Abstract: The haematopoietic system manifests several age-associated phenotypes including anaemia; loss of regenerative capacity, especially in the face of insults such as infection, chemotherapy or blood loss; and increased risk of clonal haematopoiesis and blood cancers. The cellular alterations that underpin these age-related phenotypes, which typically manifest in individuals aged over 70, remain elusive. We aimed to investigate whether changes in HSC population structure with age might underlie any aspects of haematopoietic system ageing. We sequenced 3579 genomes from single-cell-derived colonies of haematopoietic stem cell/multipotent progenitors (HSC/MPPs) from 10 haematologically normal subjects aged 0-81 years. HSC/MPPs accumulated 17 somatic mutations/year after birth with no increased rate of mutation accumulation in the elderly. HSC/MPP telomere length declined by 30 bp/yr. In cord blood and adults aged & lt;65, a small proportion of HSC/MPPs had unexpectedly long telomeres, as assessed using several criteria for outliers. The proportion of cells with unexpectedly long telomeres reduced in frequency with age. Given that telomeres shorten at cell division, these outlier cells have presumably undergone fewer historic cell divisions, supporting the existence of a rare population of dormant HSCs in humans that declines in frequency with age. To interrogate changes in HSC population structure with age, we used the pattern of unique and shared mutations between the sampled cells from each individual to reconstruct their phylogenetic relationships. The frequency of branch-points (known as coalescences) in phylogenetic trees in a neutrally evolving, well-mixed population of somatic cells is primarily determined by the product of population size and time between symmetric self-renewal cell divisions (Nt). Smaller populations and more frequent symmetric divisions both increase the density of coalescences. Specific clones can come to dominate either through neutral drift or positive selection. We found that haematopoiesis in adults aged & lt;65 was polyclonal, with high indices of clonal diversity. The number and pattern of coalescent events in the phylogenies showed that a stable population of 20,000 to 200,000 HSC/MPPs was contributing evenly to blood production in young adult life. In contrast, haematopoiesis in individuals aged & gt;75 showed profoundly decreased clonal diversity. In each elderly subject, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before age 40, but only 22% had known driver mutations. We used the ratio of non-synonymous to synonymous mutations (dN/dS) to identify any excess of non-synonymous (driver) mutations in the dataset. This genome-wide selection analysis estimated that 1/34 to 1/12 non-synonymous mutations were drivers, occurring at a constant rate throughout life, such that the set of 300 - 400 HSC/MPPs sampled from each adult individual harboured around 100 driver mutations, over 10-fold higher than the number of known drivers we could identify. Novel drivers affected a wider pool of genes than identified in blood cancers. The genes DNMT3A, ZNF318 and HIST2H3D were identified as being under significant positive selection in HSC/MPPs, despite ZNF318 and HIST2H3D not being enriched in the setting of myeloid malignancies. Loss of Y chromosome conferred selective benefits on HSC/MPPs in males. Simulations from a simple model of haematopoiesis, with constant HSC population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure observed in the elderly, which could not be explained by neutral models incorporating drift alone. Our data supports the view that dramatically decreased clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently known. By old age the majority of HSCs harbour at least one driver mutation. With such ubiquity of driver mutations, selected purely for their competitive advantage within the stem cell compartment, and with the wholesale rewiring of cellular pathways they induce, it is feasible that they may contribute to age-related phenotypes beyond the increased risk of blood cancer. Disclosures Spencer: Wugen, Inc.: Consultancy, Other: Stock Options. Vassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy. Kent: STRM.bio: Research Funding. Campbell: Mu Genomics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Abstract: Abstract 3074 Children with high-risk NB often receive using multiple treatment modalities including autoHCT. Disease remains the most common cause of treatment failure and the most recent cooperative group studies report survival rates approaching 45%. Prior reports showed comparable outcomes of patients with NB receiving auto- and allo-transplants, despite differences in relapse risk and treatment-related mortality (TRM). To update these data the CIBMTR conducted a retrospective review of 143 transplants reported 1990–2007 at 61centers, the largest group reported to date. For this analysis, patients were categorized into 2 groups for comparison: those without (Group 1; n=97) and those with a prior auto HCT (Group 2; n=46). Of the patients in Group 1, 31% were in first remission, 25% in partial response (PR) or very good PR (VGPR), and 24% with no response or with persistent/progressive disease. Of the patients Group 2, 17% were in first remission, 26% in PR or VGPR, and 28% with no response or with persistent/progressive disease. Median ages at alloHCT were similar for Group 1 (5y, range 〈 1 to 55) and Group 2 (7y, range 2–32), while the median time from diagnosis to alloHCT was shorter (9 mo) for Group 1 than Group 2 (27 mo). In Group 1, 57% received grafts from HLA-matched related donors, 27% from mismatched related donors and 16% from unrelated donors. In Group 2, 39% received grafts from matched related donors, 9% from mismatched related donors and 52% from unrelated donors. In Group 1, stem cell sources were marrow (71%), peripheral blood (10%) and cord blood (16%). In Group 2, stem cell sources were marrow (35%), peripheral blood (28%) and cord blood (37%). There was no difference in the prevalence of acute (p=.15) or chronic GVHD (p=.24) between the two groups. Median follow-up was 84 months (range, 〈 1 to 191) and 45 months (range 1 to 58) for those without and with prior autologous HCT. 1 year and 5 year OS were 59% and 29% for Group 1 and 50% and 7% for Group 2. Table 1 outlines the univariate analysis of some outcomes for the groups:Group 1: No prior AutoGroup 2: prior Autop-ValueTRM 100 days 1-year3% (1–9)2% (0–11)0.905825% (16–35)24% (12–37)0.8916Relapse 1-year 5-year27% (17–38)57% (41–70)0.001246% (34–58)70% (53–82)0.0123EFS 1-year 5-year48% (36–59)19% (9–32)0.000627% (17–38)6%A (1–17)0.0018OS 1-year 5-year59% (48–68)50% (35–64)0.343929% (20–39)7% (1–18)0.0005 Univariate analysis by donor-type also indicated higher relapse rates after unrelated donor transplants and lower relapse rates after HLA-mismatched related transplants compared with HLA-identical related donor transplants (p 〈 0.0001). EFS and survival were significantly lower for unrelated donor transplants 1 and 3 years but not 5 years post-HCT. Patients in 1st CR at transplant had lower relapse rates and better EFS and survival compared to those not in 1st CR. The presence of acute and/or chronic GVHD did not correlate with outcome in univariate analyses. The most common causes of death in Groups 1 vs. 2 were relapse (64%/75%), GVHD (5%/0%), infections (11%/5%), and organ toxicity (11%/13%). Our analysis indicates that alloHCT can cure some NB patients, with lower relapse rates and improved survival in patients without a history of autoHCT compared with those patients who had undergone auto HCT first. AlloHCT for patients after autoHCT does not seem to offer benefit. However, the reasons for Group 1 patients not receiving autoHCT as part of their upfront therapy are unclear, and this group could include patients who were curable without alloHCT. Disease recurrence remains the most common cause of treatment failure while TRM is low. Future studies comparing OS after autoHCT and alloHCT for patients in CR1 should be considered. Disclosures: No relevant conflicts of interest to declare.

Abstract: Although some trials have allowed matched or single human leukocyte antigen (HLA)–mismatched related donors (mmRDs) along with HLA-matched sibling donors (MSDs) for pediatric bone marrow transplantation in early-stage hematologic malignancies, whether mmRD grafts lead to similar outcomes is not known. We compared patients 〈 18 years old reported to the Center for International Blood and Marrow Transplant Research with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome undergoing allogeneic T-replete, myeloablative bone marrow transplantation between 1993 and 2006. In total, patients receiving bone marrow from 1208 MSDs, 266 8/8 allelic-matched unrelated donors (URDs), and 151 0-1 HLA-antigen mmRDs were studied. Multivariate analysis showed that recipients of MSD transplants had less transplantation-related mortality, acute graft-versus-host disease (GVHD), and chronic GVHD, along with better disease-free and overall survival than the URD and mmRD groups. No differences were observed in transplant-related mortality, acute and chronic GVHD, relapse, disease-free survival, or overall survival between the mmRD and URD groups. These data show that mmRD and 8/8 URD outcomes are similar, whereas MSD outcomes are superior to the other 2 sources. Whether allele level typing could identify mmRD recipients with better outcomes will not be known unless centers alter practice and type mmRD at the allele level.

Abstract: INTRODUCTION: Cancer pathogenesis is usually characterized by a long evolutionary process where genomic driver events accumulate over time, conferring advantage to distinct subclones, allowing their expansion and progression. METHODS: To investigate the multiple myeloma (MM) evolutionary history, we characterized the mutational processes' landscape and activity over time utilizing a large cohort of 89 whole genomes and 973 exomes. To improve the accuracy of mutational signatures analysis, we analyzed both the 3' and 5' nucleotide context of each mutation and we developed the novel fitting algorithm mmSig, which fits the entire mutational catalogue of each patient with the mutational signatures involved in MM pathogenesis. The contribution of each mutational signature was then corrected based on the cosine similarity between the original 96-mutational profile and the reconstructed profile generated without that signature. To reconstruct the genetic evolutionary history of each patient's cancer, we integrated two approaches. First dividing all mutations into clonal (early) or subclonal (late), then subdivided the clonal mutations into duplicated mutations (present on two alleles and therefore acquired before the duplication) or non-duplicated mutations (detected on a single allele), reflecting either pre-gain and post-gain mutations on the minor allele, or post-gain mutations acquired on one of the duplicated alleles. RESULTS Eight mutational signatures were identified, seven of which showed significant similarity with the most recent mutational signature catalogue (i.e SBS1, SBS2, SBS5, SBS8, SBS9, SBS13 and SBS18). The new mutational signature (named SBS-MM1) was observed only among relapsed patients exposed to alkylating agents (i.e melphalan). The etiology of this specific signature was further confirmed by analyzing recent whole genomes public data from human-induced pluripotent stem cells exposed to melphalan (Kucab et al, Cell 2019). Reconstructing the chronological activity of each mutational signature, we identified four different routes to acquire the full mutational spectrum in MM based on the differential temporal activity of AID (SBS9) and APOBEC (SBS2 and SBS13). Our data indicate that AID activity is not limited to the first contact with the GC, but persists in the majority of patients, behaving similarly to a B-memory cells, capable of re-entering the germinal center upon antigen stimulation to undergo clonal expansion several times before MM diagnosis. Next, we confirmed the clock-like nature (i.e constant mutation rate) of SBS5 in MM and other post-germinal center disorders such as chronic lymphocytic leukemia and B-cell lymphomas. Based on the SBS5 mutation rates and the corrected ratio between duplicated and non-duplicated mutations within large chromosomal gains, we could time the acquisition of the first copy number gain during the life history of each MM patient. Intriguingly, the first MM chromosomal duplication was acquired on average 38 years (ranges 11-64) before sample collection. In 23/27 (85%) cases the first multi gain event occurred before 30 years of age, and in 13/27 (48%) before 20 years reflecting a long and slow process potentially influenced and accelerated by extrinsic and intrinsic factors. DISCUSSION Our analysis provides a glimpse into the early stages of myelomagenesis, where acquisition of the first key drivers precedes cancer diagnosis by decades. Defining the time window when transformation occurs opens up for new avenues of research: to identify causal mechanisms of disease initiation and evolution, to better define the optimal time to start therapy, and ultimately develop early prevention strategies. Disclosures Bolli: CELGENE: Honoraria; JANSSEN: Honoraria; GILEAD: Other: Travel expenses. Corradini:Janssen: Honoraria, Other: Travel Costs; Jazz Pharmaceutics: Honoraria; KiowaKirin: Honoraria; Servier: Honoraria; Takeda: Honoraria, Other: Travel Costs; Kite: Honoraria; Novartis: Honoraria, Other: Travel Costs; Gilead: Honoraria, Other: Travel Costs; Roche: Honoraria; Sanofi: Honoraria; BMS: Other: Travel Costs. Anderson:Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board. Moreau:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Papaemmanuil:Celgene: Research Funding. Avet-Loiseau:takeda: Consultancy, Other: travel fees, lecture fees, Research Funding; celgene: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy. Landgren:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: B-other ALL represents a working definition for patients with B cell precursor (BCP) ALL without a known primary chromosomal abnormality. In this study we use whole genome sequencing (WGS) to characterize adult B-other cases (age ≥ 25yrs) from the UKALL14 trial (NCT01085617). Of 652 patients aged 25-65yrs enrolled onto UKALL14, 333 (51%) had B-other ALL. Sufficient material was available to screen 156/333 B-other cases for recurrent Ph-like fusion events (CLRF2, JAK2, ABL1, ABL2PDGFRB) using FISH and MLPA (kit P335). This identified 28 (18%) Ph-like fusion events (21 CRLF2, 5 ABL-class fusions and 2 JAK). Of the remaining 128 B-other cases 57 had available samples for tumor normal paired WGS (read depth 60x and 30x respectively). Bioinformatic analysis was performed to determine small somatic mutations (SSMs); single nucleotide variants (SNVs) and insertion/deletions (INDELs) as well as copy number aberrations (CNA) and structural variants (SV). We also undertook de novo motif analysis to identify RAG mediated deletions. We present data for 30/57 cases (median age of diagnosis 44, range 25-65), the remainder are undergoing sequencing. Within this cohort we identified 784 SVs, 49,244 SNVs and 2,881 INDELs, with each case having a median representation of 23 SVs, 1,674 SNVs and 86 INDELs. The Median SSM burden was 0.55 per megabase (range 0.31-0.81), which is in the upper third of previous ALL estimates (median 0.26 range 0.03-2.9) but low compared to most other cancer types (Alexandrov et al. 2013 Nature). Fusion gene analysis identified 11/30 (37%) cases with recurrent rearrangements (2 ZNF384r, 2 PAX5r, 5 DUX4r and FGFR1r); this identified a novel 5' partner for ZNF384r (AKAP8) and MYO18A-FGFR1 a fusion previously seen in a single case of 8p11 myeloproliferative syndrome. A further 7 clonal coding drivers were identified, 3 PAX5alt, 3 Ph-like candidates and a single ETV6-RUNX1-like candidate (ETV6 R399C, a dominant negative mutation). We classified our cohort into 6 driver subgroups as shown in table 1 using Gu et al. as a framework (Gu et al. 2019 Nat Gen). We found no evidence of driver subgroups clustering with age, although the Ph-like candidates were all identified in patients 39yrs or older. There was evidence that known driver subgroups have differing mutation burdens but these were not significant in this preliminary cohort; the single MEF2Dr case had a high SV burden compared to all other known subgroups (137 vs. cohort median 22); Favorable outcome subgroups (ZNF384r and DUX4r n=7) had a lower SNV coding mutation burden (median 14 vs. 20; U = 60, p = 0.057; Mann-Whitney U test); PAX5alt (n=5) cases had a higher INDEL burden (median 174 vs. 82; U = 16.5, p =0.057). As expected we found recurrent CNA in CDKN2A/B (63%; 19/30), PAX5 (33%; 10/30), IKZF1 (27%; 8/30), ETV6 (11%; 3/30) and BTG1 (7%; 2/30) across the cohort. IKZF1 deletions were enriched in the Ph-like candidate (n=4) subgroup compared to all other known groups (75% vs. 12%; p = 0.028; Fisher's Exact). RAG mediated deletions have been established as an oncogenic driver mechanism in childhood ALL, so we sought to ascertain its role in adults. We identified 54% (128/236) of the breakpoint resolved deletions had a RAG heptamer site. There was a significant difference (U = 167.5, p = 0.021) in RAG deletion burden between patients over 40yrs and under 40yrs at diagnosis (median 1.5 vs 3). Within the unknown driver subgroup; three cases carried ZEB2 H1038R mutations, two with concurrent IGH-CEBPA/B fusions, identifying them as likely belonging to the G12 cluster identified by Li et al. (Li et al. 2018 PNAS); one case had a high translocation burden (37 cohort median 2) and a single case had a high RAG deletion burden (45, cohort median 2); of the remaining 7 cases, four had either a NRAS or FLT3 subclonal hotspot mutation. Here we present the WGS analyses of 30 cases classified as B-other on the basis of no cytogenetic findings by standard clinical assays. The landscape of adult B-other ALL is highly heterogeneous but with WGS we were able to find at least one disease defining event in 60% of our cohort. These events often encompass novel fusion partners of established genomic subtypes or a cytogenetically cryptic lesion such as IGH-DUX4. Taken together our findings demonstrate the clinical utility of WGS as a diagnostic assay to inform and improve the management of adult B-other ALL patients in the future. Disclosures Fielding: Pfizer: Consultancy; Incyte: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Papaemmanuil:Celgene: Research Funding.

Abstract: Myeloid leukemia of Down syndrome (ML-DS) is a tractable human model of acute myeloid leukemia. A preleukemia phase, transient abnormal myelopoiesis (TAM) and silent TAM, occurs in 28% of neonates with Down Syndrome (Roberts et al. Blood 2013). TAM is caused by trisomy 21 and acquired mutations in GATA1 that result in a N-terminal truncated protein, GATA1s, in hematopoietic stem and progenitor cells (HSPCs) of fetal origin. ML-DS evolves from TAM by acquisition of additional genetic lesions. The nature of these lesions and the mechanism of transformation are incompletely understood. We performed exome sequencing and targeted resequencing of 141 ML-DS and 111 TAM patients to characterize the evolving mutational landscape from TAM to ML-DS. On average 1.6 acquired mutations were detected in ML-DS (in addition to GATA1 mutations), significantly more than in TAM (0.4 mutations per sample). Additional anticipated loss-of-function mutations acquired in ML-DS mainly affected cohesin components including CTCF (43% of patients), PRC2 components (13%), KANSL1 and other epigenetic regulators (14%). Conversely, anticipated gain-of-function mutations were most prevalent in signaling pathways, e.g. JAK kinases, MPL, KIT and RAS family members (40%). Importantly, we detected a novel recurrent hotspot mutation in 4% of patients (6/141 cases) in CSF2RB encoding the IL3-, IL5-, GM-CSF-receptor common beta chain. To test if the A455D/T variant in the CSF2RB transmembrane domain is a putative oncogenic driver, we ectopically expressed CSF2RBA455D in TF1 cells. Cells expressing CSF2RBA455D exhibited cytokine independent growth and STAT5 autonomous phosphorylation. In a CD34+-HSPC megakaryocytic differentiation assay, CSF2RBA455D blocked terminal megakaryocytic differentiation whilst increasing proliferation by 30-fold (P=0.046). Moreover, the median survival of NSG mice transplanted with CSF2RBA455DTF1 cells was shortened by 30 days compared to wild type TF1 cells (23 days compared to 53 days, P=0.0097). To experimentally test the potential of loss-of-function mutations to transform TAM to ML-DS, we performed an in vivo murine isogenic transplantation screen using Gata1s expressing fetal hematopoietic cells from Cas9-knockin mice. We tested variants in 22 genes, recurrently detected in ML-DS, with a pool of prevalidated gRNAs. This resulted in short latency (n=18 mice; median survival 36 days) and high penetrance (100%) leukemia. Leukemia was not detected in mice infected with control gRNAs. Leukemias had a typical ML-DS megakaryoblastic phenotype (CD117+ and CD41a+). Amplicon sequencing revealed on average 2.9 gRNAs per leukemia and high representation (61% of all leukemias) of gRNAs directed to the tumor suppressor Trp53, which was alone sufficient to induce leukemia with 100% penetrance. When excluding the Trp53 gRNA from pools, leukemic cells from moribund mice contained gRNAs against negative regulators of the RAS and JAK-STAT signaling cascade, such as Nf1, Cbl and Sh2b3 (70% of the mice), Ezh2, Asxl1, Kdm6a,Bcor and other epigenetic modifiers (85%) or Ctcf (15%), closely resembling the mutational landscape of ML-DS. In contrast to ML-DS, gRNAs targeting cohesion components, such as Rad21 and Stag2, were not present in any of the leukemias. In summary, we performed the largest genetic analysis of transforming events in ML-DS that cooperate with trisomy 21 and GATA1s and uncovered a previously undescribed activating mutation in CSR2B. We experimentally validated many of the loss-of-function mutations in a novel murine fetal leukemia assay for ML-DS. The field is now well-placed to study mechanisms of oncogenic cooperativity and identify novel therapeutic approaches for this leukemia. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Abstract: Current evidence supports a model in which the low-affinity state of the platelet integrin αIIbβ3 results from αIIbβ3 adopting a bent conformation. To assess αIIbβ3 biogenesis and how αIIbβ3 initially adopts the bent conformation, we mapped the conformational states occupied by αIIb and β3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that αIIbβ3 complex formation was not limited by the availability of either free pro-αIIb or free β3, suggesting that other molecules, perhaps chaperones, control complex formation. Five β3-specific, ligand-induced binding site (LIBS) mAbs reacted with much or all free β3 but not with β3 when in complex with mature αIIb, suggesting that β3 adopts its mature conformation only after complex formation. Conversely, 2 αIIb-specific LIBS mAbs directed against the αIIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-αIIb, raising the possibility that pro-αIIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-αIIb adopts a bent conformation soon after synthesis, and then β3 assumes its bent conformation by virtue of its interaction with the bent pro-αIIb.

Abstract: The first steps in the biogenesis of αIIbβ3 involve the individual synthesis of each subunit within the endoplasmic reticulum, followed by complex formation. Previous studies performed in HEL cells and in megakaryocyte-lineage cells cultured from cryopreserved progenitor cells of patients with CML have led to conflicting data with regard to whether αIIb or β3 is produced in limiting amounts that might then control complex formation. To study this question in greater detail, we conducted immunoprecipitation studies on HEK293 cells transfected with αIIb and β3 cDNAs and megakaryocyte-lineage cells derived from human umbilical cord blood grown in the presence of thrombopoietin and IL-11. Cells were labeled for 15 min with 35S-methionine/cysteine, and then lysed after 2 hr with a 1% triton X-100 buffer. The antibodies employed included murine monoclonal antibodies 10E5 (anti-αIIbβ3 complex, which, reacts with αIIb cap domain), 7E3 (anti-αIIbβ3 + αvβ3, which reacts with β3 specificity-determining loop and α1 helix), AP-5 (LIBS anti-β3, which reacts with β3 amino acids 1 - 6), 7H2 (anti-β3, which reacts with PSI domain near C13), 1990 (anti-αIIb, which reacts at unknown site), and LIBS2 (LIBS anti-β3, which reacts with a site within amino acids 602 – 690). The relative amounts of pro-αIIb, mature αIIb, and β3 immunoprecipitated by this panel of antibodies is shown in Table 1. Results were similar in both the HEK293 cells and in megakaryocyte-lineage cells derived from human umbilical cord blood. These data indicate that there are sizeable pools of both free pro-αIIb and free β3 in both cell types, and thus neither subunit controls complex formation by limited availability. Based on the selective precipitation of pro-αIIb + β3 by AP5, it appears that the pro-αIIbβ3 complex adopts a conformation similar to that of ligand-bound mature αIIbβ3. We conclude that αIIbβ3 complex formation is slow relative to the production of the individual subunits, and is probably controlled either by the ability of the subunits to adopt the proper fold and/or to interact with a chaperone(s) that facilitates complex formation. Assuming that AP5 selectively recognizes αIIbβ3 complexes in which the angle of the β3 βA (I-like) domain - hybrid domain interface results in separation of the αIIb and β3 leg domains, these data also suggest that αIIbβ3 head-head interactions precede αIIbβ3 leg-leg interactions during biogenesis, and that αIIb maturation occurs rapidly after αIIbβ3 leg-leg interactions. Such a model is consistent with our previous data demonstrating that pro-αIIb binds to the ER membrane-resident chaperone calnexin via the N-linked glycan at αIIb N15. Table 1 Antibody Pro- αIIb Mature αIIb β3 1990 ++++ +++ +++ 10E5 ++ +++ ++ 7E3 ++ +++ ++ 7H2 ++ +++ ++++ LIBS2 + − ++++ AP5 ++ − ++++