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Background: Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia. The spectrum of disease in PKD is broad, ranging from an incidentally discovered mild anemia to a severe transfusion-dependent anemia. Splenectomy partially ameliorates the anemia and reduces the transfusion burden in the majority of patients. Because hemoglobin poorly correlates with symptoms in PKD, transfusion requirements are typically used clinically to classify disease severity with those who are regularly transfused despite splenectomy recognized as the most severely affected subgroup. Aim: To compare demographics, complications, and laboratory results between the most severely-affected PKD patients (those that are splenectomized and regularly transfused) with non-regularly transfused splenectomized PKD patients. Methods: After ethics committee approval, patients were enrolled on the PKD Natural History Study, a prospective 30 site international study. All patients had molecularly confirmed PKD. Only splenectomized patients were included in the analysis. Transfusion frequency was observed over a 3-year period. Patients were divided into two groups based on transfusion frequency: the severe phenotype group was defined as those who receive regular transfusions (≥6 discrete red cell transfusion episodes per year) and the control group did not receive regular transfusions. Phenotype stability over the 3-year period was also assessed. Results: 154 splenectomized patients with PKD were included: 30 patients in the severe PKD phenotype group and 124 patients in the comparison PKD group. Results of the analysis comparing the two groups are described in the Table. Severely affected patients were more likely to be female (77% versus 51%, p=0.013), older at the time of splenectomy (median age: 5 versus 3.6, p=0.011), have iron overload (93% vs. 51%, p Conclusions: Patients with PKD who are regularly transfused despite splenectomy appeared to have similar rates of PKD-associated complications (except for iron overload) and similar relevant laboratory values and genotypes when compared to those who are not regularly transfused after splenectomy. The similarity observed between severe phenotype patients and comparison patients with PKD may result from a protective effect of transfusion (e.g. reduction of bone fractures and extramedullary hematopoiesis) or could suggest transfusion-dependence is an artificial signifier of disease severity, reflective of provider practices and patient symptoms rather than an actual distinct phenotype. Transfusion requirements in severe PKD appear to fluctuate significantly over time. Disclosures Al-Samkari: Dova: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Moderna: Consultancy. van Beers:RR Mechatronics: Research Funding; Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding. Barcellini:Agios: Consultancy, Other: Advisory board; Apellis: Consultancy; Incyte: Consultancy, Other: Advisory board; Bioverativ: Consultancy, Other: Advisory board; Novartis: Research Funding, Speakers Bureau; Alexion: Consultancy, Research Funding, Speakers Bureau. Eber:Agios Pharmaceuticals, Inc.: Consultancy. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chonat:Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Kuo:Pfizer: Consultancy; Novartis: Consultancy, Honoraria; Celgene: Consultancy; Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy. Despotovic:Dova: Honoraria; Novartis: Research Funding; Amgen: Research Funding. Kwiatkowski:Celgene: Consultancy; Terumo: Research Funding; Apopharma: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Imara: Consultancy; Novartis: Research Funding. Thompson:Baxalta: Research Funding; Novartis: Consultancy, Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Ravindranath:Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding. Rothman:Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Agios: Honoraria, Research Funding. Verhovsek:Sickle Cell Awareness Group of Ontario: Membership on an entity's Board of Directors or advisory committees; Sickle Cell Disease Association of Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding; Canadian Haemoglobinopathy Association: Membership on an entity's Board of Directors or advisory committees; Vertex: Consultancy. Kunz:Novartis: Membership on an entity's Board of Directors or advisory committees. Sheth:CRSPR/Vertex: Other: Clinical Trial Steering committee; Apopharma: Other: Clinical trial DSMB; Celgene: Consultancy. London:United Therapeutics: Consultancy; ArQule, Inc: Consultancy. Grace:Novartis: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

In this issue of Blood, Pievani et al have identified a potential solution to the remaining barrier to the success of hematopoietic stem cell transplantation (HSCT) in children with severe phenotype Hurler syndrome (mucopolysaccharidosis type I [MPS I]).

To the editor: We read with interest the description by Hoeller et al of an exon 2 GATA-1 mutation leading to severe transient myeloproliferative disease (TMD),[1][1] in which the authors speculated that the severe phenotype might reflect loss of both full-length (FL) GATA-1 and the shorter isoform

Abstract 1190 Introduction: The goal of this study was to evaluate the efficacy and safety of two prophylaxis regimens of reformulated recombinant coagulation factor IX (rFIX-R), 100 IU/kg once weekly and 50 IU/kg twice weekly, compared with on-demand treatment in subjects with severe hemophilia B. Patients and Methods: Pharmacokinetic data were collected for 47 subjects aged 6 to 64 years with severe hemophilia B. Factor IX activity (FIX:C) measurements were made immediately before rFIX-R administration and at 0.5 hours post-administration of either 100 IU/kg or 50 IU/kg doses, to assess recovery before the initiation of each weekly regimen. Another FIX:C sample was collected at least 72 hours after dosing during each regimen. All samples were analyzed at local laboratories. Results: The mean prophylactic doses of rFIX-R administered were 86 ± 29 IU/kg in the 100 IU/kg once-weekly group and 53 ± 14 IU/kg in the 50 IU/kg twice-weekly group. The treatment comparison between the two prophylaxis regimens for annualized bleeding rate was not significant (LS mean = 2.0; 95% CI, −1.2 – 5.2; n=43), although both were significantly different from the on-demand treatment period (both, P The number (%) of observed trough plasma concentrations (Ctrough) that fell into the mild (>5%) range for hemophilia were 18 (45%) and 19 (48%) for the r-FIX-R 100 IU/kg once-weekly and 50 IU/kg twice-weekly groups, respectively. However, the severe phenotype was observed in 4 (10%) and 3 (7%) subjects, respectively. Conclusions: The pharmacokinetics of r-FIX-R are dose proportional. The C0.5hr increased in proportion to the dose administered. Recovery was consistent between the two prophylaxis regimens. Both regimens provided similar therapeutic results for Ctrough. The apparent difference in time after dose of new bleeding events between the 2 regimens is intriguing and deserves further study. Disclosures: Korth-Bradley: Pfizer Inc: Employment. Valentino:Inspiration Bioscience: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center, Honoraria; CSL Behring: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center; Pfizer: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center; NovoNordisk: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center, Honoraria; GTC Biotherapeutics: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center, Honoraria; Bayer Healthcare: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center, Honoraria; Baxter Bioscience: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center, Honoraria; Biogen: Educational Grants on behalf of Dr. Valentino for Rush University Medical Center; Hemophilia and Thrombosis Research Society: Membership on an entity's Board of Directors or advisory committees, Past-President. Rendo:Pfizer Inc: Employment. Shafer:Pfizer Inc: Employment. Smith:Pfizer Inc: Employment. Baumann:Pfizer Inc: Employment. Charnigo:Pfizer Inc: Employment.

Thrombospondin 1 (TSP-1) has been shown to activate latent TGF-beta1 in vitro, but its in vivo role is unclear because TSP-1-null mice have a much less severe phenotype than TGF-beta1-null mice. TSP-1 is stored in platelet alpha-granules and released, along with latent TGF-beta1, with activation. Thus, it is strategically located to contribute to TGF-beta1 activation at sites of platelet deposition. We recently demonstrated that stirring and/ or shear could activate latent TGF-beta1 released from platelets, probably in part through thiol-disulfide exchange and we have identified TSP-1 in human platelet releasates by immunoblotting and observed that it labeled with a thiol-reactive biotin-maleimide probe (MPB) by LC/MS/MS analysis, indicating the presence of free thiols. Remarkably, we found that the amount of TSP-1 detected by immunoblotting in human platelet releasates decreased with stirring or shear and that pretreatment of platelet releasates with the thiol-reactive compounds MPB, NEM or BMCC largely prevented the loss of TSP-1 with shear or stirring. To further assess the role of TSP-1 in TGF-beta1 activation, we have now studied the effect of stirring and shear on TGF-beta1 activation in samples from WT and TSP-1-null mice. Unstirred sera or platelet releasates from WT and TSP-1-null mice had similar levels of total TGF-beta1 [serum, 91 ± 15 ng/mL in WT (n=23) and 108 ± 15 ng/ mL in TSP-1-null mice (n=23; p=0.13); platelet releasates, 58 ± 14 ng/mL in WT (n=14) and 53 ± 16 ng/mL in TSP-1-null mice (n=14; p=0.45)]. With either stirring or shear, the TSP-1-null samples demonstrated less activatibility, but the defect was only partial [stirring increased active TGF-beta1 in serum from 0.6 ± 0.2 ng/mL to 2.2 ± 0.8 ng/mL in WT mice and from 0.5 ± 0.2 ng/mL to 1.6 ± 0.5 ng/mL in TSP-1-null mice (n=23) (p=0.05 using ANOVA analysis for the linear model comparing two-way interactions of stirred samples between WT and TSP-1-null mice); stirring increased active TGF-beta1 in platelet releasates from 0.3 ± 0.2 ng/mL to 2.4 ± 1.5 ng/mL in WT mice (n=14) and from 0.2 ± 0.2 ng/mL to 1.1 ± 0.6 ng/mL in TSP-1-null mice (n=14) (p=0.005)]. To assess in vivo TGF-beta1 activation we induced carotid artery thrombi with FeCl3 in WT and TSP-1-null mice. When thrombi were removed 5 min after total occlusion, the thrombus extracts from WT and TSP-1-null mice contained similar amounts of total and active TGF-beta1 [total TGF-beta1, 5.6 ± 2.4 ng/mL in WT and 5.5 ± 1.6 ng/mL in TSP-1-null mice (n=9) (p=0.9) and active TGF-beta1 (expressed as a percentage of total), 1.7 ± 0.7% in WT and 2.5 ± 1.4% in TSP-1-null mice (n=9) (p=0.15)]. When thrombi were removed 120 min after total occlusion, however, the percentage of active TGF-beta1 in the thrombus extracts was higher in WT than TSP-1-null mice [4.5 ± 2.0% in WT mice (n=9) and 3.3 ± 1.5% in TSP- 1-null mice (n=9) (p=0.02 using ANOVA analysis comparing interactions between time and type of mice)]. We conclude that: TSP-1 released from human platelets undergoes a shear-induced, thiol-dependent conformational change, TSP-1 contributes partially to shear or stirring-dependent TGF-beta1 activation in vitro in mice, and Time-dependent activation of TGF-beta1 released from thrombi formed in vivo in mice is also partially dependent on TSP-1. These data suggest a significant, but minor role of TSP-1 in TGF-beta1 activation in vivo and thus provide an explanation for the less severe phenotype of TSP-1-null mice than TGF-beta1-null mice.