ALBERT

Description: New mechanisms behind blood cell formation continue to be uncovered, with therapeutic approaches for hematological diseases being of great interest. Here we report an enzyme in protein synthesis, known for cell-based activities beyond translation, is a factor inducing megakaryocyte-biased hematopoiesis, most likely under stress conditions. We show an activated form of tyrosyl-tRNA synthetase (YRSACT), prepared either by rationally designed mutagenesis or alternative splicing, induces expansion of a previously unrecognized high-ploidy Sca-1+ megakaryocyte population capable of accelerating platelet replenishment after depletion. Moreover, YRSACT targets monocytic cells to induce secretion of transacting cytokines that enhance megakaryocyte expansion stimulating the Toll-like receptor/MyD88 pathway. Platelet replenishment by YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from induced pluripotent stem cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. We suggest megakaryocyte-biased hematopoiesis induced by YRSACT offers new approaches for treating thrombocytopenia, boosting yields from cell-culture production of platelet concentrates for transfusion, and bridging therapy for hematopoietic stem cell transplantation.

Description: Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody–induced ITP.

Description: Abstract 552 Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib/IX complex. The main treatment for BSS is platelet transfusion but it is often limited to severe bleeding episodes or surgical interventions due to the risk of developing allo-immunization. Ware and colleagues have developed a murine model of BSS by targeting GPIbα (GPIbαnull), and have shown that the BSS phenotype was rescued by transgenic expression of hGPIbα. We have previously reported successful expression of human GPIbα in human megakaryocytes using a lentiviral vector (LV) encoding hGPIbα under the transcriptional control of integrin aIIb promoter (2bIbα). In this study, we examined the efficacy of this strategy for the gene therapy of BSS using GPIbαnull as a murine model of BSS. GPIbαnull hematopoietic stem cells (HSC) transduced with 2bIbα LV were transplanted into lethally irradiated GPIbαnull littermates. After bone marrow (BM) reconstitution, mice were analyzed. The presence of 2bIbα transgene in recipients was confirmed by PCR amplification of white blood cell derived genomic DNA. Flow cytometry demonstrated that 84.5% ± 9.5% (n = 9) of platelets expressed hGPIbα at 6 weeks after transplantation and stable expression was maintained through the entire observation period of 7 months. Immunofluorescent confocal microscopy demonstrated that transgene protein was expressed on the cell surface of transduced platelets. Tail bleeding times were corrected to normal levels in the GPIbαnull recipients who received LV transduced GPIbαnull HSC (2.3 ± 2.9 minutes, n = 9). On the other hand, recipients who received untransduced GPIbαnull HSC exhibited prolonged bleeding times (8.8 ± 2.4 minutes, n = 4) that were similar to GPIbαnull mice. Macrothrombocytopenia improved with significantly increased platelet counts and decreased platelet sizes in LV transduced GPIbαnull HSC recipients compared to untransduced GPIbαnull HSC recipients (platelet counts; 4.9 ± 1.3×105/μ l, n = 9 vs. 1.8 ± 0.1×105/μ l, n = 4, and mean platelet volume; 6.9 ± 0.7 fL, n = 9 vs. 9.3 ± 0.1 fL, n = 4, respectively). As expected, expression levels of hGPIbα correlated with platelet counts and inversely correlated with the platelet size. Immunoprecipitation followed by Western blot analysis showed that hGPIbα expressed on platelets associated with endogenous murine GPIbβ and GPIX. No antibody to hGPIbα was detected in these recipients. Furthermore, BM mononuclear cells from the primary recipients were transplanted into the secondary GPIbαnull recipients and the results showed sustained expression of hGPIbα leading to the correction of bleeding phenotype as well as macrothrombocytopenia. These results demonstrate that lentivirus mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a promising strategy for gene therapy of BSS in human. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2022 Introduction: Sitosterolemia is a rare, autosomal recessive disorder characterized by the accumulation of plant sterols in blood and tissues, and is caused by mutations in one of the adenosine triphosphate-binding cassette (ABC) transporter ABCG5 or ABCG8 genes. Patients with mutations in either of these sterol transport proteins, which normally form a heterodimer sterol egress channel, frequently develop tendon and cutaneous xanthomas and, most importantly, are at risk of developing premature coronary atherosclerosis. Other clinical manifestations include hematological abnormalities such as hemolytic anemia, macrothrombocytopenia, and loss of ristocetin-induced platelet agglutination – a measure of the ability of platelet glycoprotein (GP) Ib to function as a adhesion receptor for von Willebrand factor (VWF). Mice genetically deficient in ABCG5 or ABCG8 fully recapitulate the macrothrombocytopenia and loss of platelet function seen in human sitosterolemia, a condition that can be corrected by treatment with the sterol-absorption inhibitor, ezetimibe. Because the mechanism by which accumulated plant sterols affects platelet size, production, and function is incompletely understood, we further analyzed these traits in Abcg5- and Abcg8-deficient mice in an animal model of sitosterolemia. Methods: Blood was collected every 1–2 weeks and CBC monitored in Abcg5- and Abcg8-deficient mice that had been fed either a high or low plant sterol diet. Expression of platelet receptors was analyzed by both flow cytometry and Western blotting. GPIbα null mice were used as controls since they have similarly enlarged platelets. Platelet microparticles were analyzed by labeling with a mAb specific for GPIIb and Annexin V. Plasma samples were analyzed for von Willebrand factor (VWF) antigen and multimer patterns. Results: Following onset of a high sterol diet, the platelet count decreased over a two week period to less than 30% of that of normal controls in both Abcg5-/- and Abcg8-/- mice. At the same time, the mean platelet volume gradually increased over a 4 week period from a normal value of approximately 7 fl to approximately 10 fl. In addition, an increased number of platelet-derived microparticles were detected in Abcg5-/- and Abcg8-/- mice kept on high sterol diet over a 9 week period, suggesting that low-grade platelet activation was occurring in these mice. Unexpectedly, the surface expression of the GPIIb integrin subunit was decreased in both Abcg5-/- and Abcg8-/- mouse platelets compared to that of similarly large platelets derived from GPIbα-null (Bernard-Soulier) platelets. Intracellular staining revealed that the GPIIb in Abcg8-/- platelets was being internalized and retained within the cell. Both surface-bound and total platelet fibrinogen was increased in Abcg5-/- and Abcg8-/- mice fed a high sterol diet. Finally, plasma VWF antigen levels decreased by approximately 50% within two weeks following switching Abcg5-/- mice from a low-fat to a high-fat diet. Multimer analysis showed loss of high molecular weight multimers in some of the plasma samples. Conclusions: This study demonstrates a heightened state of platelet activation in a murine model of sitosterolemia, leading to VWF and fibrinogen binding, with concomitant internalization of GPIIb. This might explain, at least in part, the cause of macrothorombocytopenia and absence of ristocetin-induced platelet aggregation in sitosterolemia patients. Platelet activation may also lead to calpain activation, resulting filamin A and talin degradation and microparticle production. Future studies aims at identifying the mechanism by which plant sterols or their metabolites elicit these effects on platelets may provide important clues to the ability of dietary lipids to affect platelet activation and the development of atherosclerosis. Disclosures: Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.

Description: Aminoacyl-tRNA synthetases (aaRSs) are enzymes with a key role in the first step of protein synthesis by catalyzing the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. During evolution, eukaryotic aaRSs have acquired additional domains and motifs conferring non-canonical functions beyond translation, such as expressing multiple cytokine activities. Repurposing aaRSs often requires an activation step and the first reported example was for human tyrosyl-tRNA synthetase (YRS), which is abundant in platelets and released from their α-granules upon thrombin or arachidonic acid stimulation. As shown by previous work, activated YRS (YRSACT) - created by natural proteolysis, alternative splicing, or rational mutagenesis - can express the activity of different cytokines. In the current study, we demonstrate that recombinant YRSACT rendered active by the gain-of-function mutation Tyr341Ala exhibits a previously unrecognized role in megakaryocytopoiesis and thrombocytopoiesis. When administered in vivo in C57BL/6 wild type (WT) mice, recombinant YRSACT caused platelet increase both under baseline conditions as well as in a model of immune-mediated thrombocytopenia in which mice are made thrombocytopenic by injection of rat anti-mouse glycoprotein (GP) Ib monoclonal IgG. When WT mouse bone marrow (BM) cells were cultured ex vivo for 3 days, YRSACT treatment increased the number of megakaryocytes by 3.0-fold, particularly of megakaryocytes with 16N ploidy. This effect was independent of thrombopoietin (TPO) signaling because YRSACT could support the expansion of c-mpl-/- (TPO receptor knock-out) mouse megakaryocytes. YRSACT had no effect on purified mouse CD41+ or Sca1+ hematopoietic progenitor cells, indicating that YRS-dependent stimulation likely required the contribution of other cells present in BM cultures. When mouse BM cells were stimulated with different doses of YRSACT, the number of F4/80+ monocyte/macrophages as well as of megakaryocytes increased in a dose-dependent manner. Mechanistic analysis revealed YRSACT targets the Toll-like receptor (TLR) pathway signaling through MyD88 in monocyte/macrophages, thereby enhancing release of cytokines that influence megakaryocyte development. In vitro binding assay showed that YRSACT is capable of binding to TLR2 and TLR4. The effect of YRSACT was attenuated in the BM cells derived from TLR2-/- mice and was abolished in MyD88-/- mice. Among the cytokines with synthesis induced by YRSACT, IL-6 plays a pivotal role in megakaryocyte development. Thus, we tested the effect of YRSACT on megakaryocytes obtained by culturing BM cell derived from IL-6-/- mice and found that no effect was apparent. The stimulatory effect of YRSACT on megakaryocytopoiesis was confirmed with human CD41+ megakaryocyte progenitors differentiated from CD34+ hematopoietic stem cells derived from peripheral blood. In conclusion, we have documented a previously unrecognized activity of YRSACT that results in enhanced megakaryocytopoiesis and platelet production. These studies document a mechanistically distinct aaRS-directed hematological activity that highlights new potential approaches to stimulating platelet production for treating thrombocytopenia and for improving ex vivo preparation of platelet concentrates for transfusion. Disclosures Belani: aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties. Do:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Yang:aTyr Pharma: Consultancy, Patents & Royalties, Research Funding. Schimmel:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Description: Key Points Plant sterol accumulation in platelet membrane induces platelet hyperreactivity. Internalization of the αIIbβ3 complex and filamin A degradation cause macrothrombocytopenia and bleeding phenotype.

Description: Abstract 3340 Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP)Ib/IX complex. Platelet transfusion is the primary treatment for hemorrhage but is often limited because of the potential for provoking allo-immunization and refractoriness. We have previously shown that the macrothrombocytopenia and prolonged bleeding of murine BSS (GPIbαnull) can be corrected by myeloablative total body irradiation (TBI) and transplantation of hematopoietic stem cells (HSCs) transduced with a lentivirus that expresses hGPIbα under integrin αIIb promoter control (2bIbαLV). For application of this gene therapy approach to the treatment of human patients, it is important to minimize treatment-related adverse effects. In this study, we established a transgenic mouse that expresses hGPIbα at levels similar to the average levels observed in 2bIbαLV transduced HSC recipients that can be used as a consistent source of bone marrow HSCs (hGPIbαtg+). We undertook two approaches for evaluation of HSC transplantation using hGPIbαtg+ mouse bone marrow (BM) cells; 1) determine the percentage of hGPIbαtg+ HSCs necessary for therapeutic benefit and 2) determine if non-myeloablative strategies can achieve hGPIbα expression levels sufficient to correct the bleeding phenotype of GPIbαnullmice. In order to determine the percentage of HSCs required to achieve therapeutic effects, mixed BM chimeras were generated by reconstituting lethally irradiated GPIbαnull mice with variable mixtures (5% - 100%) of BM cells isolated from hGPIbαtg+ and GPIbαnull mice. Tail bleeding time assays performed after BM reconstitution demonstrated that 10% hGPIbαtg+ BM mononuclear cells mixed with 90% GPIbαnull BM mononuclear cells were sufficient to correct the tail bleeding time (n=5). Platelet analysis showed that the bleeding phenotype was rescued in recipients having higher than 40 × 103/μl of hGPIbαtg+ platelets, which corresponds to 6.5% of wild type mouse platelet counts (630 ± 57 × 103/μl, n = 8). These results suggest therapeutic potential for non-myeloablative conditioning regimens for the treatment of murine BSS. Thus we next tested the transplantation of hGPIbαtg+ mouse BM cells into GPIbαnull recipients conditioned with two non-myeloablative doses of busulfan (25 mg/kg on days -2 and -1). Analysis of platelets by flow cytometry showed that, as expected, the percentage of hGPIbα-positive platelets in busulfan-conditioned recipients was lower than those transplanted after lethal TBI conditioning (63.2% ±36.6%, n = 15 vs. 95.2% ±1.7%, n = 8). However, tail bleeding time assays showed that bleeding times of busulfan-conditioned recipients were corrected and not significantly different from lethally irradiated recipients (2.7 ± 3.1 minutes, n = 15 vs 1.9 ± 1.5 minutes, n = 8), while no untreated GPIbαnull mice (n=8) stopped bleeding within 10 minutes. Platelet counts were also significantly increased in busulfan-conditioned recipients compared to untreated GPIbαnull mice (381 ± 132 × 103/μl, n = 11 vs. 181 ± 35 × 103/μl, n = 11). Thus non-myeloablative doses of busulfan conditioning and transplantation of hGPIbαtg+mouse BM cells were shown to achieve relevant levels of engraftment in murine BSS with significantly corrected bleeding times and platelet counts. Antibody response to hGPIbα and immune-mediated thrombocytopenia was documented in 3 of 15 recipient mice, suggesting immunogenicity of hGPIbα transgene protein in GPIbαnullmice partially immunosuppressed with busulfan. However, immunotolerance was identified without treatment and antibody disappeared in all three mice 3 to 7 months after BM transplantation. In conclusion, non-myeloablative doses of busulfan conditioning may be a reasonable alternative to TBI for the BMT-based treatment of BSS and could be utilized in non-myeloablative autologous gene therapy in human BSS. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: It has been shown that megakaryocytes (MKs) can develop from a subset of MK-biased hematopoietic stem cells (HSCs). We also demonstrated that an active form of tyrosyl tRNA synthetase (YRS) has ex-translational activities that stimulate rapid platelet production in thrombocytopenic mice. Remarkably, YRS stimulates the development of MKs expressing the stem cell marker, Sca-1, and the monocyte/macrophage marker, F4/80. Thus, we hypothesized that YRS treatment mimics stress-induced megakaryopoiesis and Sca-1 may be a marker for MKs/platelets derived from MK-biased HSCs under inflammatory stress conditions. Accordingly, YRS does not induce Sca-1+ MKs in type I interferon (IFN-I) receptor knockout mice, suggesting a role of IFNs in the induction of Sca1+ MKs. Methods: We used a transgenic mouse strain expressing EGFP under the transcriptional regulatory elements of the Sca-1 gene (Sca-1-EGFP Tg) as a sensitive marker of Sca-1+ MKs. To compare transcriptional profiles, we sorted from mouse bone marrow (BM) EGFP+F4/80+CD41+mGPIbα+ and EGFP-F4/80-CD41+mGPIbα+ cells - representing Sca-1+ MKs and Sca-1- (conventional) MKs, respectively - and performed RNA-Seq analysis. To investigate the mechanism of Sca-1+ MK induction, mouse BM cells were treated with a synthetic TLR7 agonist and MKs were analyzed by flow cytometry. To elucidate the role of Sca-1+ MKs in thrombopoiesis, we infected Sca-1-EGFP Tg mice with lymphocytic choriomeningitis virus (LCMV) Armstrong strain, and analyzed the percentage of EGFP+ platelets and expression of IFN-stimulated genes. Results: We found that expression of MK-specific mRNAs is ~10-fold higher in Sca1+ than Sca1- MKs. As shown in Figure 1A, Sca1+ MKs highly expressed myeloid-related genes (Mpo, Elane, and Ctsg) and stem cell-related genes (Cd34 and Kit); in contrast, Sca1- MKs highly expressed erythroid lineage genes, consistent with origin from a common megakaryocyte-erythroid progenitor (MEP). Expression of IFN-stimulated genes, such as Ifitm1/2/3, is upregulated in Sca-1+ MKs, suggesting the involvement of IFN-I signaling in Sca-1+ MK induction. TLR7 typically recognizes single-stranded viral RNA and stimulates IFN-I production. When Sca-1-EGFP Tg BM cells were cultured in the presence of Gardiquimoid (1 µg/ml), a synthetic TLR7 agonist, Sca1- MKs were reduced and Sca1+ MKs were markedly increased compared to control vehicle treatment (9442 ± 1465 vs 4201 ± 1100). Notably, the proportion of high-ploidy cells was greater in Sca1+ thanSca1- MKs. Moreover, when Sca-1-EGFP Tg mice were infected with LCMV, which cause IFN-I-dependent thrombocytopenia, the percentage of EGFP+ platelets was markedly increased on day 3 (40.0 ± 3.7%) and peaked at day 7 (89.2 ± 5.4%) post-infection as compared to before (3.0 ± 1.0%) (Figure 1B), with 100-fold increase in the EGFP mean fluorescence intensity. Expression of Ifitm3 in platelets was also increased at day 10 post-infection. These results are consistent with our original hypothesis that YRS mimics inflammatory stress conditions and Sca1+ MKs are induced as a consequence of TLRs activation and IFN-I signaling. Conclusions: Our findings indicate that TLR7 activation and IFN-I signaling shift MK generation from Sca-1- to Sca-1+ progenitors, which rapidly develop to high-ploidy Sca-1+ MKs that may accelerate recovery from thrombocytopenia. Accordingly, under inflammatory stress conditions, such as viral infection, the majority of circulating platelets originate was from Sca-1+ MKs. The high expression of Ifitm proteins, which are known to inhibit cellular entry by viruses, in Sca-1+ MKs/platelets may contribute to their role in host defense. Disclosures Aiolfi: MERU-VasImmune, Inc: Other: Stock option. Kanaji:MERU-VasImmune, Inc: Other: Stock option. Schimmel:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder. Kanaji:MERU-VasImmune, Inc: Other: Stock option.

Description: Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collagen-induced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VInull platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VInull or FcR-γnull animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VInull platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils.