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There is a Blood Commentary on this article in this issue., Key Points SARS-CoV-2 spike proteins bind heparan sulfate and activate the alternative complement pathway on cell surfaces. Factor D inhibitor (ACH145951) blocks the complement activation induced by SARS-CoV-2 spike proteins., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins., Visual Abstract

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies, including anti-β2-glycoprotein-I (anti-β2GPI), that are considered central to APS pathogenesis. Based on animal studies showing a role of complement in APS-related clinical events, we used the modified Ham (mHam) assay (complement-dependent cell killing) and cell-surface deposition of C5b-9 to test the hypothesis that complement activation is associated with thrombotic events in APS. A positive mHam (and corresponding C5b-9 deposition) were present in 85.7% of catastrophic APS (CAPS), 35.6% of APS (and 68.5% of samples collected within 1 year of thrombosis), and only 6.8% of systemic lupus erythematosus (SLE) sera. A positive mHam assay was associated with triple positivity (for lupus anticoagulant, anticardiolipin, and anti-β2GPI antibodies) and recurrent thrombosis. Patient-derived anti-β2GPI antibodies also induced C5b-9 deposition, which was blocked completely by an anti-C5 monoclonal antibody, but not by a factor D inhibitor, indicating that complement activation by anti-β2GPI antibodies occurs primarily through the classical complement pathway. Finally, patients with CAPS have high rates of rare germline variants in complement regulatory genes (60%), compared with patients with APS (21.8%) or SLE (28.6%) or normal controls (23.3%), and have mutations at a rate similar to that of patients with atypical hemolytic uremic syndrome (51.5%). Taken together, our data suggest that anti-β2GPI antibodies activate complement and contribute to thrombosis in APS, whereas patients with CAPS have underlying mutations in complement regulatory genes that serve as a “second hit,” leading to uncontrolled complement activation and a more severe thrombotic phenotype.

Introduction : Terminal complement inhibition with a C5 inhibitor (eculizumab or ravulizumab) is the standard of care for treatment of atypical hemolytic uremic syndrome (aHUS) and leads to rapid resolution of thrombotic microangiopathy and improvement in renal function. The ideal duration of complement inhibition in aHUS is not established. Indefinite therapy is standard, though recent studies suggest that many patients with aHUS may safely discontinue therapy. Here, we present the outcomes of a physician-directed eculizumab discontinuation and monitoring protocol in a prospective cohort of 32 patients with aHUS. Methods: Consecutive patients with aHUS enrolled in the Hopkins Complement Associated Disease Registry between January 2014 and December 2019 who initiated eculizumab therapy during an acute episode of aHUS were included in the study. Patients starting eculizumab in the setting of renal transplant were excluded from the analysis. Physician directed discontinuation and monitoring was done according to the protocol outlined in Figure 1. We also evaluated relapse rate and renal outcomes and examined clinical and genetic risk factors for relapse. Results: Thirty-two consecutive patients who initiated eculizumab therapy for acute aHUS. Median age at initial diagnosis of aHUS was 44 (IQR 25, 53.5) years, 78.1% were female, and complement gene variants were present in 59.0% (13 of 22 patients with sequencing data available). Of these 32 patients, 25 (78%) discontinued eculizumab therapy after a median duration on therapy of 2.37 (IQR 1.06, 9.70) months. Six (18%) patients continued receiving it and 1 patient died during the first episode of aHUS. Of the 25 patients that discontinued eculizumab, 18 (72%) discontinued under physician direction (17 per protocol due to TMA remission including two with no renal recovery and end stage renal disease, and 1 due to non-response and a metabolic TMA thought more likely). Seven (28%) patients interrupted or discontinued therapy due to non-adherence. Overall relapse rate after eculizumab discontinuation was 20.0% (5 of 25); however, the relapse rate was higher in the case of non-adherence versus a physician directed cessation protocol (42.8% versus 11.1%, P=0.074) (Figure 2). Of the five patients who relapsed, four were re-treated and achieved remission without a decline in renal function with eculizumab; one patient died due to recurrent TMA and hypertensive emergency in the setting of non-adherence. On univariate analysis, non-adherence with therapy [OR 8.25 (95% CI 1.02 - 66.19) P=0.047] was associated with relapse while underlying complement gene variant [OR 1.39 (95% CI 0.39-4.87), P=0.598] and presence/absence of a trigger for aHUS [OR 0.66 (95% CI 0.09-4.79), P=0.687] were not significantly associated with relapse. We examined relapse rates in patients with CFH or MCP variants (40%, 2 of 5), other variants (33.3%, 2 of 6), no variants (0%, 0 of 6), and sequencing not completed (11.1%, 1 of 9) (P=0.217). These analyses are limited by the small sample size and limited number of events (5 relapses), which precluded multivariable regression analysis. Among patients that discontinued eculizumab, there was no significant decline in mean estimated glomerular filtration rate (eGFR) from the date of stopping eculizumab (47.09 + 28.28 ml/min/1.73 m2) until the most recent follow-up (56.95 + 28.02 ml/min/1.73 m2, one sided t-test P = 0.987). As seen in figure 3, improvement in eGFR (changed to a less severe eGFR category between stopping eculizumab and end of follow up) was seen in 9 of 25 patients (36%) and stable eGFR (no change in eGFR category) was seen in 15 of 25 patients (60%). Conclusion: Eculizumab discontinuation with close monitoring is safe in the majority of patients with aHUS (with native kidneys), with low rates of TMA recurrence and nearly complete salvage with eculizumab retreatment in the event of a recurrence. CFH and MCP variants may be associated with higher risk of relapse, which needs to be evaluated in larger, multicenter studies. Disclosures Moliterno: Pharmessentia: Consultancy; MPNRF: Research Funding. Chaturvedi:Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Publisher's Note: There is an Inside Blood Commentary on this article in this issue.

Introduction: Atypical Hemolytic Uremic Syndrome (aHUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. aHUS is usually caused by a predisposing germline variant in a complement regulatory gene, and a second hit of a complement amplifying condition such as infection, pregnancy, or inflammation.However, only approximately 50% of aHUS patients have identifiable genetic variants, with variants in factor H (CFH) accounting for 20%-30% of the genetic predisposition, C3 for 7%, membrane cofactor protein (MCP)/CD46 for 8%, factor B (CFB) for 2% and factor I (CFI) for 6%. For the other 50% of the patients, the genetic predisposition remains elusive. CFH binds to α2,3 sialic acid (SA) linked glycans on host cell surfaces and protects against attack by the alternative pathway of complement (APC). We hypothesized that the biosynthesis of SA is essential to complement regulation and SA biosynthesis defects predispose to aHUS. Methods: We performed targeted sequencing on 34 aHUS patients and 43 healthy controls for 4 genes that are responsible for the de novo biosynthesis of sialic acid: GNE, NANS, NANP, and CMAS. Then we used CRISPR-Cas9 and lentivirus systems to manipulate these genes in TF1 or TF1 PIGA-null cells, which lack the glycophosphatidylinositol-linked cell surface complement regulators CD55 and CD59, and allow the APC cascade to proceed once activated. α2,3 SA levels on the cell surface were measured with Maackia Amurensis lectin II (MAL II). Finally, we studied the functional consequences of these genetic changes. Normal human serum (NHS) was used to activate the APC on cells, and factor D (CFD) depleted serum (D-Dpl) was used to specifically block APC activation. C5b-9 deposition and CFH binding capacity on cells were detected by flow cytometry, and complement induced cell killing was detected via a WST-1 cell viability assay (mHam). Results: i) Rare germline variants found in SA biosynthesis genes Two rare germline variants (minor allele frequency < 0.005; data from GnomAD) were identified via targeted sequencing. An NANS M117I variant was found in an aHUS patient, while an NANP A153V variant was found in a control. The aHUS case did not harbor any variants in known complement genes. ii) Loss ofNANS but not NANP decreasesα2,3 SA on TF1 cells NANS knockout TF1 cells showed decreased α2,3 SA, demonstrating that this gene is essential for de novo SA biosynthesis. Conversely, NANP knockout TF1 cells showed no α2,3 SA level change. iii) NANS knockout increases the susceptibility to the APC TF1 PIGA-null cells with NANS knockout (TF1 DKO) had significantly higher C5b-9 deposition when treated with NHS compared to deletion of either gene alone (Figure A), demonstrating the formation of membrane attack complex (MAC). Cell viability assays also showed that TF1 DKO cells had significantly higher complement-induced cell killing when treated with NHS. Both C5b9 deposition and killing were rescued by APC-specific inhibition targeting CFD, demonstrating SA biosynthesis defects will increase the susceptibility to the APC specifically. iv) NANS knockout decreases the binding capacity of CFH To investigate CFH binding to the cell surface, C3b was first evenly loaded onto cells, followed by the addition of recombinant CFH. NANS knockout cells displayed significantly reduced CFH binding capacity, providing a mechanism for APC activation in the NANS knockout. v) NANS M117I decreases de novo biosynthesis of SA To assess the NANS variant identified in a patient with aHUS, TF1 DKO cells were transduced with a lentivirus containing the either wild type or M117I-mutated NANS, and cell-sorted to select individual clones. After treatment with sialidase to remove all SA, cells with NANS M117I had significantly lower SA level recovery compared to NANS wild type cells (Figure B). Conclusion: Targeted sequencing of 34 patients with aHUS identified a germline NANS variant in one case, suggesting an association between aHUS and SA biosynthesis defects. Functional studies showed that NANS knockout and the NANS M117I variant decreased cell surface SA levels. Loss of NANS also caused a decrease in CFH binding capacity and uncontrolled APC activation with increased cell death. Disclosures Chaturvedi: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction: The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity along with persistent antiphospholipid antibodies (aPL). Despite adequate anticoagulation, 10-30% of patients have recurrent thrombosis. Catastrophic APS (CAPS) is associated with approximately 40% mortality despite treatment. The pathogenesis of APS complications is incompletely understood. Recent animal studies indicate that complement is required for aPL-associated thrombosis, and complement has emerged as an attractive therapeutic target for refractory thrombotic APS and CAPS. Methods: We first evaluated complement activation in sera of patients with thrombotic APS by ISTH criteria (N=53), catastrophic APS (CAPS; N=8, sera available for 6), and systemic lupus erythematosus (SLE; N=74) who presented to our institution from June 2015 to June 2019 (and four patients with CAPS from other institutions). We used the modified Ham (mHam) test, a functional assay for complement activation as described previously (Gavriilaki et al. Blood 2015). The mHam assay is based on the principle that a PNH cell line (PIGA-null TF-1 cells) lacking the cell surface complement regulators CD55 and CD59 undergoes lysis in serum containing activated complement. Cell death (measured by a cell viability assay) is a measure of complement activation. Cell surface deposition of complement products (C3c, C5b-9) is also detected by flow cytometry. We then evaluated whether adding purified patient-derived aPL (anti-β2 glycoprotein IgG) to normal serum induced complement activation. Finally, we performed targeted sequencing of 15 complement genes in the study subjects, as well as 22 patients with aHUS and 36 healthy individuals as positive and negative controls, respectively. Results: (A) Complement activation is associated with thrombotic APS. A positive mHam assay (>20% cell killing) was detected in 32.1% (17 of 53) patients with thrombotic APS and 100% (6 of 6 with available sera) of CAPS compared with 6.8% (5 of 74) with SLE, (P (B) aPL from patients activate complement in vitro. Patient-derived anti- β2 glycoprotein IgG from all four patients induced complement activation in the mHam assay (Fig 2A). Flow cytometry confirmed cell surface deposition of complement activation products (C4d, C5b-9), which was inhibited by adding anti-C5 monoclonal Ab or a factor D inhibitor (representative sample in fig. 1B). (C) Catastrophic APS is associated with complement mutations. Rare (minor allele frequency Conclusions: APS serum activates complement in vitro shown by a functional assay (mHam) and increased C5b-9 deposition on the cell surface. A positive mHam test strongly associates with both recent thrombosis and triple positive APS. Purified human anti-B2GPI antibody from APS patients activates complement when added to normal human serum, suggesting that complement activation plays a pathophysiologic role in APS associated thrombosis. Finally, CAPS patients have a high rate of mutations in complement genes, which likely serves as a 'second-hit' (in addition to aPL) leading to uncontrolled complement activation and a more severe phenotype (Figure 3). Taken together, our results provide a rationale for complement inhibition as a therapeutic strategy in patients with CAPS and refractory thrombotic APS. Disclosures Chaturvedi: Shire/Takeda: Research Funding; Sanofi: Consultancy; Alexion: Consultancy. Streiff:Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Portola: Consultancy, Honoraria; Roche: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria. Petri:Astellas: Consultancy; Novartis: Consultancy; Exagen: Consultancy, Research Funding; Glenmark Pharmaceuticals: Consultancy; EMD Serono: Consultancy; Bristol-Myers Squibb: Consultancy; IQVIA: Consultancy; Janssen Pharmaceuticals: Consultancy; Aleon Pharmaceuticals: Consultancy; Momenta Pharmaceuticals: Consultancy; Blackrock Pharmaceuticals: Consultancy; Astrazeneca: Consultancy, Research Funding; UCB Pharmaceuticals: Consultancy; GSK: Consultancy; Qiagen: Consultancy; Abbive: Consultancy; Amgen: Consultancy; Decision Resources: Consultancy; Principia Biopharma: Consultancy; Eli Lilly: Consultancy; Kezaar Life Sciences: Consultancy. McCrae:Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding.

INTRODUCTION: Atypical hemolytic uremic syndrome (aHUS) is a complement mediated thrombotic microangiopathy that predominantly affects the kidneys although extra-renal manifestations are common. In the pre-eculizumab era, 40-65% of patients either died or had end stage renal disease (ESRD) at 1 year. Long-term renal and cardiovascular outcomes are less well described in the eculizumab era. We conducted this cohort study to describe the renal and cardiovascular outcomes of adult survivors of aHUS, both on and off continued eculizumab therapy. METHODS: Patients with aHUS were identified from the prospective Complement Associated Disease Registry and through the Center for Clinical Data Analysis at Johns Hopkins University. Demographic and clinical data were abstracted, including details of aHUS diagnosis, laboratory studies, treatment, and outcomes including renal function, hypertension and echocardiographic studies. The prevalence of hypertension was compared between patients with and without chronic kidney disease (CKD) using the chi squared test. RESULTS: 45 individuals with aHUS were followed at Johns Hopkins Hospital with a median [interquartile range (IQR)] time since diagnosis of 37.4 [IQR 20.7, 62.6] months. Median age at diagnosis was 32.5 [IQR 23.2, 49.2] years and 71.1% were female. Acute kidney injury was present in 98% (44/45); however, neurologic (64.4%), gastrointestinal (68.8%), and cardiovascular (55.5%) involvement was also common (Table 1). Hypertensive urgency or emergency was present in 40% (18/45), while 13.3% (6/45) had an acute coronary syndrome during the acute episode (2 ST elevation myocardial infarctions and 4 non-ST elevation myocardial infarctions). Complement gene sequencing was completed for 34 patients, of which 8 had variants in CFH, one in CFH and CD46, 5 in other genes (MCP1, CFHR1 homozygous deletion, DGKE, THBD, and THBD with del(CFH-SCR20-CFHR1-int5)]and 20 patients had no pathogenic variants. Thirty-two (71.1%) patients were treated initially with plasma exchange (median 6 [3, 12] exchanges). Thirty-nine (86.7%) received eculizumab (5 started at the time of renal transplant after developing ESRD), and 20 of these (51%) have since discontinued therapy. Median duration of eculizumab therapy was 2.7 [0.9-11.3] months for those who stopped therapy and 29.5 [8.8-55] months for those who continued. One patient died due to a myocardial infarction during the aHUS episode. Of the 44 survivors, 15 (34.1%) had complete renal recovery, 9 (20.5%) had chronic kidney disease (CKD) stage 1-4, and 20 (45.5%) developed CKD stage 5 requiring dialysis at 3 months after the acute episode. Fifteen patients underwent subsequent renal transplantation. At the end of follow up, 23 (52.2%) had CKD [2.2% stage 2, 15.6% stage 3, 4.4% stage 4 and 28.9% stage 5) (Figure 1A). Although not statistically significant, there was a higher rate of CKD (63.1% versus 52.6%, P=0.511) among those not on eculizumab; however, this primarily reflects eculizumab being stopped after ESRD. Hypertension was present in 35 (79.5%) survivors (Figure 1B), of which 14 (40%) had incident hypertension. The prevalence of hypertension was not significantly different between patients with CKD and normal renal function (87% versus 71.4%, P=0.202). Thirty-one (70.4%) were on antihypertensive therapy, and 67% (21 of 31) of these were not controlled to CONCLUSION: Malignant hypertension and cardiac involvement are common during acute aHUS. aHUS survivors also have a high prevalence of hypertension, including a notable incidence of new onset as well as uncontrolled hypertension following aHUS diagnosis. CKD is present in the majority of survivors, and structural cardiopulmonary disease is common. Complement activation has been implicated in the pathogenesis of cardiovascular disease. Further investigation is needed to evaluate the epidemiology of cardiovascular sequelae in aHUS, their associations with specific complement mutations, and optimal management. Disclosures No relevant conflicts of interest to declare.

Introduction aHUS is thrombotic microangiopathy associated with excess activation of alternative complement pathway (AP). Virtually all germline mutations that predispose to aHUS [e.g., factor H (fH)] occur in genes that regulate the amplification loop in the AP. The modified Ham test (mHam) measures the ability of a nucleated cell to protect itself from complement-mediated cell death (in vitro) in the absence of downstream cell-surface complement regulators, CD55 and CD59. Thus, the mHam exposes defects in serum-based complement regulators/activators that involve the amplification loop. The mHam is useful in distinguishing aHUS from thrombotic thrombocytopenic purpura (TTP); limitations are the lack of a reliable positive control and the lack of a confirmatory assay. In order to develop a positive control for the mHam, three complement activators, cobra venom factor (CVF), lipopolysaccharide (LPS) and sialidase (neuraminidase) were studied. CVF and LPS can activate and deplete serum of complement. Sialidase (Sia) makes cells more susceptible to complement mediated killing by removing sialic acid residues that are critical for binding the complement regulator, fH. Here, we show that Sia is a reliable positive control for the mHam and that cell surface C5b-9 accumulation correlates with a positive mHam. Methods/Results: The mHam assay was performed utilizing the PIGAnull TF1 cell line as previously described. CVF and LPS in dosages of 1.0 to 10ug/mL were added to 20% normal human serum (NHS) individually for 15 minutes at 37°C, and then added to cells in GVB++ buffer for 30 minutes. Sia (6.25unit/mL to 200unit/mL) was added to cells in GVB++ buffer for 15 minutes at 37°C first, and then incubated with 20% normal human serum (NHS) for 30 minutes. Complement-mediated cell killing in the mHam was evaluated using WST-1 absorbance as described previously. We found that sia treatment was more effective and reliable than LPS and CVF at inducing complement-mediated killing. A sia dose of 50U/mL or greater increased complement mediated killing by more than 3-fold in the mHam. Cell killing correlated with increased C5b-9 staining and was abrogated by the addition of an anti-C5 antibody and a factor D inhibitor. Next, we loaded PIGAnull TF1 cells with increasing amounts of C3b, added 20% NHS, and simultaneously performed the mHam and stained cells with an anti-C5b-9 monoclonal antibody for FACS staining to see if C5b-9 accumulation correlates with mHam killing. Briefly, PIGAnull TF1 cells in GVB+ buffer were incubated with 17.5ug C3, 4ug factor B and 0.1ug factor D for 20 minutes at room temperature (RT). Cells were washed and then incubated with 2ug factor B and 0.1ug factor D at RT for 3 minutes first, and treated with 0.25M EDTA in GVB0 buffer. Then 17.5ug C3 was added to cells for 15 minutes at 37°C. After three consecutive cycles of C3b loading, 20% NHS was added and complement activity was measured in the mHam and by flow cytometry. Cells loaded with one or two cycles of C3b showed minimal C5b-9 staining and minimal cell death ( Conclusion We describe an AP pathway specific flow cytometric assay that correlates with the mHam and may further aid in the diagnosis of aHUS. Sialidase treatment of TF1PIGAnull cells serves as a reliable positive control for the mHam by making cells more susceptible to AP killing. Disclosures No relevant conflicts of interest to declare.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by excessive activation of the alternative pathway of complement (APC). Atypical HUS is frequently a diagnosis of exclusion. Differentiating aHUS from other TMAs, especially thrombotic thrombocytopenic purpura (TTP), is difficult due to overlapping clinical manifestations. We sought to develop a novel assay to distinguish aHUS from other TMAs based on the hypothesis that paroxysmal nocturnal hemoglobinuria cells are more sensitive to APC-activated serum due to deficiency of glycosylphosphatidylinositol- anchored complement regulatory proteins (GPI-AP). Here, we demonstrate that phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells are more susceptible to serum from aHUS patients than parental EA.hy926 and TF-1 cells. We next studied 31 samples from 25 patients with TMAs, including 9 with aHUS and 12 with TTP. Increased C5b-9 deposition was evident by confocal microscopy and flow cytometry on GPI-AP-deficient cells incubated with aHUS serum compared with heat-inactivated control, TTP, and normal serum. Differences in cell viability were observed in biochemically GPI-AP-deficient cells and were further increased in PIGA-deficient cells. Serum from patients with aHUS resulted in a significant increase of nonviable PIGA-deficient TF-1 cells compared with serum from healthy controls (P < .001) and other TMAs (P < .001). The cell viability assay showed high reproducibility, sensitivity, and specificity in detecting aHUS. In conclusion, we developed a simple, rapid, and serum-based assay that helps to differentiate aHUS from other TMAs.

Introduction: Antiphospholipid antibody syndrome (APS) is characterized by recurrent thrombosis and pregnancy loss, in the presence of antibodies directed against phospholipid-binding proteins. Although persistence of antiphospholipid antibodies (aPL), including lupus anticoagulant (LA), anti-cardiolipin (aCL) or anti-β2-glycoprotein (aβ2-GPI) antibodies, is a key diagnostic feature of APS and its severe variant, catastrophic APS (CAPS), the mechanism by which aPL contribute to the clinical manifestations of these disorders remains unclear. There is evidence that excessive activation of complement plays a role in the pathophysiology of APS and CAPS, and limited case reports suggest a therapeutic role for complement inhibition; however, a clear mechanistic understanding of this process has not been elucidated. The modified Ham (mHam) is a functional assay for detection of systemic activation of the alternative pathway of complement (APC). Here, we show that the APC is active in serum from patients with APS and CAPs using the mHam assay, and provide evidence that inhibition of complement factor H (CFH), a homolog of β2-GPI, by aβ2-GPI antibodies is a plausible mechanistic link between APS/CAPS and excessive APC activation. Methods: The patient population studied included adults with APS who presented to our institution between January 2015 and January 2016. A diagnosis of APS or CAPS was based on diagnostic criteria from 2002 and 2006. Patients with a CAPS-like phenotype, who did not meet all criteria for definite or probable CAPS (Asherson et al. 2003), were also included. Patient serum was evaluated in the mHam using the PIGA-null TF1 cell line and percentage of cell killing was measured using a cell proliferation assay. Additional studies testing the addition of purified phospholipids or commercially available β2-GPI antibodies (and isotype controls) also utilized the mHam. Serum from 5 patients with atypical hemolytic uremic syndrome (aHUS) was used as a control in the phospholipid study. It was hypothesized that CFH, the principal negative regulator of the APC, may be the target of pathogenic aPL given its homology to β2-GPI. To test this hypothesis, competitive binding assays were performed with commercially available recombinant β2-GPI and purified CFH. Results: Serum from 18 patients with APS, 1 patient with definite CAPS, 3 patients with CAPS-like phenotype, 5 healthy volunteers and 14 patients with other hypercoagulable states (9 patients with lupus who did not meet criteria for APS, 4 patients with TTP and 1 patient with DIC) was analyzed in the mHam. APC-mediated cell killing in the mHam was higher in the serum from patients with APS and CAPS, compared to normal controls and patients with other hypercoagulable states (mean of 27.9% and 36.2% versus 5.2% and 12.2%, respectively; see Figure). Addition of purified phospholipids to serum from patients with APS reduced APC-mediated cell killing, but had no effect on serum from patients with aHUS, consistent with divergent mechanisms for APC activation in these disorders. Addition of a β2-GPI IgG antibody to normal human serum resulted in increased APC-mediated cell killing in the mHam, relative to addition of a β2-GPI IgM antibody or isotype controls, suggesting that antibodies directed against β2-GPI may directly activate the APC. Finally, the addition of CFH blocked binding of a commercially available β2-GPI IgG antibody to purified β2-GPI, supporting the hypothesis that aβ2-GPI antibodies may promote excessive activation of the APC in APS and CAPS by binding to and inhibiting CFH. Conclusions: Systemic activation of the APC can be demonstrated by the mHam test in serum from patients with APS and CAPS. This suggests that, in at least a subset of patients with APS and CAPS, excessive activation of the APC may be an important driver of the disease manifestations. Further study is required to determine whether there is a relationship between seropositivity for LA, aCL and aβ2-GPI and activation of the APC. Autoantibodies directed against β2-GPI may directly activate the APC by inhibiting CFH, providing further rationale for the potential use of therapies aimed at complement inhibition in these devastating disorders. Figure Figure. Disclosures Streiff: Janssen: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Portola: Research Funding; Roche: Research Funding. Brodsky:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Achillion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.

Introduction: The complement system can be activated via three pathways: classical pathway (CP), lectin pathway (LP) and alternative pathway (AP). While the CP and LP are triggered in the solid phase upon interaction of a pattern-recognition molecule with a target surface, the AP can be activated in the fluid phase. In fact, under normal physiological conditions, the AP is constitutively activated at a low level in the fluid phase via a mechanism of "C3 tickover" which leads to production of the C3b that can bind covalently to adjacent target cells or its binding activity is lost very rapidly. On the normal red cells, the bound C3b molecules are rapidly inactivated by an array of membrane-expressed or fluid phase-recruited complement regulators. However, due to the deficiency of two membrane-expressed negative regulators CD55 and CD59, the bound C3b molecules on the PNH red cells are not only amplified via the AP loop but also proceed to form the C3 convertase, the C5 convertase and ultimately the membrane attack complex, which causes hemolysis of PNH red cells. Hence, the blockade of both AP activation in the fluid phase and AP amplification on the surface of PNH red cells with a complement factor D (CFD) inhibitor is expected to be efficacious for PNH indication since hemolysis is due to the constitutive fluid phase AP activation. In this report, we evaluated whether hemolysis of PNH red cells occurs under the conditions in which the CP, LP and/or AP are activated by various pathogens in vitro. Our objective was to determine whether there is "bystander" hemolysis due to massive pathogen-driven complement activation and the potential for resulting C3b to bind PNH red cells and lead to complement-mediated hemolysis through AP amplification. Methods: Blood was obtained from PNH patients with written informed consent. Pathogen inoculums, such as E. coli and Neisseria meningitidis(NM), were prepared by standard methods. Hemolysis of PNH red cells was assessed with ~80% ABO blood type-matched pooled normal human serum (NHS) in GVB0-Mg-EGTA buffer (pH6.4) as well as in GVB++ buffer (pH7.3) in the presence and absence of a small molecule CFD inhibitor (ACH-4471). The extent of bystander hemolysis of PNH red cells was assessed with ~80% ABO blood type-matched pooled NHS in GVB++ buffer (pH7.3) for all pathogens for all pathogens except the NM isolates, where individual sera with bactericidal activity were used. Parallel evidence of complement activation by the pathogens was evaluated by 1) bactericidal activity of E. coli with pooled NHS-depleted of C1q, C2, CFD or C5 in GVB++ buffer (pH7.3), 2) bactericidal activity of NM isolates with the sera in GVB++ buffer (pH7.3) in the presence of ACH-4471, a CFD inhibitor; 3) measurement of the complement components and their activation products. Results: The extent of hemolysis of red cells harvested from PNH subjects was approximately equal to the Type II/III clone size when assessed with NHS in GVB0-Mg-EGTA buffer (pH6.4) ranging from ~30% to 90%; hemolysis was effectively blocked by ACH-4471, consistent with the data shown previously(Gavriilaki et. al. ASH 2015, Abstract No 275). No significant hemolysis of red cells harvested from PNH subjects was seen when assessed with NHS in GVB++ buffer (pH7.3) and furthermore hemolysis was not increased upon addition of bacteria (Fig. A). For E. coli, the bactericidal activity was unaffected by disruption of the CP (C1q depletion), the AP (CFD depletion), the CP and LP (C2 depletion) although, as expected, bactericidal activity was abrogated by disruption of the terminal pathway (C5 depletion) (Fig. B), confirming that complement activation was initiated via multiple pathways by E. coli. For NM isolates, the bactericidal activity was unaffected by disruption of the AP (Fig. B), confirming that the complement activation is initiated via CP or LP by NM isolates. Complement activation for other pathogens will be presented. Conclusion: We demonstrated that PNH red cells were not subjected to theoretical "bystander hemolysis" when incubated with the bacteria tested herein, suggesting no increased risk of pathogen-induced hemolytic breakthrough in PNH patients if treated with a complement alternative pathway inhibitor. Figure Figure. Disclosures Yang: Achillion: Employment, Equity Ownership. Thanassi:Achillion: Employment, Equity Ownership. Galvan:Achillion: Employment, Equity Ownership. Podos:Achillion: Employment, Equity Ownership. Huang:Achillion: Employment, Equity Ownership. Brodsky:Achillion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Factor D is a highly specific serine protease that cleaves factor B as its only substrate. It is the rate-limiting step of the alternative pathway of complement (APC). Therefore, factor D is a promising therapeutic target in diseases of excess activation of the APC, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Terminal complement inhibition by eculizumab is currently the treatment of choice for PNH and aHUS. Eculizumab must be administered intravenously and indefinitely in PNH; moreover, up to 20% of PNH patients treated with eculizumab have symptomatic hemolysis due to extravascular hemolysis. Glycosylphosphatidylinositol (GPI) anchor protein deficient erythrocytes from PNH patients or erythrocytes from animals can be used to test novel complement inhibitors for PNH; however, there are no in vitro assays available to model complement-mediated attack in aHUS. Here, we demonstrate that the modified Ham test (PIGA-null reagent cell line exposed to serum from aHUS patients) is the first reliable in vitro model to test complement inhibition for aHUS. In addition, we demonstrate that small molecule factor D inhibitors specifically block the APC in PNH and aHUS. Method: Three factor D inhibitors (ACH-3856, ACH-4100, ACH-4471) that reversibly bind factor D and are being developed for oral administration were studied. We obtained blood samples from 5 PNH and 4 aHUS patients after written informed consent. Serum and erythrocytes from PNH patients on eculizumab were collected within 60 minutes of eculizumab administration. All 3 compounds were tested in half-log dilutions ranging from 0.001 ìM to 10 ìÌ. The binding affinity to factor D was determined by surface plasmon resonance (Biacore) analysis. The inhibitory effect on factor D protease was evaluated biochemically using a natural substrate consisting of C3b and the complement factor B. The inhibition of APC-mediated hemolysis was first evaluated with rabbit erythrocytes incubated with 8% normal human serum (NHS). The inhibition of APC-mediated C3 fragment deposition was also initially evaluated with rabbit erythrocytes incubated with 20% C5-depleted NHS (to mimic the effect of eculizumab) by flow cytometry. To confirm the observation with rabbit erythrocytes, erythrocytes from PNH patients were also tested in the hemolytic assay (20% acidified NHS). Regarding aHUS, the inhibitory effect of the compounds was evaluated in the recently described modified Ham test. Briefly, PIGA-null TF-1 cells were incubated with 20% of aHUS serum for 30 minutes at 37 o C and complement-mediated cell killing was evaluated using the cell proliferation reagent (WST-1). Absorbance was measured and expressed as a ratio to the heat-inactivated control (% non-viable cells) Results: All 3 small molecules showed high binding affinity to human factor D and effectively inhibited factor D proteolytic activity in a dose-dependent manner (mean IC50 9.8-20 nM). Similarly, we observed a dose-dependent inhibition of hemolysis on rabbit erythrocytes (EC50 9-17 nM). C3 fragment deposition on rabbit erythrocytes after incubation with C5-depleted serum was reduced to undetectable levels with all 3 compounds from concentrations of 0.1 ìÌ and above. Next, we studied samples from 5 PNH patients (2 treatment naïve and 3 on eculizumab) and 4 aHUS patients (1 on eculizumab, 1 in acute phase and on eculizumab and 2 in remission). When tested on the hemolytic assay with erythrocytes from PNH patients and acidified NHS, factor D inhibitors significantly reduced hemolysis in a dose-dependent manner from concentrations as low as 0.01 ìM. Lastly, cell killing was observed as previously reported in the presence of the serum from aHUS patients in the modified Ham test and addition of factor D inhibitors caused significant dose-dependent reduction in the cell killing at concentration as low as 0.01 ìÌ. Mean percentage of non-viable cells before and after addition of compound ACH-4471 is shown in Figure 1. Conclusions: We demonstrate that factor D inhibitors, potentially the first-ever oral complement inhibitors, efficiently block hemolysis of PNH cells in vitro and mitigate the accumulation of C3 fragments. Importantly, our findings indicate that the recently described modified Ham test can be used as a preclinical model to test complement inhibitors in aHUS. Figure 1. Figure 1. Disclosures Thanassi: Achillion Pharmaceuticals: Employment. Yang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Brodsky:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a disease of excessive alternative pathway of complement (APC) activation that can be treated with eculizumab. Clinicians have been hesitant to administer eculizumab because of the lack of a diagnostic assay for aHUS and the expense of the drug. Mutations in genes that regulate the APC (factor H/CFH, I/CFI, CD46, etc.) underlie up to 50% of aHUS patients, but the clinical relevance of these mutations is often unclear. We developed the modified Ham test to distinguish aHUS from thrombotic thrombocytopenic purpura (TTP). This functional assay measures cell viability (WST-1) of a PIGA mutant cell line following exposure to patient serum. The cells are more sensitive to APC killing because complement regulators CD55 and CD59 are absent from the cell surface. CD46 is a transmembrane APC regulator that remains on the surface of the PIGA- null TF1 cells. Here, we show that the modified Ham test predicts response to eculizumab and can be adapted to assess the contribution of APC mutations to clinical phenotype. Method: We studied adult patients with thrombotic microangiopathies (TMA) who presented to our hospital from July 2014 through June 2015 and two patients from outside institutions. aHUS was defined by the presence of a TMA with platelet count 2.25 mg/dL, and ADAMTS13 activity >10%. TTP was defined as TMA in association with an ADAMTS13 ≤ 10%. Response to eculizumab was defined as TMA event-free status for at least 12 weeks and normalization of hematologic values. The modified Ham test was performed utilizing PIGA-null myeloid TF1 (PIGA only knockout/KO) as previously described. We additionally knocked-out CD46 / membrane cofactor protein (MCP) gene in the PIGA-null TF-1 cell line (PIGA/CD46 double KO), and wild-type TF1 cells (CD46 only KO) using CRISPR/Cas9 technology. Results: The modified Ham test was performed using PIGA-null TF1 reagent cells with 37 serum samples from 29 patients with TMAs. 13 patients were diagnosed with aHUS, 15 with TTP, and 1 with TMA of unknown etiology. APC-mediated cell killing was significantly increased in patients with aHUS compared to TTP (Figure 1A, grey shapes symbolize patients on eculizumab). In the ROC analysis, the cut-off value of 20.5% killing suggests an aHUS diagnosis with 100% specificity and 94.1% sensitivity. One patient presented with a TMA of unknown etiology (normal ADAMTS13 levels, and did not satisfy criteria for aHUS due to a creatinine of 12 aHUS patients received eculizumab and all responded to treatment. Eculizumab was discontinued in 6 patients after a median 11 doses (range 5-37) and all remain in remission for 8 months (range 4-15). The modified Ham test was positive in all aHUS patients that responded to eculizumab (positive predictive value 100%) and negative in 1 responder with a heterozygous variant of unknown significance in DGKE. Among 12 patients with aHUS, 7 patients had mutations in genes previously associated with aHUS (3 CFH, 1 DGKE, 1 CFH and CD46, 1 CFH and ADAMTS13, and 1 CFH and thrombomodulin). Six of those patients had additional heterozygous deletions in the CFHR3-CFHR1 gene region. To study the impact of CD46 insufficiency on complement regulation we exposed PIGA/CD46 double KO TF1, and CD46 only KO lines to aHUS serum and measured cell viability in the modified Ham test. The CD46 KO line was no more susceptible to killing than wild-type TF1 cells and the PIGA/CD46 double KO cells were no more sensitive to killing than the PIGAonly KO cells, suggesting that CD46 is a weak complement regulator compared to the combination of CD55 and CD59 (Figure 1B). Conclusions: The modified Ham test reliably distinguishes most forms of aHUS from TTP and predicts response to eculizumab. Mutations in CD46 do not impact susceptibility to APC killing in the modified Ham test. The modified Ham test may be adapted to inform investigators and clinicians on the correlation between genotype and phenotype. Disclosures Moliterno: Incyte: Membership on an entity's Board of Directors or advisory committees. Sperati:Alexion Pharmaceuticals: Other: Principal investigator for the international aHUS registry. Brodsky:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Abstract 2358 Background: The PIG-A gene is an X-lined gene required for biosynthesis of glycosylphosphatidylinositol anchor proteins (GPI-AP). The PIG-A mutations can be acquired or inherited. Acquired mutations in hematopoietic stem cells leads to paroxysmal nocturnal hemoglobinuria (PNH). Germ line null mutations are embryonic lethal but hypomorphic mutations cause severe developmental abnormalities, intractable seizures and early death. To better understand the consequence of PIG-A mutations in human development and hematopoiesis, we established human induced pluripotent stem cell lines (hiPSC) that lack PIG-A expression and are therefore GPI-AP deficient. We established an inducible system for conditional expression of the PIG-A gene within PIG-A null hiPSCs. This system allowed us to generate GPI anchor deficient (GPI-AP-) blood cells in multiple lineages. Method: We used a gene-targeting technology to knock out the PIG-A gene in male (XY) hiPSC derived from human somatic cells. F5HR (PIG-Anull, GPI-AP−) cells were transduced with pLV-PIG-A lentiviral vector (LV) containing the full-length human PIG-A transgene. F5HR (PIG-Anull, GPI-AP−) cells were co-transduced by two LV vectors: the regulator of pLV-tTR-KRAB (tTS/RFP) containing the tetracycline (tet)-SUPER inducible transgene expression system and the other of pLV-PIG-A expressing human PIG-A cDNA controlled by the human EF1α promoter. Once Dox is added, PIG-A gene expression is turned on. This inducible cell line (F5HKP) allows us to explore the function of the PIG-A gene during embryoid body (EB) generation and blood cell differentiation. Results: We examined the potential of F5 cells and F5HR cells (PIG-Anull) to form EBs and generate hematopoietic colonies. F5 hiPSC, but not F5HR hiPSC, cultured in serum free medium with BMP-4, VEGF and SCF formed morphologically normal EBs with evidence of blood-like cells surrounding the EB after 12–15 days in culture. Transduction of the F5HR cells with PIG-A restored the ability of the F5HR cells to form normal EBs and produce blood-like cells. Cells derived from the day 15 EB were further analyzed for the expression of the hematopoietic cell surface proteins (CD34 and CD45) and for the ability to generate hematopoietic colonies. The PIG-A mutant F5HR cells did not express CD59, CD34, or CD45 and were unable to generate hematopoietic colonies. We next induced mesodermal differentiation in the F5 and F5HR hiPSC and assessed cell surface expression of CD56, KDR and CD34 on days 0, 3, 7, and 10 during mesoderm differentiation. Surface expression of CD56 and KDR was virtually absent from the day 3 EBs derived from the F5HR iPS cells. CD34 expression by day 10 of mesoderm differentiation was negligible. Restoration of the PIG-A gene into F5HR cells restored the normal expression pattern of all three early mesodermal markers. Next, we transduced the F5HR iPS cells with our inducible PIG-A expression system to establish the F5HKP hiPSC. CD59+ undifferentiated F5HKP cells were cultured in mesoderm inducing medium with or without Dox for up to 14 days and the percentage of EB with blood-like cells in 96-well plates were counted. F5HKP hiPSC cultured without Dox lost GPI-AP expression, formed abnormal EBs, and did not generate blood-like cells. However, in presence of Dox for 14 days, F5HKP cells remained GPI-AP+ and made morphologically normal EB with blood-like cells. When Dox was removed from the culture conditions after 14 days myeloid and erythroid lineages were shown to be GPI-APneg. GPI-APnegblood cells derived from F5HKP cells were show to be resistant to proaerolysin, a toxin that uses GPI-APs as its receptor. Conclusion: Using PIG-A gene targeting in hiPSCs and an inducible PIG-A expression system, we have established a conditional PIG-A knockout model that allows for the production of GPI-APneg human blood cells. These studies show that PIG-A null hiPSCs are unable to form blood. The initial block is at the generation of mesodermal progenitors that express CD56, predating the deficiency of precursor cells expressing KDR and CD34. However, transient expression of GPI-APs during hematopoietic differentiation allowed us to generate GPI-APneg blood cells in multiple hematopoietic lineages. This conditional PIG-A knockout system should be a valuable tool for disease modeling PNH and other congenital diseases associated with GPI-AP deficiency. Disclosures: No relevant conflicts of interest to declare.

The technology of preimplantation genetic diagnosis (PGD) screens IVF-derived cleavage-stage embryos or oocytes from parents with inherited disorders, and is routinely used to avoid births with severe genetic disorders. More than one hundred testable genetic conditions, including severe hematologic diseases such as beta thalassemia, sickle cell anemia, and Fanconi anemia can be PCR-screened from either a micro-manipulated blastomere, or a pre-fertilization ovarian polar body. Derivation of human embryonic stem cell (hESC) lines from diseased IVF blastocysts has recently been reported, and these PGD-hESC are untested yet potentially valuable tools for investigating cellular and molecular events of human embryogenesis in diseased states. For example, a great deal of interest has recently been generated in treating hematologic diseases with genetically-corrected hematopoietic stem cells (HSC) derived from patient-specific pluripotent stem cells. Generation of hematopoietic progenitors from PGD-hESC affected with genetic syndromes may thus provide novel opportunities for testing cell-based and gene therapeutic strategies. An important candidate for such cell-based therapy includes sickle cell disease (SSD) hemoglobinopathy, a classic inherited single gene disorder resulting from the substitution of glutamic acid by valine at position 6 of the beta chain of hemoglobin. Human pluripotent stem cells derived from PGD-selected blastocysts or induced pluripotency (iPS; e.g., using patient’s somatic cells), will serve as critical reagents for testing such therapeutic strategies. In these studies, we report the characterization and erythropoietic differentiation of a novel PGD-hESC line affected with SSD hemoglobinopathy. The sickle point mutation was confirmed in this PGD-hESC line with direct genomic sequencing of the beta globin locus. This hESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SSD-hESC can be efficiently differentiated to the hematopoietic lineage under serum-free conditions, and gave rise to robust primitive and definitive erythropoieses. The expression of embryonic, fetal and adult globin genes in SSD PGD-hESC derived erythroid cells was confirmed by qRT-PCR, intracytoplasmic FACS, and in situ immunostaining of PGD-hESC teratoma sections. Moreover, we defined culture conditions for massive, long-term liquid culture expansion of sickle affected erythroid progenitors that remained in an undifferentiated erythroblastic phenotype for at least two months. These sickle erythroblasts were continuously maintained as a primary cell line that could be frozen and thawed without loss of viability. In vitro-expanded sickle erythroblasts expressed CD71+CD36+ and CD71+CD235a+ phenotypes, and underwent developmentally appropriate embryonic, fetal, and adult hemoglobin switching over a period of several months. Moreover, hESC-derived erythroblasts were readily infected with Plasmodium falciparum malaria parasites, thus demonstrating their potential utility in studying the effects of this important pathogen in normal and diseased erythropoiesis. These data demonstrate the utility of using patient-specific, hemoglobinopathic hESC for generating significant numbers of erythroid progenitors for molecular, developmental, gene therapeutic, pharmacologic, and microbiological studies. We are currently conducting a comparative erythropoietic differentiation study using SSD PGD-hESC vs. SSD iPS that were generated from somatic fibroblasts using defined pluripotency factors.

Glycosylphosphatidylinositol is an important means of anchoring many cell surface proteins. Glycosylphosphatidylinositol anchored proteins (GPI-AP) are distributed on all hematopoietic lineages, but are absent on hematopoietic cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). The absence of GPI-AP in PNH is due to mutations in PIG-A, whose product is necessary for the 1st step in GPI biosynthesis. A small percentage of GPI-AP deficient cells can be found in cell lines and that these GPI-APlo/neg cells do not harbor PIG-A mutations. The significance and mechanism of the GPI-AP deficiency in these cells are unclear. We found that 25% of the Burkitts’ lymphoma cell line, Ramos, is GPI-APlo/neg after staining with FLAER and that these GPI-APlo/neg cells do not harbor PIG-A mutations. After 2 days cultured in regular medium, the GPI-APlo/neg Ramos cells reverted to a mix of GPI-APlo/neg and GPI-APpos cells demonstrating that the GPI-APlo/neg cells appear to be precursors to the GPI-APpos cells. An in vitro assay for early steps in GPI anchor biosynthesis using UDP-[3H]GlcNAc found that the GPI-APlo/neg cells generated reduced amounts of the 1st and 2nd GPI intermediates, GlcNAc-PI and GlcN-PI. RT-PCR of the 24 known genes involved in GPI anchor biosynthesis revealed silencing of PIGL and to a lesser extent, PIGY. Methylation specific PCR demonstrated that PIGL was hypermethylated in the FLAERlo/neg Ramos cells. Furthermore, culturing the FLAERlo/neg Ramos cells in 5-Aza-2′ deoxycytidine greatly increased the percentage of cells displaying surface GPI-AP, suggesting that demethylating PIGL and perhaps PIGY may restore surface expression of GPI-AP. We hypothesized that primitive HSC may also enriched for GPI-APlo/neg cells. Thus, we isolated small lineage depleted Fr25Lin− cells from C57Bl6/NCR (Ly 5.2) mice as described1 and found that 30% were GPI-APlo/neg. Fr25lin−GPI-APlo/neg cells were highly enriched for HSC/progenitor cells using hematopoietic colony forming assays. We next transplanted lethally irradiated female recipients with either 100 Fr25Lin−FLAERlo/neg or Fr25Lin−FLAERpos, respectively. 3/4 animals transplanted with Fr25lin−FLAERlo/neg cells survived 17 weeks and revealed 81%, 85% and 90% engraftment; 2/4 animals transplanted with 100 Fr25lin−FLAER pos cells survived 17 weeks with 17% and 48% engraftment. Similar to the Ramos cells, RT-PCR analysis of the Fr25lin−GPI-APlo/neg cells revealed silencing of pigl and to a lesser extent, pigy. In this study, we found that reduced surface expression of GPI-AP is a common feature of cancer cell lines and primitive hematopoietic progenitor cells. In contrast to the fixed GPI-AP deficiency in HSC from PNH patients, the absence of GPI-AP in certain cancer cell lines and normal HSC is not fixed; the progeny of these cells acquire GPI-AP upon cellular differentiation. Furthermore, our data suggest that silencing of the genes required for early steps in GPI anchor biosynthesis, most notably PIG-L and PIG-Y, are responsible for the paucity of GPI-AP on the HSC/progenitor cells.