Heme Potently Stimulates Tissue Factor Expression By Peripheral Blood Monocytes: A Novel Mechanism For Thrombosis In Intravascular Hemolytic Diseases

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.