Catalyst Biosciences Research Articles

Genetic Variants In PLG and SIGLEC14 Contribute To Plasma Plasminogen Level Variation

The conversion of plasminogen (PLG) to plasmin is a critical step in the activation of the fibrinolytic system. Due to its broad substrate specificity, plasmin plays a pleotropic role in the circulation and extravascular space, controlling the degradation of fibrin clots as well as potential functions in regulating cell migration and angiogenesis. PLG activation is regulated through the interaction of tPA or uPA with plasminogen activator inhibitor type one (PAI-1). Genome-wide association studies (GWAS) have identified polymorphisms associated with the plasma levels of tPA and PAI-1, but to date, no studies have directly examined the control of PLG levels.In order to identify genetic determinants of PLG levels we performed GWAS in a cohort of 3244 young healthy individuals (557 sibships) who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS).(Desch KC et al. PNAS 2013, 110:588-593) All subjects provided written informed consent. Details of the genotyping and data cleaning process for these cohorts are as previously described. (Desch KC et al. PNAS 2013, 110:588-593) PLG levels were measured in both cohorts by AlphaLISA (Perkin-Elmer, Waltham, MA). Levels were natural log transformed and adjusted for participant age, gender and BMI (TSS only) prior to use in heritability estimates and GWAS, which were performed using the PLINK, EMMAX, MERLIN, GCTA and METAL software packages.The median PLG levels were 101.7 IU/dL and 104.7 IU/dL in the GABC and TSS cohorts respectively and had a 1.7 fold variation between the 5th and 95th percentiles. The narrow-sense heritability using intra-class correlation was 59.7% based on 1277 siblings in GABC and TSS, which was similar to the estimate of 60.2% based on the genotype data and the MERLIN software package. GWAS for PLG in the European only subset of GABC (n=940) did not reveal any genome-wide significant signals. However, GWAS for PLG levels in TSS (n=2304) revealed 9 significant SNPs on chromosome 6q26 at the PLG (plasminogen) and LPA (lipoprotein A) locus that together explained 7.8% of the variance in PLG levels according to the GCTA method, Figure 1. The top SNP was rs4252129 (p = 3.47E-25) which results in a single amino acid substitution (R523W) in PLG. The next strongest associations were with two SNPs near LPA, rs1084651 and rs783149 (p = 1.60E-13 and 1.07E-12 respectively). These SNPs are located between LPA and PLG (Figure 1). To determine if the LPA SNPs were independent from the top PLG SNP, a conditional analysis was performed using rs4252129 as a covariate. Rs1084651 and rs783149 remained significant (p= 7.73E-16 and 6.48E-15 respectively), verifying that they represent a second, independent association signal. Meta-analysis of both cohorts (using 723,715 common SNPs) confirmed the 6q26 signal and revealed another significantly associated region on 19q13.41, Figure 2. Two SNPs, rs10412972 and rs11084102 (p = 2.16E-09 and 1.14E-08, respectively) are intergenic variants 4.8 and 12.6 Kb downstream from SIGLEC14. The significant SNPs in the meta-analysis, 6 on 6q26 and 2 on 19q13.41, explained 5.8% of the variance for PLG. The top SNP in the TSS GWAS, rs4252129, had a minor allele frequency (MAF) of 1.4% in GABC and was not included in the GABC GWAS or meta-analysis.In summary, our data identify two loci (at PLG/LPA and SIGLEC14) that harbor genetic variants contributing to the variation of plasma PLG levels in healthy young adults. The strongest association was observed for a non-synonymous SNP in the PLG gene itself, with a MAF of 1.3% in the HapMap CEU population and 2.2% in TSS. The minor allele (W523) is associated with a 14.1% decrease in mean plasma PLG level. This low frequency, non-synonymous SNP, or another SNP in LD with it, may be the functional mutation leading to decreased PLG levels through unknown mechanisms. Whether the significant SNPs near LPA affect lipoprotein A or PLG is not clear. However, the association with causal variants at LPA is possible and consistent with previous biochemical studies demonstrating competitive inhibition of PLG binding to endothelial surfaces by lipoprotein A, or an effect of lipoprotein A on endothelial PAI-1 expression or the PAI-1 interaction with tPA/uPA. Finally, these studies identified two significant SNPs near SIGLEC14. This gene encodes a sialic acid lectin type receptor protein, closely related to SIGLEC 5 recently implicated in the clearance of von Willebrand factor. [Display omitted] [Display omitted] Disclosures:Ginsburg:Shire plc: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaeuticals: Membership on an entity's Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity's Board of Directors or advisory committees; Merck pharmaceuticals: Consultancy; Boston Children's Hospital: From Baxter to Children's Hosp (VWF), From Baxter to Children's Hosp (VWF) Patents & Royalties; University of Michigan: From Baxter to University of Michigan (ADAMTS13) Patents & Royalties.

A Sensitized Whole Genome ENU Mutagenesis Screen Identifies an Arp2 Missense Mutation As a Novel Suppressor of Lethal Thrombosis in the Factor V Leiden Mouse

Abstract 493Only ∼10% of individuals carrying the common risk factor, Factor V Leiden (FVL), will develop venous thrombosis. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen based on the perinatal synthetic lethal thrombosis observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the lethal FVQ/Q Tfpi+/− genotype. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Whole exome sequencing was performed on a progeny tested member of 8 of the 14 lines using the Agilent SureSelect mouse whole exome capture kit resulting in ∼100 fold coverage. Variant analysis revealed a small number of high confidence novel heterozygous (dominant) single nucleotide variants (SNVs) in each sample. Sanger re-sequencing of all 11 SNVs in a cohort of mice from line 1 confirmed that the G to C mutation (chromosome 11 base 19,977,300) in the Actr2 gene (present in 15/16 re-sequenced progeny p<0.0001) is the dominant FVL modifier in this line. This mutation resulted in an R286G substitution in a highly conserved amino acid in the Arp2 protein, which is essential for the function of the Arp2/3 complex. Arp2/3 is responsible for intracellular actin branching and polymerization, which is critical for the regulation of cell shape. Analysis of 31 progeny from an Arp2 R/G × Arp2 R/G cross revealed only one live Arp2 G/G mouse (p<0.05), suggesting that R286G is a loss of function mutation and that homozygous deficiency is lethal. Complete blood counts (Advia 2120) performed on 14 Actr2 heterozygous and 10 wildtype littermates revealed no significant differences in platelet count, red and white blood cell counts, hematocrit or hemoglobin. However, measurements of platelet size and size distribution including mean platelet dry mass, platelet volume distribution width and the platelet component distribution width were significantly altered in Actr2 heterozygous mutant mice (p<0.05 for each measure). Thus, partial deficiency of Arp2 appears to alter platelet structure/function resulting in a shift in hemostatic balance facilitating survival of the otherwise lethal FVQ/Q Tfpi+/− phenotype. These results suggest that variation in Arp2 or related genes could potentially modify thrombosis risk in humans, and might also identify novel therapeutic targets for the treatment of this class of disorders. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Partial in Vivo FVIII Stabilization by VWF Fragments

Abstract 15Plasma factor VIII (fVIII) circulates in complex with von Willebrand factor (VWF) and is rapidly cleared in the absence of VWF. Previous in vitro studies have 1) localized the fVIII-binding region of VWF to the N-terminus, comprised of the contiguous D’ and D3 domains and 2) observed reduced affinity for fVIII upon alterations to the tertiary structure of VWF. To gain insight into the structure-function of VWF for fVIII stabilization, we tested VWF fragments for in vivo fVIII stabilization and investigated the architecture of a VWF fragment in complex with fVIII. For the in vivo study, fragments of murine Vwf cDNA were cloned into the hepatic-specific expression vector, pLIVE, and modified to fuse tandem E and FLAG tags to the C-terminus. The following VWF fragments were expressed in vivo by hydrodynamic tail vein injection into Vwf−/− mice: 1) a monomer of the VWF D’D3 domains (monoD’D3; M1-C22, S764-P1274); 2) a truncation of monomeric D’D3 (truncD’D3; M1-C22, S764-R1035); 3) dimers of D’D3 (diD’D3; M1-P1274); 4) multimers of D’D3 (multiD’D3; M1-P1274, G2713-K2813); 5) dimers of mature VWF subunits (DPro, M1-C22, S764-K2813); or 6) full length, multimeric VWF (wtVWF, M1-K2813). Expression of all VWF fragments persisted throughout the period of observation (4 weeks) with peak antigenic levels at 1 or 3 days post-injection. Prolonged elevation of plasma fVIII activity (fVIIIa) from ∼10% to ∼50–200% were observed (100% defined as the fVIIIa level of pooled platelet poor plasma from 10 wild type C57BL/6 mice) for all but the truncated monomer of D’D3 (Figure 1). Significantly increased fVIIIa levels (p<0.05, relative to pre-injection) were first observed at 1 day, peaked at 3 days, and persisted for the duration of observation. A minimal VWF fragment (S764-R1035, truncD’D3) reported to bind fVIII in vitro significantly increased plasma fVIIIa to 34% only at 3 days post-injection. Clearance of VWF fragments from circulation were determined from injections of pooled platelet poor plasma containing recombinant VWF fragments derived from hydrodynamically injected mice into naïve Vwf−/− mice. Nonlinear regression estimated the half-life for monoD’D3 (3.4hr), diD’D3 (2.1hr), multiD’D3 (2.3hr), DPro (2.8hr), and wtVWF (3.5hr). To examine how dimers of D’D3 bind fVIII, diD’D3 from HepG2 conditioned media was purified either alone or with recombinant fVIII, and negative stained samples were visualized by electron microscopy (EM). Single-particle EM analysis revealed that each subunit of the dimer binds 1 fVIII molecule. 3D EM reconstructions indicate that the light chain of fVIII directly interacts with, and potentially induces torsion in the flexible D’D3 domains of VWF. Together, these results emphasize the importance of VWF’s tertiary structure in fVIII stabilization and that the N-terminal D’D3 alone is sufficient to support fVIII survival in vivo. These findings could lead to improved methods of recombinant fVIII production and the development of novel approaches to treatment for hemophilia and von Willebrand disease. [Display omitted] Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Disparate SEC23B Deficient Phenotypes in Humans and Mice

Abstract 974SEC23 is a core component of the COPII coated vesicle that mediates the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. Mutations in human SEC23B, one of two mammalian SEC23 paralogs, results in the autosomal recessive disease, congenital dyserythropoietic anemia type II (CDAII). In contrast, SEC23B deficient mice were recently reported to die perinatally with massive pancreatic degeneration, with no evidence of anemia at birth (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9.) To examine the impact of SEC23B deficiency on hematopoietic function in mice surviving beyond the immediate perinatal period, lethally irradiated C57BL/6J mice were transplanted with fetal liver cells (FLC) collected from E17.5 embryos that were either wild-type (WT) or homozygous for the previously reported Sec23b gene trap allele (Sec23bgt/gt). Recipients of Sec23bgt/gt FLC were indistinguishable from their WT transplant controls and exhibited none of the phenotypic characteristics of CDAII (anemia, increased bi/multi-nucleated RBC precursors, narrower band size and increased shift of the membrane protein band 3 on SDS gel electrophoresis, or RBC double membrane appearance by electron microscopy). To test for a more subtle defect in hematopoietic reconstitution, Sec23bgt/gt FLCs were co-transplanted with WT GFP+ FLC in a 1:1 ratio, with no competitive difference observed over the course of 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. To rule out an incidental mutation in a nearby gene (“passenger gene”) as the cause of the pancreatic phenotype in SEC23B-deficient mice, two Sec23b bacterial artificial chromosome transgenes were crossed into the Sec23bgt line, with both demonstrating complete rescue of the Sec23bgt/gt pancreatic phenotype, with normal survival to adulthood. Sec23bgt/gt murine embryonal fibroblasts express a SEC23B/βGEO fusion protein consistent with the gene trap insertion into Sec23b intron 19, and this fusion protein co-immunoprecipitates with SEC24A (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9). To rule out a contribution of the SEC23B/βGEO fusion protein to the disparate human and mouse SEC23B-deficient phenotypes, a second, conditional SEC23B allele was analyzed, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 results in frame shift and stop codon in exon 7. Mice with an erythroid-specific deficiency of SEC23B were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice exhibit no anemia compared to their WT litter mates. Transplant recipients of FLCs from E16.5 embryos homozygous for a germline deletion of Sec23b exons 5 and 6 (Sec23b−/−) were also indistinguishable from mice receiving WT FLCs. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre transgene confirmed the pancreatic phenotype observed. This suggests that the perinatal lethality in Sec23b deficient mice may be the result of the loss of pancreatic Sec23b expression. To explore the mechanism for the disparate human and mouse SEC23B-deficient phenotypes, the expression patterns for SEC23A and SEC23B were examined in mice and humans by RT PCR and western blotting. The ratio of SEC23B/SEC23A expression is higher in mouse pancreas (12.7) compared to bone marrow (2.6), whereas in humans, the ratio is higher in the bone marrow (7.8) relative to pancreas (5.5) (normalized to liver). Taken together with the high degree of sequence similarity between SEC23A and SEC23B (∼ 85% identity at the amino acid level), we hypothesize that these 2 SEC23 paralogs overlap extensively at the level of protein function, with the disparate deficiency phenotypes due primarily to differences in tissue and developmentally-specific gene expression programs that have shifted extensively during recent mammalian evolution. These findings have important implications for the comparative function of other closely related paralogous genes. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII and potential therapeutic approaches. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Genetic Determinants of Plasma Von Willebrand Factor (VWF) and VWF Propeptide Levels

Abstract 3313Plasma VWF levels vary by approximately five-fold in healthy populations and are influenced by both environmental and inherited factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis. Increased levels of VWF occur with age, may rise acutely due to inflammation or infection, and may serve as a biomarker for endothelial dysfunction and atherosclerosis. Genetic factors are thought to account for 60 – 70% of the variance in VWF antigen levels with ∼30% of this effect due to ABO blood type. VWF multimers are assembled in the Golgi apparatus where the VWF propeptide is cleaved to form mature VWF. Because the half life of plasma VWF propeptide (ppVWF) is ∼5X faster than the mature VWF antigen, the ratio of ppVWF/VWF:Ag in plasma primarily reflects clearance rates of mature VWF while alterations in biosynthesis and/or secretion of VWF affect the level of both ppVWF and VWF:Ag. In order to identify additional quantitative trait loci (QTL) for VWF antigen variation and altered clearance of VWF:Ag, we performed genetic analyses in a healthy young sibling cohort, the Genes and Blood Clotting Study (GABC, n=1,152). The cohort was SNP genotyped with an Illumina Omni-1 array resulting in 763,195 SNPs available for analysis after extensive QC. Plasma VWF antigen (VWF:Ag) and VWF propeptide levels were determined by AlphaLISA (Perkin Elmer, Waltham, MA), using polyclonal anti-human VWF antibodies (DAKO, Glostrup, Denmark) or monoclonal anti-ppVWF antibodies (Blood Center of Wisconsin, Milwaukee, WI). The median levels were 108.1 IU/dL, 99.97 IU/dL and 1.0 for VWF:Ag, ppVWF and ppVWF/VWF:Ag respectively. The estimated heritability (h2) was 64.5%, 55.72%, and 64.26% for VWF:Ag, ppVWF, and ppVWF/VWF:Ag respectively. A genome-wide association study (GWAS) for VWF:Ag in this cohort revealed significant signals at the ABO and VWF loci, consistent with previously published reports. However, GWAS for ppVWF levels demonstrated a signal only at 12p13, overlying the VWF gene itself. The top SNP, rs1800378 (beta 4.297, p-value 1.58E–11), encodes a nonsynonymous substitution (His484Arg) in the VWF propeptide. In contrast, GWAS for ppVWF/VWF:Ag showed a strong signal at 9q34, the ABO locus, (top SNP rs687289, beta −0.2131, p-value 1.88E–62) and no significant signal at 12p13. These results suggest that ABO is the major common determinant of variability in VWF clearance rates and a common SNP, rs1800378, is associated with VWF propeptide levels. We next utilized the sibling structure in this cohort to perform linkage analyses. Linkage analysis for VWF:Ag identified a novel locus at 2p12-2q13 in addition to the expected signal at ABO. The effect size of this chromosome 2 locus on VWF variation (19.2% variance explained) was comparable to the effect of the ABO locus (24.5%). Linkage analysis for ppVWF demonstrated novel loci at 7q21 (LOD score 5.03) and at 11q21 (LOD score 4.12), with no significant signal on chromosome 2. In contrast, linkage analysis for ppVWF/VWF:Ag identified signals at 2p12 (LOD score 3.17), 8p22 (LOD score 3.48) and 11q22 (LOD score 3.41). Taken together, these results identify a number of potential candidate loci for genetic regulation of plasma VWF through alteration of synthesis, storage, secretion or clearance mechanisms. We hypothesize that signals present in linkage but not association are due to VWF regulatory genes with high allelic heterogeneity, harboring many genetic variants that are individually rare, but in aggregate present in a large portion of sibships. Segregation of these variants among sibs could then explain the strong signal by linkage, but lack of signal by association in unrelated individuals. Replication of the strong 2p12-2q13 signal in both VWF:Ag and ppVWF/VWF:Ag linkage studies suggests that the responsible variants at this locus affect VWF levels through altered clearance. The novel QTLs for ppVWF/VWF:Ag levels on chromosome 8p22 and 11q22 are also likely to work through altered clearance mechanisms while the novel signals for ppVWF levels on 7q21 and 11q21, not significant in the VWF:Ag linkage study, may affect efficiency of cleavage or clearance of ppVWF. Identification of the underlying genes at these novel loci and their mechanism of action could lead to improved understanding of the severity, penetrance and treatment for von Willebrand Disease as well as the risk for venous thromboembolic disease associated with elevated VWF. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Discrepancies Between ABO Genotype and ABO Glycan Phenotypes

Abstract 274The ABO(H) carbohydrate blood group system is highly polymorphic in humans, with >200 ABO alleles described to date. The ABO gene encodes the glycosyltransferase, ABO, which has two main isoforms which generate blood group A or blood group B. Blood group O is the result of a non-functional ABO glycosyltransferase. ABO blood group is commonly imputed in genome-wide association studies where ABO is associated with phenotypes such as von Willebrand Factor level or myocardial infarction. We sought to determine the accuracy of a common ABO genotyping algorithm in a repository of 1092 individuals from a healthy sibling cohort study, the Genes and Blood Clotting Cohort (GABC). We utilized three published single nucleotide variants (SNVs) to assign ABO blood group (A2, B, and O) relative to the A1 reference allele: rs8176704 (G/A) to annotate A2, rs8176749 (C/T) to annotate B, and rs687289 (A/G) to annotate O. We detected ABO glycans in red blood cell-rich buffy coat samples using an adaptation of published ABO forensic dried blood spot techniques. Briefly, samples were diluted and applied to a nitrocellulose membrane, blotted with murine monoclonal anti-A (Immucor) or murine monoclonal anti-B (Immucor) antibodies followed by and streptavidin-conjugated donkey anti-mouse IgG HRP secondary (Jackson ImmunoResearch). Blots were developed, imaged using ImageQuant 350 (GE), and scored semi-quantitatively by two blinded independent observers relative to red blood cell-rich buffy coat reference panels from normal blood donors.Comparison of ABO imputed genotype to detected ABO glycans in red blood cell-rich buffy coat identified discrepancies in the predicted major ABO blood group assignment (i.e. blood group O, A, B, or AB) in 4.4% of individuals. Interestingly, A glycan density was generally low in individuals with a discrepant A phenotype, suggesting that these individuals harbor a less robust blood group A enzyme variant. There was no common pattern for genotype-phenotype discrepancies. Multiple types of errors (both presence and absence of ABO glycans) were observed, and divergent patterns of ABO glycans were observed between homozygotes for ABO SNVs, suggesting multiple ABO alleles or other genetic modifiers underlie the discordant calls. Interrogation of the Exome Variant Server (http://evs.gs.washington.edu/EVS/) found one-third of ABO nonsynonymous polymorphisms in the exome data set are novel, indicating a large proportion of the genetic variation at ABO has not been previously described. Furthermore, the majority of nonsynonymous variants in the EVS database for the genes encoding H antigen (the ABO acceptor structure), FUT1 and FUT2, were also novel.In summary, ABO(H) is more variable genetically than previously suspected. A commonly applied ABO SNV genotyping algorithm resulted in 4.4% frequency of major ABO blood group glycan discrepancy as detected in red blood cell-rich buffy coats, likely due in large part to the underlying genetic diversity at ABO. Further work is needed to deeply characterize the genetic diversity at ABO, FUT1, and FUT2 and correlate the spectrum of allelic variants with ABO(H) carbohydrate phenotypes. Such high resolution ABO and H classification would likely advance our understanding of the complex physiologic processes influenced by ABH blood groups, including disorders of hemostasis and thrombosis, vascular disease, infectious disease susceptibility, complications of pregnancy, and allogeneic exposures. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

A Novel FVIIa Variant with Increased Potency and Duration of Effect Compared to Wildtype FVIIa. A Study in a Dog Model of Hemophilia A

Abstract 2252Hemophilia patients are treated by replacement therapy, receiving the plasma-derived or recombinant clotting factor in which they are deficient. A significant number of hemophilia patients, however, develop inhibitory antibodies against the factor they receive, becoming refractory to replacement therapy. In these inhibitor patients, hemostasis can be achieved using bypass agents, activated plasma-derived prothombin complex concentrates (aPCC) or recombinant activated Factor VIIa (FVIIa). However, these treatments do not match the efficacy seen in traditional replacement therapy. To overcome this limitation, 813, a modified recombinant human Factor VIIa with enhanced biological properties was developed using a rational protein design approach. 813 was selected from a series of FVIIa variants and is characterized by increased catalytic activity and prolonged duration of effect in vivo. Compared to recombinant wild-type FVIIa, 813 has 7-fold increased catalytic activity, measured by the rate of Factor Xa generation in vitro, both in the presence and absence of tissue factor (TF).813 was studied in Factor VIII (FVIII)-deficient dogs with NovoSeven RT (wt-FVIIa) as comparator. The FVIII-deficient dogs exhibit a severe hemophilia A phenotype, with less than 1% normal coagulant activity. Dogs were dosed intravenously with 813 or wt-FVIIa and blood and plasma were collected at various time points post dosing. Plasma FVIIa levels were measured using a FVIIa-specific clotting assay. Starting from initial peak plasma levels, a time-dependent decrease in FVIIa plasma concentration was observed for wt-FVIIa and variant. Compared to wt-FVIIa, 813 exhibited a smaller volume of distribution (35–48 mL/kg vs. 108–134 ml/kg), a lower rate of clearance (10–18 mL/hr/kg vs. 45 mL/kg/hr) and consequently an increased dose adjusted exposure. Hemophilia A dogs have a prolonged activated partial thromboplastin time (aPTT); treatment with wt-FVIIa or 813 led to a dose-dependent decrease in aPTT. At equivalent doses the peak effect of 813 on decreasing aPTT was approximately two-fold compared to wt-FVIIa. Similar dose-dependent effects were observed when studying whole blood clotting profiles by thromboelastography (TEG). Treatment effects were seen for all TEG parameters monitored (time to clotting onset, rate and strength of clot formation). However, compared to wt-FVIIa, 813 given at equivalent doses caused significantly stronger effects on all TEG parameters; acute peak effects on TEG parameters measured for 813 at 10 ug/kg, were similar to those seen with 50 ug/kg wt-FVIIa. This difference became even more pronounced when monitoring treatment effect over time. All dogs used in this study were monitored throughout the study for possible treatment-related changes; both 813 and wt-FVIIa were well tolerated. No overt adverse events, no significant changes in plasma clinical chemistry or cellular blood counts were observed.In conclusion, 813 is a novel FVIIa protein with increased potency, prolonged duration of effect and a preclinical safety profile comparable to wt-FVIIa. 813 is currently in early clinical development for acute and prophylactic treatment of hemophilia patients with inhibitors. Disclosures:Pittman:Pfizer: Employment. Weston:Pfizer: Employment. Shields:Pfizer: Employment. Parng:Pfizer: Employment. Arkin:Pfizer: Employment. Madison:Catalyst Biosciences: Employment. Nichols:Pfizer: Research Funding. Fruebis:Pfizer: Employment.

Whole-Exome Sequencing to Identify Mutations in Thrombosis Modifier Genes Isolated From a Factor V Leiden-Dependent Sensitized ENU Suppressor Screen in the Mouse

Abstract 1183Only ∼10% of individuals carrying the common venous thrombosis risk factor, Factor V Leiden (FVL) will develop venous thrombosis in their lifetime. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen, based on the perinatal synthetic lethal thrombus previously observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The genome-wide ENU mutagenesis screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the otherwise lethal FVQ/Q Tfpi+/− genotype. As proof of concept, we demonstrated that reduced tissue factor (Tf+/−) suppresses the lethal FVQ/Q Tfpi+/− phenotype, suggesting that mutations at Tf should be among the suppressor genes identified by our screen. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Extensive genotyping of mice from an expanded genetic cross from one of these lines mapped a candidate suppressor locus to a chromosome 3 region encompassing the TF gene (LOD=4.93). With continued improvements in next generation sequencing technologies, we have now applied whole exome sequencing to analysis of 8 of the remaining 13 lines. The entire DNA coding region (the “exome”, totaling 49.6 Mb of DNA sequence) from a progeny-tested member of each line was captured using the Agilent SureSelect mouse exome capture system. Whole-exome sequencing using the Illumina HiSeq high-throughput sequencer yielded 12–15 gigabases of sequence data per sample, corresponding to an average of ∼200 fold sequencing coverage for each nucleotide position. Variant analysis using the Gene Analysis Toolkit revealed the presence of a small number of high confidence novel heterozygous (dominant) variants in each sample. Each of these heterozygous variants is a candidate suppressor mutation and these are presently being tested in remaining FVQ/Q Tfpi+/− mice from each respective line. Based on previous studies where ENU-induced mutations in mice have been identified, we anticipate the identification of putative exomic mutations in approximately 80% of tested suppressor lines. Identification of these mutations should provide candidate modifier genes for FVL and other human hemostatic disorders. Disclosures:Ginsburg:Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Catalyst Biosciences Signs Two Key Deals

Catalyst Biosciences, inventor of Alterase™ therapeutics, has secured two big deals: one with Wyeth for hemophilia products including the lead product, CB 813, and another deal with MedImmune to develop drug candidates against two targets, of which one will address inflammatory and autoimmune diseases.