Sickle cell disease is a hereditary condition resulting from a genetic mutation in HBB, which encodes the β-subunit of hemoglobin. It affects approximately 300,000 to 400,000 neonates worldwide each year (16). Current evidence suggests that hemoglobin containing mutant β-globin subunits tends to polymerize, leading to the sickling of erythrocytes, rendering them susceptible to hemolysis. The severity of SCD varies significantly and is often reflected in serum HPX levels. HPX has been identified as a mitigating factor in the pathophysiological processes contributing to the SCD phenotype, including microvascular stasis, NO scavenging, vaso-occlusion, increased levels of proinflammatory cytokines/chemokines, proinflammatory macrophages, acute kidney injury (AKI), silent cerebral infarction, immune system activation, and more. Recent studies have provided compelling evidence of HPX’s beneficial effects on SCD, as outlined in Table 1 . For example, Vinchi F et al. discovered that HPX plays a role in preventing heme-iron accumulation in the cardiovascular system, thereby limiting the production of reactive oxygen species (ROS), increasing endothelial nitric oxide synthase (eNOS), promoting heme detoxification, and more (22). These molecular-level changes were associated with increased blood pressure and ventricular remodeling. Exogenous administration of HPX was found to nearly normalize blood pressure and improve cardiac function (22). Pulmonary hypertension (PH) is a common complication in SCD, occurring in 6-10% of the SCD population and often resulting in right ventricular dysfunction (17). Buehler et al. used an SCD murine model to simulate PH and right ventricular dysfunction, characterized by oxidation and fibrosis in SCD patients (18). They confirmed that HPX could alleviate cardiopulmonary disease in SCD mice by preventing heme-driven oxidative protein modification in the vasculature and ventricle (18). Another study revealed that chlorine inhalation triggered acute hemolysis and induced Acute Chest Syndrome in SCD mice, while post-exposure HPX administration reduced mortality and mitigated lung injury (23). In a clinical study, lower HPX levels were observed in SCD patients experiencing vaso-occlusive crises compared to patients in a steady state, further supporting the potential benefits of HPX administration in SCD (19). An additional preclinical study found that intravenously injected HPX effectively alleviated vascular stasis induced by either Hb injection or hypoxia-reoxygenation in a dose-dependent manner (20). Notably, pre-complexed HPX with heme also resolved stasis, suggesting inherent protective effects of the HPX-heme complex beyond reducing free heme levels (20). Researchers have disclosed that HPX deficiency promoted AKI in SCD, whereas HPX supplementation protected SCD mice from AKI (24, 25). Increased free heme and depletion of HPX led to the deposition of complement factor 3 (C3) and membrane attack complex in the glomeruli of patients with SCD (21). Furthermore, Poillerat V et al. found that HPX prevented heme-mediated complement activation in both plasma and the kidneys (26). In line with previous research, a recent study revealed that hemopexin could protect factor I activity in vitro, facilitating the degradation of soluble and surface-bound C3b, thus inhibiting complement activation and subsequent vascular and organ injury (27). Notably, Gotardo et al. developed a chronic hemolysis model in 2023, which demonstrated a decrease in Hp and HPX in accordance with chronic hemolytic anemia, thus enhancing our understanding of animal models in this field (28).

During transfusions, a portion of RBCs may undergo hemolysis within macrophages. Stored RBCs for transfusion undergoing “storage lesion” may compromise the quality of the blood bag, causing Hb and heme release, and adverse vascular effects including hypoperfusion and impaired endothelial function (29, 30). Transfusing RBCs stored for over 14 days has been associated with adverse outcomes in hospitalized patients (31). Despite the clear need for blood transfusions, several clinical trials and pre-clinical studies have shown that RBC administration can have detrimental effects (32, 33). Exposure to toxic substances resulting from hemolysis, including Hb, heme, and iron, is the primary cause of transfusion reactions. This results in the saturation of transferrin and reduced plasma levels of HPX and Hp, leading to the accumulation of labile plasma iron, heme, and Hb (15, 34). Brant M. Wagener et al. highlighted the crucial role of heme in the adverse effects associated with transfusions of stored RBCs (33). Restoring sequestration proteins has been suggested as a potential method to protect against tissue injury post-transfusion. A preclinical study investigated the effects of exogenous HPX on disease pathology in mice with hemorrhagic shock after transfusing stored red blood cells (SRBCs). The findings supported the protective effect of HPX in improving the survival rate and dampening the inflammatory response, although HPX did not prevent SRBC-induced hemoglobinuria and kidney injury (35). The study by Brant M. Wagener et al. demonstrated that HPX improved the survival rate of mice subjected to massive resuscitation with stored RBCs and infected with P. aeruginosa K-strain, offering evidence for HPX administration as a potential therapy to enhance the safety of SRBC transfusions. While Hp prevented hemoglobinuria, HPX showed no substantial effect (33). It is worth noting that the administration of HPX pre- or post-transfusion has been minimally investigated in clinical trials.

Iron is a crucial element for redox chemistry and represents a fundamental requirement for pathogens (36, 37). Transfusions of RBCs can result in increased concentrations of non-transferrin-bound iron, which can enhance pathogen proliferation and exacerbate existing infections, thus complicating the condition (33, 38). Pathogens such as Staphylococcus aureus, Haemophilus influenzae, and Candida albicans have developed mechanisms to release hemolytic factors that enable them to assimilate iron from hemoproteins or free heme (39, 40). They have also evolved other means to acquire iron from the host, including the expression of siderophores, hemophores, and heme uptake systems (41). For instance, Haemophilus influenzae can dislodge heme from hemopexin, a process facilitated by the Hxu operon, which encodes genes responsible for the expression of HxuA/HxuB/HxuC and is essential for extracting heme from hemopexin (42–45). Porphyromonas gingivalis employs HmuY as a hemophore with a distinct structure that resists proteolysis by proteases secreted for heme utilization (46). In contrast to extracting heme from host hemoproteins, Porphyromonas gingivalis secretes proteases to degrade hemoglobin, haptoglobin, and hemopexin, releasing heme for binding to HmuY (47). In this case, hemopexin promotes pathogen proliferation ( Figure 2 ).

However, for pathogens that are unable to utilize hemopexin, it hinders iron acquisition. For example, the HasA/HasR system, which directly binds to heme, has been identified in pathogens like Serratia marcescens, Pseudomonas aeruginosa, and Yersinia species, potentially competing with hemopexin for free heme. A recent study found that IL-22-induced hemopexin contributes to nutritional immunity against Citrobacter rodentium by limiting iron acquisition by pathogens ( Table 2 ) (50). In a study by Brant M. Wagener, hemopexin improved P. aeruginosa-induced pulmonary edema formation, post-pneumonia survival in mice undergoing massive resuscitation with stored RBCs, and reduced lung bacterial colony-forming units (CFUs) (33). HPX is also suspected of participating in the nutritional defense against Yersinia pestis. Higher levels of HPX induced by EV67 have been reported to prolong survival and reduce pathogen proliferation in mice challenged with a virulent strain of Y.pestis, making HPX a promising adjuvant therapy. The utilization of hemoglobin iron depends on a relay network of extracellular hemophores, namely, Csa2, Rbt5, and Pgt7. Specifically, Csa2 binds to hemin, while Rbt5 and Pgt7 bind to heme, facilitating heme extraction from hemoglobin and its transfer across the cell envelope (48, 49, 51). Mariel Pinsky et al. revealed that hemopexin could inhibit hemin utilization by Candida albicans only when the hemin concentration was lower than an equimolar concentration (52). The inhibitory effect of hemopexin was mitigated by the presence of human serum albumin (HSA), and hemin was gradually transferred to hemopexin from HSA rather than initially binding to hemopexin when released in a 1:50 HPX-HSA mixture.

Sepsis arises from the dysregulation of the host’s immune responses to infection, leading to life-threatening organ dysfunction. Recent estimates indicate that nearly 50 million cases of sepsis were recorded worldwide, with 11.0 million sepsis-related deaths reported, accounting for 19.7% of all global deaths in 2017 (53). Although age-standardized incidents and mortality rates have decreased significantly from 1990 to 2017, sepsis remains a significant global health burden (53).

It is now understood that sepsis exposes red blood cells to various physiological stressors, increasing the risk of hemolysis and the release of cell-free hemoglobin into circulation. One of the most recognized causes of hemolysis during sepsis is disseminated intravascular coagulation (DIC), a common complication of sepsis (54). During sepsis, red blood cells also undergo deformation, driven by cytokines and pathogen-specific factors such as hemolysins produced by Staphylococcus aureus, Enterococcus spp, and Escherichia coli (55–57). Consistent with the process of sepsis-induced hemolysis, the plasma level of cell-free hemoglobin significantly increases in sepsis patients, positively correlated with mortality (58, 59). Accordingly, septic patients with lower hemopexin levels tend to experience death earlier than those with higher hemopexin levels. It has been found that the concentrations of hemopexin in the plasma of non-survivors are significantly lower than those in survivors with sepsis (60, 61). In pre-clinical studies (62). Larsen et al. investigated the role of heme and HPX in a polymicrobial sepsis murine model. They administered exogenous heme to low-grade polymicrobial infection mice, which markedly increased markers of organ dysfunction and sepsis severity. In cases of fatal severe sepsis after high-grade infection, reduced serum concentrations of HPX have been reported. However, when HPX was administered to mice after high-grade infection, it prevented tissue damage and lethality (62).

The protective role of HPX is attributed to its ability to neutralize the oxidative and cytotoxic effects caused by heme. To carry out this beneficial process, the expression of OH-1 is required to catabolize HPX-bound heme (62). Moreover, HPX can suppress the systemic growth of E.coli in C. rodentium, shedding light on its therapeutic potential in sepsis (50). Therefore, HPX can be regarded as a potential therapeutic strategy for preventing fatal consequences in individuals with severe sepsis (63). However, there has been a scarcity of pertinent pre-clinical and clinical trials conducted to elucidate the impact of HPX replenishment in sepsis.

There were 247 million malaria cases in 2021, resulting in the loss of 61,900 lives in 2021, according to the World Health Organization (WHO). Malaria is characterized by hemolysis, which results in the release of hemoglobin and heme. The ratio of heme to HPX is inversely associated with disease severity and the 6-month mortality rate (64). Over the past 30 years, the incidence of severe dengue hemorrhagic fever cases has increased, with many patients succumbing due to a lack of timely clinical intervention. In DHF, fibronectin, HPX, and transferrin levels significantly rise in all three phases compared to Dengue Fever (DF) (65). Besides, a 2014 study revealed increased levels of HPX and vitronectin in both DF and DHF compared to healthy controls (66), suggesting HPX as a potential biomarker to distinguish between uncomplicated dengue fever and dengue hemorrhagic fever (5).

However, HPX does not always act as a protective factor in infection-related hemolysis. For instance, in a mouse model of Shiga toxin-induced hemolytic-uremic syndrome (HUS), HPX deficiency was protective in resolving HUS pathology (67). HPX^-/- mice exhibited higher survival rates when challenged with Shiga toxin, with reduced renal inflammation characterized by decreased macrophage and neutrophil recruitment and C3c deposition [66]. This finding aligns with a previous study by Spiller et al., which concluded that HPX deficiency was protective in sepsis (68).

Intracerebral hemorrhage is the most common type of hemorrhagic stroke and has the highest mortality rate of all stroke subtypes (69, 70). The rapid accumulation of blood within the brain parenchyma leads to the disruption of the normal anatomy and the increase of local pressure (71–73). Heme has been identified as a proinflammatory substance in the brain, and HPX can target it effectively (73).

HPX is present in the plasma and expressed by neurons and glia in the central nervous system. Intrathecally produced HPX represents another source of HPX in the cerebrospinal fluid (CSF) (74). However, HPX levels in the CSF are approximately tenfold lower than those in circulation, suggesting a relatively low capacity for heme binding in the brain, which can become easily overwhelmed (75). An ex vivo study supported the neuroprotective effects of HPX, as it protects neurons and glial cells from blood-related injuries through antioxidation and downregulation of HO-1 and caspase-3 (76). Preclinical research indicated that higher levels of HPX in the brain improve outcomes after ICH in a mouse model. Elevated local HPX levels were associated with smaller lesion volumes, reduced perihematomal tissue injury, and trends toward decreased hematoma volumes (77). Additionally, mice with higher HPX levels experienced no significant changes in brain iron levels and HO-1 but exhibited increased microgliosis and decreased astrogliosis and lipid peroxidation. This suggests the potential synergy of central and peripheral heme clearance (77).

Co-administration of hemopexin effectively mitigated heme-derived toxicity at both molecular and cellular levels (73). However, an in vitro study revealed increased globin-mediated neurotoxicity when Hp was absent after hemopexin treatment. In contrast, when used in combination with Hp, the neurotoxicity was notably reduced (78). This observation supports the notion that Hp-CD163 plays an important role in activating detoxification pathways. Another hypothesis suggests that hemopexin may destabilize Hb in the absence of haptoglobin, leading to globin precipitation and iron deposition, ultimately exacerbating iron-dependent oxidative cell injury ( Figure 3 ) (78). Consequently, it has been proposed that a combined therapy involving hemopexin and haptoglobin may be more favorable following intracerebral hemorrhage than a hemopexin supplement alone[77]. When administered systemically through intraperitoneal injection, hemopexin exhibited a reduction in blood-brain barrier (BBB) disruption on day 3 in ICH mouse models. However, it had no significant effect on striatal heme content on days 3 or 7, and it did not alleviate neurological deficits, inflammatory cell infiltration, or the viability of perihematomal cells on day 8 (79). This suggests that the administration of hemopexin in this manner is inadequate for allowing hemopexin to penetrate the BBB, which likely accounts for these results.

Subarachnoid hemorrhage is a type of hemorrhagic stroke primarily resulting from the rupture of saccular aneurysms, accounting for 3% of all stroke cases (80). The presence of hemoglobin and its breakdown products in the brain, combined with the toxic cascade initiated during early brain injury, has been extensively studied and is strongly linked to the development of delayed brain injury (81). In a study involving 30 SAH patients, free heme was still detectable in the cerebrospinal fluid after SAH, suggesting saturation of the HPX-CD91 system following SAH (74). Interestingly, elevated HPX levels in the CSF after SAH were associated with a higher likelihood of delayed cerebral ischemia and poorer neurological outcomes, which differed from the effects of HPX in other diseases (74). The underlying mechanism remains to be elucidated, but a possible explanation is that when the level of hemopexin in the CSF is excessively high, it may become deleterious as it impedes the efflux of heme due to its high heme affinity, resulting in intracellular heme/iron overload, which can be toxic to neurons and glia (74). It is worth noting that while bleed size and severity may be controlled, the subtypes of Hp were not in a recent review, emphasizing the need for more studies (82). In addition, CD91 is responsible for ApoE uptake, and reduced ApoE levels were associated with more severe neurological injury and worse outcomes after SAH (83–86). Thus, Hp-HPX replenishment could scavenge Hb and heme while limiting undesired ApoE uptake (82).

In various hemolytic diseases, heme overload often significantly decreases HPX plasma levels, diminishing HPX’s ability to scavenge heme effectively. Identifying an increase in plasma heme levels can aid in diagnosing and developing targeted plasma-based therapeutics (5). The level of HPX is considered a biomarker to assess heme load in many hemolytic pathologies. It was first proposed in 1975 that plasma HPX levels could indicate the severity of hemolysis, and this parameter has since been established to assess the level of hemolysis. For instance, in SCD, HPX levels in plasma increased as hemolysis levels declined in response to hydroxycarbamide treatment (105). Patients with septic shock who experienced lethal outcomes and severe organ injury were found to have reduced HPX concentrations (62, 64). In the case of malaria, the heme to HPX ratio negatively correlated with disease severity, as estimated by severe anemia, respiratory distress, stage 3 acute kidney injury, and the 6-month mortality rate (56). However, the levels of HPX increased significantly in all three phases of dengue hemorrhagic fever, which contradicts serum HPX changes in several other diseases. A higher level of HPX in the CSF was associated with better outcomes in ICH but worse prognosis in SAH (74, 77). The next step may involve measuring HPX levels at different stages of hemolytic diseases to further establish its role in prognosis and treatment in clinical scenarios.

Considering an average plasma concentration of 770 μg/ml of HPX in adults, each milliliter of plasma can bind 6.3 μg of heme, and higher heme levels can deplete HPX unless recycling or rapid compensatory synthesis occurs (1). As discussed earlier, HPX has been evaluated as a therapeutic agent in various heme overload diseases, including SCD, blood transfusions, sepsis, malaria, hemolytic-uremic syndrome, ICH, and SAH. The preponderance of data supporting HPX as a therapeutic target suggests that HPX replenishment remains an adjuvant therapy for hemolytic disorders with high heme stress ( Figure 4 ). Over the years, preclinical models have been employed to evaluate potentially applicable scenarios. Notably, a phase 1 clinical trial is investigating HPX dosage in patients with sickle cell anemia, focusing on safety, tolerability, and pharmacokinetics (NCT04285827). However, HPX administration has only been approved for SCD by the European Commission and FDA in 2020 (20).

Beyond its application in SCD, preclinical trials have provided proof of concept for using HPX in other clinical settings. For example, in transfusion-induced hemolysis, the administration of HPX upon stored RBC transfusions in mice was shown to be partially effective, as discussed earlier. In the context of ICH and SAH, HPX replenishment alone exhibited deleterious effects in several preclinical studies, promoting the administration of both Hp and HPX. Furthermore, the potential benefits of Hb/HPX replacement have been highlighted in β‐thalassemia major and intermedia and hereditary spherocytosis (106).

The limited application of HPX in clinical settings can be partially attributed to its dual effects in different pathologies. For example, renal injury and resultant impairment of renal function are common complications of various hemolytic diseases. However, the effects of HPX on renal function remain to be elucidated. A study found that HPX could prevent complement activation in the kidneys of patients (or mice) with phenylhydrazine-induced hemolysis, substantiating the beneficial effects of HPX as a therapeutic agent, given that C3 deposition is associated with kidney injury (26). However, HPX does not always serve a protective role in hemolysis-associated renal injury. While HPX deficiency promotes AKI in sickle cell mice under hemolytic stress, HPX injection did not alter hemoglobinuria (24, 25). In a Shiga-toxin–induced HUS mouse model, HPX knockout mice displayed improved survival and reduced tubular iron deposition compared to wild-type mice (67).

Aside from HPX-mediated potential harm to the kidney, HPX increases the concentration of Hb in the CNS and promotes the aggregation of Hb or globin, consistent with findings observed in the kidneys. HPX-Hb increases iron-dependent neurotoxicity, which can be attenuated by Hp. These effects have also been shown to be deleterious to the brain, resulting in memory deficits in young mice (107). Therefore, HPX supplementation alone is not indicated for certain hemolytic conditions, while HPX plus Hp supplementation is more advisable when encountering toxicity mediated by iron or heme in pathological conditions. Plasmapheresis is a symptomatic treatment for autoimmune diseases and was investigated as a treatment for SCD patients with multiple organ dysfunction. In a case report, SCD patients refractory to RBC exchange were treated with plasmapheresis. Patients with a significant rise in plasma HPX and Hp levels showed clinical relief, in contrast to patients with minimal increases in HPX and Hp levels (108).

Recently, a hemopexin-Hp fusion protein with binding affinity and detoxification capacity for both heme and Hb was produced, which may contribute to the development of hemopexin-Hp therapies (109). This bifunctional fusion protein was generated using transient mammalian gene expression of Hp integrated with the pro-haptoglobin processing protease C1r-LP co-transfection for recombinant Hp-variant generation. This technology can likely be harnessed to generate multifunctional fusion proteins involved in the entire process of Hb and heme detoxification and clearance (110). Another study focused on increasing HPX yield and enhancing the interaction between heme and HPX (110). Utilizing a recombinant production strategy in human cell lines, Elena Karnaukhova et al. produced recombinant human HPX (rhHPX) by constructing an rhHPX expression plasmid and transfecting it into an HEK293 mammalian cell line (110).

The specific patient populations for which HPX-Hp replenishment is indicated and the degree of effectiveness achievable are beyond our current knowledge. Indeed, more research should be directed towards investigations into the role of HPX in various heme-related diseases and prospective clinical studies.

Historically, early efforts in HPX replacement therapy primarily focused on protein and plasma HPX levels. However, researchers have shifted their focus in recent years toward upregulating HPX expression and generating recombinant human HPX in eukaryotic cell expression systems (111). In mouse models of SCD, upregulating the endogenous hepatic synthesis of hemopexin through gene therapy has ameliorated inflammation and vaso-occlusion. This was demonstrated by quantifying hepatic nuclear Nrf2 expression, HO-1 activity, and NF-κB levels (112). Inspired by the success of gene therapy with adeno-associated virus (AAV) in hemophilia treatment, long-term expression of human HPX (hHPX) with AAV vectors was developed (113, 114). These vectors integrated with the full cDNA sequence of hHPX to prompt continuous hHPX expression for 58 days. Subjects exposed to heme challenges induced by heme infusions and phenylhydrazine survived (114). With the application of precision medicine and genome sequencing, the relationship between HPX genes and individualized drug side effects as well as tumorigenesis needs to be further investigated (115, 116).