same doses (50 mg) except one of them who started with 25 mg. Patients only received concurrent ITP therapy (corticosteroids and intravenous immunoglobulins) when platelet counts were under 20,000/μL or bleeding occurred.8 patients needed rescue medications throughout the treatment with Eltrombopag (7 of them only once). Eltrombopag was withdrawn in a total number of 8 patients due to different reasons: 4 patients were considered as non-responders (3 of them didn't achieve a certain platelet count to prevent major bleeding and, in the fourth one, reducing or suspending the concurrent ITP therapy was not possible. One of these non-responders received Romiplostin and a good response was achieved). In one patient, the therapy was withdrawn due to liver toxicity and in another one, at patient's request. Finally, Eltrombopag was withdrawn in 2 patients because a sustained platelet counts was held.TRAs are safe, effective and well-tolerated for those patients who relapse after splenectomy or when splenectomy is contraindicated. In our experience, TRAs could be also useful in other diseases like HCV or HIV associated thrombocytopenia. Eltrombopag and Romiplostin bind in different places to the TPO receptor (TPO-R). This circumstance, together with the different activation pathways, may explain that some non-responders to Eltrombopag could respond to Romiplostin and viceversa.No relevant conflicts of interest to declare.

Abstract 4658

Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopenia caused by autoantibodies against platelet proteins that lead to platelets destruction and suboptimal platelet production. Platelet count and bleeding phenotype vary widely between ITP patients. In spite of the low platelet number, some thrombocytopenic ITP patients seldom bleed which might indicate the presence of additional mechanisms that contribute to the haemostasis in such patients.Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of patients with ITP and can noticeably increase platelet count in most of the ITP patients, even in those with severe refractory thrombocytopenia. The aim of this work was to study platelet function and thrombin generation in thrombocytopenic ITP patients before treatment with TPO-RA and once they had recovered the normal platelet count as consequence of the treatment.Fourteen ITP patients with thrombocytopenia (TP-ITP, platelet count less than 100,000 platelets/microliter) were studied before starting TPO-RA treatment (4 patients with romiplostim and 10 patients with eltrombopag) and after reaching a platelet count higher than 100,000 platelets/microliter (NP-ITP). Thirty-three healthy subjects were included as the control group.Platelet activation was determined by flow cytometry through binding of FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 microM TRAP activated platelets. Immature platelet fraction was determined labeling platelets with thiazole orange. Expression of fibrinogen receptor was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry through FITC-annexin V binding to phosphatidylserine (PS).Thrombin generation was measured in platelet-free plasma by the method of Hemker (Calibrated Automated Thrombography, CAT). Activation was performed with a final concentration of 4 microM of phospholipids and 1 pM of tissue factor.Comparisons of quantitative variables were made with ANOVA and Dunn test. Results were expressed as mean±SD. Values of p≤0.05 were considered statistically significant.Platelets from TP- and NP-ITP patients showed a basal expression of activated fibrinogen receptor similar to controls. Platelets from TP-ITP patients presented a reduced ability for being activated by TRAP (binding of PAC-1 expressed as % of positive platelets: 89±19 % in controls and 56±22 % in TP-ITP, p<0.01). TPO-RA treatment did not improve platelet activation despite increasing platelet count (binding of PAC-1: 47±19 % in NP-ITP, p<0.01). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients. Platelets from either TP- or NP-ITP patients, expressed more PS than controls under basal conditions. Percentage of platelets that bound FITC-annexin V were: 47±12 % in controls, 63±13 % in TP-ITP and 60±15 % in NP-ITP, p<0.001). Percentage of immature platelets in TP-ITP patients were higher than in controls (0.8±0.9 % in controls and 6.6 ±4.0 % in TP-ITP, p<0.05) and was reduced by TPO-RA treatment (in NP-ITP patients: 2.2 ±1.7 %).CAT experiments showed that TP-ITP patients had an increased endogenous thrombin potential (ETP, p<0.001) and reached higher maximum levels of thrombin (peak height, p <0.001) than controls. This procoagulant characteristic of plasma from TP-ITP patients was unchanged after treatment with TPO-RA and the recovery of a normal platelet count.Our results showed that treatment of ITP patients with TPO-RA is effective for increasing platelet production but did not ameliorate platelet function or procoagulant conditions of the disease which may indicate that treatment with TPO-RA do not interfere with the mechanisms involved in impairment of platelet function and generation of a procoagulant state. This increased thrombin generation of plasma from ITP patients has to be taken under consideration when evaluating thrombotic risk of therapeutic treatments, and we would like to point out the need of performing more studies to elucidate causes of the increased thrombogenic potential observed in ITP patients.No relevant conflicts of interest to declare.

Abstract 2821

Abstract 1089