bluuebunny/crossref_metadata_2025 huggingface dataset:
Ibrutinib Abrogates TREM-1 Mediated Neutrophil Activation
Authors: Stadler Nicole , Hasibeder Astrid , Aranda Lopez Pamela , Desuki Alexander , Kriege Oliver , Weber Alexander N.R. , Michel Christian , Teschner Daniel , Hess Georg , Theobald Matthias , Radsak Markus
Published: November 2019

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor on neutrophils (PMN) and important in the innate host defence against microbial pathogens. Here we examined the influence of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib on TREM-1 dependent activation of human PMNs. Firstly, ibrutinib specifically inhibited TREM-1 mediated PMN activation of the oxidative burst and CD62L shedding, whereas TLR mediated activation remained unaffected. Correspondingly, ibrutinib suppressed ERK phosphorylation after TREM-1, but not after TLR ligation. To clarify whether this TREM-1 specific effect of ibrutinib was also relevant in vivo, we treated mice with ibrutinib, and analyzed fungal clearance in vivo upon pulmonary challenge with A. fumigatus and PMN activation ex vivo upon TREM-1 or TLR ligation, again finding that TREM-1, but not TLR induced activation was suppressed. Equivalent results were obtained in PMNs of six lymphoma patients under treatment with ibrutinib. In summary, BTK plays an important role in TREM-1 mediated PMN activation, providing a potential mechanism for the frequently observed infectious complications in patients treated with ibrutinib, and fosters studies using this drug as a novel tool to modulate TREM-1 activity for therapeutic purposes, i. e. in sepsis or autoimmune diseases.

Disclosures

Hess: Pfizer: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Roche, CTI, Pfizer, Celgene: Research Funding; Roche: Honoraria; Novartis: Honoraria.
Human Double-Negative Regulatory T Cells Selectively Suppress mTOR Signaling and Metabolic Reprogramming of Conventional T Cells
Authors: Haug Tabea , Bruns Heiko , Aigner Michael , Mackensen Andreas , Voelkl Simon
Published: November 2019

Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). The subpopulation of TCRαβ+ CD4- CD8- (double-negative, DN) T cells has been described to suppress immune responses in both mice and humans. Of particular interest, infusion and/or activation of murine DN T cells specifically suppressed alloreactive T cells and prevented development of GvHD after allogeneic hematopoietic stem cell transplantation (SCT). Moreover, clinical studies in patients after SCT revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD, suggesting a therapeutic potential of human DN T cells. However, the molecular mechanism of suppression still remains unclear. To gain a better understanding of DN T-cell functionality, we investigated whether human DN T cells modulate distinct TCR signaling processes in conventional T cells. We found that DN T cells selectively inhibit the mechanistic target of rapamycin (mTOR) signaling pathway but not activation of mitogen-activated protein kinases (MAPK). The crucial function of mTOR signaling was confirmed by treating effector T cells with a chemical activator of protein kinase Akt, which induces mTOR phosphorylation. Indeed, enforced activation of the mTOR pathway rendered conventional T cells unsusceptible to DN T cell-mediated suppression. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Intriguingly, DN T cells significantly diminished upregulation of the glycolytic machinery, expression of glucose transporters and glucose uptake in conventional T cells. These findings indicate that DN T cells inhibit metabolic reprogramming of conventional T cells by abrogating mTOR signaling, thereby inducing a quiescent phenotype. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for using them as a cellular-based therapy to limit alloreactive immune responses.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Marginal Zone B Cell Differentiation By microRNA-146a Via the Numb-Notch Pathway
Authors: King Jennifer K , Ung Nolan , Paing May , Contreras Jorge R. , Alberti Michael O. , Fernando Thilini , Zhang Kelvin , Pellegrini Matteo , Rao Dinesh S.
Published: November 2019

B cell development in bone marrow is followed by specification into spleen subsets, including marginal zone (MZ) cells. MZ require elaboration of distinct gene expression programs for development. Given their role in gene regulation, its not surprising that microRNAs (miRNAs) influence cell development. Recent work demonstrated that deficiency of NF-κB feedback regulator, Mir146 (miR-146a), led to a range of hematopoietic phenotypes, but B cells have not been extensively characterized. Here, we found miR-146a deficient mice demonstrate a reduction in MZ B cells, likely from a T cell independent developmental block. Utilizing comparative analysis of developmental stage-specific transcriptomes, we show MZ cell differentiation was impaired due to decreases in Notch2 signaling. Further, we discovered that the cell-fate regulatory protein, Numb, is a direct target of miR-146a, and its derepression in miR-146a deficient B cells underlies the decreases in Notch2. Our studies reveal miR-146a-dependent B cell phenotypes regulated by the Numb-Notch2 pathway.

Disclosures

No relevant conflicts of interest to declare.
The Critical Roles of SIN1 in Platelet Activation and Myocardial Infarction
Authors: Xu Yanyan , Chen Xue , Liu Junling
Published: November 2019

Mammalian stress-activated protein kinase interacting protein 1 (SIN1) is an essential subunit of the mTORC2 complex, which regulates Akt activation by phosphorylation of Akt at Ser473 residue. Despite the function of Akt in platelet activation and thrombosis was well studied, the role of SIN1 in platelet activation and thrombosis remains unknown. In this study, we observed that megakaryocyte/platelet specific SIN1 deficiency caused 30% reduction of platelet counts in peripheral blood probably by blockage of megakaryocyte differentiation and enhancing platelet apoptosis, suggesting that SIN1 had an important role in thrombopoiesis. More importantly, SIN1 deficiency caused a defect in platelet aggregation in response to low level of thrombin, U46619, ADP and collagen. SIN1 deficiency also exhibited diminished ability of platelet to spread on immobilized fibrinogen and the decreased rate of clot retraction in platelet-rich plasma containing SIN1 deficient platelets. mTORC2 complex analysis revealed that the expression levels of Rictor, another mTORC2 component, were significantly diminished in SIN1 deficient platelet. And SIN1 deficiency attenuated agonist-induced phosphorylation of Akt at Ser473, Thr308 and Thr450, and Gsk3β at Ser9 in platelet. SIN1 could be phosphorylated at Thr86, which correlated with the phosphorylation of Akt at Ser473 in activated platelets. Further study demonstrated that the phosphorylation levels of SIN1 at Thr86 and Akt at Ser473 and Thr450, but not at Thr308 were enhanced in the platelets collected from ST-segment elevation myocardial infarction (STEMI) patients, indicating that SIN1 activation correlated with myocardial infarction process. A mouse model of chronic myocardial infarction (MI) was performed and the results demonstrated that platelet-specific SIN1 deficient mice had less platelet activation, reduced MI size, and improved post-MI heart function. In conclusion, SIN1 plays critical roles in platelet activation, MI and post-MI heart failure, therefore serves as a target for therapeutic intervention in the thrombosis and myocardial infarction.

Disclosures

No relevant conflicts of interest to declare.
Aberrant Phosphorylation of STAT3 Protein of the CD4+ T Cells in Patients with Primary Immune Thrombocytopenia
Authors: Li Feng , Ke Yang , Cheng Yunfeng , Zhan Yanxia , Wu Boting
Published: November 2019

Background: Primary immune thrombocytopenia (ITP) is an autoimmune mediated bleeding disorder characterized by decreased platelet count. Dysfunctional cellular immunity is considered important in the pathogenesis of ITP. Signal transducer and activators of transcription 3 (STAT3) is critical for the differentiation of T cells, abnormal activation of STAT3 pathway has been confirmed in a variety of autoimmune diseases. However little is known in its role in ITP. The present studiesaimed to analyze the STAT3 protein phosphorylation of CD4+ T cells before / after therapy, response group versus no response group, in newly diagnosed ITP patients.

Methods: Twenty-nine adult patients with newly diagnosed ITP and sixteen healthy controls enrolled in this study. The IL-6 level was measured by ELISA; the phosphorylation STAT3 protein and the percentage of Th17 cells of the CD4+ T cells were analyzed by flow cytometry; the STAT3 mRNA expression was analyzed by Real-time PCR.

Results: The levels of IL-6 in the response group were higher than that in the controls. The basal level of pSTAT3 in the response group was higher than that in the no response group. The basal level of pSTAT3 correlated with the response. There was no statistical difference between the three groups both in the STAT3 mRNA expression and the percentage of Th17 cells of the CD4+ T cells.

Conclusion: The higher basal level of pSTAT3 might have good response with the corticosteroids therapy. This suggested the level of pSTAT3 might has prognostic role in ITP patients.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Recombinant Fixfc-Xten Bleeding Efficacy in Hemophilia-B Mouse Models
Authors: Liu Zhan , van der Flier Arjan , Ismail Ayman , Simpson Allison , Kurt Lutfiye , Schellenberger Volker , Loh Christine , Peters Robert
Published: November 2019

Introduction: Prophylactic treatment for hemophilia has demonstrated clear benefit over on demand treatment in hemophilic patients. Alprolix (rFIXFc) is the first of a new generation of long acting clotting factors we made to reduce the frequency of intravenous infusions (Powell, NEJM, 2013). Nevertheless, intravenous injections can be particularly challenging for young patients and patients with limited venous access and are time consuming. We are developing a novel rFIXFc fusion protein, using XTEN recombinant technology, which is suitable for prophylactic subcutaneous dosing in hemophilia B. In the current studies we explore the in vivo bleeding efficacy of subcutaneously dosed rFIXFc-XTEN in hemophilia B mouse model.

Methods: recombinant rFIXFc-XTEN was produced in HEK293 cells and affinity purified. The subcutaneous PK profile of rFIXFc-XTEN was compared to that of intravenously dosed rFIXFc in hemophilia B mice. The prolonged and acute bleeding efficacies were evaluated in HemB mouse tail-vein transection (TVT) and tail-clip bleeding models.

Results and conclusions: Subcutaneous dosing of rFIXFc-XTEN shows a tmax around 20 hours post dosing in mice, and improved plasma activity levels compared to similar (IU/kg) intravenously dosed rFIX or rFIXFc. Using the TVT bleeding model in HemB mice we show that at 72 hours post dosing, subcutaneously dosed rFIXFc-XTEN has improved in vivo efficacy compared to intravenously dosed rFIXFc at all tested doses. Similarly, acute efficacy testing in the HemB mouse tail-clip bleeding model showed improved efficacy of intravenously dosed rFIXFc-XTEN compared to rFIXFc. These data support the potential of once weekly or less frequent subcutaneous prophylactic dosing of rFIXFc-XTEN in humans.

Disclosures

Liu: biogen: Employment, Equity Ownership. van der Flier:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment, Equity Ownership. Simpson:Biogen: Employment, Equity Ownership. Kurt:Biogen: Employment, Equity Ownership. Schellenberger:Amunix: Employment, Equity Ownership. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder.
Bleeding in Factor XI Deficient Mice
Authors: Ay Cihan , Hisada Yohei , Cooley Brian C. , Mackman Nigel
Published: November 2019

Introduction: Factor XI (FXI) is a key component of the intrinsic or contact pathway of coagulation. It can be activated by either FXIIa or thrombin and amplifies thrombin generation during clot formation. Humans with congenital FXI deficiency, a rare bleeding disorder also referred to as hemophilia C, have a mild hemostatic defect with rare spontaneous bleeding but bleeding after hemostatic challenge, as occurs with some instances of surgery or trauma (e.g. dental surgery, tonsillectomy, prostate surgery or in the postpartum period). In previous studies, FXI deficient mice did not have increased bleeding in a tail transection model. In contrast, we observed increased surgical wound bleeding in FXI deficient mice compared to wild-type mice, which promted us to further investigate the effect of an absence of FXI in mice on bleeding in different models.

Methods: We assessed the hemostatic capacity of FXI-/- mice using two quantitative bleeding models (tail transection and saphenous vein injury). In the saphenous vein bleeding model, the saphenous vein was partially transected, clots that formed were removed and the total number of clots formed within a 30 minutes period (i.e. number of hemostatic events) were recorded under the microscope. In this model, a larger number of reforming clots corresponds to a higher hemostatic capacity.

Results: We found that tail bleeding times of FXI-/- mice were similar to wild-type mice (median [range]: 63 [42 - 90] vs. 70 [60 - 114] seconds, p=0.119), which is consistent with previous studies. In contrast, we observed an impaired hemostatic capacity in FXI-/- mice compared with wild-type controls in the saphenous vein bleeding model (median number of hemostatic events [range]: (19 [9 - 26] vs. 25 [21 - 30], p=0.003). However, this phenotype was milder than the bleeding observed in FIX-/- mice (median number of hemostatic events [range]: 2 [0 - 4], p<0.001).

Conclusions: Our results indicate that FXI-/- mice have a mild hemostatic defect after laparotomy and injury to the saphenous vein but not after tail transection. The mild increase in bleeding in FXI deficient mice after hemostatic challenge is similar to the increased bleeding observed in patients with FXI deficiency. Therefore, these mice may be useful in studying bleeding associated with FXI deficiency and in the presence of FXIa inhibitors.

Disclosures

No relevant conflicts of interest to declare.
Escalating Single Doses of PF-05230907 (Recombinant Factor Xa Variant FXaI16L) Are Safe and Demonstrate Hemostatic Pharmacology in Healthy Volunteers
Authors: Arkin Steven , Hua Fei , Kantaridis Constantino , Li Gang , Parsons-Rich Dana , Pittman Debra D
Published: November 2019

PF-05230907 (FXaI16L) is a zymogen-like variant of recombinant activated coagulation factor X (FXa), which is more resistant to inactivation by plasma protease inhibitors (compared to endogenous FXa). Previous studies showed that its cofactor, Factor Va (FVa), facilitates its transition to an active conformation, promoting hemostatic activity. The properties of PF-05230907 are consistent with its use as a short acting hemostatic agent to control bleeding and it currently is in clinical development for treatment of individuals with intracerebral hemorrhage (ICH).

We conducted a Phase 1 First-in-Human study to determine if single escalating doses of PF-05230907 were safe and tolerable and to characterize pharmacokinetics (PK) and pharmacodynamics (PD). The study was conducted in healthy adult volunteers aged 18-35 years with no risk factors for thrombosis and was approved by the local ethics committee. Subjects were assigned to one of six ascending single dose cohorts, each with 2 placebo treated subjects and 6 subjects treated with PF-05230907 (0.1, 0.3, 1, 2, 3, or 5 µg/kg). Assessments included safety monitoring, PD, PK, and immunogenicity testing. All placebo treated subjects were to be included in the safety and PD analyses. All PF-05230907 treated subjects were to be included in the safety, PK and PD analyses.

Forty-nine male subjects were enrolled after informed consent. One subject from the 2 µg/kg cohort received less than 80% of the planned PF-05230907 dose and was replaced. This subject was included in the safety analysis, but excluded in PK and PD analysis. PF-05230907 exhibited a fast clearance rate with a terminalhalf-life of approximately 4 minutes. Exposure of PF-05230907 appeared to increase proportionally with dose. Treatment-related PD changes were observed for activated partial thromboplastin time (aPTT), thrombin-anti-thrombin III complexes, prothrombin fragment 1+2, D-dimer and thrombin generation assay (lag time and peak thrombin). PF-05230907 was safe and well tolerated at all dose levels tested with no dose limiting toxicity. There were no serious adverse events. One subject treated with PF-05230907 had a weak and transient non-neutralizing anti-drug antibody response, which did not cross react with native FX or native FXa.

PF-05230907 was safe and tolerated and exhibited pharmacologic effect in healthy adults when administered at doses up to 5 µg/kg. The safety profile, PK, and PD observed in this study support further development of PF-05230907 for hemostatic treatment in individuals with acute hemorrhagic conditions.

Disclosures

Arkin: Pfizer Inc: Employment. Hua:Pfizer Inc: Employment. Kantaridis:Pfizer Inc: Employment. Li:Pfizer Inc: Employment. Parsons-Rich:Pfizer Inc: Employment. Pittman:Pfizer: Employment.
Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation
Authors: Lollar Pete , Parker Ernest T. , Healey John F. , Doering Christopher B.
Published: November 2019

Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes.

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
Exome Sequencing in Venous Thromboembolic Disease Identifies Excess Mutation Burden in PROS1, PROC, SERPINC1 and STAB2
Authors: Desch Karl C. , Ozel Ayse Bilge , Halvorsen Matt , Jacobi Paula M. , Germain Marine , Tregouet David Alexandre , Reitsma Pieter H. , Goldstein David , Ginsburg David
Published: November 2019

Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, and seven other loci are associated with VTE. Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 have been linked to VTE in family studies. In order to identify new genetic variants altering the risk for VTE, we performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with unprovoked VTE and compared results to a previously sequenced control cohort of 5784 unrelated Europeans. To avoid variant calling bias, only SNVs from exons with >10X coverage and less than 5% difference in coverage between cases and controls were included, removing 11,813 of 188,689 intervals. We used an emerging framework for a "collapsing" analysis on genes, defining qualifying variants on the basis of annotation and minor allele frequency <0.05%, and assumed a dominant model of inheritance. Tests were performed via a Fisher's exact test for a total of 11,585 CCDS genes. Strikingly, ranked by p-value, the top 4 genes were PROS1 (P= 2.01E-09, OR 11.8), STAB2 (P=2.70E-7, OR 3.37), PROC (P=3.24E-05, OR 11.0) and SERPINC1 (P=1.10E-04, OR 8.5). We detected 29 qualifying variants in 373 cases and 106 qualifying variants in 5784 controls in STAB2. This gene encodes Stabilin-2, which is a transmembrane glycoprotein scavenger receptor. Common variants at ABO are associated with VTE and are also known to regulate von Willebrand Factor (VWF) and coagulation Factor VIII (F8) plasma levels. Common variants in STAB2 are also associated with VWF/F8 levels in a large GWAS and with VTE risk in a smaller candidate gene study, suggesting that haploinsufficiency for Stabilin-2 may increase VTE risk through elevated levels of VWF/F8. Although replication and functional testing of these findings is warranted, this study demonstrates the utility of collapsing analyses using WES data to identify multiple loci harboring an excess of rare variants in individuals with a common complex disease trait.

Disclosures

Ginsburg: Shire: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: recombinant VWF and recombinant ADAMTS13; Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
Coagulation Profile of Children on Extracorporeal Membrane Oxygenation (ECMO) - Is FXIII the Missing Link?
Authors: Haas Thorsten , Doell Carsten , Schmugge Markus , Cushing Melissa M. , Cannizzaro Vincenzo
Published: November 2019

Background

Published data about bleeding management on extracorporeal membrane oxygenation (ECMO) in children is sparse and to date no global transfusion algorithm has been established. Viscoelastic testing can be effective for determining the etiology and management of coagulopathic bleeding during cardiothoracic procedures, but data regarding its usefulness in ECMO patients are scarce. Recently, low factor XIII levels were determined to be a frequent finding in adult ECMO patients(Kalbhenn et al; Perfusion 2015;30:675-82).

Methods

This is a retrospective analysis of thromboelastometry (ROTEM®) and factor XIII data obtained in children (ages 0 to 18 years) undergoing ECMO since 2013 in a single center children's hospital. Acute bleeding treatment was based on daily ROTEM testing, complete blood count and routine plasmatic coagulation testing. The transfusion algorithm targeted a hemoglobin level >13g dL-1, a Quick's value >50%, a plasma fibrinogen level >1.5g L-1, and a platelet count of >100,000 µL-1. Red blood cells (RBC), solvent detergent (S/D) plasma and platelet apheresis concentrates were exclusively used to maintain hemostasis. Measurement of FXIII levels is not part of routine testing, but was assessed when unexplained bleeding was observed.

Results

Laboratory and transfusion data from sixteen patients, age 4 (1-15) months [median(IQR)] with a body weight of 6 (3-8) kg were included. Median time on ECMO was 7 (4-9) days. Large volumes of allogeneic blood were transfused to all children, meeting criteria for massive transfusion each individual day on ECMO (Tab.1). Overall, median daily ROTEM measurements were within reference ranges (Tab.2), while median levels of FXIII were decreased despite massive transfusion [FXIII levels 42% (28-51%)].

Conclusion

Pediatric ECMO was almost always combined with daily massive transfusion, which led to correction of overall ROTEM values. Notably, despite transfusion of large amounts of plasma, decreased FXIII levels were noted. This finding is supported by results of a study in adult ECMO patients, where FXIII levels <50% were observed in 88% of all patients. Although inherited homozygous FXIII deficiency is usually defined by levels <5%, even mildly to moderately reduced FXIII levels have been reported to contribute to increased bleeding after cardiac surgery(Ternström et al; Thromb Res 2010;126:e128-33). Further studies should be performed to assess the impact of FXIII substitution in pediatric ECMO patients and to investigate whether substitution of FXIII may decrease bleeding without increasing thrombotic complications.

Disclosures

Haas: CSL Behring: Speakers Bureau; TEM International: Speakers Bureau; Octapharma: Consultancy.
Perioperative Management for Congenital Factor XI Deficiency; A Systematic Review
Authors: Chai-Adisaksopha Chatree , Rattanathammethee Thanawat , Drakulic Milana , Matino Davide , Iorio Alfonso
Published: November 2019

Background: Congenital Factor XI (FXI) deficiency is the most common rare bleeding disorder, characterized by production defect of FXI. Bleeding manifestation is usually mild and associated with trauma or surgery. The treatment options during surgery include plasma, FXI concentrate, recombinant activated factor VII (rFVIIa), etc. This study aimed to investigate the efficacy of hemostatic control and thrombotic complications in patients with congenital FXI deficiency who underwent surgery or an invasive procedure.

Methods: We conducted a systematic search in MEDLINE, EMBASE and CENTRAL database from inception to March 2016. We included the case reports and case series of patients with congenital FXI deficiency. Studies must report the perioperative treatment regimen, hemostatic outcome and thromboembolic complication. The pre- and post-FXI levels were collected. The primary outcome was the efficacy of hemostatic control and the safety outcome was thromboembolic complication.

Results: There were 87 studies included in this review, involving 310 patients with congenital FXI deficiency. Median age was 49.5 years (range 0.3 to 94). Forty percent of the patients were male. Median baseline FXI level was 6.0% (range <1 to 75.0). FXI concentrate was the most common treatment (48.39%), following by plasma (21.61%), rFVIIa (12.90%) and others (FEIBA, DDAVP, tranexamic acid and no treatment; 17.1%). Hemostatic control was found to be good or excellent in 118 patients (88.06%) who had major surgery, 65 patients (92.85%) in minor surgery and 47 patients (97.91%) in dental procedure, Table 1. Thromboembolic events were reported in 7/150 (4.67%) patients treated with FXI concentrate and 4/34 (11.76%) patients treated with rFVIIa, Table 2. None of patients receiving plasma, FEIBA, DDAVP or tranexamic acid had thrombosis.

Conclusion: Hemostatic control during perioperative periodwas achieved in majority of patients with congenital FXI. Peak plasma FXI should be obtained in patients treated with plasma or FXI concentrate with the target FXI of 60-70% for major surgery and 40-50% for minor surgery. With respect to the higher risk of thromboembolic complication, we recommend plasma over FXI concentrate and rFVIIa for perioperative treatment in patients with FXI deficiency.

Disclosures

No relevant conflicts of interest to declare.
Risk of Bleeding from Primary Thromboprophylaxis (PTP) in Patients with Solid Cancer Receiving Chemotherapy: A Systematic Review and Meta-Analysis of Randomized Controlled Trials (RCT)
Authors: Thein Kyaw Zin , Tun Aung M , Yeung Sai-Ching J , Oo Thein H.
Published: November 2019

Introduction: Thrombosis is the second leading cause of death in cancer patients. Hypothetically, prognosis might be improved by preventing thrombotic events by PTP. PTP has to be outweighed with the bleeding risk from PTP benefit. Moreover, PTP in ambulatory cancer patients remains uncertain. We performed a systematic review and meta-analysis of RCTs to determine the risk of major bleeding (MB) and clinically relevant non-major (CRNM) bleeding from PTP with low-molecular weight heparins (LMWH) in solid cancer patients receiving chemotherapy.

Methods: We systematically conducted a comprehensive literature search using MEDLINE and EMBASE databases through May 31, 2016. The RCTs with MB and CRNM bleeding as safety outcomes were included in the analysis. Mantel-Haenszel method was used to calculate the estimated pooled risk ratio (RR), and risk difference (RD) with 95% confidence interval (CI). Random effects model was applied.

Results: 12 RCTs with a total of 8643 patients were eligible for analysis. Dalteparin, nadroparin, certoparin, semuloparin and bemiparin were used in the studies. 9 studies utilized prophylactic doses, while one used intermediate doses and 2 employed therapeutic dose. The PTP duration was from 6 weeks to 6 months. MB events were reported in 67 (1.486%) patients on PTP compared to 46 (1.112%) in control group. The pooled RR for MB was statistically nonsignificant at 1.341 (95% CI: 0.917 to 1.960, P = 0.130). CRNM bleeding events were noted in 209 (4.637%) in PTP group and 106 (2.563%) in control group. The RR for CRNM bleeding was statistically significant at 1.729 (95% CI: 1.017 to 2.938, P = 0.043). The absolute RD in CRNM bleeding was 0.020 (95% CI: 0.004 to 0.035, P = 0.012) with an estimated number needed to harm (NNH) of 48 to cause one CRNM bleeding event.

Conclusions: Approximately 60 patients are needed to be treated with PTP to prevent one symptomatic venous thrombosis among all ambulatory unselected cancer patients on chemotherapy in a previous meta-analysis. Our meta-analysis revealed that PTP contributed an increase in CRNM bleeding events with NNH of 48. The risk of bleeding should not be underestimated and more RCTs are required before making any recommendations.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

Oo: Daiichi Sankyo: Research Funding.
A Systematic Review and Meta-Analysis of Randomized Controlled Trials (RCT) in Ambulatory Thromboprophylaxis (ATP) in Patients with Lung Cancer Receiving Chemotherapy
Authors: Thein Kyaw Zin , Yeung Sai-Ching J , Oo Thein H.
Published: November 2019

Introduction: Lung cancer (LC) is the commonest cause of cancer mortality in USA. Thromboembolism (TE) is the second leading cause of death in cancer patients. The ambulatory thromboprophylaxis (ATP) in solid cancer patients remains uncertain. However, LC is at least an intermediate risk for TE according to Khorana scoring system. We conducted a systematic review and meta-analysis of RCTs to determine the benefit and risk of ATP with low-molecular weight heparins (LMWH) in LC patients receiving chemotherapy.

Methods: We performed a comprehensive literature search using MEDLINE and EMBASE databases through June 30, 2016. The RCTs with reduction in TE as a primary or secondary endpoint and the major bleeding (MB) as a safety outcome were included in the analysis. Mantel-Haenszel method was used to calculate the estimated pooled risk ratio (RR), and risk difference (RD) with 95% confidence interval (CI). Fixed effects model was applied.

Results: A total of 4315 patients with LC from 4 RCTs and a subgroup of another 2 RCTs were included in our analysis. The prophylactic doses of dalteparin, nadroparin, certoparin, semuloparin and bemiparin were used in the studies. The ATP duration ranged from 3 to 6 months. The randomization ratio was 2 to 1 in PROTECHT study and 1 to 1 in other studies. The TE incidence was 89 (4.007%) in ATP group and 166 (7.927%) in control group with a RR of 0.510 (95% CI: 0.397 to 0.654, P < 0.001). The absolute RD was -0.039 (95% CI: -0.053 to -0.025, P < 0.001) with an estimated number needed to treat (NNT) of 25 to prevent one TE event. MB events were 24 (1.506%) in ATP group compared to 15 (1.019%) in control group according to an analysis of 4 RCTs. The pooled RR for MB was statistically nonsignificant at 1.468 (95% CI: 0.785 to 2.746, P = 0.229). Clinically relevant nonmajor (CRNM) bleeding events were 80 (5.571%) in ATP group and in 24 (1.674%) in control group on an analysis of 4 RCTs. The RR for CRNM bleeding was statistically significant at 3.253 (95% CI: 2.092 to 5.059, P < 0.001).

Conclusions: In our study, the relative risk reduction for TE is 49% with a NNT of 25 to prevent one TE without increasing MB. Nevertheless, CRNM bleeding episodes were significant at a ratio of 3.253. Based on the findings, selection of LC patients who are at high TE risk is important. Further studies are necessary to define a subset of LC patients who may benefit from ATP.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

Oo: Daiichi Sankyo: Research Funding.
Novel Class of Direct Thrombin Inhibitors Prevent Thrombosis By Inhibiting Fibrinogen Cleavage While Preserving Platelet Function
Authors: Sivaraja Mohan , Sizikov Sivan , Sandoval Nichole , Lin Kenneth , Igoudin Lev , Dash Subhadra , Xu Chengpei , Clemens Daniel , Estiarte Angels , Shiau Timothy , Short Kevin , Williams David , Datta Anirban , Kita David
Published: November 2019

We have developed a new class of direct thrombin inhibitors (VE-DTIs) that act through reversible covalent enzyme inhibition. These compounds inhibit thrombin with high potency (IC50 < 10 nM) and with better than 100× selectivity against related serine proteases. In rodents, VE-DTIs show dose-dependent efficacy in preventing arterial and venous thromboses respectively in arteriovenous shunt thrombosis and venous stasis thrombosis models. They also prevent thrombin-induced pulmonary embolism in rodents. Despite their strong anticoagulation properties, VE-DTIs are associated with significantly lower bleeding times than other approved anticoagulants dabigatran, argatroban or apixaban in rodent bleeding models (tail bleeding time and saphenous vein bleeding time).

We compared the effect of VE-DTIs and the DTIs argatroban and dabigatran on the coagulation cascade. In vitro, VE-DTIs inhibited thrombin-mediated fibrinogen cleavage with similar potency. Inhibition of thrombin/thrombomodulin complex-mediated activation of TAFI and protein C by VE-DTIs were also comparable. In contrast, VE-DTIs were more than 1000-fold less potent in inhibiting thrombin-mediated platelet activation than the other DTIs. Additionally, and also in contrast to argatroban and dabigatran, VE-DTIs protect rodents from the effects of thrombin-induced pulmonary embolism without significantly inhibiting platelet activation. The ability of VE-DTIs to prevent thrombosis without significantly impacting platelet function could lead to a new anticoagulant therapy with lower bleeding risk.

Disclosures

Sivaraja: Verseon Corporation: Employment, Equity Ownership. Sizikov:Verseon Corporation: Employment, Equity Ownership. Sandoval:Verseon Corporation: Employment, Equity Ownership. Lin:Verseon Corporation: Employment, Equity Ownership. Igoudin:Verseon Corporation: Employment, Equity Ownership. Dash:Verseon Corporation: Employment, Equity Ownership. Xu:Verseon Corporation: Employment, Equity Ownership. Clemens:Verseon Corporation: Employment, Equity Ownership. Estiarte:Verseon Corporation: Employment, Equity Ownership. Shiau:Verseon Corporation: Employment, Equity Ownership. Short:Verseon Corporation: Employment, Equity Ownership. Williams:Verseon Corporation: Employment, Equity Ownership. Datta:Verseon Corporation: Employment, Equity Ownership. Kita:Verseon Corporation: Employment, Equity Ownership.
The Effect of Platelet Transfusion on Death in the Intensive Care Unit
Authors: Arnold Donald M. , Ning Shuoyan , Barty Rebecca , Liu Yang , Cook Richard , Rochberg Bram , Iorio Alfonso , Shih Andrew W. , Heddle Nancy M
Published: November 2019

Background: Thrombocytopenia is a common complication of critical illness and an independent risk factor for death in the intensive care unit (ICU). Whether platelet transfusions modify the risk of death in critically ill patients is unknown.

Methods: Adult patients admitted to ICU who received one or more platelet transfusion over a 10-year period (2006 - 2015) from 3 academic hospitals in Canada were analyzed from a blood transfusion registry. Oncology patients were excluded. Contemporaneous non-transfused ICU patients were used as controls. Data from the registry were validated by integrity checks with medical records and laboratory information systems. We estimated the effect of platelet transfusion on mortality in ICU adjusted for baseline and time-varying covariates including multi-organ dysfunction score (MODS) and severity of thrombocytopenia using a stratified cox proportional hazards model. Significance was set at p<0.05 for all analyses.

Results: Of 43,234 non-oncology patients admitted to ICU, 5,621 (13.0%) received one or more platelet transfusion. Compared with non-transfused controls, transfused patients had lower platelet counts (median, 82 x109/L vs. 163 x109/L); were more often admitted after surgery (90.7% vs. 46.9%) especially cardiac surgery (86.8% of surgeries vs. 60.6%); and had higher unadjusted mortality (10.7% vs. 6.5%). Using regression analysis adjusted for covariates (nadir platelet count, red blood cell transfusion, need for hemodialysis) and stratified by age, baseline MODS score (available for 66.2% of patients) and need for invasive mechanical ventilation, platelet transfusions were associated with a lower risk of death in ICU [hazard ratio (HR)= 0.66; 95% confidence interval (CI), 0.46 - 0.96; p= 0.028; n= 26,404 with all available data]. A similar effect was observed in the subgroup of cardiac surgery patients (HR= 0.50; 95% CI, 0.26 - 0.98; p=0.044; n= 10,676) but not all surgical patients (HR = 0.73; 95% CI, 0.46 - 1.17; p= 0.188; n= 14,461).

Conclusion:After adjusting for illness severity, thrombocytopenia and other confounders common among critically ill patients, platelet transfusions were associated with improved survival in the population of mostly cardiac surgery patients. This potential protective effect of platelet transfusions requires further evaluation in prospective studies.

Disclosures

Arnold: Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; UCB: Consultancy; Amgen: Consultancy, Research Funding.
Blood Management Strategies to Reduce Transfusions after Elective Lower-Extremity Joint Arthroplasty Surgeries: One Tertiary Care Hospital's Early Experience with an Alternative Payment Model - a Total Joint 'Bundle
Authors: Kansagra Ankit J. , Dahiya Saurabh , Andrzejewski Chester , Krushell Robert , Lehman Andrew , Greenbaum Jordan , Ehresman Alice , Visintainer Paul , McGirr Joan , Mahoney Kathleen , Cloutier Darlene , Stefan Mihaela
Published: November 2019

Background and objective: Blood loss associated with total lower-extremity joint arthroplasty (TJA) often results in postoperative anemia and need for red blood cell transfusions (RBCT).

We report the results of a quality improvement initiative to improve blood management and decrease transfusions in patients undergoing TJA in one tertiary hospital.

Methods: Pre and post analysis after the implementation of a multifaceted intervention which included preoperative assessment for anemia, use of tranexamic acid, discouragement of autologous pre-operative blood collection and institution of more restrictive RBCT protocols. The results were stratified into three periods: I - pre-interventional (01/01/2013 -09/30/2013); II - peri-interventional (10/01/2013 -04/30/2014); and III - post-interventional (05/01/2014 -12/31/2014). We used fractional logistic regression with robust standard errors and regression modeling was configured using a segmented, or "piecewise", approach in which slope coefficients in each period were estimated.

Results: During the study period 2511 patients underwent surgery. Compared with the pre-intervention period, the total number of RBC units transfused decreased from a total of 587 in the pre- to 107 in the post-intervention period (81.8% decrease). The percentage of patients receiving transfusion declined from 36.7% in pre-implementation period to 8.8% to post-intervention period. Depending upon the costing methodology used, annualized savings in RBC expenditure between time period 1 and 3 ranged from a low of $108,000 using the acquisition cost per unit (~$225/unit) to $480,000 when using activity based costing (~$1000/unit). Mean length of stay (days) and 30-day readmission rates remained stable during the study period.

Conclusions: A multidisciplinary approach with proactive involvement of all the interested parties can be successful and sustainable in reducing RBCT and its associated costs, in patients undergoing TJA.

Disclosures

No relevant conflicts of interest to declare.
Inhibiting IL-2 Signaling and the Regulatory T-Cell Pathway Using Computationally Designed Novel Peptides
Authors: Price-Troska Tammy , Diller David , Bayden Alexander , Jarosinski Mark , Audies Joseph , Yang Zhi-Zhang , Ansell Stephen M
Published: November 2019

Regulatory T-cells (TREG) are the gateway to immune function and typically regulate immune cell activation. Cytokines, including interleukin-2 (IL-2), induce T-cell differentiation and promote a regulatory phenotype. Once activated via the IL-2 receptor (IL-2R), a cascade of events in T-cells initiate signal transducer and activator of transcription 5 (STAT5) and Forkhead box P3 (FOXP3) activation which appear to function as important regulators of this immunologic pathway and promote the development and function of TREG cells. In non-Hodgkin lymphoma (NHL), we have found that intratumoral TREG cells are increased in number and suppress immune function. In previous work, we have found that TREG cells inhibit T-cell proliferation, suppress cytokine production and limit effector cell cytotoxicity. We have also shown that increased serum levels of soluble sIL-2Rα is a prognostic factor in NHL and that sIL-2Rα can bind to IL-2 and promote its signaling thereby increasing TREG cell numbers. In this study, we developed a strategy to inhibit the binding of IL-2 to sIL-2Rα with the goal of suppressing the induction of FOXP3 and decreasing TREG cell numbers. To do this, we developed peptides designed to disrupt the interaction between IL2 and sILRα.

In collaboration with CMDBioscienceSM, we developed and analyzed 22 peptide compounds derived by structure-based computational design. Initially, we screened each peptide at increasing concentrations using an ELISA assay to test the inhibition of IL-2/IL-2Rα binding by the solubilized peptide. Candidate peptides were then further tested using upregulation of pSTAT5 and FOXP3 in T-cells measured by flow cytometry as a measure of inhibition of IL-2 signaling. The peptides were developed according to different design hypotheses and grouped into different families; the screening ELISA results indicated 4 promising peptides that inhibited IL2/IL2Rα binding (up to 100% inhibition; max peptide concentration of 100uM). These peptides were then used to determine their effect on STAT5 and FOXP3 expression. A lead candidate peptide consistently reduced the expression of FOXP3 and STAT5 expression compared to cells not exposed to peptide. Use of the peptide to disrupt IL-2 signaling inhibited the development of cells with a TREG phenotype.

We conclude that structure-based peptide design can be used to identify novel peptide inhibitors that block IL-2/IL-2Rα signaling and inhibit STAT5 and FOXP3 upregulation. These peptides could be used as new therapeutic agents to limit immune suppression by TREG cells and promote a more effective anti-tumor immune response in NHL.

Disclosures

No relevant conflicts of interest to declare.
Direct Targets of Epo Receptor-JAK2-pSTAT5 Signalling in Erythropoiesis
Authors: Gillinder Kevin R. , Tuckey Hugh , Bell Charles , Huang Stephen , Ilsley Melissa , Magor Graham , Perkins Andrew C
Published: November 2019

Erythropoietin (EPO) acts through the dimeric erythropoietin receptor (EpoR) to stimulate proliferation, survival and differentiation of colony-forming units-erythroid (CFU-e). We undertook two complimentary approaches to find pSTAT5-depenendent and independent target genes in erythroid cells. We performed RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5, 30 minutes after EPO stimulation of J2E cells. This is the first time genome wide pSTAT5 ChIP-seq has been successfully undertaken in hematopoietic cells. We found ~320 robust pSTAT5 occupied sites in the erythroid genome. About 15% of these reside in promoters while the rest reside in intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of peaks contained a central palindromic GAS element, TTCYXRGAA, and there was significant enrichment of GATA and CACCC-box elements, suggesting co-regulation of some EPO-induced genes by GATA1 and KLF1. Using 4sU RNA-seq and CAGE data, we found just 57 genes to be immediately transcribed in response to EPO within 30 minutes while other target genes had delayed responses. We suggest this has biological relevance for feed forward and feedback controls on EPO driven erythropoiesis. Some of the DEGs (e.g. Bcl2l2 and Cish) are known direct targets of pSTAT5, but many are novel and suggest new pathways by which EPO regulates erythropoiesis. These include mRNA splicing, epigenetic regulation via histone methylation, and adaptive changes in the composition of the EpoR. This could provide increased sensitivity of erythroid progenitor cells to anaemic stress; i.e. sensitization of erythroid cells to JAK2-STAT5 signalling. We found a significant overlap between direct STAT5 target genes in erythroid cells, mammary epithelial cells and lymphoid cells, which imply conserved generic effectors of JAK-STAT5 signalling in different cell types. Our results provide new insights into how EPO co-ordinates erythroid cell proliferation, survival and differentiation. Some of these DEGs could be used as biomarkers for monitoring disease activity in polycythaemia vera (PV) and responses to JAK2 inhibitors.

Disclosures

Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria.