Intravascular hemolytic diseases such as sickle cell anemia as well as diseases such as sepsis/DIC in which intravascular hemolysis occurs are frequently complicated by micro- and macrovascular thrombosis but mechanisms underlying this association are unclear. Plasma heme levels in sickle cell anemia are typically 5-30 µM. LPS is a prototypic and potent agonist for peripheral blood monocyte tissue factor expression and by this mechanism contributes to thrombosis in sepsis. Because heme, like LPS, signals by binding to and activating TLR4, we hypothesized that heme would also stimulate tissue factor expression in monocytes and so promote thrombosis in sickle cell anemia. We isolated human peripheral blood mononuclear cells (PBMC) and monocytes, incubated them 4 hours in the presence or absence of 10 µM hemin or 10 ng/ml LPS, made whole cell lysates by freeze-thawing and sonication and measured tissue factor activity using a one-stage clotting assay employing recalcified citrated human plasma. The assay was standardized with recombinant human tissue factor and the tissue factor dependence of the assay was confirmed using the neutralizing anti-human tissue factor antibody ATF. Hemin stimulated PBMC TF activity ca. 40-fold (range 6- to 296-fold) from 53 to 1885 pg/ml/million cells, an extent comparable to that of LPS (1939 pg/ml/million cells). In isolated monocytes heme increased TF activity ca. 70-fold (range 25-115-fold) to 9800 pg/ml/million cells, again comparable to LPS (8500 pg/ml/million cells.) Heme stimulation of monocyte TF expression did not reflect endotoxin contamination of our hemin preparation because it was 1) reproduced with a preparation of hemin made for infusion into patients with intermittent porphyria that had no detectable endotoxin; 2) unaffected by addition of 1 µg polymyxin B, which abrogated LPS stimulation; 3) blocked by 15 µM hemopexin, a high affinity heme-binding protein. qRT-PCR of TF expression in monocytes shows a 140-350-fold increase in TF mRNA levels over baseline between 2 and 4 hours after exposure to heme. Examining pathways for heme signaling, we demonstrate that heme stimulation of TF expression in monocytes is impaired > 90% by inhibitors of TLR4 (TAK242), NADPH oxidase (diphenylene iodonium), PKC (5 µM calphostin) and ERK ½ (10 µM U-0126), impaired 75% by the p50 NF-kB inhibitor 10 µg/ml andrographolide and unaffected by 100 nM wortmannin, an AKT/PI3K pathway inhibitor. We conclude that heme, at concentrations found in intravascular hemolytic diseases such as sickle cell anemia, is a highly potent stimulant of blood monocyte TF transcriptional expression dependent upon signaling through TLR4, PKC, NADPH oxidase, ERK ½ and NF-kB. By this direct mechanism heme may promote thrombosis and contribute to the pathogenesis of sickle cell anemia and other diseases characterized by intravascular hemolysis, including sepsis/DIC. Disclosures: No relevant conflicts of interest to declare.

1481 Thiocyanate (SCN-) is, unexpectedly, the principal physiologic substrate for eosinophil peroxidase (EPO) and a major (i.e., accounting for 50% of H2O2 consumed) substrate for myeloperoxidase (MPO). The product of these reactions is HOSCN, a weak, exclusively sulfhydryl-reactive oxidant that we have previously shown to be a uniquely potent (up to 100-fold) oxidant transcriptional inducer of human umbilical vein endothelial cell (HUVEC) tissue factor (TF), ICAM-1 and VCAM-1 expression via a mechanism dependent upon NF-κB p65/p50 activation. We hypothesized that oxidative activation of p38 MAPK is a necessary and proximal step in the activation of NF-κB p65/p50 at the TF, VCAM-1 and ICAM-1 promoters. To test this we utilized the p38 MAPK-specific pyridinyl imidazole inhibitor SB 203580 (SB). In HUVEC monolayers exposed 4h to 150 μM HOSCN in M199 medium containing 10% FCS, SB strongly inhibited (>90%, ED50 300 nM) TF activity, decreased VCAM-1 expression assessed by western blot by > 50% but had no discernible effect upon ICAM-1 expression. qRT-PCR analysis confirmed that the effects of SB on these three molecules was transcriptionally mediated. Immunoprecipitation of HOSCN-treated HUVEC whole cell extracts with a polyclonal anti-phospho-p38 MAPK reagent showed rapid (within 1 min) phosphorylation of p38 MAPK that lasted >30 min with consequent kinase activity documented by detection of the downstream target product phospho HSP27. Confocal immunofluorescence confirmed the rapid and durable induction of phospho p38 MAPK from undetectable to abundant, primarily cytoplasmic, but also some nuclear localization. We further hypothesized that the disparate effects of SB on these three molecules reflects differences in the sequences of their NF-κB-binding sites that affect their ability to bind the p65 NF-kB subunit. Chromatin immunoprecipitation (ChIP) analysis of HUVEC treated for 3h with HOSCN using an anti-p65 antibody revealed that SB inhibited by 75% the 10-fold increase in binding of p65 to the authentic endogenous VCAM-1 and NF-κB binding loci but had no discernible effect on p65 binding to the ICAM-1 locus. An identical pattern was seen in HUVEC exposed 30 min to the prototypical inflammatory cytokine TNF. To test the physiologic significance of these findings we assessed the effect of SB upon PMN and eosinophil binding to HOSCN-exposed HUVEC monolayers treated as described above. SB pretreatment of HUVEC blocked by > 80% the 3–8-fold increase in binding of PMN and eosinophils that occurs in HOSCN-exposed monolayers. Blocking antibodies to ICAM-1 and VCAM-1 demonstrate that both of the adhesion molecules contribute to HOSCN-induced PMN and eosinophil adhesion. We conclude that HOSCN generated by PMN and eosinophils attached or subjacent to vascular endothelium has the capacity to rapidly activate endothelial p38 MAPK-dependent activation of p65 binding to the TF and VCAM-1, but not ICAM-1, NF-κB binding loci; and these differences manifest in physiologically relevant transcriptional regulation of protein expression. Therefore, p38 MAPK inhibitors may have anti-thrombotic and anti-inflammatory potential, the latter particularly in states, such as allergic inflammation, that are most dependent on VCAM-1. Disclosures: No relevant conflicts of interest to declare.

PMN and eosinophils (EO) activated by physiologic agonist such as LPS, C5a, GM-CSF and IL-5 form extracellular traps (ET) comprised of extracellular DNA, histones, and active secondary granule constituents that retain microbicidal capacity and thus contribute to host defense but also participate in the pathogenesis of sepsis, microangiopathy, vasculitis, asthma and thrombosis. Previous studies have implicated superoxide, H2O2 and myeloperoxidase (MPO) in PMN ET formation, which reflects a novel death pathway dubbed “ETosis.” Thiocyanate (SCN-) is the principle physiologic substrate for eosinophil peroxidase (EPO) and a major substrate for MPO. Because previous studies of ET were all performed in the absence of SCN- and HOSCN, the product of SCN- peroxidation, is a weak, sulfhydryl-specific oxidant markedly less toxic than HOCl or HOBr, we hypothesized SCN- blocks PMN and EO ET formation. We also hypothesized that heme, elevated levels of which occur in intravascular hemolytic states such as sickle cell disease, induces PMN ETosis because both LPS and heme signal by engaging TLR4. We assessed ET formation by human PMN stimulated with either GM-CSF + C5a or 10 µM heme in RPMI one hour using glass slide-attached leukocytes with both cell impermeant (Sytox Orange) and permeant (Syto13) DNA stains and IF localization of secondary granule proteins using confocal microscopy. ET formation in response to to both stimuli was 5-20% of PMN in stimulated cells (vs. 0% in control). The NADPH oxidase inhibitor DPI, MPO inhibitor 4-ABAH and 1 mM SCN- all diminished ET formation by >80% in response to both stimuli. Heme-dependent ET formation was inhibited 80% by the TLR4 antagonist TAK242 (see figure below in which intracellular DNA stains green and extracellular and membrane-compromised PMN DNA stains orange). PMA-induced PMN ET formation requires singlet O2 resulting from the secondary reaction of HOCl with excess H2O2. In PMN stimulated with GM-CSF + C5a, the singlet O2 scavenger edaravone (10 µM) inhibited ETs by 80%. ETs also formed in 4-8% of EOs stimulated by IL-5 + C5a, vs. 0% in control EO and 22.5% in unstimulated EO supplemented with the alternative EPO substrate 1 mM Br-. The EPO inhibitor resorcinol decreased by >90% EO ET formation stimulated with by IL-5 + C5a or by adding Br-, as did addition of SCN- or edaravone. These studies show that both PMN and EO ETosis depend upon MPO- and EPO- generated oxidants, respectively, and the presence of an alternative physiologic substrate, SCN-, markedly antagonizes ETosis. We propose that SCN- inhibits ET formation by generating HOSCN that reacts with H2O2 to yield cyanate, not singlet O2. In addition, we also demonstrate that heme is a potent inducer of PMN ETosis through a mechanism dependent upon TLR4, NADPH oxidase and MPO, raising the possibility that this pathway may directly promote the inflammation and thrombosis that contribute of the pathogenesis of sickle cell disease. Based on these findings, we speculate that dietary augmentation of SCN- to normal or supranormal levels might have clinically beneficial therapeutic effects in a variety of inflammatory states, including sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

3205 Hypothiocyanous acid (HOSCN) is the predominant physiologic product of eosinophil peroxidase (EPO) and a major (50%) product of myeloperoxidase (MPO), a product of H2O2 oxidation of the pseudohalide substrate thiocyanate (SCN-). We have previously shown that this sulfhydryl-specific, cell-permeant oxidant, unlike HOCl, can potently induce expression of several NF-kB regulated genes, including the adhesion molecules ICAM-1 and VCAM-1, in human umbilical vein endothelial cells (HUVEC) through a transcriptional mechanism reflecting NF-κB activation. We here tested the hypothesis that HOSCN generated within activated PMN and eosinophils (EO) promotes their adhesion to ICAM-1 and VCAM-1 by enhancing the avidity for their leukocyte integrin counterligands, CD11b/18 (Mac-1) and CD49d/29 (VLA-4), respectively. We isolated human PMN and EO and tested the influence of reagent HOSCN upon their adhesion to purified human ICAM-1 and VCAM-1 immobilized in the bottom of bovine serum albumin (BSA)-blocked of tissue culture plate wells using a method based on crystal violet staining of nuclei and spectrophotometric assay of acetic acid extracts of adherent cells. 3–150 μM HOSCN enhanced adhesion to ICAM-1 of EO 10–18-fold over baseline binding and PMN 2–3-fold. This increase was inhibited >90% by both CBRM1/5, a blocking antibody against CD11b/18 and M1/70, which blocks ICAM-1, showing the specificity of this interaction. Similar concentrations of H2O2 did not significantly influence EO and PMN adhesion. HOSCN did not affect PMN or EO binding to immobilized VCAM-1. Pretreatment of EO with 33–150 μM HOSCN for 15 min prior to washing out HOSCN and layering them over TNF-α-activated HUVEC monolayers for 30 min stimulated a 5–15-fold increase in both EO and PMN adhesion that was > 75% inhibited by blocking antibodies against ICAM-1 and VCAM-1 (EO only) as well as CD11b. Assessing the potential mechanism behind HOSCN enhancing of CD11b/18-dependent EO and PMN adhesion, flow cytometry experiments showed HOSCN did not induce total cell surface expression of EO CD11b/18, but confocal microscopy showed prominent clustering of CD11b 20 min after exposure. Hypothesizing that the “stress” kinase p38 MAPK plays a role in HOSCN promotion of EO adhesion, we probed western blots whole cell lysates of EO exposed 1–10 min to 75 μM HOSCN for activated phospho-p38 MAPK and found a pronounced (10x baseline) but evanescent (1–2 min long) increase starting at 2 min. 1 μM SB203580, a potent and specific p38 MAPK inhibitor, blocked by >80% EO and PMN HOSCN stimulation of adhesion to both immobilized ICAM-1 and activated HUVEC. Collectively, these data show that HOSCN is a uniquely effective phagocyte oxidant inducer of both EO and PMN binding to ICAM-1 by a mechanism depending upon activation of p38 MAPK and enhancement of the CD11b/18-ICAM-1 interaction. This, in turn, suggests that endogenous MPO- and EPO-generated HOSCN may play a hitherto unsuspected physiologic autocrine amplification role in the regulation of adhesion and, hence, inflammation. In view of this, p38 MAPK inhibitors may have promise as therapeutic inhibitors in pathologic human inflammatory states, especially allergic diseases involving EOs. Disclosures: No relevant conflicts of interest to declare.