Heme is an essential molecule in aerobic organisms. Heme consists of protoporphyrin IX and a ferrous (Fe ²⁺ ) iron atom, which has high affinity for oxygen (O ₂ ). Hemoglobin, the major oxygen-carrying protein in blood, is the most abundant heme-protein in animals and humans. Hemoglobin consists of four globin subunits (α ₂ β ₂ ), with each subunit carrying a heme group. Ferrous (Fe ²⁺ ) hemoglobin is easily oxidized in circulation to ferric (Fe ³⁺ ) hemoglobin, which readily releases free hemin. Hemin is hydrophobic and intercalates into cell membranes. Hydrogen peroxide can split the heme ring and release “free” redox-active iron, which catalytically amplifies the production of reactive oxygen species. These oxidants can oxidize lipids, proteins, and DNA; activate cell-signaling pathways and oxidant-sensitive, proinflammatory transcription factors; alter protein expression; perturb membrane channels; and induce apoptosis and cell death. Heme-derived oxidants induce recruitment of leukocytes, platelets, and red blood cells to the vessel wall; oxidize low-density lipoproteins; and consume nitric oxide. Heme metabolism, extracellular and intracellular defenses against heme, and cellular cytoprotective adaptations are emphasized. Sickle cell disease, an archetypal example of hemolysis, heme-induced oxidative stress, and cytoprotective adaptation, is reviewed. Antioxid. Redox Signal. 12, 233–248.

Red blood cells (RBCs) demonstrate procoagulant properties in vitro, and elevated hematocrit is associated with reduced bleeding and increased thrombosis risk in humans. These observations suggest RBCs contribute to thrombus formation. However, effects of RBCs on thrombosis are difficult to assess because humans and mice with elevated hematocrit typically have coexisting pathologies. Using an experimental model of elevated hematocrit in healthy mice, we measured effects of hematocrit in 2 in vivo clot formation models. We also assessed thrombin generation, platelet-thrombus interactions, and platelet accumulation in thrombi ex vivo, in vitro, and in silico. Compared with controls, mice with elevated hematocrit (RBC^HIGH) formed thrombi at a faster rate and had a shortened vessel occlusion time. Thrombi in control and RBC^HIGH mice did not differ in size or fibrin content, and there was no difference in levels of circulating thrombin-antithrombin complexes. In vitro, increasing the hematocrit increased thrombin generation in the absence of platelets; however, this effect was reduced in the presence of platelets. In silico, direct numerical simulations of whole blood predicted elevated hematocrit increases the frequency and duration of interactions between platelets and a thrombus. When human whole blood was perfused over collagen at arterial shear rates, elevating the hematocrit increased the rate of platelet deposition and thrombus growth. These data suggest RBCs promote arterial thrombosis by enhancing platelet accumulation at the site of vessel injury. Maintaining a normal hematocrit may reduce arterial thrombosis risk in humans. Publisher9s Note: There is an Inside Blood Commentary on this article in this issue. Key Points Elevated hematocrit promotes arterial thrombus formation. During arterial thrombosis, elevated hematocrit enhances platelet accumulation at the site of vessel injury.

Heme oxygenase-1 (HO-1) enzyme plays a critical role in metabolizing the excess heme generated during hemolysis. Our previous studies suggested that during intravascular hemolysis the expression of HO-1 protein is not sufficient to reduce the oxidative burden of free heme in the vasculature. This led us to hypothesize that a post-translational mechanism of control exists for HO-1 expression. Micro-RNAs (miRNA) affect gene expression by post-transcriptional gene regulation of transcripts. We performed in silico analysis for the human HMOX1–3′ untranslated region (3′ UTR) and identified candidate miRNA binding sites. Two candidate miRNAs, miR-377 and miR-217, were cloned and co-transfected with a luciferase vector containing the human HMOX1-3′UTR region. The combination of miR-377 and miR-217 produced a 58% reduction in HMOX1–3′UTR luciferase reporter expression compared with controls. The same constructs were then used to assess how overexpression of miR-217 and miR-377 affected HO-1 levels after induction with hemin. Cells transfected with the combination of miR-377 and miR-217 exhibited no change in HMOX1 mRNA levels, but a significant reduction in HMOX1 (HO-1) protein expression and enzyme activity compared with non-transfected hemin-stimulated controls. Transfection with either miR-377 or miR-217 alone did not produce a significant decrease in HO-1 protein expression or enzyme activity. Knockdown of miR-217 and miR-377 in combination leads to up-regulation of HO-1 protein. Exposure to hemin induced a significant reduction in miR-217 expression and a trend toward decreased miR-377 expression in two different cells lines. In summary, these data suggests that the combination of miR-377 and miR-217 help regulate HO-1 protein expression in the presence of hemin.

Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Erythrocyte sickling, the primary pathologic event in SCD, results in dramatic morphological changes in red blood cells (RBCs) because of polymerization of the abnormal hemoglobin. We used a mouse model of SCD and blood samples from sickle patients to determine if these changes affect the structure, properties, and dynamics of sickle clot formation. Sickling of RBCs and a significant increase in fibrin deposition were observed in venous thrombi formed in sickle mice. During ex vivo clot contraction, the number of RBCs extruded from sickle whole blood clots was significantly reduced compared with the number released from sickle cell trait and nonsickle clots in both mice and humans. Entrapment of sickled RBCs was largely factor XIIIa–independent and entirely mediated by the platelet-free cellular fraction of sickle blood. Inhibition of phosphatidylserine, but not administration of antisickling compounds, increased the number of RBCs released from sickle clots. Interestingly, whole blood, but not plasma clots from SCD patients, was more resistant to fibrinolysis, indicating that the cellular fraction of blood mediates resistance to tissue plasminogen activator. Sickle trait whole blood clots demonstrated an intermediate phenotype in response to tissue plasminogen activator. RBC exchange in SCD patients had a long-lasting effect on normalizing whole blood clot contraction. Furthermore, RBC exchange transiently reversed resistance of whole blood sickle clots to fibrinolysis, in part by decreasing platelet-derived PAI-1. These properties of sickle clots may explain the increased risk of venous thromboembolism observed in SCD.

Vascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. We sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. We assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2^V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. We found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. By using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2^V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte-endothelial adhesion.