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Background COVID-19 related in-hospital venous thromboembolism (VTE) incidence is high but data reported vary significantly. Some studies show that up to half of the events are diagnosed early after admission. Objectives To study symptomatic VTE incidence in acute COVID-19 hospitalized patients and to describe timing of VTE diagnosis. Methods Multicenter cohort of 5966 patients hospitalized with acute COVID-19. Multicenter Registry of 844 hospitalized patients with acute COVID-19 and associated acute VTE. Results By the time of cohort data collection, 68 patients (1.14%) were still hospitalized, 19.8% had died, and 5.4% required ICU. During a median follow-up of 6 days (IQR, 4–12), 183 patients (3.07%; 95% CI, 2.64–3.55) presented a symptomatic VTE event. The cumulative incidences of VTE at 7, 14 and 21 days in wards [2.3% (95% CI, 1.9–2.7), 3.6% (95% CI, 3.0–4.3), and 4.3% (95% CI, 3.5–5.1)] were similar to the ones reported in ICU [2.2% (95% CI, 1.0–4.4), 2.9% (95% CI, 1.5–5.3), and 4.1% (95% CI, 2.2–6.8)], but at 30 and 60 days were higher in ICU [6.9% (95% CI, 4.2–10.5), and 12.8% (95% CI, 8.1–18.5)] than in wards. Eighty-eight VTE events (48%) were diagnosed early, within 48 h of admission. VTE was not associated with death (HR, 0.79; 95% CI, 0.55–1.12). Conclusions Incidence of symptomatic VTE in our COVID-19 cohort is consistent with that of other real-life studies recently published. Early VTE events are, along with COVID-19, the reason for admission rather than an in-hospital complication.

Thrombocytopenia has been identified as a common complication of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the general population. In an attempt to determine the impact of coronavirus disease 2019 (COVID-19) in patients with immune thrombocytopenia (ITP), a retrospective single-centre study was performed. Thrombocytosis was observed in patients with chronic ITP after SARS-CoV-2 infection, frequently needing treatment adjustment or even discontinuation of therapy. Relapses and newly diagnosed cases showed a fast response after initial treatment compared to ITP. Reduced immune activity due to lymphopenia during COVID-19 could explain this paradoxical effect, although further studies are needed.

INTRODUCTION: We present the first registry of patients with congenital bleeding disorders and COVID-19. The study has been carried out in the Community of Madrid, which has the highest number of cases in Spain. The objective is to understand the incidence of COVID-19, the course of the disease if it occurs and the psychosocial and occupational impact on this population. METHODS: We included 345 patients (246 of haemophilia, 69 of von Willebrand Disease, two rare bleeding disorders and 28 carriers of haemophilia). A telephone survey was used to collect the data. RESULTS: Forty-two patients presented symptoms suggestive of infection by COVID-19, and in six cases, the disease was confirmed by RT-PCR. The cumulative incidence of our series was 1.73%. It is worth noting the complexity of the management of COVID-19 in two patients on prophylaxis with non-factor replacement therapy. Adherence to the prescribed treatment was maintained by 95.5% of patients. Although 94% were independent for daily living activities, 42.4% had a recognized disability and 58% required assistance, provided by the Madrid Haemophilia Association (Ashemadrid) in 75% of cases. Only 4.4% of consultations were held in person. CONCLUSIONS: Patients with congenital bleeding disorders infected with SARS-CoV-2 presented a mild course of the disease that did not require admission. Their identification and treatment by a specialist team from a Haemophilia Treatment Center are essential to make a correct assessment of the risk of haemorrhage/thrombosis. COVID-19 had a major impact on the psychosocial aspects of these patients which must be remedied with recovery plans.

It is important to investigate which factors are associated with the development of arthropathy in people with mild haemophilia (PWMH), in order to prevent it and to limit its effect on function and quality of life.To examine the risk factors associated with arthropathy and to predict its presence and degree of involvement in our PWMH population.This was an observational, cross-sectional cohort study of 85 PWMH under follow-up in our centre. Patient variables (age, body mass index, haemophilia type, genetic mutations, baseline factor levels, age at diagnosis, history of inhibitor, level of physical activity and years of practice, history of haemarthrosis and muscle haematoma) were analysed and related to the presence or absence of arthropathy and the degree of arthropathy (measured with HJHS and HEAD-US). Multivariable models were performed.Some 36.5% of PWMH had arthropathy. The variables that showed an independent association with the presence of arthropathy were patient age (median 42 years) and clotting factor levels (median 10.5 IU/dL). The risk of developing arthropathy increased by 7.9% for each additional year of age and decreased by 7.7% for each 1 IU/dL increase in clotting factor. When arthropathy developed, its degree of involvement might have been influenced by the type of physical activity performed.Patient age and factor levels appear to be the most determinant risk factors associated with the development of arthropathy in mild haemophilia. Monitoring joint health is desirable in this patient population. In patients with arthropathy, physical activities with a low risk of bleeding are the most recommended.

The treatment goal for patients with immune thrombocytopaenia (ITP) is to raise platelet counts to levels that minimize or stop bleeding. Thrombopoietin receptor agonists (TPO-RAs) have been successfully and extensively employed as second-line therapy for ITP. However, TPO-RAs have a small but significant increase in the risk of thrombosis. The aim of this study was to elucidate the mechanisms involved in the pro-coagulant effect of TPO-RAs to take them into account when considering their use in ITP patients with concomitant diseases/conditions that might increase risk of suffering thrombotic events. Eighty-two patients with chronic primary ITP (40 untreated and 42 undergoing TPO-RA therapy) and 112 healthy individuals were recruited. The patients with ITP undergoing TPO-RA therapy presented a pro-coagulant profile due to the formation of a more fibrinolysis-resistant clot because of increased plasminogen activator inhibitor-1 (PAI-1) levels. Increase in platelet content of PAI-1 might be the result of the effect of TPO-RA during megakaryopoiesis, as suggested by experiments performed in MEG-01 cells. Moreover, patients under TPO-RA treatment presented an enhanced pro-coagulant activity associated with microparticles and an increased platelet apoptosis that causes a higher exposure of phosphatidylserine and, consequently, a larger surface for the binding of the prothrombinase complex.

We describe the case of a patient with severe hemophilia A who underwent major orthopedic surgery managed postoperatively by telemedicine (TM). The case is a splendid example of the implementation of TM and good collaboration between a Comprehensive Hemophilia Treatment Center (CHTC) and a local hospital.

In times of crisis, continuous adaptation is necessary. Communication between all members of a research team is key to adapting the development of clinical trials to the context of the epidemiological crisis of coronavirus. We are accustomed to performing day‐to‐day tasks to fulfil protocol requirements with precision. To this end, we typically have had protocol‐guided face‐to‐face visits and we have followed a detailed sequenced procedure during each visit.

Etiopathogenesis of myelodysplastic syndrome (MDS) might cause per se an anomalous haemostasis that can be even more deteriorated by thrombocytopaenia. So, evaluation of haemostasis in patients with MDS rises as a necessity.This work aimed to characterize haemostasis in non-bleeder MDS patients with a platelet count similar to healthy controls to establish differences between the two groups not related to thrombocytopaenia.Thromboelastometry in samples from MDS patients suggested the existence of at least two antagonistic processes: one of them giving a hypocoagulable pattern (prolonged clotting time and lower α angle) and another conferring a procoagulant profile (decreased fibrinolysis). Hypocoagulable state might be due to a decreased ability of platelets to be stimulated and to the presence in plasma of a factor/s that prolonged the time to initiate thrombin generation. This factor/s might be antibodies as this effect was observed in samples from MDS patients with an associated autoimmune-inflammatory condition.Otherwise, hypercoagulable state seemed to rely on an increased presence of red cell- and monocyte-derived microparticles and to the increased exposure of phosphatidylserine that served as scaffold for binding of coagulation factors.We concluded that haemostasis in MDS patients is a complex process influenced by more factors than platelet count.

Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count It is widely accepted the involvement of platelet autoantibodies on deterioration of platelets from patients with ITP. Moreover, an enhanced activity of neuraminidase may also reduce sialic acid from glycoside residues on platelet surface, especially from the highly glycosylated von Willebrand factor (vWF) receptor. Because controversial results regarding the functionality of platelets from ITP patients can be found in literature, we aimed to determine platelet ability to be stimulated by agonists. Moreover, we aimed to determine the way anti-platelet auto- antibodies (abs) and neuraminidase activity may affect the function of platelets derived from MKs of healthy controls. Methods: This observational, prospective and transversal study included 42 patients with chronic primary ITP and 55 healthy controls. Platelet fibrinogen and vWF receptors and activation markers (PAC1 binding to activated fibrinogen receptor and exposure of P-selectin after agonists treatment), were evaluated by flow cytometry. Presence of Antibodies (abs) against platelet's glycoproteins in ITP serum was analysed with a Luminex based assay (LifecodesPak Lx). Neuraminidase (NEU) activity in serum was determined with the substrate 20-(4-methylumbelliferyl)-a-D-N-(MUNANA). Human CD34 + cell-enriched population was obtained with CliniMACS (MiltenyiBiotec) from G-CSF mobilized peripheral blood of a healthy donor. For MK differentiation, CD34 + cells were cultured 12 days in StemSpan™ Serum-Free Expansion Medium II (SFEM II) with 50ng/ml of recombinant human thrompoietin. Then, 10% of serum from healthy controls (4) or ITP patients (4) were added to the culture of mature MKs and incubated for 3 days. Phenotypic analysis of MKs and culture derived-platelets was carried out using abs against CD34, CD41, CD42a and CD42b.Platelet-like particles were considered as CD41-positive events with a size (FSC) and granularity (SSC) scatter properties similar to blood platelets. Culture-derived platelets were stimulated with 100 µM TRAP and 10 µM ADP and activation markers were analyzed by flow cytometry. Results: Expression of fibrinogen receptor on platelets from ITP patients were similar to those from healthy controls but showed a reduced capacity to be activated. Impairment in platelet degranulation measured as exposition of P-selectin after agonist's stimulation was also observed in platelets from these patients (Figure 1). Of note, surface content of CD42b subunit of vWF receptor was reduced (Figure 1). To determine whether diminished platelet function might be due to a plasma component, we induced platelet production from MK of healthy controls as referred in Methods. Abs against platelets and neuraminidase activity were determined in serum samples. Serum from 4 healthy controls or from 4 ITP patients (1 with anti-CD42b, 1 with anti-GPIa-IIa and 2 with undetectable abs) were added to MKs culture. No differences existed in MK differentiation and platelet production between MKs incubated with serum from healthy controls or from ITP patients, but similarly as observed in platelets from ITP patients, MK-derived platelets had an impaired ability to be activated (Table 1). Platelets derived from MKs incubated with ITP serum with anti-platelet abs had also a diminished exposure of CD42b (73±8% of controls). Moreover, neuraminidase content of these samples was slightly higher than that from ITP samples without abs (130 vs 100 % of controls). Conclusion: Platelets from ITP patients had a diminished ability to be stimulated. In vitro study showed that megakaryopoiesis was normal in presence of ITP serum, but released platelets had a lower ability to be activated. Involvement of abs in this effect cannot be ruled out despite we detected abs only in 2 of the tested sera because efficiency of method to detect these abs is ~ 50%. On the other hand, reduced levels of CD42b might be due to the increased activity of neuraminidase. Reduction of sialic acid from CD42b might initiate its metalloproteinase-mediated cleavage or change affinity of the ab used for its detection. Research funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures 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.

Introduction: The management of Factor (F) VII deficiency is complex due to the lack of a clear correlation between FVII levels and the bleeding manifestations of the patients, with a variety of responses to the treatments. Additionally,the presence of FVII inhibitors may also occur. Thus, it is essential to be able to monitoring the hemostatic profile and the effect of the different therapies in each patient individually. Our aim was to perform a personalized analysis of the coagulation status of patients with FVII deficiency and to evaluate their response to new potential therapies using five different coagulation tests. Methods: Six patients were included (clinical data in Table-1):4 with bleeding symptoms on prophylaxis with recombinant activated FVII (rFVIIa), one of them with FVII inhibitors; and 2 untreated patients without bleeding episodes. Blood samples obtained from patients on prophylaxis were collected predose. Prothrombine time (PT) and activated partial thromboplastin time (aPTT)were tested using HemosIL ® RecombiPlasTin 2G, and SynthASil respectively. Rotational Thromboelastometry (ROTEM ®) was performed for monitoring the clot formation using blood with corn trypsin inhibitor (CTI) to inhibit contact activation and a low amount of Tissue Factor (TF) as trigger (dilution 1:50,000 of EXTEM reagent). The thrombin generation (TG) was tested with the Calibrated Automated Thrombogram (CAT) system using platelet poor plasma (PPP) obtained from blood with CTI and activated with only 1 pM TF plus phospholipids (PPP-Low ®, Stago). The plasmin generation (PG) was measured in citrated PPP using a comercial kit (Synapse). The total thrombus-formation analysis system (T-TAS ®, Zacros) was conducted by loading CTI blood samples in AR-chips coated with collagen and thromboplastin for assessing thrombus formation mediated by the activation of the coagulation under flow conditions (High shear). The Area under the flow pressure curve (AUC) was calculated over 30 min after starting. Effects of ex vivo spiking doses of factor replacement (rFVIIa) or non-factor replacement treatments (anti-TFPI, clone mAb2021, Creative Biolabs) were tested. Results: aPTT values remained normal in all patients. FVII deficiency significantly affected PT and patients with more severe bleeding phenotype (patient #1, 2, 3, and 4) showed much longer PT values. Similarly, ROTEM and T-TAS assays showed that FVII deficiency only caused an important delayed clotting time (CT) and very anomalous thrombus formation (AUC) in patients with severe bleeding symptoms.More prolonged lag time (LT) and an important decrease in the peak of thrombin and plasmin generation was also observed in the same patients (#1, 2, 3, and 4). Patient #1 with FVII inhibitors presented a more affected hemostatic profile according to all the coagulation parameters obtained with the five different assays. In contrast, the patients #4 and #5 with absence of bleeding complications showed most of these values within the normal reference range obtained from healthy controls. Concentrations of 1µg/ml (equivalent to 90 μg/kg) of the factor-replacement treatment rFVIIa, showed the normalization of the PT, the clotting time (CT), and the restoration of the thrombin and plasmin generation, and the regularization of the coagulation-dependent thrombus formation in all the patients. In vitro spiking with anti-TFPI (400-800 ng/ml), an alternative non-factor replacement treatment,corrected the thrombus formation (AUC) defects under high shear flow observed in the patients, and produced a significant reduction of CT and LT, and increments of thrombin generation although less effectively than the factor replacement therapy. Conclusions: All the global tests, performed with the described conditions in this study, were sensitive enough to show an abnormal hemostatic profile in the FVII-deficient patients with worst clinical symptoms, validating their use to monitor the risk of bleeding events and the responses to different treatments in this deficiency. These assays may allow to monitoring more personalized treatments to these patients. The results also pointed to the possibility that inhibition of TFPI might be useful for treatment of patients with FVII deficiency, opening the idea of its usage especially as an alternative therapy for patients with inhibitors. Research funded by ISCIII-Fondos FEDER PI19/00772 and PI19/00631 Figure 1 Figure 1. Disclosures 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.

Introduction: 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. We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. Methods: This is a single-centre, case-control, non-interventionist, prospective study. During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed: Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway. Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software. TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA). The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. Results: Those patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests. Table 1 shows the results obtained. Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI. Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI. Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. Conclusions: UBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient. Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes. TFPI inhibition might emerge as a good therapy for some of these patients. Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field. This work was supported by Novo Nordisk Pharma S.A. Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LT: lagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. Figure 1 Figure 1. Disclosures 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.

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. Aim:This work aimed to evaluate if there exists a relationship between fibrin capacity of polymerization and joint condition in SH patients. Methods:This is a prospective and transversal study that was approved by Ethics Committee from Hospital Universitario La Paz. Twenty eight SH patients (25 with SHA, 5 of them with inhibitors; 3 with SHB, 2 of them with inhibitor), median age [p25-p75]=41 [29.8-51.3] years old) were recruited. Joint condition was evaluated using the HEAD-US score. Plasma level of fibrinogen (Fib) was determined by Clauss method. Rotational thromboelastometry (ROTEM) was performed with fibTEM, an extrinsically activated test with tissue factor and the platelet inhibitor cytochalasin D. fibTEM results correlate well in many cases with the Clauss Fib assay, but is additionally influenced by fibrin polymerization ability which cannot reliably be detected with clotting tests. Fibrinogen capacity to increase the clot strength was evaluated by the ratio R= Fib/fibTEM maximum clot firmness (MCFfibTEM) which means the plasma concentration of Fib needed to increase maximum clot firmness in 1 mm. Data were analysed with GraphPrism Pad 6.0. Results:Median value of Fib was 306 [263-341] mg/dl, MCFfibTEM = 12 [10-15] mm and R= 25.0 [20.5-29.5] mg/dl/mm. Patients were classified taking into account the value of the 25th percentile of each variable (Fib, MCFfibTEM and R). Subjects with Fib ≤ 263 mg/dl were considered patients with low fibrinogen level, those with MCFfibTEM ≤ 10 mm were considered patients with low platelet independent maximum clot strength and subjects with a R < 20.5 mg/dl/mm were considered good responders to fibrinogen. Based on this analysis, neither Fib nor MCFfibTEM influenced the joint condition. However, patients with poor response to fibrinogen (R ≥ 20.6 mg/dl/mm) had a significantly greater joint damage than the good responders to fibrinogen (R < 20.6 mg/dl/mm) (Table 1). Poor responders to fibrinogen had frequently joint damage in ankles followed by elbows and knees being cartilage the most commonly affected joint compartment. Good responders to fibrinogen only presented milder joint alterations in elbows and ankles. Conclusions: Our results suggested that polymerization capacity of fibrin may be variable between patients with SH and might influence joint condition. More patients need to be recruited to confirm this finding. This work was supported by grants from FIS-FONDOS FEDER (PI19/00631 and PI19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures 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.

Introduction: Emicizumab is a humanized, monoclonal, bispecific antibody that binds factor (F) FX and FIXa allowing thrombin generation in the absence of FVIII, which is used for routine treatment of patients with Hemophilia A (HA) with and without inhibitors. Plasma level of FIX will be an important limiting factor for the formation of the FX-FIXa-emicizumab ternary complex in the absence of FVIII, suggesting the potential use of FIX concentrates to regulate the procoagulant function of emicizumab and therefore, to use it as an alternative treatment in certain circumstances to stop or prevent bleedings in patients on prophylaxis with emicizumab. At the present time there are several recombinant FIX concentrates with differences in their content of activated FIX (FIXa) and in their half-life (standard or extended) that may differ in their procoagulant effects when combined with emicizumab. Aim: The aim of this study was to evaluate if there were differences in thein vitroprocoagulant effects of two recombinant FIX concentrates, one with standard half-life (rFIX Nonacog Alfa, BeneFIX®, Pfizer) and the other with extended half-life (rFIX fused to rAlbumin, Albutrepenonacog Alfa, Idelvion®, CLS Behring) in samples from patients on prophylaxis with Emicizumab. Methods: This is a prospective and transversal pilot study that was approved by the Ethics Committee from La Paz University Hospital. Two patients with haemophilia A (HA) with inhibitors in prophylaxis with emicizumab were recruited and one haemophilia B (HB) patient was included as a control for the effects of FIX. Blood samples were collected in tubes with corn trypsin inhibitor (CTI, Haematologic Technologies, USA), to block thecontact phase and to only evaluate coagulation mediated by the extrinsic pathway. Levels of FIXa in concentrates of FIX were quantified using the Spectrozyme® FIXa chromogenic substrate (LOXO) and measuring the increase in OD at 405 nm. Rotational thromboelastometry (ROTEM) was performed using whole blood activated by a low concentration of tissue factor solution (final dilution 1:50,000 of EXTEM reagent) plus recalcification. Parameters evaluated in ROTEM were CT (cloting time), defined as time until detection of a clot firmness of 2 mm; and CFT (clot formation time), defined as time between detection of a clot firmness from 2 to 20 mm. Calibrated automated thrombogram (CAT)was performed using platelet free plasma (PFP) activated by low concentration of tissue factor plus phospholipids (PPP-Reagent LOW®, Stago). Parameters evaluated in CAT were: Peak, defined as maximum thrombin concentration reached, in nM; and ETP, defined as the total amount of thrombin generated over time, in nMxmin. Results: The presence of FIXa activity assayed by a chromogenic substrate was not detectable with 20 IU of Idelvion while BeneFIX® showed a specific concentration-dependent FIX activity that was blocked with the serine protease inhibitor EGR-chloromethylketone (Figure 1). ROTEM (Figure 2) and CAT (Figure 3) results showed that the addition of increased concentrations of both concentrates of rFIX produces an enhanced procoagulant effect of Emicizumab similar to the effect produced by the addition of the bypassing agent rFVIIa (NovoSeven®, NovoNordisk). These results also showed that it is necessary three-four times higher concentration (U/dl) of Idelvion® to get similar procoagulant effects that those obtained with BeneFIX®. The higher procoagulant effects of BeneFIX® were also observed in samples of a patient with severe HB. Conclusion: Global coagulation assays suggest that increasing endogenous FIX levels with two rFIX concentrates that have different FIXa content and half-life, produce an enhanced procoagulant effect of Emicizumab opening the idea of the use of these concentrates as an alternative treatment for bleedings in patients with inhibitors on prophylaxis with Emicizumab. Further studies need to be performed to evaluate the procoagulant activity of the concomitant use of different rFIX concentrates and Emicizumab, and to assess security of this therapeutic approach. This work was supported by grants from FIS-FONDOS FEDER (PI19/00631 and P19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures 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.

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. Aim: We aimed to assess in ITP patients whether changes in platelet glycosylation, mainly the loss of sialic acid, may condition platelet function, apoptosis and binding of prothrombinase complex. Methods: This is an observational, prospective and transversal study approved by Ethics Committee from La Paz University Hospital. One hundred and eight patients with chronic primary ITP (68 with a platelet count ≥30x103 platelets/µL and 40 with a platelet count Platelet activation markers were determined in platelet rich plasma; whereas platelet glycosylation, binding of prothrombinase, annexin V and caspase's activities were assayed in washed platelets. Samples were analyzed by flow cytometry. Table 1 shows lectins tested and their sugar-binding specificity. Data were analyzed with GraphPad Prism 6.0 software. Results: Platelets from ITP patients with a platelet count These differences in glycosylation observed in ITP patients with a platelet count Conclusion: Changes in glycoside composition of GPs on platelet's surface impaired their functional capacity, increases their apoptosis and modifies conditions for the binding of coagulation proteins. These modifications in platelet's glycoside residues seem to be related to severity of ITP. This work was supported by grants from FIS-FONDOS FEDER (PI19/00772) and and Platelet Disorder Support Association. EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures 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.

Multifactorial mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) might condition the ability of patients with ITP to respond to treatments. Examining their platelet and immune features, we aimed to detect singular characteristics of patients with ITP who do not respond to any treatment. We studied patients with chronic primary ITP who had been without treatment, or untreated (UT-ITP), for at least six months; included were responders to agonists of thrombopoietin receptors (TPO-RA), patients who showed no response to first- and second-line treatments (NR-ITP), and healthy controls. Platelets from NR-ITP patients exposed a reduced amount of sialic acid residues. Increased loss of platelet surface sialic acid residues was associated with increased platelet apoptosis. NR-ITP patients had an increased fraction of naive lymphocyte (L) B cells and a reduced LTreg (Lymphocyte T-regulator) subset. They also presented an anomalous monocyte and NK (Natural Killer) cells distribution. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls. In conclusion, our results indicate a severe deregulation of the immune system of NR-ITP. The inverse correlation between loss of sialic acid and LTreg count suggests a potential relationship between glycan composition on the platelet surface and immune response, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP.

Introduction: Mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) appear to be multifactorial: autoantibodies, autoreactive CD8+ cytotoxic T cells, enhanced apoptosis and loss of sialic acid which mediates platelet clearance through the Ashwell-Morell receptors present in hepatocytes. Differential involvement of each of them might condition the ability of patients with ITP to respond to treatments. We aimed to examine platelet features and the immunological state of patients with ITP who do not respond to any treatment to detect the unique characteristics of this group. Methods: This was an observational, prospective and transversal study. Patients with chronic primary ITP were included: 28 ITP patients without treatment for at least 6 months (UT-ITP); 36 responders to agonists of thrombopoietin receptors (TPO-RA); and 14 ITP patients who did not respond to first- and second-line treatments (NR-ITP). A healthy control group (n=104) was also included in the study. Active caspase-3, -7, -8 or -9 were determined by flow cytometry using CaspaTag kits (Millipore, Madrid, Spain) in PRP diluted with HEPES-buffer containing 2 mM Ca2+ and 2 mM Gly-Pro-Arg-Pro (Sigma-Aldrich, Madrid, Spain) to prevent fibrin formation . Platelet surface glycan exposure was analysed by determining the binding of lectins by flow cytometry. To do so, washed platelets were incubated with 1 μg/ml Alexa fluor 488-conjugated wheat germ agglutinin lectin (WGA, Invitrogen, Spain) or with 1 μg/ml FITC-conjugated Ricinus communis agglutinin (RCA, Vector Labs, UK). WGA binds to sialic acid and N-acetylglucosaminyl residues, and RCA is a galactose-specific legume lectin which binding serves as an indirect measurement of the loss of sialic acid. Peripheral blood mononuclear cells (PBMCs) subsets were analysed by flow cytometry using specific antibodies. Experimental data was analysed using SPSS 9.0 software (SPSS Inc., Chicago, IL). Results: Platelets from TPO-RA treated and from NR-ITP patients had increased caspase-3, -7, -8 and -9 activities (Figure 1A). Platelets from NR-ITP patients exposed less sialic acid and more N-acetylglucosaminyl residues than the other groups (Figure 1B). Binding of WGA and RCA correlated with caspase activities (Table 1). Distribution of lymphocytes, monocytes and natural killer cells is shown in Table 1. NR-ITP patients had an increased proportion of B lymphocyte (LB), maybe due to a significant rise in the fraction of naive LB cells, and a diminution in LTreg subset. Whereas classical monocytes was increased, nonclassical monocyte fraction was decreased in the UT-ITP and NR-ITP groups. NR-ITP patients also presented an increased CD16+CD56bright cells fraction and a diminished NK CD16+CD56dim subset. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls (monocyte and NK cells subset distribution and LTreg count similar to control group). It is of interest to note the relationship between loss of sialic acid from platelet surface glycans and Tregs count: the most reduced surface exposure of sialic acid, the less Treg count (Figure 2). Conclusions: Platelets from NR-ITP patients had more signs of apoptosis and a different composition of surface glycans, accompanied by a diminished LTreg population, a higher LB naïve percentage, and an increased CD16+CD56bright cells fraction in circulation, indicating a severe deregulation of the immune system. Since an inverse correlation was observed between loss of sialic acid and LTreg count, a potential relationship between glycan composition on the platelet surface and immune response is suggested, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP. On the other hand, TPO-RA appears to have a beneficial effect on immune response. Nevertheless, one of the limitations of our study was that patients were recruited once the response to TPO-RA was achieved; therefore, a longitudinal study would provide more information regarding TPO-RA effects. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Álvarez Roman: Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau. Fernandez-Bello:Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Martín:SOBI: Research Funding; Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. Canales:Novartis: Honoraria; Takeda: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Celgene: Honoraria; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Janssen: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

Introduction: The incidence of bleeding in patients with severe (S) haemophilia A (HA) on prophylaxis with emicizumab is similar to that seen in patients with moderate-mild HA, therefore these patients will require administration of factor (F) VIII concentrates (patients without inhibitors) or bypassing agents (patients with inhibitors) in case of acute bleeding or surgery. In the absence of FVIII, thrombin generation is guided by the FIX plasma levels that becomes the limiting factor for the formation of the FX-FIXa-emicizumab ternary complex. We hypothesize that the increment of level of FIX in patients on prophylaxis with emicizumab would increase their procoagulant function. Objectives: To measure the in vitro procoagulant effect of increasing factor IX activity in patients on prophylaxis with emicizumab. Material and Methods: We performed a study in 6 patients on prophylaxis with emicizumab (2 patients with inhibitors) and 20 healthy controls. We evaluated the in vitro procoagulant effect of therapeutic concentrations of recombinant activated FVII (rFVIIa), activated prothrombin complex concentrate (aPCC) and several concentrations of FIX using global assays as thrombin generation test (CAT) and thromboelastometry (ROTEM). Results: In correspondence with our hypothesis, our ROTEM results indicated that increasing concentration of FIX up to 110% was able to normalize the procoagulant capacity of all patients. Further increment of in vitro FIX concentrations up to 125% had a procoagulant effect similar to what would be obtained after one standard dose of 90 mcg/kg rFVIIa. In regard to aPCC, we needed to increase FIX levels up to 200 IU/dl to achieve a procoagulant effect similar to what would be obtained with a dose of 2.5 IU/kg of aPCC. CAT showed total normalization of thrombin generation in all patients with levels of 125 IU/dl of FIX which support the idea of normalization of procoagulant function of patients on prophylaxis with emicizumab in response to low increments of FIX. Moreover, similar to ROTEM, 200 IU/dl of FIX had comparable procoagulant effect to concentrations of aPCC that would be obtained after one dose of 2.5 IU/kg aPCC. With these results we might speculate on a possible good safety profile of this high level of FIX in patients on prophylaxis with emicizumab if we take into account that 2.5 IU/kg aPCC is 20 times lower than aPCC dose [50 IU/kg] used in HAVEN-1 with no associated incidence of thrombotic events, however, more studies are needed to explore this hypothesis. Conclusions: In vitro increment of FIX plasma levels enhances in vitro procoagulant function of patients on prophylaxis with emicizumab opening the idea of an alternative hemostatic treatment in this type of patient. The use of FIX concentrates with much longer half-life compared to FVIII concentrates or bypassing agents in patient on prophylaxis with emicizumab might produce longer period of time with a normalized haemostatic function which might speed up the recovery, might reduce the incidence of bleeding complication and would require much less number of administrations, this later of paramount importance in patients with limited venous access. However, more studies should be addressed to confirm these results. Moreover, and even more important, prothrombotic risk of combinatory therapy with FIX and emicizumab should be carefully studied in pre-clinical studies. NB holds a tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Fernandez-Bello: Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Alvarez Román:Novartis: Speakers Bureau; Novo Nordisk: Speakers Bureau; Bayer: Speakers Bureau; CSL Behring: Speakers Bureau; Roche: Speakers Bureau; Sobi: Speakers Bureau; Amgen: Speakers Bureau; Shire (Takeda): Research Funding, Speakers Bureau. Martín:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. García Barcenilla:SOBI: Research Funding; Bayer, Pfizer, Takeda, Novartis: Speakers Bureau. Canales:Sandoz: Honoraria; Gilead: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria, Speakers Bureau; Takeda: Speakers Bureau; SOBI: Research Funding; Celgene: Honoraria; iQone: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Novartis: Honoraria. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau.

Introduction: Efmoroctocog alfa (Elocta®) is a recombinant coagulation FVIII-Fc (rFVIIIFc), a fully recombinant fusion protein produced in human embryonic kidney cells, with an extended half-life used for the treatment and prevention of bleeding in patients with severe hemophilia A. Using rFVIIIFc for the treatment of severe hemophilia A patients received the approval of reimbursement in Spain at the end of 2016. Therefore, there are no many comparative data published about real life use of rFVIIIFc. Objective: This work aims to describe characteristics of the treatment of severe hemophilia A patients with rFVIIIFc and to compare its results with those previously obtained employing other FVIII products. Methods: This was an open-label non-interventional retrospective study reviewing patient characteristics and treatment outcomes before and after the use of rFVIIIFc. The La Paz University Hospital Ethics Committee approved the experimental protocol. Patients with severe hemophilia A without inhibitors being treated with rFVIIIFc since at least six months before study approval by Ethics Committee were included. The following data were collected for patients included in the study: dose (IU/kg) and prophylaxis treatment regimen, number of spontaneous and traumatic bleedings, annual bleeding rate (ABR) and FVIII trough level. The statistical analysis on the variables listed above comparing before and after rFVIIIFc usage was performed by the Biostatistics Unit of La Paz University Hospital with the statistical package SPSS v.18.0 (SPSS Inc., Chicago, IL, USA). Results: Twenty two severe hemophilia A patients (median age: 20 years old, ranging from 6 to 63 years) on prophylaxis with rFVIIIFc were considered to be included in this study, but two were excluded due to lack of data. Median follow-up period was 14 months (ranging from 6 to 28 months). Nineteen severe hemophilia A patients have been previously treated with rFVIII (two of them with other extended half-life product) and one with plasma-derived FVIII. Eight of the ten severe hemophilia A patients who presented an ABR greater than 0 with previous treatments reduced their ABR when treated with rFVIIIFc (Table 1). Among those patients with an ABR=0 with previously used FVIII products, only one increased to an ABR=1 when treated with Elocta® due to a traumatic bleeding. Table 1 shows ABR across all patients before and after rFVIIIFc. There was no difference in dose per injection between other FVIII products and rFVIIIFc (median dose for patients treated with other FVIII products: 46.0 IU/kg, ranging from 26 to 65 IU/kg; median dose for patients treated with rFVIIIFc: 46.5 IU/kg, ranging from 26 to 65 IU/kg). Nevertheless, a reduction was observed in administration frequency. Among the twelve patients who received treatment with other FVIII products every 48 hours, eleven came to receive rFVIIIFc 3 times a week and the one previously receiving a plasma-derived FVIII, to twice a week. Five of the patients receiving treatment 3 times a week reduced its frequency to twice per week. Three patients maintained the same schedule of administration. To note, one of the two patients receiving another prolonged half-life product maintained the schedule of treatment and the other reduced its frequency from every 48 hours to 3 times a week. FVIII trough level in plasma (% of FVIII), expressed as median (25th-75th percentile), was 1.1 (0.1-4.0) for rFVIIIFc treatment and 0.2 (0.0-1.9) for other FVIII products (p=0.06). Conclusions: 85% of the severe hemophilia A patients from our cohort reduced the weekly dose administration after beginning treatment with rFVIIIFc. Most of the patients increased plasma trough level of FVIII with rFVIIIFc. 45% of patients reduced and 40% kept their ABR=0 when they changed rFVIIIFc. These data suggest that treatment with rFVIIIFc gives a higher protection to severe hemophilia A patients. However, further research with larger sample size is required to investigate this. This work was supported by SOBI. NB holds a tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Álvarez Roman: Takeda: Research Funding; Amgen: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau. Fernandez-Bello:Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Martín:SOBI: Research Funding; Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. García Barcenilla:Bayer, Pfizer, Takeda, Novartis: Speakers Bureau; SOBI: Research Funding. Canales:SOBI: Research Funding; iQone: Honoraria; Karyopharm: Honoraria; Novartis: Honoraria; Takeda: Speakers Bureau; Gilead: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Sandoz: Honoraria. Butta:Roche, Pfizer: Speakers Bureau; Novartis: Consultancy. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau.

Despite their low platelet count some immune thrombocytopenia (ITP) patients seldom bleed, indicating the presence of factors to compensate thrombocytopenia. Moreover, ITP patients may have an increased risk for thrombosis. These facts suggest the presence of procoagulant mechanisms that have not been clarified yet. The aim of this study was to identify these possible factors. Moreover, the utility of rotational thromboelastometry (ROTEM® ) to test haemostasis in these patients was also evaluated. Patients with ITP presented a procoagulant profile due to an increased amount of platelet- and red cell-microparticles, an increased resistance to protein C and the formation of a clot more resistant to fibrinolysis due to augmented levels of plasminogen activator inhibitor-1, which might reflect an endothelial damage/activation in ITP patients. Despite increased maximum clot firmness and reduced lysis, ROTEM® profiles showed a prolonged clotting time that might rely on the presence of anti-platelet antibodies as suggested by the increased lagtime in thrombin generation test caused by plasma from ITP patients on platelets from healthy controls. These results indicate the need to individualize therapeutic treatment for ITP patients, considering their procoagulant profile and the presence of concomitant risk factors. Moreover, ROTEM® appeared to be useful for evaluating haemostasis in ITP patients.

Introduction: Obstructive sleep apnea syndrome (OSA) is a common disorder characterized by repetitive episodes of nocturnal breathing cessation due to upper airway collapse that cause intermittent hypoxia. Because of this, there is an increase in oxidative stress, endothelial damage and mortality associated with the incidence of thrombotic events. The evaluation of platelet function by flow cytometry (FCM) and hemostatic capacity through global hemostasis tests have been successfully used in the study of the prothrombotic state associated to numerous diseases. We anticipate that these techniques may help to clarify the mechanism involved in the prothrombotic state of OSA. Materials and Methods: We included 16 OSA patients diagnosed in the Pneumology Unit of the University Hospital La Paz and 59 healthy controls recruited in the Donor Unit of the same Hospital. Platelet surface receptors were analysed by FCM (FACScan, BD Biosciences) with specific monoclonal antibodies (mAbs) against CD41/αIIb and CD61/β3, CD42a and CD42b. Platelet activation was assessed by FCM through PAC1-FITC binding (mAbs that recognizes activated conformation of fibrinogen receptor), P-selectin and CD63 exposure in baseline condition and after stimulation with 100 µM TRAP (agonist of the PAR-1 receptor) and 10 µM ADP. Platelet apoptosis was analysed by FCM through FITC-annexin V (BD Pharmingen) binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions and by determination of activity of caspases -3/7, -8, and -9 (BD Millipore). The overall hemostatic capacity was determined by the evaluation of thrombin generation using the Calibrated Automatic Thrombinography (CAT) with fresh platelet-poor plasma (obtained after double centrifugation of whole blood at 2500g, 20 minutes and 24ºC) and by the assessment of kinetics of clot formation using Rotational Thromboelastometry (ROTEM® Gamma, Tem Innovations GmbH, Spain) using whole blood and activation by recalcification (naTEM® test, Tem Innovations GmbH, Spain). The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Statistical significance was established at p Results: No significant differences in age or sex were found between groups. Patients presented anxiety-depressive disorder (15%), ischemic cardiomyopathy (8%), dyslipidemias (38%), arterial hypertension (69%) and lower minimum oxygen pressures during sleep (mean±SD; patients: 76±8 % vs controls: 89±1 %; p< 0.001) indicating hypoxia. In basal conditions, an increase of CD42a subunit of the von Willebrand factor receptor was observed in patients with OSA but no differences were found in the exposure of fibrinogen receptor either at basal condition or after activation with TRAP or ADP (Table 1 and Figure 1). Platelet PS exposure before and after TRAP stimulation was higher in the patient group (Figure 1) but no differences between groups were observed in caspase activities (Table 2). Clotting time (CT), maximum clot firmness (MCF) and MP-associated thrombin generation were higher in patients with OSA (Figure 2). Conclusion: Mechanisms related to the mobilization of P-selectin to platelet surface and with the maintenance of phospholipids distribution in platelet membrane might be altered in OSA. The increment of P-selectin exposure may favor the interaction between white cells and platelets through PSGL-1 ligand from lymphocytes which in turn may produce endothelial damage explaining part of OSA's pathophysiology. A lower CT and higher MCF reflected a hypercoagulable state in patients, perhaps related to their higher MP-associated thrombin generation and, at least in part, due to the enhanced platelet-PS exposure. Our results also suggest that both ROTEM and CAT are useful tools for characterizing prothrombotic states in OSA patients. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Disclosures Álvarez-Roman: Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding; Sobi: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet count (≤100x109/L) due to platelet destruction and insufficient platelet production. There is a wide variation in the presentation of the disease, the clinical course and the response to therapeutic treatments. It has been proposed that certain autoimmune diseases might be caused by changes in glycosylation of cell surface proteins which induce the immune system to recognize cells as "non-self". The aim of this work was to evaluate whether glycosylation might be involved in ITP ethiopathogenesis. So, in this preliminary study we aimed to evaluate the glycosylation pattern of platelets from ITP patients, its relationship with platelet features and with the response to treatments. Methods: Seventy eight ITP patients were recruited: 32 without treatment (UT-ITP) for at least six months, 35 responders to thrombopoietin receptor agonists (TPO-RA, 62.8% with eltrombopag, 27.2 % with romiplostim), and 11 refractory (r-ITP). Eighty one healthy controls were also included. Human peripheral blood samples were collected in 3.8% sodium citrate. Platelet activation was determined by flow cytometry (FCM) through binding of FITC-PAC1 (a mAb that recognizes the activated conformation of fibrinogen receptor) after activation with 100 mM thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland). Apoptosis was evaluated by measurement of active caspase-3, -8 or -9 by FCM with a specific kit (Millipore, Madrid, Spain). Platelet surface exposure of lectins was analyzed with 1 µg/ml FITC-conjugated Wheat germ agglutinin lectin (WGA) or 1 µg/ml FITC-conjugated Erythrina cristagalli lectin (ECA). WGA binds to sialic acid and N-acetylglucosaminyl residues and ECA is a galactose specific legume lectin. Glycoprotein derived glycans from a healthy control and from a r-ITP patient were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion whilst O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-MS. Comparisons of quantitative variables were made with SPSS.22 software. Results: Platelet count (x109/L) was 269±55 in controls, 141±126 in UT-ITP, 111±85 in TPO-RA and 8±5 in r-ITP. ITP patients with the lowest platelet count showed the most pronounced binding of WGA and ECA (Table 1). Moreover, binding of these lectins showed a mild positive correlation with caspase activities (Table 1). No relationship was observed between platelet ability to be activated and glycosylation features. When lectins' binding was studied stratifying patients according to their response to therapeutic treatments we observed that platelets from all ITP patients bound more ECA and WGA (Figure 1). Despite no significant differences were observed between ITP groups, r-ITP seemed to bind more WGA. To increase our knowledge about protein glycosylation of platelet proteins in ITP patients we performed glycomic analyses of platelets from one healthy control and from one r-ITP patient. It was demonstrated that bi-, tri- and tetra-antennary complex type N-glycans are the dominant class of N-glycans with also some high mannose structures. Analysis of sialylated N-glycan, via digestion with sialidase, revealed lower levels of sialylation in r-ITP patient platelets compared to healthy control platelets. Platelet O-glycan data revealed the presence of mucin-type core-1 and core-2 structures. Conclusion: Glycomic analysis demonstrates that human platelet glycoproteins are modified with a complex array of both N- and O-linked glycans. Preliminary data indicates that there could be changes in glycosylation associated with immune thrombocytopenia. These changes in platelet protein glycosylation might be involved in an enhancement of apoptosis in platelets from ITP patients. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Disclosures Álvarez-Roman: Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Bayer: Consultancy, Research Funding; Sobi: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; NovoNordisk: Consultancy, Research Funding; Shire: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

Background: The treatment goal for patients with immune thrombocytopenia (ITP) is to raise platelet counts to levels that minimize or stop bleeding. Thrombopoietin receptor agonists (TPO-RAs) have been successfully and extensively employed as second-line therapy for ITP. TPO-RAs, however, have a small but significant increase in the risk of thrombosis. Aim: The aim of this study was to elucidate the mechanisms involved in the procoagulant effect of TPO-RAs. Methods: This is a prospective, observational and transversal study. Eighty-two patients with chronic primary ITP, 40 without treatment for at least six months (UT-ITP) and 42 responders to TPO-RA therapy (64.3% with eltrombopag and 35.7 % with romiplostim) were recruited. One hundred and twelve healthy participants were also included. ROTEM® (naTEM test: only recalcification) was performed on platelet rich plasma adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until 2 mm of amplitude [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2 mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded. Surface exposure of phosphatidylserine (PS), active caspase-3, -8 or -9 and prothrombinase complex binding to platelets were assessed by flow cytometry. Plasma and platelet levels of PAI-1 were determined by ELISA (eBioscience Ltd., Hatfield, United Kingdom). The effect of TPO and romiplostim on PAI-1 content of MEG-01 cells was evaluated by Western blot. Three MEG-01 cell cultures were initiated simultaneously: control without drugs and treated with either TPO (100 ng/mL) or romiplostim (53 μg/mL). Samples were collected at the start and after 24, 48 and 72 hours to determine the PAI-1 content. The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Results: The ROTEM® studies showed significant differences in the dynamics of clot formation when comparing the control with ITP samples. There was a delay in clot formation in the UT-ITP group, as observed by a prolonged CT [expressed as median (p25-p75): control: 516 (490- 633) s; UT-ITP: 938 (914-1348) s, p Higher values of MCF observed with platelets from ITP patients treated with TPO-RAs might be a consequence of their augmented apoptosis signs: platelets from this group exposed more PS than controls and this situation was accompanied by an increased activity of caspases-3,7, -8 and -9 (Figure 1 A and B). Moreover, platelets from ITP patients on treatment with TPO-RAs bound more prothrombinase complex than platelets from UT-ITP patients and healthy controls (Figure 1 C). Reduced clot lysis observed in ITP patients treated with TPO-RA was due, at least in part, to increased plasma and platelet levels of PAI-1 (Table 1). Increase in platelet content of PAI-1 might be the result of the effect of TPO-RAs during megakaryopoiesis since treatments of MEG-01 cells with TPO or romiplostim induced a 3-fold increase in their endogenous PAI-1 content after an incubation period of 48 hs. Conclusion: The patients with ITP undergoing TPO-RAs therapy presented a procoagulant profile due to the formation of a more fibrinolysis-resistant clot because of increased platelet and plasma PAI-1 levels. Moreover, platelets from this group of patients showed more signs of apoptosis that causes a higher exposure of PS and, consequently, a larger surface for the binding of the prothrombinase complex. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Disclosures Álvarez-Roman: SOBI: Consultancy; NovoNordisk: Consultancy; Shire: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Sobi: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

Introduction: The goal of treatment of patients with immune thrombocytopaenia (ITP) is to raise platelet count to a level that will minimize or stop bleeding. One of the therapeutic strategies employed is to augment platelet production with agonists of the thrombopoietin receptor (TPOR-A). Clinical trials for TPOR-A comparing their effects with placebo showed a reduction in proportion of patients reporting bleeding (Tarantino et al, Blood Coag Fibrinolysis, 2013, 24:284-296). This effect might rely on the increase in platelet count, but other factors known to compensate bleeding risk in thrombocytopenic ITP patients might be regulated by TPOR-A. Objective: We aimed to compare haemostasis in ITP patients untreated with any pharmacologic agent (UT-ITPp) and treated with TPOR-A (TPOA-Rp) using the coagulation global assays thromboelastrometry (ROTEM®) and Calibrated Automated Thrombogram (CAT). Methods: Thirty chronic UT-ITPp [platelet count: (86±60)x109 platelets/L], twenty six responders to TPOR-A [16 with Romiplostim® and 10 with Revolade®, platelet count: (132±46)x109 platelets/L] and fifty healthy controls were included. Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP). Aliquots were stored at -80ºC until analysis. Non-activated ROTEM was performed on PRP adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded. Plasma thrombin generation was measured in PPP using the CAT test at a final concentration of 1 pM tissue factor and 4 microM phospholipids. 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. Platelet activation was determined by flow cytometry through PAC1-FITC binding after stimulation with 100 micromol/L thrombin receptor-activating peptide 6 (TRAP). Fibrinolytic proteins were tested in PPP using commercialized kits. Comparisons of quantitative variables were made with SPSS.22 software. Values of p≤0.05 were considered statistically significant. Results: Platelets from all patients with ITP had a defect in their ability to be activated, as shown by the lower PAC1 binding (p No differences were observed among groups for thrombin generation except for an increase in ETP in both groups of ITP patients [control: 1240±320 nMxmin, UT-ITPp: 1464±443 nMxmin (p In ROTEM experiments, PRP from UT-ITPp and TPOA-Rp showed a prolonged CT [control: 550± 95 sec, UT-ITPp: 1090±75 sec (p To evaluate whether increased LI60 values were due to an imbalance in fibrinolysis related proteins, tPA, uPA, TAFI and PAI-1 plasma levels were measured. No differences were observed among the two groups of patients and healthy controls except for PAI-1 which level was increased in TPOA-Rp (control: 12.9±12.3 ng/ml, UT-ITPp: 17.3±12.5 ng/ml, TPOA-Rp: 46.8±20.8, ng/ml p Conclusions: As we previously described, TPOR-A treatment increased platelet count but did not ameliorate their function (Álvarez Román et al, Thromb Haemost. 2014, 112:65-72). Nevertheless, ITP patients responders to TPOR-A showed a haemostasis unbalanced on a hypercoagulable profile due, at least in part, to a hipofibrinolytic pattern mainly caused by an increase in PAI-1 plasma level. This fact, together with the increment in platelet count caused by TPOR-A, might help to protect these patients from bleeding. This work was supported by a grant from the FIS-FEDER PI15/01457 Disclosures No relevant conflicts of interest to declare.

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder in which both increased platelet destruction and insufficient platelet production are involved. Patients can have a range of bleeding manifestations from none to severe at a similar platelet count. In some cases, patients have fewer bleeding symptoms than expected considering the low platelet count that they might have. Objective: The aim of this study was to determine the procoagulant profile of platelets from ITP patients in order to determine whether any of their features may explain this observation. Methods: Twenty-five patients with chronic ITP [(68±100)x109 platelets/L, mean age: 59.6 ± 16.1 years old, 56% female)] and thirty-five healthy controls [(256±36)x109 platelets/L, mean age: 41.6 ± 13.5 years old, 51% female) were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP). To obtain washed platelets, the top two-thirds volumes of PRP were collected and centrifuged (650 g for 10 min at 23°C) after the addition of acid-citrate-dextrose (ACD, 1:10) and the pellet was resuspended in an equal volume of HEPES buffer. Platelet activation was determined by flow cytometry through binding of FITC-PAC1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 micromol/L thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) or 20 micromol/L ADP. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions. To characterize platelet ability to bind coagulation factors, washed platelets (1x108/mL) were activated with 100 micromol/L TRAP and then incubated with FVa and/or FXa (5nM each, 10 min, ambient temperature). After fixation with 2% paraformaldehyde to cross-link the platelet-bound factors Va and Xa, platelets were washed two times with Hepes Buffer. Non-specific binding sites were blocked with 8% bovine serum albumin (30 min, room temperature). Following centrifugation, platelets were first incubated with anti-CD41-PE, anti-FVa and/or anti-FXa and then with a secondary FITC-goat anti-mouse IgG and stored at 4°C until flow cytometry analyses. Results: Platelets from ITP patients showed a basal expression of activated fibrinogen receptor similar to controls and a reduced ability for being activated by agonists (% of positive platelets for TRAP-induced PAC1 binding: 60±20 % in controls and 35±23 % in ITP, p Platelets from ITP patients expressed more PS than controls under basal conditions [mean fluorescence (MF) for FITC-annexin V binding was: 336±128 in controls, 588±25 in ITP, p Since the PS is the anchor site of the prothrombinase complex, we studied the binding of FVa and FXa at baseline and after activating platelets with TRAP. The binding of these factors in both conditions was higher in the group of patients with ITP (MF for basal FVa binding: 41.4±14.4 in controls, 58.1±24 in ITP, p Conclusions: Platelets from ITP patients, despite having less capacity of activation by agonist stimulation, have an increased procoagulant surface with greater ability to bind prothrombinase complex (FXaVa) than those from healthy controls. This feature might be a procoagulant compensatory mechanism that could reduce the risk of bleeding in patients with ITP. This work was supported by a grants from the FIS-FEDER, PI12/01831 and PI15/01457 Disclosures No relevant conflicts of interest to declare.

SummarySevere manifestations of X-linked recessive disorders such as haemophilia A (HA) are rare in females. Here we describe the clinical and genetic findings in five female HA patients from two different Spanish families. Three sisters born to consanguineous parents presented moderate bleeding due to a known mutation (p.Ser1791Pro) detected in a homozygous state. In the second family, two sisters with Morris syndrome (46,XY) and mild/moderate illness were hemizygous for a novel missense mutation, p.Phe2127Ser. The mutation is predicted to impair binding to the factor VIII (FVIII) carrier protein, von Willebrand factor, and thus increased clearance of FVIII from plasma. Clinical and molecular characterisation of these patients is essential to optimise follow-up, genetic counselling and treatment of the disease.

Background: Patients with ITP have a wide variation in the presentation of the disease, platelet count and their clinical course. The decision to begin treatment is based on the hemorrhagic symptoms and platelet count. Intravenous immunoglobulin (IVIG) is usually associated with glucocorticoid administration in patients with severe bleeding or platelet counts Materials and Methods: We recruited patients with ITP before and after responding to treatment with IVIG (n = 11) and AR-TPO (4 patients with romiplostim and 10 with eltrombopag), 5 splenectomized patients and 82 healthy controls. The percentage of reticulated platelets, platelet activation and binding of annexin-V were evaluated by flow cytometry. Plasma levels of TPO and "a proliferation-inducing ligand" (APRIL) were determined by ELISA. Procoagulant activity associated microparticles (MP) and the ability of plasma to generate thrombin were determined, respectively, with Zymuphen kit and calibrated automated thrombinography (CAT) triggered by 1 pM tissue factor and 4 micromolar phospholipid (PPP-low reagent, Diagnostica Stago, Spain). Results: Patients with ITP that respond to IGIV and AR-TPO treatments recovered platelet counts without reaching the levels of the control group, whereas the platelet count in splenectomized patients did not differ from it. Plasma levels of TPO and the number of immature platelets in the first two groups were higher than in controls before responding to treatment. Despite recovering platelet count, platelet capacity of being activated by agonists such as TRAP (thrombin receptor agonist for PAR-1) was less than that of the controls in all groups. This decrease was not due to a reduction in the expression of the fibrinogen receptor on platelets from ITP patients. Platelets from ITP patients before and after responding to all treatments studied, showed more phosphatidylserine exposure and greater microparticles-associated and plasma-associated procoagulant activity. Plasma levels of APRIL, a factor that stimulates B cells and antibody production, decreased in ITP patients who responded to the AR-TPO, reaching the levels observed in the control group. In the group of splenectomized patients a decrease of APRIL was also observed, but still remained higher than in healthy controls. Conclusions: ITP patients who respond to treatment with IVIG and AR-TPO and undergoing splenectomy recovered platelet count but not its function. The treatments did not modify the microparticles- and plasma-associated thrombogenic capacity. Among all the treatments studied, AR-TPO and splenectomy had an addittional benefical effect reducing APRIL plasma levels Disclosures No relevant conflicts of interest to declare.

Background: Prophylaxis is considered as the optimal therapy for patients with severe hemophilia, but its high cost compels its individualization. Factor (F) VIII half-life (t1/2) is one of the most important variables for individualization of prophylaxis. It requires at least 5 time-points in children to be determined which constitutes a handicap due to several reason such as difficulties to obtain blood samples or those derived from the lost of hours of school or work in case of parents. The use of bayesian models to predict pharmacokinetic (PK) parameters is a promising tool for facilitating the tailoring of prophylaxis regimen in patients with hemophilia. However, software for the hemophilic population is lacking. MyPKFiT® (Baxalta®) is a novel website software based on bayesian analysis recently developed to predict PK parameters of FVIII in patients with hemophilia. This software needs only two well-selected blood samples to estimate the individual PK parameters. We hypothesized that the use of MyPKFiT® will facilitate individualization of prophylaxis and will help to detect anomalous FVIII PK profile in children and adolescents with haemophilia. Objectives: To evaluate the usefulness of the web application MyPKFiT® for the determination of individual pharmacokinetic parameters in patients under 18 years old (y) and detection of anomalous PK profiles in this patient population. Material and methods: Patients < 18 y under prophylaxis with FVIII concentrate (ADVATE®) and without inhibitors at inclusion were recruited. Washout period was not used due to it is not required for the use of the software. Joint health was determined by the Haemophilia Joint Health Score (HJHS) and evaluation of physical activity was based on the scale of Broderick. Both parameters were used together with PK parameters obtained by MyPKFiT for tailoring prophylaxis. Results: Eighteen patients were included. Mean age was 15 y (range 2-14). All patients were treated on primary prophylaxis. HJHS was zero in all the patients. PK profile was obtained with 2 samples in 14 patients and with 3 samples in 4 patients suspected to have atypical PK values. Physical activity had a mean relative risk of 1 in 15 patients and 2.7 in the rest. Based on physical activity, joint score and PK parameters provided by MyPKFit, through level was chosen to calculate the most appropriate dosage for each patient. The t1/2for FVIII was within the population curve in all patients except two in which the existence of an inhibitor were suspected. Conclusions: MyPKFit allowed us to obtain PK curve with fewer samples than required. The software helped to detect patients with anomalous PK profile. These patients were those suspected to have shorted t1/2. It is require further studies to detect the causes that might explain these altered profiles. Disclosures No relevant conflicts of interest to declare.

Background: Immune tolerance therapy (ITT) is the only available therapy for eliminating the inhibitor in patients with haemophilia and high-responding inhibitors. However, it is a very expensive treatment with an efficacy of up to 60%-75%. Factors influencing the outcome of ITT are not completely understood. Some studies have suggested that plasma derived factor VIII (FVIII) concentrates might offer certain advantages in the elimination of the inhibitor when compared with recombinant derivatives. The presence of von Willebrand factor (Auerswald et al ,Haematologica 2003;88:EREP05; Kreuz et al ,Haematologica 2001;86:16-20) and some molecules with immunomodulatory effect like anti-idiotype antibodies, transforming growth factor-β, HLA type I molecules and soluble FAS ligand in plasma derived concentrates may be involved in such effect (Berntorp et al ,Haemophilia 2001;7:109-13; Hodge et al ,Br J Haematol 2001;115:376-81; Hodge et al , Haemophilia 2006;12:133-9; Ghio et al , Thromb Haemost 2003;89:365-73). The activated prothrombin complex concentrate (FEIBA®) is a plasma derived product used for bleeding control in patients with haemophilia A and high-responding inhibitors. We hypothesize that FEIBA® may have an immunomodulatory activity due to its plasmatic origin. This effect might be observed in patients with intense treatment with FEIBA® (e.g. prophylaxis treatment) by the reduction in levels of antibodies against FVIII compared with patients on demand treatment with this product. Objective: The aim of this study was to compare levels of inhibitors in patients with severe haemophilia A and inhibitors on demand or under prophylactic treatment with FEIBA®. Methods and results: This was a prospective, observational and multicenter study. A total of 12 adult patients with severe haemophilia A and high-responding inhibitors (6 patients on demand and 6 patients under prophylaxis treatment with FEIBA®) were included. All patients had normal levels of serum creatinine, serum alanine aminotransferase and CD4+ lymphocyte counts at inclusion. Levels of inhibitors were determined by Bethesda assay. Levels of inhibitors in patients on prophylaxis with FEIBA® were lower than those observed in patients on demand treatment but differences were not statistical significant. In most of the patients on prophylaxis, levels of inhibitors went down along the prophylaxis regimen. | Patient No | Age | Type of treatment | Usual dose (IU/kg) | Frecuency | Titer (BU) | Time frame with start of prophylaxis (months)** | | ---------- | ---- | ----------------- | --------------------------------- | ------------------------- | ---------- | ----------------------------------------------- | | 480101 | 53 | Prophylaxis | 60 | Three times a week | 64* | 4.4 | | 80 | 4.1 | | 96 | 1.3 | | 250 | -1.3 | | 480102 | 58 | Prophylaxis | 55 | Three times a week | 5* | 3.3 | | 12 | 2.1 | | 7 | -3.8 | | 12 | -6.1 | | 150101 | UNK | Prophylaxis | 60 | Three times a week | 4* | 3.0 | | 5 | -6.0 | | 6 | -8.0 | | 280101 | 28 | Prophylaxis | 56 | Every other day | 109* | 7.3 | | 77 | 4.1 | | 101 | -8.3 | | 101 | -9.7 | | 280102 | 57 | On Demand | 56 x 8 doses | N/A | 174* | N/A | | 37 | N/A | | 83 | N/A | | 21 | N/A | | 25 | N/A | | 184 | N/A | | 280103 | 57 | On Demand | 55 x 6 doses | N/A | 8000* | N/A | | 64 | N/A | | 5 | N/A | | 21 | N/A | | 280104 | 58 | On Demand | 68 x 6 doses | N/A | 152* | N/A | | 280105 | 23 | On Demand | 68 x 3 doses 40 x 6 doses | N/A | 30* | N/A | | 080101 | 28 | On Demand | 50 x 5 doses | N/A | 5.2* | N/A | | 7.0 | | 7.2 | | 4.0 | | 7.8 | | 460101 | 37 | Prophylaxis | 66 | every 48h | Negative | 21,0 | | 460102 | 57 | On demand | 70 | 2 doses/ bleeding usually | 66.5* | More than 20 years | | 300001 | 50 | Prophylaxis | 85 | Three times a week | 8* | 3 | * *Titer at inclusion. **Positive values represent number of months after starting prophylaxis and negative values are referred to number of months before starting prophylaxis. For example, the patient number 480101 had 250 BU at 1.3 months before starting prophylaxis (in the table= -1.3 months) and 64 UB at month 4.4 after initiation of prophylaxis (in the table= 4.4 months). N/A: not applicable. Table. Medical history of patients. Conclusion: Prophylaxis with FEIBA® might decrease levels of inhibitors in patients with severe haemophilia A and high-responding inhibitors. However, a broader study is needed to confirm this result. Disclosures Iruin: Baxalta: Research Funding. Bonanad: Baxalta: Research Funding. Lopez Fernandez: Baxalta: Research Funding. Altisent: Baxalta: Research Funding. Garcia Candel: Baxalta: Research Funding. Rivas Pollmar: Baxalta: Research Funding. Canales: Baxalta: Research Funding.

Background: Bleeding complications can have serious outcomes in patients with myelodysplastic syndromes (MDS) and are usually related to thrombocytopaenia. In a recent work we demonstrated that platelets from MDS have impaired capacity to respond to agonist stimulation and exposed more phosphatidylserine (PS) than those from healthy controls whichever their platelet count was (Martin et al, Thromb Haemost., 2013;109:909-19). The exposure of PS in the outer layer of cell membranes supports coagulation through enhanced formation of the tenase (factors IXa, VIIIa and X) and prothrombinase (factors Xa, Va and prothrombin) complexes and thrombin generation. So, the possibility exists that these processes are increased in these patients. Objectives: The aim of this work was to study whether thrombus formation and thrombin generation is impaired in MDS patients with normal platelet count employing global coagulation tests thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT). Methods: Thirty-one MDS patients with normal platelet count and twenty healthy controls were included. Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP). For ROTEM and CAT experiments, PRP was adjusted to a platelet count of 25 x 109/L. Aliquots for ROTEM assay were tested within the two hours after obtaining blood samples. For CAT experiments, adjusted PRP and PPP aliquots were stored at -70ºC until analysis. Kinetics of clot formation, non-activated ROTEM was performed on adjusted PRP. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), and maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]), were recorded. Thrombin generation was measured in adjusted PRP without any trigger and in PPP with 1 pM tissue factor and 4 µM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands) as trigger. 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 were evaluated. Platelet activation was determined by PAC1 (BD, Madrid, Spain) binding after stimulation with 100 μM thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) and surface PS through Annexin-V binding and flow cytometry analyses. Results: Platelets from MDS patients had a reduced response to TRAP stimulation (control= 12016+6384 arbitrary units; MDS= 5829+3704 arbitrary units) and exposed more PS than controls (control= 362.1+80.5 MF; MDS= 378.5+173.5 MF). ROTEM experiments showed kinetic parameters corresponding to a hipocoagulable profile (CT: control= 550+95 sec, MDS= 922+216 sec, p In order to evaluate whether the impaired clot formation was due to a reduction in plasma-associated thrombin generation, CAT experiments were performed in PPP samples. No differences were found between MDS patients and control group (ETP: control=1223.4+257.8 nMxmin, MDS= 1224.4+344 .1 nMxmin; peak: control= 279.5+54.7nM, MDS= 265.9+64.1 nM). On the other hand, when thrombin generation experiments were performed in adjusted PRP, ETP and peak values were lower than in control group (ETP: control=1574.8+430.5 nMxmin, MDS= 1167.4+354 nMxmin,p Conclusions: Platelet dysfunction might be resposible of bleeding complications observed in patients with MDS with normal platelet counts. Increment in PS exposure on platelet surface did not seem to compensate impairment in platelet function. Disclosures No relevant conflicts of interest to declare.

Background: Patients with platelet counts less than 20 or 30 x 109/L have an increased risk of bleeding. Nevertheless, some patients with immune thrombocytopenia (ITP) have fewer bleeding symptoms than expected. In a previous communication (ASH 2014) we reported that these patients presented high microparticles (MP)-associated procoagulant activity to compensate bleeding risk and that cellular origin of these MPs were platelets and red cells. However, other mechanisms might be involved. Objective: The aim of this work was to analyse the involvement of other factors to compensate bleeding risk in thrombocytopenic ITP patients. Moreover, the feasibility of using the coagulation global assays thromboelastrometry (ROTEM) and Calibrated Automated Thrombogram (CAT) to test haemostasis in these patients was evaluated. Methods: Fifty patients with chronic ITP with platelet count less than 50 x 109/L and twenty-five 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 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP) and at 1,500 g for 15 min at 23°C for platelet-poor plasma (PPP) and aliquots were stored at -70ºC until analysis. To assess the kinetics of clot formation, non-activated ROTEM was performed on PRP adjusted to a platelet count of 25 x 109/L. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness,which reflects the maximum tensile strength of the thrombus (MCF, [in mm]) and LI60, which describes the percentage of maximum clot strength present at 60 min (in %), were recorded. Plasma thrombin generation was measured in PPP using the Calibrated Automated Thrombogram (CAT) test at a final concentration of 1 pM tissue factor and 4 mM 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. Fibrinolytic proteins and E-selectin was tested in PPP using commercialized kits. 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. Results: PRP from ITP patients showed a prolonged CT (control: 550+ 95 sec, ITP: 890+165 sec, p In order to evaluate whether increased LI60 values were due to an imbalance in fibrinolysis related proteins, tPA, uPA, TAFI and PAI-1 plasma levels were measured. No differences were observed between patients and healthy controls except for PAI-1 which level was increased in ITP patients (control: 14.7 ng/ml+11.7 ng/ml, ITP: 30.4+17.5, p Thrombin generation in PPP was also measured and a shorter time to peak (control: 9.3+1.2 sec, ITP: 8.3+1.7 sec,p Conclusions: We demonstrated that ITP patients presented a hypercoagulable profile that might be related, at least in part, to a reduced fibrinolysis mainly caused by an increase in PAI-1 level that seemed to be related to endothelial damage. Moreover ROTEM and CAT appeared to be useful tools for evaluating coagulant profile in ITP patients. Disclosures No relevant conflicts of interest to declare.

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). Objective: The aim of this study was to evaluate and characterize the microparticle-associated procoagulant activity in ITP and MDS patients with thrombocytopenia. Methods: Thirty-five patients with chronic ITP and twenty-six patients with MDS with a platelet count less than 50 x109/L and twenty-five healthy controls were included. Blood cell 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, [PFP]) 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. The identification of MP’s cell origin was determined by flow cytometry labeling MPs with Annexin-V-fluorescein and the following specific monoclonal antibodies (mAb) conjugated with phycoerythrin: anti CD41 mAb for platelets, anti CD14 mAb for monocytes, anti CD144 mAb for endothelial cells, anti CD235 mAb for red cells and anti CD45 mAb for leukocytes. APRIL plasma levels were determined by ELISA (DuoSet ELISA, R&D Systems, Minneapolis, MN, USA). Results: Ph-MP associated procoagulant capacity in MDS and ITP patients was higher than in controls (p MPs analysis by flow cytometry of seventeen controls, twenty ITP and six MDS patients showed that ITP patients had an increased percentage of MPs from platelets and red cells, whereas MDS had an increased percentage of monocytes derived MPs. Proportion of MPs derived from other cell types were similar to the control group. Increased percentage of monocyte-derived MPs in MDS patients is in accordance with the higher MP-TF-associated capacity observed in this group since monocytes are tissue factor rich cells. Nevertheless, no differences were found in monocyte count with control and ITP groups and monocyte count did not correlate with MPs associated procoagulant activity and the percentage of monocyte-derived MPs. In the ITP group neither MP-Ph-associated procoagulant activity nor the percentage of red cell-derived MPs correlated with red cell count. On the contrary, MP-Ph-associated procoagulant activity and the percentage of platelet-derived MPs inversely correlated with platelet count. ITP is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction. A proliferation-inducing ligand (APRIL) is a factor that promotes B-cell maturation and survival. All patients with ITP and thrombocytopaenia showed higher APRIL plasma levels than MDS and control groups (p Conclusion: Peripheral and central thrombocytopenias might present a MP-associated procoagulant activity to compensate bleeding risk present in these patients. Cellular origin of these MPs differed according to thrombocytopaenia etiology. Disclosures No relevant conflicts of interest to declare.

Introduction 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. Objective 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. Methods 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. Results 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 ITP-1 patients showed significantly higher APRIL plasma levels (37.60+28.73 ng/ml, p 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 Conclusion 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):4639-4645 Disclosures: No relevant conflicts of interest to declare.

Abstract 3313 Objectives: Behçet's disease (BD) is a chronic inflammatory disease of unknown etiology associated with an increased risk of venous and arterial thrombosis. To date, no studies for haemostasis have been conducted in this population by rotational thromboelastometry and thrombin generation test. These methods appear to be more sensitive and specific than routine coagulation tests in detecting defects of the coagulation system and could provide new elements for better understanding the mechanisms involved in the hypercoagulable state observed in BD patients. On this basis, this work aims to study haemostasis in patients with BD by rotational thromboelastometry (ROTEM ®) and thrombin generation test (CAT). Methods: Twenty-six patients with BD were included. Ages were between 25–86 years (mean ± SD: 48.60 ± 15.66 years) and 73% were female. Eight patients had active disease at the time of enrollment and 6 had a history of thrombosis. Control group included 20 healthy individuals aged between 28–55 years (42.85 ± 9.02 years) and 65% were female. Rotational thromboelastometry was performed in whole blood with ROTEM® coagulation analyzer (Pentapharm, Munich, Germany) at INTEM condition (activation of coagulation mainly throughout intrinsic pathway). Samples were allowed to rest 1 hour at room temperature and heated for 4 minutes at 37 ° C immediately before testing. 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 mM of phospholipids and 1 pM of tissue factor which favored the activation of coagulation mainly throughout intrinsic pathway. Normal distribution for continuous variables was assessed with the Shapiro-Wilk testComparisons of quantitative variables were made with non-paired Student's t-test or Mann-Whitney's U test as appropriate. Results: Values of thromboelastometry parameters were increased in samples from BD patients. The higher clot consistency at 5 and 10 minutes (p=0.0452 and p=0.0179 respectively), and the pronounced maximal clot firmness (p=0.0064) suggest that platelet function may be altered in our patient cohort. Moreover, values higher than control ones for a angle (p=0.0266), maximum clot formation velocity (p=0.0454) and area under the curve at 5 and 10 minutes (p=0.0206 and p=0.0220 respectively) point to an increased thrombin generation in BD patients. In order to verify this hypothesis, thrombin generation was evaluated in samples from these patients. CAT experiments showed that BD patients had an increased endogenous thrombin potential (p = 0.0251) and reached higher maximum levels of thrombin (peak height, p = 0.0119) than controls. To determine if haemostatic profile in BD patients evaluated by rotational thromboelastometry and CAT correlates with disease activity, Spearman's rank correlation coefficient was calculated. There was a significant but mild correlation between disease activity and area under the curve at 5 minutes (r=0.3818, p=0.0494) and the maximal clot firmness (r=0.3978, p= 0.0399) (thromboelastometry parameters) and the peak height (r=0.4615, p=0.0251) (CAT parameter). Conclusions: Rotational thromboelastometry at INTEM condition and thrombin generation test confirmed that there is a hypercoagulable state in Behcet's disease. Procoagulant/anticoagulant protein equilibrium and platelet function may be altered in this pathology as indicated by several parameter measured by both tests. Disclosures: No relevant conflicts of interest to declare.

Abstract 3806 The myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders with peripheral cytopenias and increased incidence of leukemic transformation. The prognosis of MDS is determined by several factors, including the presence of specific cytogenetic abnormalities, the percentage of blastoid cells in bone marrow and peripheral blood, the number of affected cell lineages, and transfusion dependency. The most commonly used risk stratification system is the International Prognostic Scoring System (IPSS). This score divides patients into a lower risk subset (low and intermediate-1) and a higher risk subset (intermediate-2 and high). Patients with MDS may have hemorrhagic complications with serious outcomes that are among the major causes of death in this population. These bleeding episodes that are often related to thrombocytopenia also occur in MDS patients with normal platelet count. The aim of this work was to study functional characteristics of platelets in MDS patients and their relationship to risk evaluated as indicated by IPSS. Eighty diagnosed MDS patients risk-stratified according to IPSS were included: 40 with low-risk, 29 with intermediate-1-risk (I-1), 8 with intermediate-2-risk (I-2) and 3 with high-risk. Eighty healthy donors were included as control group. Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-μm aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time, CT), which is expressed in seconds. Platelet activation was determined through FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 μM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (αIIb and β3 subunits) was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane phosphatidylserine (PS) exposed in basal conditions. I-2 and high-risk patients were gathered together in a high-risk group in order to analyze experimental results. Statistical analysis was performed with one-way ANOVA and Tukey test. CTs obtained with COL-EPI and COL-ADP cartridges in controls and low risk patients were similar and significantly shorter than CTs observed in I-1-risk and high-risk MDS patients (p Platelets from all MDS patients showed a reduced capability for being activated by 100 μM TRAP. This impairment was more evident in I-1-risk and high-risk patients: PAC-1 binding, in arbitrary units (AU), was 11368±1017 in controls; 7849±789 in low-risk MDS (p Platelets from MDS patients expressed more PS than controls under basal conditions. Mean fluorescence values for FITC-annexin binding were: 383±16 in controls; 444±21 in low-risk (p Our results indicated that platelets from MDS patients had less ability to be activated and were more apoptotic than control ones. These dysfunctions were more pronounced when the risk of the disease was higher according to IPSS. Disclosures: No relevant conflicts of interest to declare.

Abstract 3707 Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by isolated thrombocytopenia (platelet count less than 100,000/μL) and the absence of any obvious initiating and/or underlying cause for the thrombocytopenia. In spite of the low platelet number, some thrombocytopenic patients seldom bleed, indicating the existence of other factors that regulate haemostasis in these patients. Elevated levels of plasma microparticles (MPs) had been observed in IPT patients. MPs are vesicles with a size less than 0.5 micrometers, derived from cell membranes after their activation or apoptosis. Most MPs are highly procoagulant, expressing annexin V binding sites and tissue factor. However, relatively little is known of their specific functions in ITP. In the present study we aim to elucidate if a relationship exists between microparticle-linked procoagulant activity and haemostasis in ITP patients. Twenty-two ITP patients, 3 male and 19 female, aged between 25 to 92 years, were included. Sixteen age- and sex-matched healthy individuals were used as control group. Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-micrometer aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time), which is expressed in seconds. MP procoagulant activity was determined with ZYMUPHEN MP-Activity kit (Aniara, Mason, Ohio) and by calibrated automated thrombography (CAT) in plasma samples obtained after 2 centrifugations at room temperature (first: 15 min at 1,500 g, second: 2 min at 13,000 g). These methods measure endogenous thrombin generation. CAT evaluates four parameters of thrombin generation: the endogenous thrombin potential (ETP), lag time, time to peak (TTP) and peak height. PFA-100® determinations with COL-EPI and COL-ADP cartridges in blood samples from ITP patients with less than 50,000/μL showed longer closure times than control group (p Our results indicate that increased MP procoagulant activity in ITP patients may be protective against bleeding events that should be observed in those thrombocytopenic conditions. Three of the ITP patients included in this study had been splenectomyced and we consider of interest to point out that two of them in spite of recovering a normal platelet count still maintain a high MP procoagulant activity. This observation agrees with a recent work that postulates that MPs might contribute to an increased risk of thrombosis, progression of atherosclerosis and cardiovascular disease following splenectomy (Fontana et al, Thromb Research, 2008;122:59). Disclosures: No relevant conflicts of interest to declare.