bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharmacogenocis of PAR4: PAR4 Polymorphism Determines Platelet Response in the Presence of Dual Anti-Platelet Therapy
Authors: Tourdot Benjamin , Bray Paul F. , Edelstein Leonard C. , Holinstat Michael 
Published: October 2019

Abstract
Aberrant platelet activation contributes to the mortality associated with coronary heart disease (CHD). Our lab group has recently shown that platelets from black donors were hyper-responsive to activation of the thrombin receptor Protease-Activated Receptor-4 (PAR4) compared to platelets from white donors. The racial difference observed through PAR4 persists in platelets treated ex vivo with dual antiplatelet inhibitors (COX-1 and P2Y12 receptor inhibitors), a standard of care for many CHD patients. The racial difference in PAR4 signaling is largely attributable to one PAR4 variant more frequently expressed in blacks than whites, PAR4 T120A. Independent of race, T120 is associated with greater PAR4-mediated platelet reactivity compared to the A120 variant. The role PAR4 variation plays in regulating thrombin-mediated platelet activation in CHD has not been elucidated to date. We hypothesize that people homozygous for the T120 will have an increase in PAR4-mediated platelet reactivity compared to patients homozygous for A120. To investigate this hypothesis, we studied PAR4 signaling in platelets obtained from healthy donors. Platelet activity was assessed in each subject at the beginning of the study. Subsequently subjects were placed on a 7-day treatment of either COX-1 or P2Y12 inhibitors and on day 7 their blood was again drawn and tested for PAR4-AP-mediated platelet activation. Subjects who are homozygous for T120, have an increase in platelet reactivity as measured by aggregation, αIIbβ3 activation and granule secretion compared to individuals who are homozygous for A120. Further distal signaling nodes such as calcium and Rap1 are hyperactive in individuals who are homozygous for T120. This study reinforces the personalized medicine approach to therapeutic intervention and challenges the one size fits all approach which often leaves at risk populations without adequate protection from thrombotic events and stroke. This study was supported in part by grants MD007880, GM105671, and HL 114405 (MH) from the NIH.

Disclosures
No relevant conflicts of interest to declare.
Murine Bone Marrow-Derived Megakaryocytes Are Capable of Antigen Cross-Presentation on Major Histocompatibility Class (MHC) I Molecules
Authors: Zufferey Anne , Speck Edwin R. , Guo Li , Aslam Rukhsana , Kapur Rick , Semple John W. 
Published: October 2019

Abstract
Major Histocompatibility complex (MHC) class I is a complex composed of two proteins, the MHC-encoded alpha heavy chain and the beta2-microglobulin light chain. In the endoplasmic reticulum, it receives an antigen peptide of usually eight to eleven residues in its groove and is exported to the plasma membrane. In classical MHC class I presentation, endogenous antigens are presented to CD8+ T cells. On the other hand, cross-presentation consists in the presentation of exogenously-derived proteins in the context of MHC class I molecules to CD8+ T lymphocytes, inducing a cytotoxic response against the antigen presenting cell (APC). Platelets have been previously shown to be able to cross-present immunogenic ovalbumin (OVA) peptides, as well as bacterial peptides and to trigger CD8+ T lymphocyte activation. It is thought that the antigen cross presenting function of platelets is transmitted from megakaryocytes (MK) during thrombopoiesis, but whether megakaryocytes can cross-present is still unknown. To address this question, we developed an in vitro endocytosis model of murine bone marrow-derived megakaryocytes. C57BL/6 mouse megakaryocytes are enriched by gradient sedimentation and pulsed with fluorescent ovalbumin (OVA) for various times. In addition, we used DQ-OVA, a BODIPY FL-conjugated OVA, which becomes fluorescent upon proteolysis. Cells were then analyzed by flow cytometry or confocal microscopy to assess the OVA megakaryocyte internalization, its proteolytic degradation and its loading onto MHC class I molecules. We can show the endocytosis of exogenous OVA by megakaryocytes, in a pathway involving lysosomes. The endocytosed OVA were subsequently proteolytically cleaved into the immunogenic OVA peptide SIINFEKL and the peptide is inserted into the antigen binding groove of MHC class I molecules. The MHC class I-OVA peptide complexes are stored in the MK alpha-granules and can be translocated to the plasma membrane. Thus, we have shown that MKs can effectively process and present exogenous antigens on MHC class I molecules. This opens a new avenue toward a functional characterization of this pathway and its modulation, leading to new therapeutic approaches in autoimmune diseases targeting megakaryocytes, such as immune thrombocytopenia (ITP).

Disclosures
No relevant conflicts of interest to declare.
Acute Efficacy of a Novel Anti-Tissue Factor Pathway Inhibitor Antibody BAY 1093884 in Hemophilia A Mouse Severe Tail Bleeding
Authors: Xu Yifan , Koellenberger Maria , Laux Volker , Kauser Katalin , Sim Derek 
Published: October 2019

Abstract
Tissue factor pathway inhibitor (TFPI) is the major inhibitor of the tissue factor initiated extrinsic coagulation pathway in blood and is intact in patients with hemophilia. The inhibition of TFPI may restore hemostasis in patients with hemophilia. BAY 1093884 is a fully human monoclonal antibody against TFPI developed as a bypass agent for hemophilia patients with or without inhibitors. It restores thrombin burst for stable clot formation in hemophilic conditions in vitro. The goal of these studies was to determine the in vivo acute efficacy of BAY 1093884 in the hemophilia A (HemA) mouse.
In the first study, the acute efficacy of BAY 1093884 (3−100 mg/kg) was demonstrated and compared with full-length recombinant factor VIII (rFVIII; 10−100 IU/kg) by a HemA mouse tail clip model, in which blood loss from a severed tail tip was measured over 45 minutes after injury (n=12−27 mice/group). Naive C57/BL6 and HemA mice were used as positive and negative controls, respectively. Whereas isotype control antibody−treated HemA mice had median blood loss of 870 μL, increasing doses of BAY 1093884 to 50 and 100 mg/kg significantly reduced blood loss to a median of 55 and 5 μL. The dose required to reduce blood loss by 50% was 18 mg/kg, approximately equivalent to the efficacy of 20 IU/kg rFVIII.
In a second study, we characterized the combined action of BAY 1093884 and activated recombinant factor VII (rFVIIa; n=10−25 mice/group). Low doses of BAY 1093884 (2.5 mg/kg) and rFVIIa (0.5 and 1.0 mg/kg) with minimal efficacies were tested. Untreated HemA mice had median blood loss of 860 μL. As stand-alone treatments, 2.5 mg/kg BAY 1093884, 0.5 mg/kg rFVIIa, and 1 mg/kg rFVIIa provided minimal blood loss protection, with bleeding volume reduced to 675, 830, and 770 μL, respectively. In comparison, the combination of 2.5 mg/kg BAY 1093884 with 0.5 mg/kg rFVIIa or 1.0 mg/kg rFVIIa reduced median blood loss to 215 and 35 μL, respectively. These results showed a combination effect of BAY 1093884 and rFVIIa in this severe acute efficacy model.
These studies demonstrate that BAY 1093884 could potently reduce acute blood loss in HemA mice and may offer a new treatment option for hemophilia patients.

Disclosures
Xu: Bayer HealthCare LLC: Employment. Koellenberger:Bayer Pharma AG: Employment. Laux:Bayer Pharma AG: Employment. Kauser:Bayer HealthCare LLC: Employment. Sim:Bayer HealthCare LLC: Employment.
A Method for Deriving Pharmacokinetic Constants for Factor VIII and IX from Limited Blood Sampling
Authors: Escobar Miguel Antonio , Bond Daniel Preston , Cantini Madeline , Cannon Krishna 
Published: October 2019

Abstract
Introduction
The treatment of hemophilia A and B is based on the replacement of the deficient factor dosed by weight. However, dosing for hemophilia treatment has been arrived at by empiric assessment, essentially "trial and error" based on the pharmacokinetics (PK) of the factors and the characteristics of the replacement product. In the last decade clinical pharmacokinetics has gained popularity in hemophilia so that factor dosage can be adjusted according to the requirement of the individual patient. Factor-specific population pharmacokinetic models have been developed for individualized treatment of patients with hemophilia A and B. The math modeling technique presented in this project relies on minimal blood sampling to derive the constants necessary to predict the peak and trough levels within the commonly excepted error bounds.
Methods
Data that included FVIII, FIX levels and weight were obtained retrospectively from severe hemophilia A and B adult patients and approved by the ethics committee of the University of Texas Health Science Center. A single compartment decay model equation was used in both a custom iOS application and an Excel spreadsheet to calculate the decay constant between any 2 points on a decay curve. Using this local constant as the half-life in the standard decay equation allowed the calculation of the peak at time = 0. This peak combined with dosing information and the subject's weight allowed the calculation of the Recovery. These 2 constants in the equation: Level=(dose*Recovery/weight)*0.5^(time/half-life) allowed the calculation of the level at any point on the curve. Using this method on several example datasets showed that the model is reasonably able to predict peak and trough levels for both factor VIII and IX. Because of the time dependent nature of the local decay constant, better results are obtained using data points after the first half of the expected half-life. This method can also be used to predict steady state levels, peaks and troughs for any prophylaxis schedule.
Conclusion
Appropriate dosing of factor VIII or IX is at best an approximate calculation. The method described in this publication generates a math model that is generally as accurate as a multiple sample PK study with far fewer blood samples taken. Clinical applications of this model can be utilized to predict factor levels after a single infusion of factor VIII/IX in adults that are treated on-demand or prophylaxis. It can also be utilized in individuals that are infusing extended half-life products or in the surgical setting.

Disclosures
Escobar: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bond:Pfizer: Consultancy, Research Funding. Cantini:Pfizer: Research Funding. Cannon:Pfizer: Research Funding.
Mechanisms of Immunosuppression By FVIII-Specific TCR Engineered Human Regulatory T Cells
Authors: Kim Yong Chan , Zhang Ai-Hong , Yoon Jeong Heon , Scott David William 
Published: October 2019

Abstract
Expanded antigen-specific engineered regulatory T cells (Tregs) have been proposed for potential clinical application for the treatment of undesirable immune responses, such as inhibitor responses in hemophilia A patients and autoimmune diseases. By providing an antigen-specific T-cell receptor (TCR) to polyclonal natural Tregs, we suggested that antigen-specific engineered Tregs would migrate specifically to particular target tissues and induce antigen-specific immune tolerance in the local milieu. Previously, we developed FVIII C2-specific Tregs using a long-term stabilization protocol in vitro and demonstrated that these stabilized engineered Tregs successfully modulated FVIII-specific T-cell- and B-cell immune responses. Herein, we examined the mechanism of suppression by antigen-specific engineered Tregs compared to polyclonal normal natural Tregs.
Initially, we tested whether these FVIII-specific engineered Tregs were able to suppress neighboring activated T-cell effectors locally. We found that FVIII C2-specific Tregs strongly suppressed myelin basic protein (MBP)-specific T effectors by presentation of both specific antigens in same APC population. However, we also observed that C2-specific Tregs could suppress MBP-specific T effectors presented on different APCs. These results imply contactless suppressive function of C2-specific engineered Tregs. Using a modified trans-well suppression assay, in which physical distance and clear separation between Tregs and a set of T effectors was created, we found that C2-specific activated Tregs showed significant contactless suppression only when T effectors were also present. In addition, and confirming previous studies with polyclonal Tregs, suppression by FVIII-specific engineered Tregs could be overcome by increasing the dose of IL-2 in co-culture media. This suggests that Tregs act, in part, by usurping IL-2 needed by T effectors to proliferate. Surprisingly, neutralization of CTLA-4 did not interfere with FVIII C2-specific suppression of engineered Tregs in contrast to the reversal seen with anti-CD3e-driven non-specific immunosuppression.
Our data strongly suggest that suppressive function of FVIII-specific engineered Tregs is not restricted to cell-to-cell contact. Rather cross-talk of engineered Tregs and T effectors potentially generate a contactless suppressive mechanism to suppress other FVIII-specific multiple effector cells in the local milieu for effective immune tolerance. Understanding the mechanism of contactless suppression mechanism should provide critical clues to develop more effective engineered Tregs as a therapeutic tool in hemophilia A.
(Supported by NIH grants HL061883 and HL126727)

Disclosures
Kim: Henry Jackson Foundation: Other: patent filed. Zhang:Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.
Signs of Hypofibrinolysis in Patients with Unprovoked Idiopathic Venous Thromboembolism
Authors: Bassora Fernanda Dutra Santiago , Orsi Fernanda Andrade , Alves Graziela Silveira Araujo , Montalvão Silmara Aparecida De Lima , Zapponi Kiara , Romano Anna Virginia Calazans , Colella Marina Pereira , de Paula Erich Vinicius , Annichino-Bizzacchi Joyce Maria 
Published: October 2019

Abstract
Background: The causes for venous thromboembolism (VTE) remain undetermined in at least 30% of patients with unprovoked VTE. Hypofibrinolysis may be associated to VTE, however the occurrence of hypofibrinolysis in patients with unprovoked, idiopathic, VTE is not well stablished.
Aims: To evaluate whether hypofibrinolysis would be associated with unprovoked idiopathic VTE.
Methods: Patients with a history of unprovoked VTE without acquired or inherited thrombophilia were included. Global tests of fibrinolysis, such as euglobolin lysis time (ELT) and lysis area on fibrin plate (LAFP), and specific tests of fibrinolysis, such as plasma activity plasminogen, a-2 antiplasmin (a2AP), plasminogen activator
inhibitor-1 (PAI-1), and thrombin activatable fibrinolysis inhibitor (TAFI), were performed in patients and healthy controls. We also analyzed the plasma activity of factor (F) XIII.
Results: Thirty-one patients and fifty healthy controls were included. ELT results were higher in patients than in controls (median= 295 and IQ= 205-355 minutes vs. median=250 and IQ= 167-295 minutes, respectively, p = 0.006) and LAFP values were lower in patients compared to controls (median=81 and IQ= 56-110 vs. median= 95 and IQ 72-132 respectively, p = 0.0014), suggesting that they were experiencing a hypofibrinolytic state. Plasma activity of plasminogen (median= 131 and IQ= 119-141 vs. median=120 and IQ=111-137, respectively, p = 0.045) and FXIII (median= 103 and IQ 89-127 vs. median= 96 and IQ=80-105, respectively, p = 0.050), were higher in patients than in controls, whereas plasma activity of α-2AP was lower in patients (median= 124 and IQ 114-128 vs. median= 127 and IQ=121-133, p≤0.001). Interestingly, patient's median TAFI activity was lower than in controls (median=16 and IQ 13-18μg/mL vs. median= 19 and IQ= 17-21g/mL, p≤0.001). However, PAI-1 activity did not differ between groups.
Conclusions: Hypofibrinolysis may occur in patients with unprovoked idiopathic VTE and may be detected by global tests of fibrinolysis. It is possible that hypofibrinolysis contribute to the pathogenesis of thrombosis in these selected cases.

Disclosures
No relevant conflicts of interest to declare.
Kras Is Critical for B Cell Lymphopoiesis
Authors: Zheng Yongwei , Chen Yuhong , You Xiaona , Yu Mei , Fu Guoping , Su Xinlin , Zhu Wen , Wu Zhihong , Zhang Jing , Wen Renren , Wang Demin 
Published: October 2019

Abstract
Small GTPases of the Ras subfamily regulate multiple signaling pathways and control numerous biological functions. Although the three major Ras members, Kras, Hras and Nras, are highly homologous, individual Ras gene can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological role of Kras. Here, we generated and examined mice with hematopoietic- and B cell-specific deletion of Kras. In VavCreKrasfl/fl mice with hematopoietic deletion of Kras, the populations of bone marrow (BM) pre-, immature and mature B cells were reduced. The population of peripheral follicular (FO), marginal zone (MZ) and B1 mature B cells were also reduced in VavCreKrasfl/fl relative to control mice. In addition, BM chimeric mice with B cell-specific deficiency of Kras generated by transplantation of T- and B-null Rag1-deficient mice with a mixture of BM cells from VavCreKrasfl/fl mice and B cell-deficient μMT mice displayed a marked reduction of pre-, immature and mature B cells in the BM and mature B cells in the spleen. Thus, Kras deficiency intrinsically impairs early B cell development and late B cell maturation. Further, the effect of Kras deficiency on B-cell proliferation and survival was examined. The 3H-thymidine incorporation rate of Kras-deficient, relative to control, mature B cells in response to anti-IgM or anti-IgM plus IL-4 was markedly reduced. In addition, Kras-deficient B cells displayed a marked decrease of cell cycle entry and increase of cell apoptosis upon anti-IgM or anti-IgM plus IL-4 stimulation. Thus, Kras deficiency impairs BCR-induced B cell proliferation and survival. Lastly, the role of Kras in BCR signaling was studied. The level of total BCR-activated Ras (Ras-GTP) was largely reduced in Kras-deficient B cells. BCR-induced Ca2+ flux was comparable between Kras-deficient and control B cells. BCR-induced phosphorylation of Akt and IkBa was normal in mutant relative to control B cells. However, BCR-induced activation of ERK1/2 but not JNK or p38 was impaired in Kras-deficient relative to control B cells. Consistently, BCR-induced activation of Raf-1 and MEK1/2, the upstream activators of ERK1/2, was markedly reduced in mutant B cells. In addition, pre-BCR-induced ERK1/2 activation was impaired in Kras-deficient pre-B cells. Pre-BCR-induced activation of AP-1, the transcription factors downstream of ERK1/2, was decreased in mutant pre-B cells. Thus, Kras plays an important role in BCR- and pre-BCR-mediated activation of the ERK pathway in B cells. Taken together, these findings demonstrate that Kras is the Ras family member that critically regulates early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway.

Disclosures
No relevant conflicts of interest to declare.
The NFkB System Regulates Flt3-Mediated Hematopoiesis
Authors: Liu Yi , Hoffmann Alexander 
Published: October 2019

Abstract
FMS-like tyrosine kinase 3 (Flt3) plays pivotal roles in the survival, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPCs). However, how proliferation and differentiation via Flt3 is regulated is poorly understood. In this study, our bioinformatic meta-analysis of existing cancer microarray datasets in Oncomine correlated reduced nfkb1 expression with acute myeloid leukemia (AML). The nfkb1 gene products p105 and p50 are not transcriptional activators but regulators of NFκB activators. Examining bone marrow HSPCs, we found that nfkb1-/- mice produce much higher (~5-8X) total cell yields compared to wild type (WT) when cultured with Flt3 Ligand (Flt3L) ex vivo. Cell cycle analysis and apoptosis assay revealed that nfkb1-/- cells have profoundly increased proliferation and enhanced survival compared to WT cells. Nfkb1-/- cells show skewed differentiation towards plasmacytoid DCs and delayed differentiation of conventional DCs. To examine the molecular mechanism, we investigated how the NFκB system is assembled as HSPCs, which have little NFκB, progress through hematopoiesis to become differentiated immune cells with robust NFκB systems. In nfkb1-/- cells, the distribution of NFκB signaling complexes was altered and resulted in overall elevated RelA expression. Reducing RelA expression in compound rela+/- nfkb1-/- HSPCs partially rescued the myelopoiesis phenotype, suggesting that hyper NFκB activity is the cause. Further in vivo studies are underway to further test NFκB's role in a Flt3L injection model and a Flt3-ITD-mediated mouse leukemia model. In conclusion, our work reveals that precisely timed control of NFκB system assembly is a hallmark of hematopoiesis and that its misregulation perturbs normal Flt3-mediated hematopoiesis and can be a cause of myeloid cancer.

Disclosures
No relevant conflicts of interest to declare.
Characterizing the Function of an RNA Binding Protein, IGF2BP3, in Hematopoiesis
Authors: Tran Tiffany M , Palanichamy Jayanth Kumar , Howard Jonathan , Contreras Jorge R. , Fernando Thilini , Basso Giuseppe , Pigazzi Martina , Sanford Jeremy R , Rao Dinesh S. 
Published: October 2019

Abstract
Post-transcriptional control of gene expression plays important roles in defining normal and pathological cellular phenotypes. Amongst mechanisms of post-transcriptional regulation, RNA binding proteins (RBPs) have recently been shown to play important roles. However, in vivo roles for RBPs are not well understood. Here, we identified the RBP IGF2BP3 to be specifically overexpressed in MLL-rearranged B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and a risk of high relapse. IGF2BP3 was required for the survival of B-ALL cell lines, and knockdown led to decreased proliferation and increase apoptosis. In addition, enforced expression of IGF2BP3 in murine bone marrow transplant assays caused a proliferation of hematopoietic stem and progenitor cells and a skewing of hematopoietic development to the B-cell/myeloid lineage. Using cross-link immunoprecipitation and high-throughput sequencing, we uncovered the transcriptome regulated by IGF2BP3; including novel direct targets, MYC and CDK6. These were regulated following experimental alteration of IGF2BP3 expression in vivo, and are regulated via elements within their 3'untranslated regions. Hence, IGF2BP3 mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism operant in MLL-rearranged B-ALL, highlighting IGF2BP3 and its cognate RNA binding partners as potential therapeutic targets in this disease.

Disclosures
No relevant conflicts of interest to declare.