TMEM16F Deficiency Causes a Strain-Dependent Lethality in Mice and Reduced Phosphatidylserine Exposure on Heterozygous As Well As Homozygous Deficient Mouse Platelets

Abstract Scott Syndrome is a rare bleeding disorder characterized by a defect in platelet phosphatidylserine (PS) exposure. The syndrome has recently been linked to mutations in TMEM16F, a Ca++-activated ion channel. Tmem16f-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding (Yang et al, Cell 151: 111-122; 2012). We now report analysis of an additional gene targeted Tmem16f allele generated in C57BL/6 ES cells. JM8 ES cell were obtained from EUCOMM, and successful Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f+/gt (+/gt) intercross progeny identified no surviving Tmem16fgt/gt (gt/gt) mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryo’s exhibiting no morphological abnormalities on gross or routine histologic examination. Though complete deficiency of TMEM16F is lethal in the C57BL/6J genetic background between E17.5 and birth, an F2 intercross of +/gt mice outcrossed one generation to 129x1SvJ resulted in gt/gt mice surviving to weaning, though at reduced numbers (6/75 total progeny compared to ~19 expected, p <0.002). Progeny testing of surviving gt/gt mice suggest a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3. Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time. These data suggest the existence of a viability-determining genetic modifier of TMEM16F in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover novel functions for TMEM16F and the regulation of hemostatic function. The observation of a PS exposure and hemostatic phenotype in +/gt mice also suggests the possibility that heterozygous TMEM16F mutations may subtly influence hemostasis or thrombosis in humans. Disclosures No relevant conflicts of interest to declare.

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Abstract 42: Genetic Ablation of TMEM16F Exhibits Strain-specific Lethality in Mice

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.42 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Colin A Kretz ◽

Gary Gilbert ◽

David Ginsburg

Keyword(s):

Es Cells ◽

Genetic Modifier ◽

Proximal Region ◽

Strain Identification ◽

Progeny Testing ◽

Spontaneous Bleeding ◽

Genetic Ablation ◽

Intron 1 ◽

Total Progeny ◽

Report Analysis

Background: Scott Syndrome is a rare bleeding disorder characterized by a defect in platelet phosphatidylserine (PS) exposure. The syndrome has recently been linked to mutations in TMEM16F. Tmem16f -/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding. We now report analysis of an additional gene targeted Tmem16f allele generated in C57BL/6 ES cells. Results: JM8 ES cell were obtained from EUCOMM, and successful Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f +/gt (+/gt) intercross progeny identified no surviving Tmem16f gt/gt (gt/gt) mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryo’s exhibiting no morphological abnormalities on gross or routine histologic examination. Though complete deficiency of TMEM16F is lethal in the C57BL/6J genetic background between E17.5 and birth, an F2 intercross of +/gt mice outcrossed one generation to 129x1SvJ resulted in gt/gt mice surviving to weaning, though at reduced numbers (6/75 total progeny compared to ~19 expected, p <0.002). Progeny testing of surviving gt/gt mice suggest a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3. Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). Conclusion: These data suggest the existence of a viability-determining genetic modifier of TMEM16F in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover novel functions for TMEM16F and the regulation of hemostatic function.

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Platelet Phosphatidylserine Exposure, Survival and Blood Coagulation in Mice Lacking TMEM16F

Blood ◽

10.1182/blood.v126.23.sci-32.sci-32 ◽

2015 ◽

Vol 126 (23) ◽

pp. SCI-32-SCI-32

Author(s):

Gary E. Gilbert ◽

Colin Kretz ◽

David Ginsburg ◽

Valerie Anne Novakovic

Keyword(s):

Conflicts Of Interest ◽

Es Cells ◽

Genetic Modifier ◽

Bleeding Disorder ◽

Cation Binding ◽

Strain Identification ◽

Severe Bleeding ◽

Phosphatidylserine Exposure ◽

Cation Binding Site ◽

Responsible Gene

Abstract Scott Syndrome is a rare, moderately severe bleeding disorder caused by a defect in platelet, red cell, and lymphocyte phosphatidylserine exposure. The syndrome has been linked to mutations in TMEM16F, a Ca++-activated ion channel. A moderately severe bleeding disorder in German Shephard dogs, characterized by decreased platelet phosphatidylserine exposure, has also linked to mutations in TMEM16F. TMEM16F, is a member of a recently-identified family of calcium-activated chloride channels that are also called anoctamins. Members of the family apparently serve both as ion channels and phospholipid scrambling channels. A crystal structure of one member of the TMEM16 family shows a homo-dimeric structure of an integral membrane protein with each unit containing 10 transmembrane helices. A transmembrane hydrophilic cavity lies at the interface, and contains a cation binding site, and a slot with dimensions that could accommodate acyl chains. Tmem16f-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding. We have developed an independent gene-targeted (gt) Tmem16f allele generated in C57BL/6 ES cells and find results that both confirm prior reports and contrast with them. JM8 ES cells were obtained from EUCOMM, and Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f+/gt intercross progeny identified no surviving Tmem16fgt/gt mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryos. Blinded pathological evaluation of E17.5 gt/gt pups indicated reduction of ossification and angular limb changes, consistent with 2 prior reports. Thus, our results confirm previously reported bone changes, but in contrast with prior reports, indicate that Tmem16f deficiency is lethal in the C57BL/6 genetic background. An F2 intercross of +/gt mice outcrossed one generation to 129x16vJ resulted in gt/gt mice surviving to weaning, though at only 30% of the expected Mendelian frequency. Progeny analysis points to a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3. Further efforts to localize the responsible gene(s) are underway. Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time. Our data suggest the existence of a viability-determining genetic modifier of the TMEM16F deficiency phenotype in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover a novel regulator of hemostatic function. The observation that heterozygous deficiency leads to a PS exposure and hemostatic phenotype also suggests the possibility that heterozygous TMEM16F mutations may influence hemostasis or thrombosis in humans. Disclosures No relevant conflicts of interest to declare.

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Differential Effects of HIV Protease Inhibitors on Release and Activation of Platelet-Derived TGF-β1: Potential Association with HIV-Linked CVD

Blood ◽

10.1182/blood-2019-126225 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2341-2341

Author(s):

Kouzbari Karim ◽

Gostynska Sandra ◽

Sonia Elhadad ◽

Dube Pratibha ◽

Jeffrey Laurence ◽

...

Keyword(s):

Protease Inhibitors ◽

Low Dose ◽

Cardiac Fibrosis ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Minor Effect ◽

The Impact ◽

Tgf Β1 ◽

In Vivo Assessment

Combination antiretroviral therapies (cART) have markedly reduced mortality in HIV infection. However, cardiovascular disease (CVD), including heart failure linked to fibrosis, remains a major cause of morbidity and mortality in HIV/cART patients. The magnitude of this risk increases with use of certain protease inhibitors (PI), but the underlying mechanism remains unclear. We showed that the PI ritonavir leads to increased plasma levels of the pro-fibrotic cytokine TGF-β1, cardiac dysfunction, and pathologic cardiac fibrosis in wild-type (wt) C57BL/6 mice. Mice with targeted depletion of platelet TGF-β1 had reduced cardiac fibrosis and partially preserved cardiac function following ritonavir exposure (Laurence, et al. PLoS One 2017;12:e0187185). Several groups have examined the effects of a variety of cART agents on agonist-induced platelet aggregation, but correlations with clinical CVD are weak. Since platelets are a rich source of TGF-β1, we hypothesized that ritonavir and other PIs linked clinically to an increased CVD risk directly activate platelets to release TGF-β1 and activate latent (L)TGF-β1 to initiate signaling for organ fibrosis. We examined the impact of clinically relevant doses of ritonavir, alone and in combination with two other contemporary PIs, atazanavir and darunavir, which are currently used along with low dose ritonavir in so-called PI-boosted cART regimens. We incubated human platelet-rich plasma and washed platelets with PIs alone or in combinations at various doses for 10 min at 37°C in a platelet aggregometer (BioData. Corp). Total and active TGF-β1 levels were measured by ELISA. For in vivo assessment, we treated wt mice with a low dose of ritonavir, as used in PI-boosted cART, and measured the levels of plasma TGF-β1 by ELISA, and TGF-β1 signaling in tissues by immunofluorescence imaging for pSmad2. We found that ritonavir dose-dependently increased total TGF-β1 release from freshly-isolated platelet-rich plasma and washed human platelets. This release was blocked by ceefurin-1 and MK517, potent inhibitors of the ATP binding cassette transporter ABCC4. Darunavir alone did not cause release of TGF-β1, and did not alter significantly ritonavir-induced TGF-β1 release (Figure-1A). Atazanavir alone did induce release of TGF-β1 from platelets and did not affect the extent of such release induced by ritonavir (Figure-1A). Since total TGF-β1 released from platelets must be activated in order to signal, we tested whether these PIs could activate LTGF-β1. Ritonavir alone, in low dose, activated TGF-β1 by 4-5-fold (Fig-1B). Darunavir alone did not activate LTGF-β1, and had only a minor effect on ritonavir-induced TGF-β1 activation (Fig-1B). In marked contrast, while atazanavir also did not activate LTGF-β1, it significantly inhibited ritonavir-induced LTGF-β1 activation (Fig-1B). For in vivo assessment, wt mice were injected daily for 8 weeks with ritonavir, which dose-dependently increased plasma TGF-β1 levels (mean levels with vehicle 2.1 ng/ml; 6.4 ng/ml with 5 mg/kg ritonavir; 8.5 ng/ml with 10 mg/kg ritonavir). Increased TGF-β1 levels correlated with development of pathologic fibrosis and increased phosphorylated Smad signaling in hearts of ritonavir-treated vs. vehicle-treated mice. Clinical correlations with these in vitro and in vivo mouse studies are important. The fact that ritonavir effected both release and activation of platelet TGF-β1 is consistent with its ability to induce cardiac fibrosis and dysfunction in mice, and its association with accelerated CVD in HIV-infected individuals. Our findings that low dose ritonavir in combination with darunavir induced release and activation of platelet TGF-β1, whereas atazanavir blocked TGF-β1 activation, are consistent with the strong association of ritonavir-boosted darunavir, but not ritonavir-boosted atazanavir, with CVD in the setting of HIV (Ryom, et al. Lancet-HIV 2018;5:e291-e300). Future work will examine the effects of other contemporary cART agents, including cobicistat, which is currently replacing ritonavir in many PI-boosted therapies and some integrase-boosted regimens, on TGF-β1 release and activation, for which correlations with clinical CVD are not yet available. Identification of the mechanism of pathologic fibrosis in the heart, and potentially other organs affected by certain cART regimens, such as the kidney, may suggest specific therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

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The Effects of Anti-A and Anti-B On Platelet Function: An in Vitro Model of ABO Non-Identical Transfusion.

Blood ◽

10.1182/blood.v114.22.2120.2120 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2120-2120

Author(s):

Majed A. Refaai ◽

Neil Blumberg ◽

Charles W. Francis ◽

Richard Phipps ◽

Sherry Spinelli ◽

...

Keyword(s):

Normal Saline ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Type A ◽

P Value ◽

Blood Product Transfusion ◽

Trauma Patients ◽

Type B ◽

Average Percentage

Abstract Abstract 2120 Poster Board II-97 Background: Transfusion of ABO non-identical red blood cells (RBCs) can cause immune mediated hemolytic transfusion reactions. Therefore, only ABO identical RBCs are transfused, except in emergencies, when group O RBCs are transfused. Use of exclusively ABO identical plasma and platelet (PLT) transfusions is not uniformly practiced nor always feasible despite reports of hemolytic reactions. Since PLTs and soluble plasma proteins possess A and B antigens, ABO non-identical PLTs could, theoretically, be activated and/or rendered hypofunctional by anti-A and anti-B antibodies (Abs) in transfused or recipient plasma. Recent findings demonstrate that transfusion of ABO non-identical PLTs is associated with increased bleeding in surgical patients and patients with leukemia. Blunt trauma patients who received at least one ABO non-identical blood product transfusion demonstrated a significantly higher RBC usage (12.3 ± 6.9 SD versus 8.4 ± 9.9 SD, p-value 0.0011) compared to those patients who received only ABO identical transfusions (Transfusion. 2007;47:192A). ABO identical PLT transfusions in leukemia patients were a significant predictor of survival (Leukemia. 2008;22:631-5). In a multi center retrospective analysis of more than one million cancer patients over a period of 9 years, Khorana et al. demonstrated an overall venous thromboembolism (VTE) rate of 4.1%. In multivariate risk factor analysis, the association between blood transfusions and VTE had an odds ratio of 1.35 (1.31-1.39, 95% CI) with a p value of < 0.001 (Arch Intern Med. 2008;168:2377-81). We hypothesized that PLTs activated by ABO Abs might have altered function. Methods and Materials: PLT function was evaluated by testing aggregation in platelet rich plasma (PRP). Aggregation was performed with PRP from 7 type A and 6 type B normal blood donors following a 10 min incubation period at 37°C with either normal saline, group O or AB plasma. PLTs were activated by 20 mM ADP and aggregation quantitated from the maximum change in OD. Similar experiments were repeated utilizing different titration of the commercial anti-A and anti-B anti-sera. Results: Following incubation with O plasma, PLT aggregation was inhibited by a mean of 38% and 18% for group A and B PLTs, respectively (P ≤ 0.005) (Figure). A trend toward inhibition was observed when type A PLTs were incubated with control AB plasma (average of 14%, P = 0.187), whereas type B PLT showed no inhibition when incubated with AB plasma (P = 0.939) (Table 1). PLT aggregation with the anti-sera showed gradual inhibition correlated with the antibody titer (Table 2). Conclusion: Mediators in group O plasma, most likely anti-A and anti-B Abs, cause impaired PLT aggregation in ABO non-identical PLTs. These in vitro findings may explain, at least in part, clinical observations that patients receiving ABO non-identical PLT transfusions experience more bleeding than recipients of ABO identical PLT transfusions. Table 1: PLT aggregation of A and B PRP with saline, O and AB plasma. Blood Donor Type N Average Percentage of Platelet Aggregation (SD) Normal Saline “O” Plasma P value* “AB” Plasma P value A 7 92 (7.4) 54 (9.9) < 0.005 78 (2.9) 0.187 B 6 85 (6.8) 67 (9.8) 0.005 85.3 (7.9) 0.939 P value < 0.05 is considered statistically significant. Figure: PLT function of type A PRP incubated for 10 min at 37°C with O or AB plasma, or normal saline. Figure:. PLT function of type A PRP incubated for 10 min at 37°C with O or AB plasma, or normal saline. Table 2: PLT aggregation of A and B PRP with different titration of the commercial anti-A and anti-B anti-sera. Anti-sera/Plasma Type A PRP P value Type B PRP P value Baseline 93.7 (3.1) — 83.4 (11) — 1:1024 48.7 (8.5) 0.006 36.3 (7.8) 0.0005 1:512 57.3 (2.5) 0.0001 47.7 (7.5) 0.002 1:256 59.5 (3.5) 0.008 59.5 (0.7) 0.002 1:128 55.5 (3.5) 0.006 67 (2.8) 0.027 AB plasma 87.7 (3.2) 0.08 81.2 (16) 0.88 Disclosures: No relevant conflicts of interest to declare.

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The Rho GTPase Rac1 Is Not Required for Definitive Hematopoietic Specification in ES-Derived Hematopoiesis.

Blood ◽

10.1182/blood.v114.22.1494.1494 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1494-1494

Author(s):

Michael D. Milsom ◽

Akiko Yabuuchi ◽

George Q. Daley ◽

David A. Williams

Keyword(s):

Rho Gtpase ◽

De Novo ◽

Conflicts Of Interest ◽

Embryoid Body ◽

Es Cells ◽

Yolk Sac ◽

Embryoid Bodies ◽

Hematopoietic Progenitors ◽

The Impact

Abstract Abstract 1494 Poster Board I-517 Rac1 is a Rho GTPase involved in integrating signaling pathways that regulate numerous cellular processes including adhesion, migration, proliferation and HSC engraftment. Homozygous deletion of Rac1 is lethal in the murine embryo prior to E9.5 and Rac1−/− embryos demonstrate defective gastrulation associated with reduced epiblast adhesion and motility. We have recently demonstrated using lineage-specific conditional deletion that Rac1 insufficiency results in severely impaired hematopoiesis in the embryonic sites of hematopoiesis (AGM, aortic clusters and fetal liver) in the setting of normal hematopoietic development in the yolk sac (YS) and reduced HSC and progenitors in the fetal circulation. This data appears to support the controversial hypothesis that YS derived HSC seed embryonic sites, but an alternative explanation is that Rac1 is essential for some aspect of the induction of intraembryonic hematopoiesis in situ. Another possibility is that Vav1-Cre-mediated excision of Rac1 occurs prior to the onset of hematopoiesis in the embryo proper but not early enough to affect yolk sac hematopoiesis. To test whether Rac1 insufficiency perturbs the normal early differentiation of hematopoietic cells in vitro, we used a lentivirus expressing a Rac1-specific shRNA to knock down expression in an ES line previously characterized to have good hemogenic potential. We observed that the de novo knockdown of Rac1 expression appeared to have no impact upon derivation of hematopoietic progenitors. To demonstrate that this was not the result of inefficient knockdown of Rac1, we derived Rac1−/− ES lines from blastomeres resulting from the mating of Rac1+/− mice. Rac1−/− ES lines were produced in normal Mendelian ratios (4 Rac1+/+: 9 Rac1+/−: 3 Rac1−/−) and did not demonstrate any evidence of abnormal expansion on murine embryonic fibroblasts. In order to assess the impact of Rac1 deficiency on the hemogenic potential of ES cells, standard in vitro differentiation via embryoid body formation was utilized. Neither Rac1 haploinsufficiency nor complete absence of Rac1 had any impact on the production of CD41+/c-Kit+ hematopoietic progenitors within embryoid bodies (Table 1). Furthermore, colony forming assays demonstrated that Rac1 insufficiency did not alter the relative frequency of hematopoietic progenitor compartments (Table 2). We conclude that in the absence of a requirement for vascular migration of HSC, Rac1 is not required for the specification of definitive hematopoiesis. These data, together with our previously published in vivo data continue to support the hypothesis that HSC migration from the YS to the embryo may be required for development of hematopoiesis in the embryo proper. Disclosures: No relevant conflicts of interest to declare.

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Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.1462.1462 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1462-1462

Author(s):

Michael Tallack ◽

Thomas Whitington ◽

Brooke Gardiner ◽

Eleanor Wainwright ◽

Janelle Keys ◽

...

Keyword(s):

Regulatory Networks ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Es Cells ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Gene Promoters ◽

Erythroid Cells ◽

Red Cell Membrane ◽

Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

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Specific Hematopoietic and Erythroid Differentiation Defects in Mouse Embryonic Stem (ES) Cells with Abortive Ribosome Assembly.

Blood ◽

10.1182/blood.v114.22.1088.1088 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1088-1088

Author(s):

Tracie A. Goldberg ◽

Adrianna Henson ◽

Sharon Singh ◽

Abdallah Nihrane ◽

Jeffrey Michael Lipton ◽

...

Keyword(s):

Cell Lines ◽

Colony Formation ◽

Conflicts Of Interest ◽

Es Cells ◽

Bone Marrow Failure ◽

Embryonic Stem ◽

Gene Trap ◽

Embryoid Bodies ◽

Ribosome Assembly ◽

Trap Cell

Abstract Abstract 1088 Poster Board I-110 Background Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes, characterized by erythroid hypoplasia, congenital anomalies and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, RPL35a), which somehow triggers apoptosis of erythroid precursors. There is a marked variation in phenotype among members of the same family and also between subsets of patients with different mutations. Methods We studied primary and secondary in vitro differentiation of two murine ES gene trap cell lines with mutations in Rps19: S17-10H1, in which Rps19 is disrupted by insertion of the ROSAFARY gene trap vector between exons 2 and 3; and YHC074, in which the pGT0Lxf gene trap vector is inserted between exons 3 and 4 and whose growth is feeder cell-independent. For primary differentiation and generation of embryoid bodies (EBs), the ES cells were cultured in a serum-supplemented methylcellulose-based medium containing stem cell factor (SCF). After 7 days, the cultures were fed with a medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 13 for hematopoietic percentage. Secondary (hematopoietic) differentiation was performed on day 9 EBs. EBs were harvested and disrupted with collagenase, and the disrupted cells were suspended in a serum-supplemented methylcellulose-based medium with SCF, IL-3, IL-6 and epo. Hematopoietic colonies were counted on day 10. Results Decreased expression of Rps19 protein was confirmed by Western blot analysis in both S17-10H1 and YHC074 gene trap cell lines. We focused on YHC074 because its growth is feeder-independent, and it expresses approximately 50% of normal Rps19 levels. By polysome analysis, we found a selective reduction in the 40S subunit peak in mutant YHC074 cells as compared to parental controls. By Northern blot assays, we also found a relative increase in the 21S pre-rRNA to 18S rRNA ratio in mutant YHC074 cells. The viability of undifferentiated ES cells was not significantly different from parental control cells in the first 72 hours of culture; however, there was a significantly decreased number of EBs, particularly hematopoietic EBs, following primary differentiation (Fig. 1). Furthermore, when day 9 EBs were induced to secondary (hematopoietic) differentation, there was a significant decrease in the ratio of erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation in mutant YHC074 cells. In order to confirm these results in an isogenic background, we stably transfected S17-10H1 cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones were found to overexpress Rps19 and were further studied in secondary differentiation experiments. There was a significant decrease in erythroid and myeloid colony formation and in BFU-E size from mutant S17-10H1 cells when compared to the Rps19-overexpressing clone, suggesting a direct relationship between the levels of Rps19 protein and hematopoietic growth and differentiation. Conclusion Using two ES cell lines with slightly different Rps19 mutations and genetic backgrounds, we have recapitulated the major DBA erythroid growth and differentiation defect, as well as the defect in ribosome assembly and rRNA processing caused by Rps19 haploinsufficiency. Disclosures No relevant conflicts of interest to declare.

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Increased Thrombin Generation in Severe Hemophiliacs with Mild Clinical Phenotype

Blood ◽

10.1182/blood.v112.11.515.515 ◽

2008 ◽

Vol 112 (11) ◽

pp. 515-515

Author(s):

Elena Santagostino ◽

Maria Elisa Mancuso ◽

Armando Tripodi ◽

Veena Chantarangkul ◽

Gianluigi Pasta ◽

...

Keyword(s):

Thrombin Generation ◽

Clinical Phenotype ◽

Platelet Rich Plasma ◽

Gene Mutations ◽

Clinical Score ◽

Bleeding Tendency ◽

Spontaneous Bleeding ◽

Chromogenic Assay ◽

Bleeding Episodes ◽

The Impact

Abstract Introduction: Some severe hemophiliacs (FVIII/FIX<1%) exhibit a mild bleeding tendency, but the basis for this clinical heterogeneity is poorly understood. This study investigated the relationship between the values of endogenous thrombin potential (ETP) and clinical phenotype in severe hemophiliacs. The impact of FVIII/FIX gene mutations and thrombophilic polymorphisms was also evaluated. Methods: severe hemophiliacs older than 18 years without inhibitor history and treated on demand were eligible. Mild bleeders (MB) and severe bleeders (SB) were defined as follows: spontaneous bleeding episodes per year ≤2 (MB) or 25 (SB) and concentrate consumption <500 (MB) or >2000 (SB) IU/Kg/year. Patients who did not fit these criteria were considered as intermediate bleeders (IB). FVIII was measured by chromogenic assay and ETP was measured in platelet-rich plasma after addition of tissue factor. Results: 22MB, 22SB and 28IB were enrolled. MB had lower clinical and radiological scores when compared with both IB and SB (p<0.005). MB showed an older age at first bleed compared to SB (p < 0.005) and p for trend among the 3 groups was also significant (p < 0.05). The prevalence of severe FVIII/FIX gene defects (null mutations) was lower and ETP values were higher in MB compared with both IB and SB (p<0.05; table 1). Conclusions: our results indicate an extremely low prevalence of null mutations in severe hemophiliacs with mild bleeding diathesis. The measurement of thrombin generation in platelet-rich plasma may allow to identify this subgroup of patients, not otherwise distinguishable by conventional functional assays. SB (#22) IB (#28) MB (#22) p Age (yr) 38 (21–76) 38 (23–62) 32 (22–73) NS Age 1st bleed (yr) 1 (0–4) 2 (0–6) 3 (1–10) < 0.005 Bleeding episodes/yr 36 (25–60) 10 (3–20) 0 (0–2) < 0.0005 Factor use (IU/Kg/yr) 2207 (2040–8696) 1068 (207–2400) 60 (25–487) < 0.0005 Clinical score 18 (10–35) 10 (0–34) 3 (0–17) < 0.005 Pettersson score 44 (14–62) 28 (0–48) 17 (3–40) < 0.0005 Null mutations (%) 59 70 6 < 0.005 PTG20210A (%) 0 7 5 NS FV Leiden (%) 5 7 0 NS Median ETP (nM) 414 478 850 < 0.05

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Fucosyltransferase VI Induces Platelet Activation: A Novel Property of a Plasma Glycosyltransferase.

Blood ◽

10.1182/blood.v114.22.4016.4016 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4016-4016

Author(s):

José-Tomás Navarro ◽

Shwan Tawfiq ◽

Roland Wohlgemuth ◽

Karin M. Hoffmeister ◽

Robert Sackstein

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Light Transmission ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Surface Protein ◽

Surface Flow ◽

Platelet Surface ◽

Biochemical Studies

Abstract Abstract 4016 Poster Board III-952 A number of glycosyltransferases are present in human plasma with the α(1→3) fucosyltransferase, Fucosyltransferase VI (FTVI), having the highest plasma concentration. Notably, elevated plasma levels of FTVI are associated with a variety of cancers and correlate with tumor load/progression. The well-known association of neoplasia with thromboembolic complications prompted us to examine whether FTVI has direct effect(s) on platelet function. We obtained human platelets from blood of healthy donors and separated from platelet-rich plasma by differential centrifugation. Freshly isolated platelets (x108/ml) were stirred and exposed at 37°C to varying concentrations (20, 40, 60 and 80 mU/mL) of glycosyltransferases FTVI, β-1-4-galactosyltransferase-I (βGalT-I), or α,2-3-N-sialyltransferase (α2,3-N-ST), or to 1 U/mL thrombin. Platelet aggregation and activation was assessed by aggregometry (light transmission) or by flow cytometry of FSC/SSC characteristics and of surface expression of P-Selectin, respectively. FT-VI reproducibly induced platelet aggregation and activation, whereas other glycosyltransferases (β4GalT-I and α2,3-N-ST) had no effect on platelets. FTVI activation of platelets was concentration-dependent, and the aggregation curve for FTVI was one wave, similar to that for thrombin. FTVI-induced platelet activation was independent of catalytic conversion of surface glycans, but was inhibited by FTVI denaturation, indicating that FTVI-induced platelet activation is a lectin-mediated process. To determine the membrane target(s) mediating FTVI-induced platelet activation, biochemical studies were performed after catalytic exofucosylation of the platelet surface. Flow cytometry after platelet exofucosylation showed formation of the carbohydrate structure sLex, detected by the mAb Heca452, but no formation of Lex (CD15). Western blot showed that enforced fucosylation induced sLex on a single platelet surface protein, and further biochemical studies revealed that this protein is GPIbα. These findings unveil a previously unrecognized property of FTVI as an activator of platelets, mediated via a specific lectin/carbohydrate interaction on GP1ba, and offer novel perspectives on the pathobiology of tumor-associated thrombogenesis. Disclosures: No relevant conflicts of interest to declare.

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Regulation of Gata1 Expression by HS+3.5

Blood ◽

10.1182/blood.v116.21.3864.3864 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3864-3864

Author(s):

Julia E Draper ◽

William G Wood ◽

Catherine Porcher ◽

Paresh Vyas

Keyword(s):

Bone Marrow ◽

Colony Formation ◽

Transgene Expression ◽

Conflicts Of Interest ◽

Es Cells ◽

Embryonic Stem ◽

Regulatory Elements ◽

Haematopoietic Stem Cell ◽

Wild Type ◽

Foetal Liver

Abstract Abstract 3864 Precise regulation of Gata1 expression is required in order to control the balance between lymphoid/granulomonocytic (GM) and megakaryocytic-erythroid (MegE) specification, as well as to ensure correct differentiation of the MegE lineages. Transcriptional control is conferred in part by cis regulatory elements. An upstream enhancer, HS-3.5, and the erythroid first exon IE of Gata1 are necessary and sufficient to direct transgene expression in primitive but not definitive erythroid cells. Transgene expression in definitive red blood cells is restored by inclusion of an intronic DNaseI hypersensitive site, HS+3.5. Here we report the characterization of the HS+3.5 null embryonic stem cells and the HS+3.5 knockout mouse. In vitro differentiation of HS+3.5 null ES cells resulted in reduced myeloid and megakaryocytic colony formation compared to wild type. The ΔHS+3.5 ES cells retained normal primitive erythroid colony formation. ΔHS+3.5 definitive erythroid colony progenitors displayed a decreased sensitivity to Interleukin 3 (IL3) signalling compared to wild type. ΔHS+3.5 mice were viable and had normal blood counts and films. GM and erythroid progenitors also developed normally. However, there was a mild expansion of the E14.5 foetal liver Megakaryocytic Progenitor (MkP) compartment and an increase in Gata2 expression in both the bone marrow and foetal liver MkPs. Turning to Gata1, a decrease in Gata1 expression was observed in the following compartments: the bone marrow long term haematopoietic stem cell (LT-HSC) and the foetal liver common myeloid progenitor (CMP). The relationship between the effect of the HS+3.5 deletion on Gata1 expression and the haematopoietic phenotype will be discussed. Disclosures: No relevant conflicts of interest to declare.

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