ALBERT

Description: Platelet transfusions are widely used for patients with severe thrombocytopenia. There are, however, practical problems in the current donor-dependent platelet transfusions, such as the limited supply and risk of serious immune reactions. Thus, the development of new strategies for generating platelets for transfusion is crucial. Platelets have been differentiated from hematopoietic stem cells, fetal liver cells, embryonic stem cells, induced pluripotent stem cells, NF-E2-transduced fibroblasts, and preadipocytes. Here, among these cells preadipocytes, especially in the subcutaneous adipose tissue, could be ideal candidate cells for manufacturing megakaryocytes (MKs) and platelets, because (1) they are relatively easy to obtain large quantities and have ability to proliferate in vitro, (2) their differentiation does not require gene transfer, as they possess genes in relation to megakaryopoiesis and thrombopoiesis, such as p45NF-E2 and c-mpl, and (3) they differentiate into MKs and platelets using an endogenous thrombopoietin. Thus, to clarify the usefulness of preadipocytes as a donor-independent source for platelet transfusion, we compared both number and function between platelets derived from mouse subcutaneous preadipocytes and those from bone marrow mononuclear cells (BMMNCs), the established cell source for manufacturing platelets. First, BMMNCs were not feasible for their expansion in vitro and therefore the cells were directly seeded in MK lineage induction media. In contrast, preadipocytes were to be passaged 6 times without any morphological changes, and then cultured in MK lineage induction media for their differentiation into platelets. Thus, as assessed by CD41-positive platelet-sized cells, 106.2±5.0 ×105 or 3.9±1.0 ×105 platelets were obtained from 106 preadipocytes or 106 BMMNCs, respectively (p

Description: Abstract 3199 The platelet αIIbβ3 integrin changes its conformation to increase the affinity for ligands upon platelet activation. It has been shown that binding of the intracellular proteins to the cytoplasmic tails activate αIIbβ3 by disrupting the endogenous interaction between the two cytoplasmic tails in inside-out signaling. However, the mechanism how separation of the cytoplasmic tails initiate structural rearrangement of the extracellular domains has not been investigated. In αIIbβ3 crystal structure, the α-extracellular tail consisting of thigh, calf-1, and calf-2 domains makes large interface with the β-extracellular tail consisting of EGF-2, -3, -4, and βT domains. We have previously shown that clasp formation between the two extracellular tails completely abrogated αIIbβ3 activation induced by the cytoplasmic tail truncation. This suggests that separation of the two extracellular tails is essential for activation induced by inside-out signaling. To examine whether this is the case, N-glycan binding sites consisting of the N-X-T/S motif were introduced in αIIb amino acid residues located in the thigh/EGF-2, calf-1/EGF-3, or calf-2/βT interfaces. Attachment of a bulky N-glycan to these sites would prevent endogenous interface formation, thus forces the tails to separate from each other. When expressed in CHO cells, Q513NKT mutation in the thigh, M660NRT mutation in the calf-1, V760NVT mutation in the calf-2 domain all induced robust fibrinogen binding without any activators. Attachment of N-glycan was confirmed by SDS-PAGE analysis on the αIIb fragments carrying these mutations. This activation was inhibited by stabilizing the β-head/β-tail interface or by limiting the αIIb movement with artificially introduced disulfides, both of which prevents integrin from assuming the extended conformer. Prevention of the hybrid domain swing-out also completely inhibited activation. These results suggest that separation of the tails activate αIIbβ3 by inducing integrin extension. To confirm if endogenous α-tail/β-tail interaction indeed keeps integrin in low affinity state, αIIb amino acid residues that participate in the interface formation were mutated and the effect on fibrinogen binding was examined. Among the mutants, the K514A in the thigh, R671A in the calf-1, and R751A in the calf-2 domains induced only weak activation. However, combining these mutations resulted in robust activation. This activation was completely inhibited by the C-terminal clasp formation. Taken together, these results suggest that the α-tail/β-tail interface is kept by a group of key interdomain interaction that individually is not strong enough to do so. In conclusion, the α-tail/β-tail interface interaction regulates integrin activation by keeping integrin in low affinity state. Disruption of the interaction induces separation of the extracellular tails and activates integrin by initiating the structural rearrangement from the low affinity bent conformer to the high affinity extended conformer. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3321 Background: Edoxaban is a direct inhibitor of factor Xa (FXa), and its efficacy as an oral anti-coagulant agent is less likely to be affected by food intake or drug-drug interaction. This profile of edoxaban suggests a good compliance in clinical use.However it is not clear whether genetic variations of FX influence the efficacy of edoxaban. Objectives: To investigate the possible inter-patient variability in the efficacy of edoxaban stemming from SNPs in the FX gene, we characterized the enzyme activity of FXa derived from wild type FX (FX-WT) and from FX with two known mutations, A152T (FX-A152T) and G192R (FX-G192R). The impact of FX mutations were also tested on the pharmacological activity of edoxaban. Methods: Among known FX SNPs in the NCBI dbSNP database, two non-synonymous SNPs are located inside mature FX, rs3211772 (allele frequency: 0.006) and rs3211783 (allele frequency: 0.022), corresponding to A152T and G192R. The former located inside the light chain and the latter located inside the activation peptide of FX. We selected these two SNPs and examined whether they might influence on the efficacy of edoxaban. We prepared recombinant FX proteins of FX-WT, FX-A152T and FX-G192R. We measured the enzyme activities of these FXa and the anti-FXa and anticoagulant effects of edoxaban on these FXa. Recombinant FX proteins were activated with Russell's viper venom factor × activator and FXa activity was measured using a chromogenic substrate S-2222. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured using FX-deficient plasma supplemented with recombinant FX proteins with a coagulometer CA-50. Results: Km values of FX-WT, FX-A152T and FX-G192R FXa were 0.55, 0.53 and 0.54 nM, respectively, Vmax of FX-WT, FX-A152T and FX-G192R FXa were 21.0, 21.8 and 21.4 mOD/min, respectively. PTs of plasma containing these mutations were 25.2 (FX-WT), 24.2 (FX-A152T) and 24.1 (FX-G192R) seconds. aPTTs of plasma containing the mutated FXs were 76.7 (FX-WT), 77.3 (FX-A152T) and 72.6 (FX-G192R) seconds. These data indicated that these mutations do not affect the basal FXa catalytic activity and coagulation activity. The Ki values of edoxaban for the mutated Fxas, the concentrations of edoxaban required to double the PT (PTCT2) and aPTT (aPTTCT2) in plasma containing the mutated FXs did not affected by two FX mutarions (Table 1). These data demonstrated that those mutations have no impact on the anticoagulant activity of edoxaban. Conclusions: Two FX mutations, A152T and G192R, do not affect the basal FXa catalytic activity and coagulation activity. Edoxaban acts equally on FX-WT, FX-A152T and FX-G192R. It is suggested that edoxaban has little inter-patient variability stemming from SNPs in FX gene. Disclosures: Noguchi: Daiichi Sankyo Co., Ltd.: Employment. Takahashi:Daiichi Sankyo Co., Ltd.: Employment. Ishihara: Daiichi Sankyo Co., Ltd.: Employment. Morishima:Daiichi Sankyo Co., Ltd.: Employment. Shibano:Daiichi Sankyo Co., Ltd.: Employment.

Description: Abstract 260 Platelet αIIbβ3 integrin undergoes structural rearrangements to increase the affinity for ligands. Two major changes in integrin three-dimensional structure are critical for this activation process. One is the straightening of integrin legs (extension), and the other is the swing out movement of the β3 hybrid domain (swing-out). In this study, we have found that this structural requirement for activation differs depending upon the divalent cations present. We previously reported that the swing-out-defective mutant αIIbD319C/β3V359C did not bind fibrinogen in the presence of Ca2+/Mg2+. However, the same mutant bound fibrinogen in the presence of Mn2+, when activated by monoclonal antibody PT25-2 or by forcing the integrin legs to extend by concomitant αIIbQ595NTT mutation. We engineered another mutation that was designed specifically to prevent the swing-out. Likewise, this β3G327C/β3V419C mutation blocked fibrinogen binding in the presence of Ca2+/Mg2+, but not in the presence of Mn2+. On the other hand, when integrin extension was specifically prevented by αIIbD464C/αIIbS728C mutation, fibrinogen binding was observed neither in the presence of Ca2+/Mg2+ nor in Mn2+. Thus, swing-out is not essential for activation in Mn2+, as long as integrin legs are in extended state, while both swing-out and extension are required in Ca2+/Mg2+. To explore the underlying mechanism originating this difference, three distinct cation-binding sites in the βA domain were mutated. Among the three sites, MIDAS is occupied by Mg2+, while ADMIDAS and LIMBS/SyMBS are occupied by Ca2+ in Ca2+/Mg2+ condition. When amino acid residues composing these sites were mutated to Ala, none of them bound fibrinogen, except for D126A and D127A in the presence of Ca2+/Mg2+. Notably, these mutations restored fibrinogen binding in the swing-out-defective mutant. Amino acid residues D126 and D127 compose ADMIDAS together with S123 and M335. Swing-out of the hybrid domain disrupts M335 from ADMIDAS. This allows Ca2+ to move toward MIDAS, bringing the β1-α1 loop closer to ligand to provide contact site. However, similar rearrangement of the cation/ligand binding sites has been shown to take place in Mn2+ without the swing-out. The results suggest partial disruption of ADMIDAS is the key event for activation in Ca2+/Mg2+, while it is not so in Mn2+. Our results may reconcile the dispute over the apparently contradicting findings in the crystal structure that integrin with a closed head with Mn2+ represents an active conformation as one with an open head with Ca2+/Mg2+. Disclosures: Kawai: Daiichi Sankyo: Consultancy; Bayer: Consultancy; Toyama Chemical: Consultancy.

Description: Platelets are released from terminally differentiated megakaryocytes (MKs) through lineage commitment, although the underlying mechanisms of megakaryopoiesis and subsequent thrombopoiesis are incompletely understood. MKs were reportedly generated from not only hematopoietic stem cells (HSCs), but also pre-adipocytes, without any gene transfer in an in vitro culture system (Matsubara et al, 2012 Methods Mol Biol). Because of high efficiency of platelet production from pre-adipocytes, much interest has been attracted to clarify the mechanisms of megakaryopoiesis from pre-adipocytes. Here we demonstrate a novel mechanism that megakaryopoiesis from pre-adipocytes is regulated by the inducible production of endogenous TPO via transferrin receptor CD71. NF-kB pathway in pre-adipocytes might modulate the TPO production through the stimulation of transferrin. Previously, we reported that pre-adipocyte cell line OP9 has the gene expressions of thrombopoietin (TPO) and its receptor c-MPL. This study first examined the TPO protein levels in supernatant from human (Cell Applications) and mouse pre-adipocytes cultured with MK lineage induction (MKLI) media (Ono et al, 2012 Blood, Matsubara et al, 2012 Methods Mol Biol) in the absence of recombinant TPO (rTPO). The TPO levels were 36.6 ± 9.2 pg/mL in mouse pre-adipocytes on Day 8 and 36.1 ± 5.8 pg/mL in human pre-adipocytes on Day 8, and undetectable levels of TPO were observed in both cells before MK induction. Differentiation efficiency (approximately 20%) of MKs from pre-adipocytes cultured with rTPO was similar to that without rTPO. The function of pre-adipocyte-derived platelets cultured with rTPO was similar to that without rTPO when assessed by incorporation of platelets into thrombus formation on the collagen-coated surface under flow conditions. The number of MKs produced from pre-adipocytes was markedly decreased in the presence of anti-c-MPL blocking antibody, AMM2. Regarding the MK differentiation from HSCs, it is well known that HSCs do not differentiate into MKs in the absence of rTPO. We did not observe the TPO secretion from human bone marrow CD34(+) cells and mouse bone marrow Lin(-)Sca1(+)c-kit(+) cells in a culture condition using MKLI media in the absence of rTPO. The observations suggest that the MK differentiation from pre-adipocytes has a distinct mechanism of megakaryopoiesis from HSCs. These findings indicate the unique mechanism of megakaryopoiesis from pre-adipocytes; the endogenous TPO stimulation via c-MPL in pre-adipocytes has activity for promoting MK differentiation and subsequent production of functional platelets. To elucidate the molecular mechanism of TPO production during MK differentiation from pre-adipocytes, we examined which components of MKLI media were responsible for TPO production. We found iron-saturated transferrin, but not apo-transferrin, included in MKLI media is the critical factor to induce TPO production and MK differentiation from pre-adipocytes. The effects of transferrin were not observed when pre-adipocytes were cultured in the presence of anti-CD71 blocking antibody, 8D3, or deferoxamine mesilate, an iron-chelating agent. The effects of transferrin on TPO production and MK differentiation were also abolished in pre-adipocytes transfected with siRNA-CD71, and the TPO levels were 44.8 ± 15.9 pg/mL for siRNA-control and undetectable levels for siRNA-CD71 on Day 6. We next investigated a mechanism of inducing TPO production via CD71 in pre-adipocytes. The 5’ region of human TPO gene contains transcription factor-binding sequences, such as GATA1, GATA2 and NF-kB. Since NF-kB is a downstream target of CD71, we then examined whether NF-kB involved in MK differentiation derived from pre-adipocytes. Nuclear translocation of NF-kB in pre-adipocytes was observed when cells were stimulated with transferrin. Both TPO secretion and MK production were decreased in the presence of a peptide to inhibit nuclear translocation of NF-kB. Taken together, it is concluded that NF-kB pathway in pre-adipocytes modulates megakaryopoiesis through endogenous TPO production via CD71. The present findings suggest that pre-adipocytes have a unique mechanism of megakaryopoiesis through lineage commitment. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2287 The TARGET system is an online database that can be easily accessed by physicians. The TARGET system is operated by the Japanese Society of Hematology. The registration of one's own chronic myeloid leukemia (CML) patients in the TARGET system makes it possible to share experiences among physicians, and, thus, may facilitate appropriate treatment for patients. Patients participating in clinical trials are usually selected according to strict eligibility criteria. Previous publications have questioned the use of other overly restrictive exclusion criteria in clinical oncology trials. In practical situations, however, the clinical features of patients are much more heterogeneous than those defined by the selection criteria in clinical trials. From this point of view, the TARGET system might provide more practical and general features compared with the IRIS study. Patients were registered in the TARGET system from October 2003 to March 2010 in Japan. A total of 1,236 patients from 176 hospitals were registered in Japan. We analyzed data from 639 CML chronic phase patients not receiving prior therapy registered in this system. After 90 months follow-up, high survival rates were demonstrated for imatinib-treated newly diagnosed CML patients, with event-free survival (EFS), progression-free survival (PFS), and overall survival (OS) rates of 79.1, 94.8, and 95.1%, respectively. A landmark analysis of 296 patients who showed a complete cytogenetic response (CCyR) at 12 months after the initiation of imatinib treatment revealed that, at 90 months, 99% of patients (95% CI, 98 to 100) had not progressed to accelerated phase (AP) or blastic crisis (BC). The patients showing a CCyR and a reduction of at least 3 log levels of BCR-ABL transcripts after 18 months of treatment had an estimated survival rate without CML progression of 100% at 84 months.By 84 months, 315 patients had achieved an MMR. A total of 226 of 315 patients (71.7%) achieved undetectable BCR-ABL by 84 months. We therefore analyzed the probability of achieving undetectable BCR-ABL based on the molecular response at 12 and 18 months. The probability of achieving undetectable BCR-ABL by 72 months in patients with an MMR at 12 months was 86.5%, compared with 64.7% for those without an MMR (P

Description: Abstract 1180 Background: Although peripheral blood stem cell (PBSC) donation has been considered safe and less stressful, certain fetal or life-threatening acute (within 30 days of post donation) adverse events as well as late occurrence of hematological malignancies have been reported among donors. Since the Japan Marrow Donor Program requires the confirmation of safety and risk of PBSC donation at family donors prior to applying this technique for volunteer donors, the Japan Society for Hematopoietic Cell Transplantation (JSHCT) performed nation-wide consecutive pre-registration and follow up for PBSC family donors from 2000 to 2010. This time, we report the comprehensive outcome of this project. Methods: The JSHCT mandated the registration of every PBSC family donor at the donor registration center then, issued donor identification number to each donor. The society required every harvest center to observe the JSHCT standards for donor eligibility, stem cell mobilization and harvest. The society also required to notify it of any severe adverse events, the results of the day30 clinical and laboratory check and of the annual health check for five years. Findings: Among 3,264 pre-registered donors, 47 emergency reports were submitted for 5 years and it was revealed that acute unexpectedly severe adverse events such as interstitial pneumonitis or anginal attack occurred at 0.58 % of donors although no mortality cases within 30 days of post donation were found. The relationship between donors' basic information such as age or gender and clinical and laboratory abnormalities obtained from 2,882 day 30 reports was studied and it revealed the followings; the risk factor for fatigue, headache, insomunia, anorexia and nausea was female, the risk factors for prolongation of hospitalized period were older age, low body weight, high total dose of granulocyte colony stimulating factor (G-CSF), the presence of past or current health problems and the episode of past stem cell donation, the risk factors for thrombocytopenia were older age and high total dose of G-CSF, the risk factors for splenomegary were older age and high total dose of G-CSF, the risk factors for poor CD34+ cell mobilization were older age, female and the episode of past stem cell donation, the risk factor for over CD34+ stem cell mobilization was younger age. The information from 6,233 annual health checks from 1,708 donors for 5 years showed the followings; the incidence of non-malignant but significant health problems was 1.5%, the incidence of non hematological malignancies was 0.7%, the incidence of hematological malignancy was 0.06%. It was also confirmed that the incidence of hematological malignancies among PBSC donors was not high compared with that among retrospectively surveyed bone marrow family donors. Interpretation: The consecutive donor pre-registration and annual follow up system that sets strict standards for donor eligibility, cell mobilization and harvest is effective in preventing real life-threatening acute adverse events and also is useful to know the real figures on PBSC donors and to assure donor safety. Such a system should be applied to all hematopoietic stem cell (family and volunteer, bone marrow and peripheral blood) donors. Disclosures: No relevant conflicts of interest to declare.

Description: Determinant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid–derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.

Description: Abstract 4742 The regulation of megakaryopoiesis and thrombopoiesis are incompletely understood. Better understanding of the underlying mechanisms would be of biological import, but may also lead to novel approaches for generating of platelets for clinical application. One cell line that undergoes terminal differentiation into megakaryocytes is pre-adipocytes. These cells represent a potential candidate cell for ex vivo generation of megakaryocytes. Here we demonstrate that pre-adipocytes differentiate into MKs and platelets by using an endogenous paracrine loop involving TPO, the primary cytokine involved in megakaryopoiesis, and its receptor c-mpl. We previously reported that pre-adipocytes differentiate into MKs and form platelets when the culture medium is switched from maintenance medium to MK lineage induction (MKLI) medium. Neither media include TPO. Based on these observations, the present study tested the hypothesis that pre-adipocytes might differentiate into MKs and platelets by endogenous TPO and c-mpl expression of sufficient magnitude to drive megakaryopoiesis. We used primary mouse pre-adipocytes (CD45-, CD117-, Sca1+, CD29+, CD73+, CD90+, CD105+, CD106+) from subcutaneous adipose tissues and also the mouse pre-adipocyte line 3T3-L1 cells. The TPO levels, as assessed by ELISA, in the supernatant from 106 pre-adipocytes cultured in 2 ml MKLI medium without exogenous TPO (TPO-) were unmeasurable level on Day 0, 29±14 pg/ml on Day 7 and 8±2 pg/ml on Day 12. Similar results were obtained in the supernatant from 3T3-L1 during MK differentiation. We did not observe measureable TPO in supernatants from pre-adipocytes and 3T3-L1 cells cultured in maintenance medium on Days 0, 7 and 12. We then compared MK differentiation from pre-adipocytes in MKLI media in the absence (TPO-) or addition (final concentration, 50 ng/ml; TPO+) of TPO. The frequency of CD41-postive pre-adipocytes in culture after 6 days was 58±11% for TPO+ and 70±7% for TPO- (p〉0.05), consistent with endogenous TPO being sufficient under these circumstances to stimulate MK differentiation of pre-adipocytes. DNA ploidy and c-mpl expression assessed by immunohistochemistry were also similar with or without added exogenous TPO. We next examined the number of CD41-expressing large-sized (〉15 μm) cells beginning with 1.2 × 104 preadipocytes in the absence or presence of the c-mpl inhibitor rat anti-mouse c-mpl inhibitor AMM2 (final concentration, 10 μg/ml). In the absence of AMM2, 10-fold more CD41-positive, large cells were seen (6.8±1.7×103) than in its presence (0.6±0.2×103, p