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Aim: To analyze the perception of anticoagulation with dabigatran in patients with nonvalvular atrial fibrillation previously treated with vitamin K antagonists over a 6-month period. Materials & methods: This is a prospective, noninterventional, noncontrolled, multicenter study. To assess patients’ perceptions, PACT-Q2 questionnaire was completed. Results: Six hundred and fifty nine patients (73.1 ± 9.4 years, CHA 2 DS 2 -VASc 3.6 ± 1.6) were included. At baseline, the convenience and satisfaction scores were 60.9 ± 24.9 and 49.9 ± 17.7, respectively. The scores significantly increased along the study. Convenience score was higher in males and in patients with low–moderate thromboembolic risk. Satisfaction score was higher in females. Only 8.0% of patients discontinued dabigatran (3.7% due to side effects). Conclusion: Convenience and satisfaction scores for nonvalvular atrial fibrillation patients treated with dabigatran at 6 months were significantly better than with previous vitamin K antagonists.

Summary The influence of driver mutations on leukaemic transformation was analysed in 1747 patients with polycythaemia vera or essential thrombocythaemia. With a median follow-up of 7·2 years, 349 patients died and 62 progressed to acute leukaemia or myelodysplastic syndrome. Taking death as a competing risk, CALR genotype was associated with a lower risk of transformation [subdistribution hazard ratio (SHR): 0·13, 95% confidence interval (CI): 0·2–0·9, P = 0·039], whereas JAK2 V617F showed borderline significance for higher risk (SHR: 2·05, 95% CI: 0·9–4·6, P = 0·09). Myelofibrotic transformation increased leukaemic risk, except in CALR-mutated patients. Next generation sequencing of 51 genes at the time of transformation showed additional mutations (median number: 3; range: 1–5) in 25 out of 29 (86%) assessable cases. Mutations (median: 1; range: 1–3) were detected in 67% of paired samples from the chronic phase. Leukaemia appeared in a JAK2 V617F negative clone in 17 (58%) cases, eleven of them being previously JAK2 V617F-positive. JAK2 V617F-mutated leukaemia was significantly associated with complex karyotype and acquisition of TP53 mutations, whereas EZH2 and RUNX1 mutations were more frequent in JAK2 V617F-negative leukaemia. Survival was longer in JAK2 V617F-unmutated leukaemia (343 days vs. 95 days, P = 0·003). In conclusion, CALR genotype is associated with a lower risk of leukaemic transformation. Leukaemia arising in a JAK2 V617F-negative clone is TP53 independent and shows better survival.

Background Direct oral anticoagulants (DOACs) have emerged as a treatment of choice in patients with chronic atrial fibrillation (AF) or for secondary prevention of venous-thromboembolism (VTE). In myeloproliferative neoplasms (MPN) very small series have been reported and robust data reporting the safety/efficacy profile of these drugs is not available. We conducted an international, multi-country, retrospective study involving 19 hematologic centers from Europe, US and Canada, with the aim to describe in a large cohort of MPN patients the incidence of thrombosis and bleeding complications associated with DOAC use in real word clinical practice. Methods Centers reported in an electronic CRF data on 442 patients (M/F: 221/221; median age: 65 years) with a WHO diagnosis of polycythemia vera (PV, n=178), essential thrombocythemia (ET, n=172) and primary myelofibrosis (PMF, n=92) who had received DOACs (Rivaroxaban n=187; Apixaban n=157; Dabigatran n=50; Edoxaban n=48) for either primary and secondary antithrombotic prophylaxis in atrial fibrillation (AF, n=203) or secondary prophylaxis of venous thromboembolism (VTE, n=239). Eighty-two patients (18.6%) shifted to DOAC after a previous exposure to a vitamin K antagonist (VKA) mainly due to patient preference (8.4%), bleeding/thrombosis (5.2%) or INR instability (4.1%). In 60 patients, DOAC was discontinued after a median duration of 1.1 years; reasons included completion of a pre-determined duration (2.9%), patient decision (0.9%), major bleeding (2.5%) or thrombosis (2.9%), minor bleeding (0.9%), thrombocytopenia (2.0%), surgery (0.9%), or other (0.5%). Results Median time from MPN diagnosis to DOAC initiation was 4.4 years (range: 0-34.7 years). Concomitant therapies included antiplatelet agents (31%) and cytoreductive drugs in 90% (hydroxyurea in 87%of cases). After a median follow-up of 1.83 years, 32 major thrombotic events (rate: 3.3% pts/yr) and 26 major bleeding events (rate: 2.6% pts/yr) were reported. 1. AF. Ten thrombotic events (rate: 2.1% pts/yr) occurred in patients receiving DOACs: 4 TIA, 3 MI and 3 DVT of the lower extremities (LE); the rate was remarkably different in primary prevention (1.5% pts/yr) vs. secondary prophylaxis (i.e. after a previous thrombosis, mainly arterial: 4.6% pts/yr). This rate was almost double that reported in secondary prophylaxis in non-MPN population in randomized clinical trials (RCTs) (2.1-3.2% pts/yr). Previous arterial thrombosis was the only significant risk factor for recurrences in this subset (HR: 3.89, p=0.035). 2. VTE. Twenty-two recurrences while receiving DOACs were reported (5 arterial, 10 DVT of the LE +/- PE, 3 splanchnic vein and 4 others venous; rate: 4.5% pts/yr) and irrespective of initial site of thrombosis and type of DOACs. This rate was similar to MPN patients receiving VKAs (5.3% pts/yrs - De Stefano V et al, Leukemia 2016) but higher than in non-MPN population in RCTs (1.4-1.9% pts/yr). In univariate analysis, significant factors for recurrences were: previous arterial thrombosis (HR: 3.55, p=0.023), hypertension (HR: 2.88, p=0.021) and diabetes (HR: 3.33, p=0.018). 3. Among 26 major bleeding events, 12 were gastrointestinal, 7 intramuscular, 1 CNS and 6 in other sites. The overall rate was 2.6% patients/year, which is comparable to that reported in MPN patients under VKA (2.4% pt/yrs, De Stefano V et al, ibidem) and in non-MPN population of RCTs (1.60-3.11% pt/yrs). The frequency of major bleeding was similar in AF (3% pt/yrs) and VTE (2.3% pt/yrs) setting. In univariate analysis, significant risk factors were PMF diagnosis (HR: 2.95, p=0.007) and the use of Dabigatran in comparison to the other DOACs (HR: 2.91, p=0.016) whereas no increase was due to antiplatelet drugs. Conclusions This is the largest observational study describing vascular events in MPN patients receiving DOACs for prevention of thrombosis in AF or secondary prevention of VTE. Overall, the rate of re-thrombosis is similar to that reported with warfarin in MPN and double the rate of non-MPN population. The highest rate was found in patients with a previous history of arterial thrombosis and with cardiovascular risk factors. In regard to bleeding, we highlight the significant bleeding tendency in PMF and treatment with Dabigatran. In conclusion, the risk/benefit profile of DOACs in MPN is similar to that of Warfarin. Disclosures Barbui: AOP-Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. De Stefano:Novartis: Other: Personal fee, Research Funding; Amgen: Other: Personal fee; Bayer: Other: Non-financial support; Celgene: Other: Non-financial support, personal fee; Janssen Cilag: Other: Non-financial support. Vannucchi:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Palandri:Novartis: Consultancy, Honoraria. Harrison:Celgene: Honoraria, Research Funding, Speakers Bureau; Gilead Sciences: Honoraria, Speakers Bureau; Incyte Corporation: Speakers Bureau; Janssen: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Promedior: Honoraria; Roche: Honoraria; Sierra Oncology: Honoraria; CTI Biopharma Corp: Honoraria, Speakers Bureau. Griesshammer:Novartis: Honoraria, Speakers Bureau; AOP Orphan: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; CTI: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau. Koschmieder:Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding; Geron Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Incyte/Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; CTI Biopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding; Promedior: Other. Benevolo:Amgen: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Gupta:Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Mascarenhas:Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution); Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy.

Patients with polycythemia vera (PV) or essential thrombocythemia (ET) presenting with splanchnic vein thrombosis (SVT) might have a specific clinico-biological profile. To investigate this hypothesis, 3705 PV/ET patients from three national registers, 118 of them presenting with SVT, were reviewed. After correction for age and sex, PV/ET patients with SVT showed an increased risk of death (HR 2.47, 95% CI 1.5–4.01, p

We analyzed 597 patients with myeloproliferative neoplasms (MPN) who presented transient ischemic attacks (TIA, n = 270) or ischemic stroke (IS, n = 327). Treatment included aspirin, oral anticoagulants, and cytoreductive drugs. The composite incidence of recurrent TIA and IS, acute myocardial infarction (AMI), and cardiovascular (CV) death was 4.21 and 19.2%, respectively at one and five years after the index event, an estimate unexpectedly lower than reported in the general population. Patients tended to replicate the first clinical manifestation (hazard ratio, HR: 2.41 and 4.41 for recurrent TIA and IS, respectively); additional factors for recurrent TIA were previous TIA (HR: 3.40) and microvascular disturbances (HR: 2.30); for recurrent IS arterial hypertension (HR: 4.24) and IS occurrence after MPN diagnosis (HR: 4.47). CV mortality was predicted by age over 60 years (HR: 3.98), an index IS (HR: 3.61), and the occurrence of index events after MPN diagnosis (HR: 2.62). Cytoreductive therapy was a strong protective factor (HR: 0.24). The rate of major bleeding was similar to the general population (0.90 per 100 patient-years). In conclusion, the long-term clinical outcome after TIA and IS in MPN appears even more favorable than in the general population, suggesting an advantageous benefit-risk profile of antithrombotic and cytoreductive treatment.

It is unclear whether anticoagulation guidelines intended for the general population are applicable to patients with polycythemia vera (PV) and essential thrombocythemia (ET). In the present study, the risk of thrombotic recurrence was analyzed in 150 patients with PV and ET treated with vitamin K antagonists (VKA) because of an arterial or venous thrombosis. After an observation period of 963 patient-years, the incidence of re-thrombosis was 4.5 and 12 per 100 patient-years under VKA therapy and after stopping it, respectively (P

To investigate the characteristics and clinical course of cerebral vein thrombosis (CVT) in patients with myeloproliferative neoplasms (MPN) we compared 48 patients with MPN and CVT (group MPN-CVT) to 87 with MPN and other venous thrombosis (group MPN-VT) and 178 with MPN and no thrombosis (group MPN-NoT) matched by sex, age at diagnosis of MPN (±5 years) and type of MPN. The study population was identified among 5,500 patients with MPN, from January 1982 to June 2013. Thrombophilia abnormalities were significantly more prevalent in the MPN-CVT and MPN-VT than in MPN-NoT group (P = 0.015), as well as the JAK2 V617F mutation in patients with essential thrombocythemia (P = 0.059). Compared to MPN-VT, MPN-CVT patients had a higher rate of recurrent thrombosis (42% vs. 25%, P = 0.049) despite a shorter median follow-up period (6.1 vs. 10.3 years, P = 0.019), a higher long-term antithrombotic (94% vs. 84%, P = 0.099) and a similar cytoreductive treatment (79% vs. 70%, P = 0.311). The incidence of recurrent thrombosis was double in MPN-CVT than in MPN-VT group (8.8% and 4.2% patient-years, P = 0.022), and CVT and unprovoked event were the only predictive variables in a multivariate model including also sex, blood count, thrombophilia, cytoreductive, and antithrombotic treatment (HR 1.97, 95%CI 1.05-3.72 and 2.09, 1.09-4.00, respectively).

Criteria of response and definition of resistance and intolerance to hydroxyurea (HU) in polycythemia vera (PV) were proposed by the European LeukemiaNet (ELN). Such criteria were evaluated in 261 PV patients (median follow-up, 7.2 years) treated with HU for a median of 4.4 years. Complete response, partial response, and no response were observed in 24%, 66%, and 10% of patients, respectively. Achieving ELN response (complete or partial) or hematocrit response did not result in better survival or less thrombosis and bleeding. On the contrary, having no response in leukocyte count was associated with higher risk of death (HR, 2.7; 95% confidence interval [CI], 1.3%-5.4%; P = .007), whereas lack of response in platelet count involved a higher risk of thrombosis and bleeding. Resistance and intolerance to HU was registered in 11% and 13% of patients, respectively. Resistance to HU was associated with higher risk of death (HR, 5.6; 95% CI, 2.7%-11.9%; P < .001) and transformation (HR, 6.8; 95% CI, 3.0%-15.4%; P < .001). In summary, fulfilling the ELN definition for response to HU was not associated with a benefit in the clinical outcome in PV, whereas response in platelet and white blood cell counts were predictive of less thrombohemorrhagic complications and better prognosis, respectively. Resistance to HU was an adverse prognostic factor.

Patients with essential thrombocythemia (ET) have an increased frequency of thrombosis, but the relationship of both thrombosis and JAK2 V617F allele burden with platelet turnover, acquired activated protein C resistance (aAPCR), and levels of coagulation factors and soluble markers of platelet, and endothelial activation is not well known. In 53 ET patients (26 with a history of thrombosis), reticulated platelets (RP) percentage, aAPCR, platelet tissue factor (TF) expression, and plasma levels of TF, coagulation factors, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer and prothrombin fragment 1 + 2 were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 mutational load. ET patients with thrombosis had significantly higher values for RP percentage, aAPCR, and levels of factors V and VIII, VWF:Ag, sP-selectin, and sCD40L than patients without thrombosis and controls. At multivariate study, RP percentage, factor V levels, and aAPCR were independently associated with an increased risk of thrombosis. Patients with JAK2 mutation had significantly lower levels of free protein S (PS) and higher levels of TF, sP-selectin, sCD40L, VWF:Ag, and sTM than those with wild-type allele. A mutant allele dosage effect (>or= 12%) was observed for TF, sP-selectin, sCD40L, VWF:Ag, and PS levels. These results support a role for platelet turnover, factor V, and aAPCR in the thrombosis of ET as well as the association between JAK2 V617F allele burden and either decreased free PS or increased TF and soluble markers of platelet and endothelial activation.

Platelet and leukocyte activation has been demonstrated in polycythemia vera (PV) and essential thrombocythemia (ET), but such information is limited in primary myelofibrosis (PMF). Platelet, leukocyte, endothelial, and coagulation activation status was assessed in 26 PMF patients and compared with data from 22 age- and sex-matched healthy individuals. Study included flow cytometry assessment of platelet P-selectin expression [at baseline and after adenosine diphosphate (ADP), thrombin and arachidonic acid stimulation], platelet-neutrophil and platelet-monocyte complexes, and CD11b expression in neutrophils and monocytes. Additionally, soluble P-selectin, sCD40L, tissue factor, thrombomodulin, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer were measured by enzyme-linked immunosorbent assays. The above parameters were correlated with the patients' clinical data and presence of the JAK2 V617F mutation. Compared with controls, PMF patients had increased baseline platelet activation, as shown by significantly higher levels of soluble and platelet P-selectin expression, and also higher percentages of platelet-monocyte complexes. Neutrophil and monocyte CD11b expression was significantly higher in patients with the JAK2 mutation than in those with wild-type allele or the controls. Endothelial and coagulation activation, as demonstrated by increased plasma levels of thrombomodulin and F1 + 2, was also found in PMF, with patients with the JAK2 mutation showing significantly higher values of F1 + 2 than those with wild-type allele. In conclusion, PMF patients have platelet, leukocyte, endothelial, and coagulation activation similar to that in PV and ET. CD11b overexpression and F1 + 2 are correlated with the presence of the JAK2 mutation.

BACKGROUND: Despite the good safety of rivaroxaban, there is limited information on strategies for urgent reversal of its antihemostatic effects.Methods and Results:Alterations of hemostasis induced by rivaroxaban (230 ng/ml) were assessed by using several tests applied to steady and circulating human blood. Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were measured. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with circulating blood. The potential reversal of prothrombin complex concentrates (PCCs; 50 IU/kg), activated PCCs (aPCCs; 75 IU/kg), or recombinant factor VIIa (rFVIIa; 270 μg/kg) was evaluated. Impairment of TG parameters induced by rivaroxaban were corrected by the different concentrates (aPCC≥PCC>rFVIIa). Prolonged clotting times and reduced clot firmness caused by rivaroxaban on TEM tests were improved by different concentrates (rFVIIa≥aPCC>PCC). Rivaroxaban significantly reduced platelets and fibrin interactions with damaged vascular surfaces in perfusion studies. While alterations of platelet interactions were favourably counteracted by rFVIIa or aPCCs, reductions in fibrin formation were only partially restored by the different factor concentrates (rFVIIa>aPCC≥PCC). CONCLUSIONS: Rivaroxaban-induced alterations on coagulation parameters measured through assays performed under static conditions were easily reversed by the different concentrates. Studies under flow conditions revealed that these concentrates normalized the action of rivaroxaban on platelets, and significantly improved fibrin formation; although in the later case, levels were not restored to the pre-treatment value. (Circ J 2015; 79: 331-338).

This study investigates whether the response criteria proposed by the European LeukemiaNet (ELN) to evaluate cytoreductive therapies in essential thrombocythemia (ET) correlate with clinically relevant outcomes in patients receiving anagrelide. We evaluated 154 ET patients treated with anagrelide (upfront in 87) for a median of 2.9 years. Complete response (CR), partial response, and no response were observed in 56, 30.5, and 13.5 % patients, respectively. Only 38 patients (25 %) achieved a sustained CR. Overall, the aggregated time on CR and without CR was 200.1 and 333.6 person-years, respectively. The incidence rate of thrombosis and hemorrhage was independent of the CR status. The only factor associated with shorter survival after anagrelide start was the patient's age, whereas achieving a CR with anagrelide had no predictive value for subsequent survival. In conclusion, CR according to the ELN definition is not associated with any measurable clinical benefit in ET patients treated with anagrelide.

Hydroxyurea (HU) is frequently given as treatment for myelofibrosis (MF), but data on its efficacy and tolerability are scarce. The results of HU therapy were evaluated in 40 patients with hyperproliferative manifestations of primary (n = 32), post-polycythemia vera (n = 6), or post-essential thrombocythemia (n = 2) myelofibrosis. Median interval between diagnosis and HU start was 6.2 months (range 0–141.7). Reasons for treatment were constitutional symptoms (55%), symptomatic splenomegaly (45%), thrombocytosis (40%), leukocytosis (28%), pruritus (10%), and bone pain (8%). The starting dose was 500 mg/day, subsequently adjusted to the individual efficacy and tolerability. Response was bone pain 100%, constitutional symptoms 82%, pruritus 50%, splenomegaly 40%, and anemia 12.5%. According to the International Working Group for Myelofibrosis Research and Treatment criteria, clinical improvement was achieved in 16 patients (40%). Median duration of response was 13.2 months (range 3–126.2). Worsening of the anemia or appearance of pancytopenia were observed in 18 patients, requiring administration of erythropoietin-stimulating agents (n = 17) and/or danazol (n = 9). Oral or leg ulcers appeared in five patients and one had gastrointestinal symptoms. HU is an effective and generally well-tolerated therapy for the hyperproliferative manifestations of MF. The accentuation of the anemia often induced by HU is usually manageable with concomitant treatment.

The effectiveness of antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia (ET) is not proven. In this study, the incidence rates of arterial and venous thrombosis were retrospectively analyzed in 300 low-risk patients with ET treated with antiplatelet drugs as monotherapy (n = 198) or followed with careful observation (n = 102). Follow-up was 802 and 848 person-years for antiplatelet therapy and observation, respectively. Rates of thrombotic events were 21.2 and 17.7 per 1000 person-years for antiplatelet therapy and observation, respectively (P = .6). JAK2 V617F–positive patients not receiving antiplatelet medication showed an increased risk of venous thrombosis (incidence rate ratio [IRR]: 4.0; 95% CI: 1.2-12.9; P = .02). Patients with cardiovascular risk factors had increased rates of arterial thrombosis while on observation (IRR: 2.5; 95% CI: 1.02-6.1; P = .047). An increased risk of major bleeding was observed in patients with platelet count greater than 1000 × 109/L under antiplatelet therapy (IRR: 5.4; 95% CI: 1.7-17.2; P = .004). In conclusion, antiplatelet therapy reduces the incidence of venous thrombosis in patients with JAK2-positive ET and the rate of arterial thrombosis in patients with associated cardiovascular risk factors. In the remaining low-risk patients, this therapy is not effective as primary prophylaxis of thrombosis, and observation may be an adequate option.

The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes.We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63(+) basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor.We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63(+) basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63(+) basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden.These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells' abnormal function and, putatively, in the pathogenesis of pruritus.

Vascular events and evolution to either myelofibrosis (MF) and acute leukemia (AL) are the main causes of morbidity and mortality in individuals with essential thrombocythemia (ET). However, the frequency of these complications in young ET patients is not well known. The objective of the present study was to assess the frequency of vascular events and the incidence of MF and AL in young patients with ET and to identify the factors associated with the development of such complications. In 126 subjects diagnosed with ET at a median age of 31 years (range: 5–40), overall survival and probability of survival free of either thrombosis, bleeding, MF, AL, and polycythemia vera (PV) were analyzed by the Kaplan-Meier method, followed by the log-rank test. With a median follow-up of eleven years (range: 4–25) three patients have died, being the probability of survival 98% at ten years. A total of 31 thrombotic events were registered in 25 patients; thrombosis-free survival (TFS) was 84% at ten years. Tobacco use was the only factor associated with an increased thrombotic risk, since TFS at 10 years was 72% in smokers versus 90% in non-smokers (p=0.03). Severe hemorrhagic complications were observed in 11 patients, and the estimated probability of bleeding-free survival was 92% at ten years. Evolution to MF was seen in 6 patients, four of whom had never received treatment for ET. MF-free survival was 97% at 10 years, with the risk being higher in patients showing an increased reticulin network in the bone marrow biopsy performed at diagnosis of ET (p=0.005). Transformation to AL was registered in one patient. JAK2 was mutated in 33 out of the 87 assessable patients (38%) and the mutation was associated with higher Hb values at diagnosis (p = 0.001). ET evolved into PV in five patients, being the probability of evolution into PV of 15% in JAK2 V617F positive patients versus 0% in JAK2 V617F negative patients (p=0.01). In conclusion, severe vascular complications are not infrequent in young subjects with ET, whereas transformation to MF or AL is a rare event.

The mechanisms accounting for the increased risk of thrombosis in patients with essential thrombocythemia (ET) are not well known. The aim of the present study was to ascertain the role of platelet and leukocyte activation in the thrombosis of ET.The activation status of platelets and leukocytes was assessed by flow cytometry studies in 49 patients with ET (22 with previous thrombosis and 27 without a history of thrombosis) and in a group of age- and sex-matched healthy individuals. The assessment included platelet P-selectin expression (measured both at baseline and after stimulation with ADP, thrombin, arachidonic acid (AA), and collagen), platelet-neutrophil and platelet-monocyte complexes, determination of CD11b in the neutrophils and monocytes, and expression of tissue factor in the monocytes (mTF). The JAK2 V617F mutation was studied and correlated with platelet and leukocyte activation.As compared with controls, ET patients had significantly higher values of baseline P-selectin and thrombin- and AA-induced platelet P-selectin expression, as well as higher platelet-neutrophil and platelet-monocyte complexes, neutrophil CD11b expression and baseline mTF expression. Platelet P-selectin, monocyte CD11b, and lipopolysaccharide-induced mTF expression was significantly higher in ET patients with a history of thrombosis than in patients without thrombosis. Patients with the JAK2 V617F mutation or thrombosis showed higher baseline and AA-induced platelet P-selectin expression than did those without thrombosis.These results would support a role for platelet and monocyte activation in the thrombosis of ET. In these patients, the presence of the JAK2 V617F mutation is associated with higher platelet activation.

Thrombosis is a frequent complication of polycythemia vera and essential thrombocythemia, but its incidence and predisposing factors in idiopathic myelofibrosis (IM) are unknown. In 18 (11.6%) of 155 patients diagnosed with IM in a single institution, 31 thrombotic events (19 arterial, 12 venous) were registered after a mean follow-up of 4.2 (s.d.: 4.5) years. In six patients, the thrombosis was simultaneous to or appeared a few months before IM diagnosis and 14 had one or more thrombotic episodes. When compared with the general population, a significant increase was observed in the incidence of venous thrombosis (odds ratio 17.5, 95% confidence interval: 10.3–31.4). At multivariate analysis, the initial variables associated with an increased risk of thrombosis were thrombocytosis (platelets >450 × 109/l, P=0.001), presence of one cardiovascular risk factor (arterial hypertension, smoking, hypercholesterolemia, or diabetes, P=0.003), cellular phase of myelofibrosis (P=0.005), and Hb >11 g/dl (P=0.02). Considering post-diagnosis events, the 5-year thrombosis-free survival probability was 90.4% in the series, 80.6% for patients with platelets >450 × 109/l, 82.6% for patients with one cardiovascular risk factor, and 85.1% for those in cellular phase. These results indicate an increased thrombotic risk for IM patients with hyperproliferative features and/or coexistent cardiovascular risk factors.

Severe retroperitoneal hemorrhage represents an infrequent and serious complication of bone marrow biopsy. A 53-year-old man, diagnosed with polycythemia vera 12 years earlier, was submitted to a bone marrow biopsy due to the appearance of anemia with clinical and hematological features suggesting myelofibrotic transformation, a diagnosis that was confirmed by the marrow study. At 2 h of a right anterior iliac bone marrow trephine biopsy, the patient suddenly developed severe pain in the area of the biopsy, with antialgic flexion of the right leg. Computed tomographic (CT) scan of the abdomen showed a 5 x 9.5 cm hematoma in the right iliac and psoas muscles. The patient was initially managed with analgesics and transfusional support, but the pain persisted and a continuous fall in the hematocrit was observed in the following days. Angiographic examination of the right external iliac artery showed contrast extravasation arising from the circumflex iliac branch, which was embolized using polivinyl alcohol particles and one coil. Following such procedure, the patient recovered uneventfully and was discharged in good condition a few days later. This case illustrates the effectiveness of an endovascular approach in providing a fast and minimally invasive treatment for this life-threatening complication of bone marrow trephine biopsy.

Autologous stem cell transplantation is a therapeutic option for chronic myeloid leukemia (CML) patients who are not candidates for allogeneic transplant. To reduce the risk of post-autografting disease recurrence, different strategies of stem cell selection have been attempted. The results of using recombinant human granulocyte colony-stimulating factor (rHuG-CSF) for harvesting hematopoietic progenitors in CML patients treated with interferon-a (IFN) are reported.Twenty-one CML patients who received IFN for a median of 21 (8-68) months were mobilized with rHuG-CSF (10 mg/kg/day). Twelve were in complete (CCR) or major (MCR) cytogenetic response. Complete success was considered a sufficient harvest (1 x 10(6)/kg CD34(+) cells/kg) without Philadelphia (Ph)+ metaphases in at least one apheresis; a partial success was a sufficient harvest with 1-35% Ph(+) cells.A total of 78 aphereses were performed. No patient had major side-effects. The median number (range) of mononuclear and CD34(+) cells obtained was, respectively, 8.6 x 10(8)/kg (0.9-22.6) and 3.3 x 10(6)/kg (0.4-26.3) per patient. A sufficient cell yield was collected in all but three patients. A complete/partial success was achieved in seven CCR/MCR patients (63%) and in three (33%) with other responses. Four patients underwent successful autografting using the stem cells obtained after rHuG-CSF mobilization.Mobilization of IFN-treated patients using rHuG-CSF is safe and provides a significant proportion of Ph-negative progenitors in CML patients in complete or major cytogenetic response.

BACKGROUND: Despite the proven efficacy and safety profile of dabigatran as compared to warfarin, bleeding remains a concern as with all anticoagulants and the reversal of dabigatran’s anticoagulant effect for emergency procedures remains controversial. Recently, idarucizumab, a specific antidote for dabigatran, has been functionally characterized and its efficacy demonstrated in animal models and healthy volunteer studies. AIMS: We explored the effects of dabigatran on hemostasis in human blood focusing on possible interference with platelet and coagulation responses to vessel injury under flow conditions. We also compared the potential efficacy of idarucizumab with procoagulant strategies such as prothrombin complex concentrates (PCC), activated PCC (aPCC) or rFVIIa at reversing the antithrombotic action of dabigatran to better understand local processes in response to injury. METHODS: Concentrations of dabigatran equivalent to the Cmax reported at steady state after therapy with 150 mg twice daily (184 ng/mL) were added in vitro to blood aliquots from 11 healthy donors. Whole blood samples were used to evaluate modifications in different coagulation biomarkers: 1) fibrin and platelet deposition on damaged vascular segments with whole blood under flow conditions at a shear rate of 600 s-1, 2) dynamics of thrombin generation (TG) in plasma using a fluorogenic assay (Technothrombin TGA) and 3) viscoelastic parameters of clot formation in whole blood using by thromboelastometry (ROTEM) The efficacy of specific reversal with idarucizumab 0.3, 1 and 3 mg/mL was compared with that of non specific procoagulant concentrates such as aPCC 25 and 75 IU/kg, PCC 70 IU/kg, or rFVIIa 120 µg/kg. RESULTS: Dabigatran (184 ng/mL) caused a pronounced 85% reduction of fibrin coverage on the damaged vessel from 67.2±9.8 to 9.5±1.3 % (p CONCLUSIONS: Dabigatran (184 ng/mL) added to blood from healthy volunteers caused evident alterations in hemostasis parameters related to its recognized anticoagulant action. Procoagulant concentrates significantly compensated for the overall anti-hemostastic action of dabigatran. Overall, 75 U/kg aPCC seemed the more efficient nonspecific reversal therapy. In clear contrast with non specific procoagulant strategies, idarucizumab, the specific antidote to dabigatran completely reversed all alterations in coagulation parameters evaluated in circulating human blood and in assay systems. (Supported by SAF 2011-2814 and PI13/00517, Spanish Gov & FEDER) Disclosures van Ryn: Boehringer Ingelheim Pharma: Employment. Escolar:Boehringer Ingelheim Pharma: Investigator Sponsored Research Funding Other.

Background Patients with Philadelphia-negative myeloproliferative neoplasms (MPN) can develop venous thrombosis and MPN are the leading cause of splanchnic vein thrombosis. Cerebral vein thrombosis (CVT) is a rare life-threatening disease that in approximately 3% of cases encounters MPN among risk factors and tends to recur in 2-4% of patients as CVT and in 4-7% as venous thrombosis at other sites. Little or no information is available on patients with MPN who develop CVT. Objective and design To investigate the characteristics and clinical course of CVT in patients with MPN we carried out a multicenter (n=11), observational, retrospective cohort study. Patients Centers were asked to provide information on index patients with MPN who developed CVT (group MPN-CVT). For each of them, 2 patients with MPN and venous thrombosis other than CVT (group MPN-VT) and 4 patients with MPN and no venous thrombosis (group MPN-NoVT) were provided, matched by sex, age at diagnosis of MPN (+/-5 years) and type of MPN (polycytemia vera, essential thrombocytemia, myelofibrosis) with index patients. Results From January 1982 to June 2013, 48 MPN-CVT, 87 MPN-VT and 178 MPN-NoVT patients were identified in a population of 5,500 patients with MPN. Diagnosis of MPN and thrombosis coincided in 46% of MPN-CVT and 29% of MPN-VT patients (p=0.046). Compared to MPN-NoVT, MPN-CVT and MPN-VT patients had a higher prevalence of thrombophilia abnormalities (40% and 35% vs 21%, p=0.015) and, among those with essential thrombocytemia, of the JAK2 V617F mutation (76% and 78% vs 55%, p=0.059). Compared to MPN-VT, MPN-CVT patients had a higher rate of recurrent thrombosis (42% vs 25%, p=0.049) that in two-third of patients in both groups was venous, with a similar site distribution. This difference occurred despite a shorter median follow-up period (6.1 vs 10.3 years, p=0.019), a higher proportion of patients on long-term antithrombotic treatment (94% vs 84%, p=0.099) and a similar proportion of patients on cytoreductive treatment (75% vs 72%, p=0.745) among MPN-CVT than MPN-VT patients. The incidence of recurrent thrombosis was 8.8% patients/year in MPN-CVT and 4.2% patients/year in MPN-VT patients (log-rank test, p=0.022) and CVT was the only variable in a multivariate model including blood counts, thrombophilia, cytoreductive and antithrombotic treatment, that was predictive of recurrent thrombosis (HR 1.86, 95%CI 1.00-3.58). Conclusions Patients with MPN develop recurrent thrombosis in a much higher proportion than those without, particularly if they had a CVT. Patients with MPN and CVT have an approximately 2-fold increased probability to develop recurrent thrombosis than those with MPN and venous thrombosis at other sites, independently of other risk factors. Disclosures: No relevant conflicts of interest to declare.

Abstract 2261 BACKGROUND: Bleeding is still a frequent complication for patients on treatment with new oral anticoagulants. There is a lack of information on therapies that could reverse the effects of new oral anticoagulants in patients that require a rapid restoration of their impaired hemostatic mechanisms. OBJECTIVES: We present preliminary results of an ongoing clinical trial (Eudra CT2010–022985–29; ClinicalTrials.gov Identifier:NCT01478282) with focus on: 1) assessment of the effects of new oral anticoagulants rivaroxaban and dabigatran on hemostasis; 2) evaluation of the impact of prothrombin complex concentrates (PCCs) and rFVIIa to reverse the alterations of hemostasis induced by these new anticoagulants. METHODOLOGY: Ex vivo studies were performed using blood samples from healthy individuals (n=6, as of July 2012) subjected to treatments with 20 mg/day for rivaroxaban and 150 mg/12 h for dabigatran doses separated by a washout period of 14 days. Blood samples were spiked in vitro with: a) prothrombin complex concentrates (PCCs) (50 IU/kg); b) activated PCCs (aPCCs) at 75 IU/kg, or c) rFVIIa at 270 μg/kg. A series of laboratory tests were performed to explore modifications in hemostatic mechanism using technologies implying steady and flow conditions. Effects on thrombin generation (TG), thromboelastometry parameters (TEM) and standard coagulation parameters were assessed. Special attention was paid to studies evaluating modifications on platelets and fibrin deposition on damaged subendothelial surfaces performed under flow conditions. RESULTS: Standard coagulation parameters (PT, INR and APTT) were variably affected by rivaroxaban and dabigatran. aPCCs and rFVIIa totally reversed the effects of rivaroxaban in these routine tests, whereas only aPCC were capable to reverse the effects of dabigatran on APTT. Rivaroxaban caused a moderate reduction of TG with delayed time to peak and decreased velocity index (prFVIIa). Viscoelastic properties of clot were affected after rivaroxaban or dabigatran treatments. All the coagulation concentrates were able to reverse the effects of rivaroxaban (aPCC=rFVIIa>PCC; prFVIIa=PCC. Alterations in fibrin formation following dabigatran treatment were only reverted by aPCCs (p CONCLUSION: Rivaroxaban and dabigatran treatments resulted in alterations of the different laboratory tests related to their recognized anticoagulant action. These alterations were variably compensated or even reversed by the different factor concentrates. Reversal strategies may differ depending on the anticoagulant agent and its specific mechanism of action. Results of studies with blood circulating through vascular surfaces, that would more closely reproduce the bleeding situation, indicate that while alterations in hemostasis induced by rivaroxaban were reversed to a variable extent by all the concentrates tested (PCCs, aPCCs and rFVIIa), reversion of dabigatran effects was only observed with aPCCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 2263 Background: Newly developed oral anticoagulants require less frequent controls, have fewer interactions with other medications and effects are more reliable. Despite these advantages, bleeding is still a frequent complication for patients on treatment with oral anticoagulants and reversal of their effects remains an unsolved issue for the newer agents. Apixaban is a new oral anticoagulant with inhibitory action on FXa. It has showed a very good efficacy/safety profile in studies in prophylaxis of thrombotic complications after mayor orthopaedic surgery and in atrial fibrillation (AF). Aims: To explore the effects of concentrations of apixaban in excess of those achieved after standard approved dosage, on platelet and coagulation mediated mechanisms of hemostasis. To evaluate the effectiveness of different factor concentrates at reversing the alterations of hemostatic mechanism induced by apixaban. Methods: Apixaban was added in vitro to blood alliquots from healthy donors. Concentrations of apixaban tested (200 ng/ml) doubled the average Cmax reached at steady state with standard approved treatment for AF. Modifications in platelet reactivity towards surfaces were measured at elevated shear rates in a cone and plate analyzer (Impact R®). Effects on thrombin generation (TG) and on thromboelastometry parameters (TEM) were also assessed. Changes in platelet adhesive, aggregating and procoagulant activities were additionally evaluated in perfusion studies through vascular surfaces in a system producing results that correlate with clinical situations (Transfus Med Rev 15:144, 2001). The potential action of prothrombin complex concentrates (PCCs) (50 IU/kg), activated PCCs (aPCCs) at 75 IU/kg, or rFVIIa at 270 μg/kg reversing the anticoagulant actions of apixaban were evaluated. Results: Apixaban did not affect the reactivity of platelets to surfaces in the Impact R®. Apixaban caused a moderate reduction in TG as confirmed by delayed lag phase, prolonged time to peak and reduced velocity index. Thrombin peak and velocity indexes in TG were improved by concentrates with the following order of efficacy PCC≥aPCC>rFVIIa. Apixaban prolonged clotting time (CT) and reduced maximal clot firmness (MCF) in TEM studies using tissue factor as activator. Alterations in these parameters were corrected by the different concentrates according to the following order of efficacy rFVIIa≥aPCC>PCC. Apixaban quantitatively reduced fibrin and platelet interactions with damaged vascular surfaces (pPCC>aPCC. Conclusions: Apixaban at 200 ng/ml did not show a direct antiplatelet effect in studies in the cone plate analyzer, but demonstrated evident alterations in hemostasis related to its recognized anticoagulant action. These alterations were variably compensated or even reversed by the different factor concentrates. Effects of these concentrates were not homogeneous in all the tests, with PCCs showing more efficacy in TG and rFVIIa being more effective on TEM and perfusion tests. Studies with flowing blood that reproduce conditions closer to the bleeding situation indicate that current coagulation factor concentrates can effectively restore the impairement in fibrin formation on damaged vascular surfaces resulting from apixaban overdose. Disclosures: Escolar: BMS: Honoraria, Research Funding; Bayer: Honoraria; Boehringer Ingelheim: Honoraria.

Abstract 2828 Antiplatelet therapy with low-dose aspirin is commonly used in combination with cytoredution for the prevention of thrombosis in patients with high-risk essential thrombocythemia (ET), as determined by age > 60 years and/or previous history of thrombosis. However, it is uncertain whether low-dose aspirin adds any benefit to cytoreductive therapy in patients without a history of thrombosis. In this study, the probability of thrombosis and bleeding was retrospectively analyzed in 248 patients with ET (median age: 66 years; 83 males, 165 females) treated with cytoreduction plus low-dose aspirin (n=170) or with cytoreduction only (n=78). Patients with a history of thrombosis or bleeding, as well as those receiving anticoagulant therapy, were excluded from the study. The indication of cytoreduction was age > 60 years (n=198), extreme thrombocytosis (n=37), microvascular disturbances (n=6), and others (n=7) First-line cytoreductive therapy consisted of hydroxyurea (n=216), anagrelide (n=27), interferon-α (n=4), and busulfan (n=1). During a median follow-up of 7.4 years (range: 0.1–26), a total of 28 thromboses (arterial, n=22; venous, n=6) were registered. At 5 years, the probability of thrombosis in patients receiving cytoreduction plus low-dose aspirin or cytoreduction only was 8% and 17%, respectively (p=0.006). No significant differences were observed depending on gender, presence of cardiovascular risk factors, JAK2 V617F mutational status, WBC count at diagnosis or type of cytoreductive therapy. At multivariate analysis, patients not receiving antiplatelet therapy were found to have a higher risk of thrombosis (HR: 3.3, 95%CI: 1.3–7.9, p=0.009). During the study period, a total of 13 major hemorrhagic events were registered (digestive, n=7, intracranial, n=3, others, n=3). At 5 years, the probability of major bleeding in patients receiving cytoreduction plus low-dose aspirin or cytoreduction as monotherapy was 6% and 4%, respectively (p=0.1). In conclusion, in patients with ET receiving cytoreductive therapy as primary prophylaxis of thrombosis, the addition of low-dose aspirin reduces the risk of thrombosis without increasing the risk of bleeding. Disclosures: No relevant conflicts of interest to declare.

There is increasing evidence that platelet and leukocyte activation plays an important role in the thrombotic complications of patients with essential thrombocythemia (ET), but the relationship of both thrombosis occurrence and JAK2 V617F mutation status with the levels of circulating tissue factor (TF) and of soluble markers of platelet and endothelial activation is not known. In 53 ET patients (26 of whom had a previous history of thrombosis), platelet TF expression and plasma levels of TF, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer, and prothrombin fragment 1+2 (F1+2), measured by ELISA, were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 V617F mutation status. ET patients with thrombosis had significantly higher levels of sP-selectin than patients without thrombosis and the controls, whereas ET patients without thrombosis had significantly higher levels than the controls (99.8 ± 47.1 ng/mL versus 70.6 ± 37.8 ng/mL versus 32.4 ± 11.9 ng/mL; p= 0.0001 for all comparisons). The same applied to sCD40L levels (226.7 ± 104.7 pg/mL in patients with thrombosis, 186.4 ± 92.1 pg/mL in patients without thrombosis, and 81.3 ± 22.0 pg/mL in controls; p= 0.0001 for all comparisons). Circulating VWF:Ag and F1+2 levels were higher in ET patients than in controls, but no significant difference was observed between patients with and without thrombosis. No differences in TF platelet expression, TF and sTM plasma concentrations were found between patients and controls. A positive correlation was observed between sP-selectin and F1+2, a marker of thrombin generation (r= 0.378, p= 0.01). Patients with the JAK2 mutation (22 out of 52 assessable patients), as compared with those with the wild-type allele, had significantly higher levels of sP-selectin (p= 0.002), sCD40L (p= 0.03), TF (p= 0.016), VWF:Ag (p= 0.0001), and sTM (p= 0.032). These results support a role for soluble markers of platelet activation in the thrombosis of ET as well as their potential to identify ET patients at greater risk of thrombosis. The association between JAK2 mutation and increased levels of TF and soluble markers of platelet and endothelial activation would suggest that the mutation could promote an enhanced prethrombotic state in ET.

The mechanisms accounting for the increased risk of thrombosis in patients with essential thrombocythemia (ET) are not well known. Recently, a possible role for the granulocytes in the development of thrombosis in ET has been suggested. To analyze the possible relationship between granulocyte activation and a history of thrombosis in ET, granulocyte activation was studied in 53 ET patients (26 with a previous history of thrombosis and 27 without) and 26 healthy controls. Neutrophil and monocyte CD11b expression, monocyte tissue factor (mTF) expression, and platelet-neutrophil (PNC) and platelet-monocyte (PMC) complexes were studied using whole blood cytometry and the results were expressed as percentages and in MESF (molecules of equivalent soluble fluorochrome) units. Granulocyte CD11b and mTF expression were measured at baseline and after activation with lipopolysaccharide (LPS). The results are as follows: 1) ET patients had significantly higher baseline percentages of circulating PNC and PMC as well as neutrophil CD11b MESF expression than the controls (p=0.0001); 2) monocyte CD11b expression was higher in patients with ET and thrombosis (mean 151241 MESF) than in those without (127191 MESF) and in the controls (65717 MESF) (p= 0.0001); 3) mTF baseline surface expression was increased in ET patients as compared with the controls (15635 vs 9385 MESF, p= 0.0001); 4) following LPS activation, patients with ET and previous thrombosis had significantly higher mean percentage of mTF expression than those without thrombosis and the controls (48%, 36% and 25%, respectively) (p= 0.0001). No differences were noted when the results were analysed according to the treatment that the patients were receiving at the time of study. In conclusion, patients with ET and a history of thrombosis show a marked increase in granulocyte activation, especially in monocyte activation, and therefore such alteration might play a role in the pathogenesis in these patients.

Essential thrombocythemia (ET) is a myeloproliferative disorder associated with an increased tendency to thrombosis, the pathogenesis of which is not well known. The aim of the present study was to analyze the relationship between platelet turnover and activation and a history of thrombosis in patients with ET. The expression of platelet P-selectin (measured both in unstimulated platelets and after agonist-stimulation with ADP, thrombin, arachidonic acid (AA), collagen or epinephrine) and the percentage of reticulated platelets (RP) were measured in 53 ET patients with (n=26) or without (n=27) previous thrombosis and in 26 healthy controls. Both variable were assessed using quantitative whole blood cytometry and the results were expressed as percentages and in MESF (molecules of equivalent soluble fluorochrome) units. The results were as follows: 1) patients with thrombosis showed a significantly higher basal platelet P-selectin expression (6.1%, 95% CI: 4.6–6.8) than those without thrombosis (4.6%, 95% CI: 3.7–5.5)) and the healthy controls (3%, 95% CI: 2–4.2) (p= 0.0001 for all comparisons); 2) following thrombin activation, ET patients had a higher mean percentage of platelet P-selectin expression than the controls (61% versus 46%, p = 0.01), but no difference was seen between the two groups of patients; 3) after AA activation, patients with ET and thrombosis also showed higher platelet P-selectin values (78%, 95% CI: 69–86) than those without thrombosis (63%, 95% CI: 55–76) and the controls (46%, 95% CI: 33–58) (p=0.001 for all comparisons); 4) ET patients with thrombosis had a significantly higher mean percentage of RP (18%, 95% CI: 13–20) than patients without thrombosis (13%, 95% CI: 11–18) and the controls (8%, 95% CI: 5–10) (p = 0.0001 for all comparisons). No differences were observed when the patients were separated on the basis of the treatment that they received at the time of study. The above results point at a possible role of AA-induced platelet acitvation in the development of thrombosis in ET and give conceptual support to the prohylactic use of aspirin in such patients.

Young patients (age < 60 years) with essential thrombocythemia (ET) and no history of thrombosis are considered at low risk of thrombosis and therefore managed on a conservative approach with antiplatelet therapy or even without any treatment. JAK2 V617F and CALR exon 9 mutations are the most frequent molecular alterations observed in ET, with CALR-positive ET being considered a distinct clinical entity due to its higher platelet counts and lower incidence of thrombosis as compared with JAK2 V617F-positive ET. There is some evidence supporting a role for antiplatelet therapy in JAK2 V7617F-positive neoplasms. However, the role of antiplatelet therapy in CALR-positive ET has not been studied. The aim of the present study was to assess the effect of antiplatelet therapy in the primary prevention of thrombosis in patients with CALR-positive ET without indication of cytoreductive therapy. For such purpose, 240 patients (107 males, 133 females) diagnosed with ET at a median age of 42 years (range 13-59) were included in a multicenter retrospective study. Initial treatment consisted of antiplatelet therapy (n=109) or careful observation (n=108), whereas 23 patients received cytoreduction since diagnosis and were excluded. During a median follow up of 8 years, 137 patients were started on cytoreductive therapy because of the following indications: age > 60 years (n=10), thrombosis (n=10), bleeding (n=2), microvascular symptoms (n=18), extreme thrombocytosis (n=89), and others (n=8). Median time free of cytoreductive therapy was 3.2 years. Thrombosis-free survival restricted to the time of cytoreductive therapy abstention was calculated using the Kaplan-Meier method. Variables attaining a significant level at the univariate analysis were included in a Cox proportional hazard model. During the period of abstention of cytoreductive therapy, a total of 10 thrombotic events and 8 major bleeding episodes were registered. The probability of thrombosis at 3 years was 5% in patients managed with careful observation and 1% in those receiving antiplatelet therapy (p = 0.2). At multivariate analysis, antiplatelet therapy did not result in a lower risk of thrombosis after correction for age, sex and presence of cardiovascular risk factors. Interaction studies did not identify any subgroup of patients that benefited from antiplatelet therapy in thrombosis prevention. Regarding major bleeding, patients receiving antiplatelet therapy experienced a higher rate than those managed on observation (3-year probability of major bleeding, 5.5% and 0%, respectively, p=0.05). At multivariate analysis, antiplatelet therapy was associated with a tendency towards and increased risk of major bleeding (HR: 7.7, 95%CI: 0.9-66.2, p=0.06) independently of platelet count at diagnosis, age and gender. In conclusion, CALR-mutated low-risk ET patients under cytoreductive therapy abstention may not obtain a clear benefit from antiplatelet therapy since the increase in the rate of bleeding may offset the reduction in the rate of thrombosis Disclosures García-Gutierrez: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Harrison:CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Gilead: Honoraria; Sanofi: Honoraria, Speakers Bureau; Shire: Speakers Bureau. Cervantes:Sanofi-Aventis: Consultancy; Novartis: Consultancy, Speakers Bureau; CTI-Baxter: Consultancy, Speakers Bureau. Vannucchi:Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Apixaban is a new oral anticoagulant with a specific inhibitory action on FXa. No information is available on the reversal of the antihemostatic action of apixaban in experimental or clinical settings. We have evaluated the effectiveness of different factor concentrates at reversing modifications of hemostatic mechanisms induced by moderately elevated concentrations of apixaban (200 ng/ml) added in vitro to blood from healthy donors (n = 10). Effects on thrombin generation (TG) and thromboelastometry (TEM) parameters were assessed. Modifications in platelet adhesive, aggregating and procoagulant activities were evaluated in studies with blood circulating through damaged vascular surfaces, at a shear rate of 600 s(-1). The potential of prothrombin complex concentrates (PCCs; 50 IU/kg), activated prothrombin complex concentrates (aPCCs; 75 IU/kg), or activated recombinant factor VII (rFVIIa; 270 μg/kg), at reversing the antihemostatic actions of apixaban, were investigated. Apixaban interfered with TG kinetics. Delayed lag phase, prolonged time to peak and reduced peak values, were improved by the different concentrates, though modifications in TG patterns were diversely affected depending on the activating reagents. Apixaban significantly prolonged clotting times (CTs) in TEM studies. Prolongations in CTs were corrected by the different concentrates with variable efficacies (rFVIIa≥aPCC>PCC). Apixaban significantly reduced fibrin and platelet interactions with damaged vascular surfaces in perfusion studies (p