ALBERT

Description: High doses of recombinant factor VIIa (FVIIa) have been found to bypass factor IX or factor VIII deficiency and ameliorate the bleeding problems associated with hemophilia patients with inhibitors. Recent studies show that FVIIa also acts as an effective hemostatic agent in other categories of patients, and thus has become a promising candidate for prevention and treatment of excessive bleeding associated with many other diseases/injuries. Although recombinant FVIIa has proven to be a very effective and safe drug in the treatment of bleeding episodes in hemophilia patients with inhibitors and other indications, a small fraction of patients may be refractory to FVIIa treatment. The reason for this is unclear at present, but it is possible that administration of very high pharmacological doses of FVIIa or use of genetically modified FVIIa molecules with increased potencies may circumvent the problem. The most dramatic effect on the activity (a 40-fold increase in proteolytic activity) of FVIIa was obtained by occupying the corresponding positions in thrombin/factor IXa for those positions 158, 296 and 298 of FVIIa (FVIIaDVQ). A FVIIa mutant in which the hydrophobic residue Met 298 was replaced with Gln (FVIIaQ) has 7-fold higher proteolytic activity. In the present study, we investigated the interactions of FVIIaQ and FVIIaDVQ with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT) in solution and at the vascular endothelium. Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type FVIIa in the absence of TF. In the presence of TF, TFPI, TFPI-Xa and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the wild-type FVIIa failed to generate significant amounts of factor Xa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of factor Xa on unperturbed endothelium (1 nM of factor VIIa generated 0.3 ± 0.15 nM factor Xa/h whereas FVIIaQ and FVIIaDVQ generated 1.26 ± 0.1 nM/h and 9.48 ± 1.32 nM/h, respectively). Annexin V fully attenuated the FVIIa-mediated activation of factor X on unperturbed endothelial cells whereas anti-TF IgG had no effect. On stimulated HUVEC, FVIIa and FVIIa variants activated factor X at similar rates (30–40 nM/h). AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. AT inhibition of FVIIa bound to stimulated endothelial cells requires exogenous heparin. Interestingly, TFPI-Xa was found to inhibit the activities of both FVIIa and FVIIa analogs bound to unperturbed endothelial cells. Despite significant differences observed in factor Xa generation on native endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions, probably due to limited availability of anionic phospholipids and/or putative factor Xa and Va binding sites on their cell surface. Over all, our present data suggest that although FVIIa variants may generate factor Xa on native endothelium, the resultant factor Xa does not lead to enhanced thrombin generation on native endothelium compared to FVIIa. These data should reduce potential concerns about whether the use of FVIIa variants triggers unwanted coagulation on native endothelium, and may facilitate the development of FVIIa analogs as effective therapeutic agents in near future for treatment of patients with bleeding disorders.

Description: Acute chest syndrome (ACS) is a major pulmonary complication of sickle cell disease (SCD) with currently no mechanistic-based therapies. Although ACS is more frequent in childhood, survival among children 1-9 years is ~10-fold higher compared to adults 20 years and older. Hitherto, mechanisms responsible for the high ACS survival in children have not been defined. We previously reported that acute elevation of circulating heme causes a TLR4-dependant lethal acute lung injury (ALI) in transgenic mice with homozygous SCD (SS) reminiscent of severe ACS (Ghosh et al., J Clin Invest, 2013). In the current study we discovered that young (4-6 weeks) and adult (12 weeks) SS mice suffer comparable severity of hemolytic crises when challenged with intravenous hemin (35 μmoles/kg bw). However, virtually none of the young mice develop ALI while nearly all the adults succumb with respiratory failure. We discovered that young SS mice rapidly clear excess heme from the circulation suggesting this was the reason for their resistance to intravenous hemin. Interestingly, young SS mice have significantly lower plasma hemopexin levels than adult SS mice, which excludes the classical heme scavenging pathway as the reason for their heme resistance. Previous studies have linked genetically high heme oxygenase-1 (HO-1) expression with low-risk of ACS, and raised steady-state levels of plasma heme with high-risk ACS in children. To explore these associations further, we studied a large cohort of patients and discovered for the first time that the concentration of plasma HO-1 in SCD children 1-9 yrs is 2-fold higher than adults 20 yrs and older (23.6±1.1, n=191 versus mean 10.7±0.6, n=67); these findings were phenocopied in transgenic SS mice. Plasma fractionization experiments revealed that HO-1 co-localizes with the enzymatic and co-factor machinery required for heme degradation indicating that heme can be degraded in the blood circulation. To test this idea ~3 weeks old SS mice were treated with the HO-1 inhibitor tin protoporphyrin (SnPP) or vehicle, and challenged with intravenous hemin five days later to induce ALI/ACS. Plasma HO activity declined by ~76% in SnPP-treated mice and was unchanged in the vehicle treated animals (n=12-14, p

Description: Abstract 3480 Poster Board III-417 Frequent spontaneous joint bleedings in severe hemophilia leads to chronic arthopathy, which significantly reduces the quality of life of these patients. Recent clinical studies suggest that the prophylactic use of rFVIIa markedly reduced the number of bleeding incidents, hospitalization time and days of immobility in hemophilic patients with inhibitors. Interestingly the prophylactic effect of rFVIIa was maintained during the 3-month post-prophylactic follow up (Konkle et al., J Thromb Haemost 2007; 5:1904-13). Given the short biological half-life rFVIIa (∼2 to 3 h), it is unclear how rFVIIa prophylaxis continues to reduce the number of hemorrhagic events in the post-treatment period. It had been hypothesized that rFVIIa administered pharmacologically may reach the extravascular spaces where it could be retained for extended time periods and therefore continue to be available at the site of injury to contribute to the hemostatic plug (Hedner, J Thromb Haemost 2006; 4:2498-500). The present study was aimed to examine the extravascular distribution of pharmacologically administered rFVIIa. Recombinant mouse FVIIa was tagged with AF488 fluorophore (AF488-FVIIa) and the AF488-FVIIa was administered into anesthetized male C57BL/6 mice through the tail vein (120 mg/kg b.w.). At different time intervals following AF488-FVIIa administration (from 10 min to 7 days), mice were exsanguinated and various tissues were collected. The tissues were fixed, processed, sectioned and examined for rFVIIa by immunohistochemistry by using specific antibodies directed against AF488. We also examined the distribution of AF488-rFIX under similar experimental settings. The immunohistochemistry data revealed that rFVIIa given at a pharmacological dose enters the extravascular compartment and accumulates differentially in different tissues. The tissue and cell localization appear to be specific for rFVIIa as we found significant differences between rFVIIa and rFIX distribution following their administration to mice. rFVIIa, immediately following administration, was found associated with the endothelium lining of large blood vessels. This may reflect rFVIIa binding to EPCR as FVIIa has been shown to bind to EPCR and endothelial cells lining large blood vessels have been shown to express EPCR. In contrast to rFVIIa, rFIX failed to associate with the endothelial cells. Within one hour, rFVIIa bound to endothelial cells was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. Although we have noted rFVIIa association with the endothelium in many vascular beds (e.g., bone joints, liver, skin, heart and kidney), it is conspicuously absent in the lung and brain. In liver, rFVIIa was localized initially to the portal vein and the sinusoidal capillaries. rFVIIa was also accumulated in hepatocytes. Although rFVIIa levels in the liver were decreased 6 h after administration, traces of rFVIIa were still seen at both 3 and 7 days following rFVIIa administration. In the bone, large amounts of rFVIIa accumulated in the zone of calcified cartilage for 6 h following administration and were decreased only slightly thereafter. A significant amount of rFVIIa was retained in the zone of calcified cartilage even a week following the administration. The common finding of the present study is that rFVIIa in extravascular spaces was mostly localized to the regions that contain TF expressing cells. For example, in the skin, the sebaceous gland and hair follicles that are rich with TF were shown to sequester FVIIa for a longer time period than other regions where TF is absent. In the kidney, FVIIa was retained in glomeruli that express high levels of TF. Overall, the present study suggests that pharmacologically administered rFVIIa readily associates with the endothelium, probably by binding to EPCR, and then enters into extravascular spaces probably by EPCR-mediated transcytosis where it likely binds to TF and is consequently retained for extended time periods. This may explain the prolonged pharmacological effect of rFVIIa. Disclosures: Hedner: Nono Noridsk: Consultancy. Rao:Novo Nordisk: Research Funding.

Description: Abstract 4836 Sickle cell disease (SCD) is a chronic hemolytic and inflammatory disorder characterized by repeated episodes of vaso-occlusion and hemolysis, resulting in oxidative stress and endothelial dysfunction. We have recently demonstrated that the heme scavenging capacity in SCD is severely impaired, highlighting the danger posed by excess heme in this disorder. Paradoxically, heme induces expression of several cyto-protective enzymes including the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in glutathione (GSH) synthesis, which is a crucial antioxidant in the lung. While the induction of cytoprotective enzymes is thought to attenuate the deleterious effects of heme in SCD the somatic origin of this protection has not previously been defined. Using transgenic mouse models we show for the first time that the level of GCLM in the sickle lung is markedly up-regulated due primarily to enhanced expression of the enzyme in the epithelium and blood mononuclear cells, but not in the endothelium. Based on these findings, we tested the hypothesis that leukocyte-derived GCLM was sufficient to protect the sickle lung from oxidative stress. Thus, bone marrow chimeric SCD mice with GCLM deficiency were generated by transplanting bone marrow from Berkeley SCD transgenic mice into GCLM null mice recipients. We confirmed that the chimeric GCLM-null-SCD mice had a SCD phenotype as determined by 〉95% engraftment of donor white blood cells, reticulocyte counts, urine osmolality and hemoglobin gel electrophoresis. Whole lung GCLM and total GSH levels in the chimeric mice were identical to the levels in the wild-type SCD mice. Moreover, lung function, as determined by oxygen saturation and breath rate, were identical in the two mouse strains. These results show that loss of GCLM expression in resident lung cells does not compromise production of GSH or the function of the lung in SCD. Disclosures: Ofori-Acquah: Emory University: Patents & Royalties.

Description: Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa, and the formation of TF-VIIa complexes on cell surfaces trigger the coagulation cascade and cell signaling. It is a well-known fact that only a small fraction of TF at the cell surface is coagulantly active whereas a majority of TF on the cell surface is non-functional (cryptic). However, it is unclear, at present, how the coagulant active TF differs from the cryptic form, and mechanisms involved in TF activation. Recent studies show that a thiol oxidizing agent, HgCl2, increases TF coagulant activity on the surface of HL-60 cells by several fold (Chen et al., Blood vol 106, abstract #684, 2005). Further, TF is shown to associate with protein disulfide isomerase (PDI) in HaCaT cells (Ahamed et al., Blood vol 106, abstract #685, 2005). Based on these and other observations, it has been proposed that switching between cryptic and coagulant TF involves cleavage and formation of allosteric disulfide bond (Cys186-Cys209) and PDI has been implicated in controlling the conversion of cryptic TF to the coagulant form and to act as a switch between TF-mediated signaling and coagulation. Although these data are interesting and novel, there is no fail-proof evidence that disulfide switching alone and not other potential changes, such as exposure of anionic phospholipids, at the cell surface is responsible for the TF activation associated with various treatments. Therefore we have examined the effect of HgCl2 and other treatments on TF activation in MDA 231 cells in relation to anionic phospholipids and also characterized the cellular expression of PDI in this and other cell types. As reported earlier, the HgCl2 treatment increased the cell surface TF coagulant activity (5-fold or higher). However, the HgCl2 treatment also increased the prothombinase activity by 3-fold. More importantly, annexin V, which binds to anionic phospholipids, markedly reduced the increased TF coagulant activity associated with the HgCl2 treatment whereas it had only minimal and insignificant effect on TF activity of the control cells. Further, pretreatment of cells with 5,5′-dithio-bis(2-nitronezoic acid) (DTNB), a sulfhydryl reagent that reacts with thiol groups and thus can block disulfide switching, failed to prevent the increase in TF activity associated with the HgCl2 treatment. Interestingly, we found that treatment of MDA 231 cells with glutathione (5 to 100 mM), a cell impermeable reducing agent, also increased the surface TF activity by about 2- to 3-fold. In additional studies, we found that PDI antibodies had no effect on either the TF coagulant activity or TF-mediated cell signaling. Further, we found no evidence for the expression of PDI at the cell surface in immunofluorescence confocal microscopy as both monoclonal and polyclonal PDI antibodies failed to stain nonpermeabilized cells whereas they brightly stained intracellular PDI in permeabilized cells. In contrast, TF antibodies stained intensely the surface of both nonpermeabilized and permeabilized cells. Exposure of tumor cells to various proteases failed to transport the intracellular PDI to the cell surface. The present data raise a valid question whether disulfide switching by PDI plays the predominant and general regulatory role in controlling the TF coagulant activity and signaling functions. Our data also suggest that other cellular changes, including increase in anionic phospholipids, may be responsible for increased TF coagulant activity associated with the thiol oxidizers and other treatments.

Description: Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates the cellular defense mechanism by mediating a coordinate induction of cytoprotective antioxidant responsive element-driven genes. Nrf2 agonists augment fetal hemoglobin expression in hematopoietic progenitors and have emerged as a new class of drugs for therapeutic induction of fetal hemoglobin in sickle cell anemia (SCA). However, the cytoprotective effect of Nrf2 on the pathobiology of SCA has not been previously defined. To investigate the role and mechanism of Nrf2 in SCA independent of globin gene modulation, we generated chimeric mice with disruption of Nrf2 in non-hematopoietic tissues. A total of twenty-six Nrf2-/- mice were transplanted with bone marrow harvested from the Berkeley sickle and hemizygous (Hemi) mice and the Townes sickle (SS) and control (AA) mice. All sickle/Nrf2-null chimeras (SSNHNrf2-/-; n=13) developed classical hematological and intravascular hemolysis features of SCA after eight weeks of transplantation. Despite the presence of erythrocyte Nrf2, SSNHNrf2-/- chimeras developed more severe intravascular hemolysis than SS wild-type (SSWT) donor-litter-mates. The concentration of plasma cell-free Hb (p

Description: Transition from pediatric to adult care is associated with poor prognosis and a sharp rise in mortality in sickle cell disease (SCD). Hitherto, mechanisms that promote the severe adult phenotype of SCD have not been defined. We performed a longitudinal cohort study in thirty-two Townes transgenic mice (SS; n=16, AA; n=16) aged 1 month through to 10 months to identify prognostic factors of SCD severity. Thirty-eight percent of the SS mice died during this period while all the control AA mice survived (Log-rank Mantel Cox test, p

Description: Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilia patients with inhibitors. However, rFVIIa is cleared from the circulation relatively fast, with circulating half-life of about 2–4 h, requiring repeated administration of rFVIIa for the effective treatment. Therefore, development of FVIIa analogs that could remain in the circulation for a longer period of time would be of a great value for improving the treatment options of rFVIIa. e.g., by prophylaxis. PEGylation of plasma proteins was shown to extend their circulatory half-lives but the PEGylation may also disrupt macromolecular interactions. In the present study we characterized the interaction of two glycoPEGylated analogs of rFVIIa, rFVIIa-10K PEG and rFVIIa-40K PEG, with its cofactor tissue factor (TF), substrate factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). Both the PEGylated FVIIa analogs exhibited similar amidolytic activity as of wild-type rFVIIa (wt-rFVIIa) in the absence or presence of relipidated TF. The analogs were as effective as wt-rFVIIa in activating FX in the absence of TF. No significant differences were found between the PEGylated rFVIIa analogs and wt-rFVIIa in TF-dependent FX activation at saturating concentrations of rFVIIa, however, at lower concentrations of rFVIIa (10 to 50 pM), rFVIIa-10K PEG and rFVIIa-40K PEG activated FX at a slightly lower rate, 50% and 75%, respectively, of wt-rFVIIa. Further studies revealed that both AT/heparin and TFPI inhibited the PEGylated rFVIIa-TF complexes effectively but slightly at a lower rate compared to that was noted for wt-rFVIIa-TF. TFPI-Xa inhibited the PEGylated rFVIIa-TF and wt-rFVIIa-TF at a similar rate. On unperturbed HUVEC, wt-FVIIa (10 nM) could activate FX, albeit slowly, (1.7 nM/h) and the PEGylated rFVIIa activated FX even at much lower rates (0.23 nM/h for rFVIIa-10K PEG and 0.15 nM/h for rFVIIa-40K PEG). On stimulated HUVEC expressing TF, the PEGylated rFVIIa variants were slightly less effective at lower concentrations compared to wt-rFVIIa in activating FX, but no significant differences were found among them in activating factor X at saturating concentrations of rFVIIa (80–100 nM/h). The PEGylated rFVIIa analogs bound to cell surface TF were inhibited by TFPI-Xa complex at a similar rate as that was observed for wt-rFVIIa (IC50 in nM: 0.102 ± 0.032 for wt-rFVIIa, 0.111 ± 0.024 for rFVIIa-10K PEG, and 0.096 ± 0.019 for rFVIIa-40K PEG). AT/heparin inhibited rFVIIa-10K PEG bound to endothelial cell TF at a similar rate as it inhibited wt-rFVIIa (IC50 in μg/ml: wt-rFVIIa, 3.42 ± 068; rFVIIa-10K PEG, 3.56 ± 0.073), but the inhibition rate was slightly lower for rFVIIa-40K PEG bound to TF (IC50 5.92 ± 0.44 μg/ml). Overall, our present data suggest that long-acting PEGylated FVIIa analogs retain full enzymatic activity and can interact TF and FX effectively, and are inhibited by AT/heparin and TFPI-Xa as for wt-rFVIIa. Although the pegylated rFVIIa variants exhibited somewhat lower affinity towards TF, this may not critically affect the TF-driven FXa generation. Further work is needed to fully characterize these molecules.

Description: Erythropoiesis occurs in specialized niches in the bone marrow consisting of a central macrophage, surrounded by differentiating erythroblasts. This central macrophage has been identified by several markers including, CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G. These CD169+ macrophages support erythropoiesis both at steady state and during stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of the cellular oxidative defense system. It modulates hematopoietic stem cells but its loss produces no visible phenotype in steady state hematological parameters. However, the importance of Nrf2 and macrophage subsets has not been fully characterized during recovery from stress erythropoiesis. We examined specific subsets of CD169+ macrophage populations in Nrf2 knockout (Nrf2−/−) mice as well as the role of Nrf2 in recovery from stress erythropoiesis in vivo. We quantified the expression of CD169, F4/80 and CD11b, markers of central macrophages, in the BM and spleen of Nrf2+/+ and Nrf2-/- mice at steady state. Surprisingly, Nrf2-/- mice showed a phenotype characterized by lower percentages of cells expressing known macrophage markers. We observed a significant decrease of 47% (p≤0.01), 24% (p≤0.01) and 50% (p≤0.01) in BM macrophage subpopulations expressing F4/80hiCD169hi, F4/80hiCD11bhi and CD169hiCD11bhi respectively, in age-matched Nrf2-/- mice compared to Nrf2+/+ control mice (Fig. 1a). In the spleen, we also observed a similar significant deficiency in BM macrophages (p≤0.01). Further validating this phenotype, immunofluorescence staining of isolated spleen tissue showed that expression of CD169+ macrophages was dramatically lower in spleen sections of Nrf2-/- mice than in Nrf2+/+ control mice. We hypothesized that our macrophage-deficient mice would display a defect in recovery from blood loss. Five to seven days after acute blood loss, immature erythroid progenitors (CD71hiTer119hi) increased in marrow by about 5-fold in Nrf2+/+ mice (p≤0.001, Fig. 1b), and mature erythroid progenitors (CD71loTer119hi) increased in marrow by 12-fold (p≤0.05) but the erythroid marrow response was impaired significantly in the macrophage deficient Nrf2-/- mice. To extend our observations regarding macrophage deficiency and impaired erythroid response, we chose a more functional outcome of recovery from anemia after high-grade blood loss produced by daily phlebotomies over 3 consecutive days to induce stress erythropoiesis in Nrf2+/+ and Nrf2-/- mice. We found significantly lower packed cell volume values specifically on Days 2, 4 and 10, implying delayed erythroid recovery (p≤0.05, two-way ANOVA). The Nrf2-/- mice also showed a significant decline in total hemoglobin than the Nrf2+/+ mice (p≤0.05). Additionally, peripheral blood reticulocyte response to blood loss is delayed in Nrf2 deficient mice compared to age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p≤0.001). We analyzed expression of heme-oxygenase 1 (HO-1), a well-known Nrf2-regulated gene. HO-1 mRNA expression increased 3-fold and 23-fold in Nrf2+/+ mice animals subjected to phlebotomy and hemin treatment compared to 2-fold and 12-fold expression in Nrf2-deficient mice (p≤0.05). We demonstrate for the first time that Nrf2-deficient mice have a deficiency of macrophages that includes subsets considered erythroblastic island (EI) macrophages, and that this deficiency is associated with impaired erythroid response to induced stress. Secondly, our multiple phlebotomies data in aggregate demonstrate that Nrf2-/- mice deficient in BM macrophages have significant delay in functional erythroid response and recovery from experimentally-induced anemia. Thirdly, impaired inducibility of HO-1 is a known feature of Nrf2-/- mice, which we confirmed in our results, could be contributing to the impairment in erythroid response. However, it is not likely that restricted iron trafficking to erythroid progenitors occurs in Nrf2-/- mice, since there is no characteristic alteration of mean corpuscular volume and mean corpuscular hemoglobin in peripheral blood. This is an area worthy of additional investigation. We conclude that the Nrf2 gene plays a previously unappreciated role in erythroid biology that appears to be mediated through macrophage function. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Bayer: Research Funding; Novartis, Global Blood Therapeutics: Consultancy, Research Funding.