Management of hemolytic transfusion reactions

Delayed hemolytic transfusion reactions (DHTRs) in patients with sickle cell disease are underappreciated and potentially fatal. Patients with DHTRs typically have symptoms of pain or dark urine days to weeks following a red blood cell (RBC) transfusion. In instances of DHTRs with hyperhemolysis, the patient's hemoglobin (Hgb) may be significantly lower than it was pretransfusion, and the Hgb A may drop by more than 50%. In most cases, at least 1 RBC alloantibody and sometimes multiple RBC alloantibodies can be identified during the DHTR, with those antibodies presumably having fallen below the level of detection at the time of the implicated transfusion. However, in up to one-third of cases, no new RBC alloantibodies can be identified posttransfusion. Complement is increasingly being appreciated to play a role in DHTRs and hyperhemolysis, not only due to classic pathway activation (with complement fixed antibody bound to RBCs) but also due to alternative pathway activation (resulting in part from plasma free heme). As such, anti-C5 inhibition has recently been reported to be effective at mitigating hemolysis in the setting of some severe DHTRs. Transfusion avoidance during DHTRs is recommended if possible, with long-term transfusion support advice being less clear; for example, a history of a severe DHTR may lead to questions regarding the safety of transfusions prior to curative therapies such as stem cell transplantation or gene therapy. A better understanding of antibody-positive and antibody-negative DHTRs, including patient- or disease-specific risk factors, is necessary to improve transfusion safety.

Oxidative Stress and Pathogenesis in Malaria

Malaria is a highly inflammatory and oxidative disease. The production of reactive oxygen species by host phagocytes is an essential component of the host response to Plasmodium infection. Moreover, host oxidative enzymes, such as xanthine oxidase, are upregulated in malaria patients. Although increased production of reactive oxygen species contributes to the clearance of the parasite, excessive amounts of these free radicals can mediate inflammation and cause extensive damage to host cells and tissues, probably contributing to severe pathologies. Plasmodium has a variety of antioxidant enzymes that allow it to survive amidst this oxidative onslaught. However, parasitic degradation of hemoglobin within the infected red blood cell generates free heme, which is released at the end of the replication cycle, further aggravating the oxidative burden on the host and possibly contributing to the severity of life-threatening malarial complications. Additionally, the highly inflammatory response to malaria contributes to exacerbate the oxidative response. In this review, we discuss host and parasite-derived sources of oxidative stress that may promote severe disease in P. falciparum infection. Therapeutics that restore and maintain oxidative balance in malaria patients may be useful in preventing lethal complications of this disease.

Successful Use of Plasma Exchange Preceding RBC Exchange in Sickle Cell Disease Hyperhemolytic Crisis with Hepatic Sequestration: A Case Report

Introduction Hyperhemolytic crisis is an uncommon complication of SCD that may cause multiorgan failure and lead to significant mortality. There are no current national or international guidelines for management of hyperhemolytic crisis and associated complications. There have been limited number of case reports and series that demonstrated utility of plasma exchange in the patients with multiorgan failure resulting from hemolysis complications (Zaidi GZ et al.,2020). We are presenting the case where hyperhemolytic crisis was complicated by hepatic sequestration and acute liver failure, that was dramatically reversed by 2 plasma exchange treatments followed by RBC exchange. Case report We present a case of a 35-year-old African American male with SCD and beta thalassemia trait with frequent hospitalizations for sickle cell pain crisis. He presented with pain typical for his acute pain crises and was admitted for intravenous hydration and pain control. The next morning, lab work showed bicytopenia with a drop in hemoglobin from 10.5 to 5.8 g/dL and platelets (PLT) from 100 to 22 X10E3/uL. Lactate dehydrogenase (LDH) increased from 434 to 2848 U/L, haptoglobin was 36 mg/dL, but disseminated intravascular coagulation (DIC) and Heparin-induced Thrombocytopenia (HIT) antibody panel were negative. The blood urea nitrogen (BUN) creatinine (Cr) ratio was also elevated (30.6) suggesting renal damage as well. He was transferred to the intensive care unit and started on Intravenous Immunoglobulin (IVIG) 0.4 grams/kilogram daily for 5 days and methylprednisolone 500 mg daily for 2 days followed by a prednisone taper. Liver enzymes continued to trend upward with AST of 19,866 U/L and ALT of 3,675 U/L on day 3 of hospitalization. Ultrasound of abdomen demonstrated mild splenomegaly with a spleen measuring 13.3 cm. The clinical presentation and hepatocellular pattern of injury was consistent with hepatic sequestration crisis. Despite receiving 1 unit of platelet 3 units of pRBC, there was little improvement and apheresis service was consulted. Plasma exchange was initiated for 2 procedures on consecutive days followed by RBC exchange with rapid improvement in clinical status and laboratory findings with a reduction of LDH (1304), AST (129), ALT (204), Hgb (8.0), PLT (41), BUN/Cr (20.0). He was discharged on day 7 at baseline status. Discussion Although the mechanism of development of hyperhemolysis in SCD is not fully understood, the hemolysis leads to release of free hemoglobin (Hb) and free heme that activate neutrophils, and vascular endothelial cells via TLR-4. This ultimately leads to inflammatory, coagulative, and cytotoxic damages and decreased nitric oxide (NO) bioavailability which further contributes to SCD complications such as pulmonary and systemic vasculopathy, pain crisis and acute chest syndrome and multi organ failure (Louie JE et al., 2018). This provides a rationale for plasma exchange - removal of free heme from the patient plasma and replenishing exhausted haptoglobin and hemopexin reserves from donor plasma. Hemolytic crisis causing visceral organ damage is relatively rare. There are no current guidelines for management of such patients. In 1996 Betrosian et al. discussed the first case of liver failure in a SCD with vasa-occlusive crisis treated with RBC and plasma transfusions (Betrosian A et al., 1996). Since then, there have been case reports/series of plasma exchange/plasma transfusions in SCD with multi organ failure (Geigel EJ et al., 1997, Louie JE et al., 2018) but reports about use of plasma exchange in SCD patients with hepatic sequestration have not been identified by our literature review. Our case demonstrates that plasma exchange in hyperhemolysis and hepatic sequestration is: Safe Leads to quick and significant improvement in hemolysis laboratory values. Results in quick and durable reversal of hepatic sequestration and associated liver failure. Adds plasma exchange as therapeutic apheresis modality in addition to previously accepted RBC exchange. Provides data about priority of plasma exchange over RBC exchange in this clinical situation. Disclosures No relevant conflicts of interest to declare.

Heme-Induced Platelet Mitochondrial Oxidant Production Regulates Platelet Granule Release

Background: Patients with Sickle Cell Disease (SCD) are at greater risk for thrombosis and the development of chronic vasculopathy, both of which are major contributors to morbidity and mortality in these patients. While thrombosis and vasculopathy are associated with hemolysis in SCD, the molecular mechanisms by which hemolysis propagates these conditions remains unclear. At the cellular level, we and others have shown that hemolytic components, including hemoglobin and its degradation product, free heme, directly activate platelets. Notably, activated platelets are not only central to thrombosis, but are also implicated in vasculopathy through their degranulation, which results in the release of vasoactive molecules. Though heme and hemoglobin-induced platelet activation has been widely studied, the effect of hemolytic products on platelet degranulation and the identity of the resulting platelet secretome remains less clear. We hypothesize that free heme activates a platelet signaling cascade resulting in platelet degranulation and the release of specific "secretome" molecules. Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate (n=6). Platelets were treated with heme (2.5µM) in the presence or absence of MitoTEMPO (10 µM) - a mitochondrial oxidant (mtROS) scavenger, or ARQ092 (10 µM), a small molecule that prevents phosphorylation of the serine/threonine kinase Akt at S473. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. Thrombospondin-1 (TSP1), CXCL7, Fibroblast Growth Factor (FGF) basic, TGFβ, IL-1β, PDGF-B, angiostatin, kininogen, CD40L and PAI-1 were quantified in heme treated platelet releasates in the presence and absence of MitoTEMPO by dot blot. The enzymatic activity of mitochondrial electron transport complex V was measured spectrophotometrically by kinetic assay. Results: We found that heme stimulated the release of a specific set of molecules from the α-granule of platelets, including TSP1, CXCL7, FGF basic, TGFβ, IL-1β, PDGF-B, angiostatin, and kininogen; but did not stimulate the release of CD40L or PAI-1. Mechanistic studies demonstrate that the release of several of these molecules was dependent on heme-induced activation of platelet Akt which inhibits mitochondrial complex V, resulting in mtROS production. Consistent with this mechanism, the heme-stimulated release of TSP1, CXCL7, FGF basic, IL-1β, PDGF-B, and angiostatin were significantly attenuated by preventing Akt phosphorylation at Ser473 with ARQ092, which also prevented complex V inhibition and mtROS production. Direct scavenging of mtROS with MitoTEMPO also attenuated heme-induced release of these molecules. Conclusion: These data, for the first time, begin to characterize the platelet secretome released in response to free heme. Further, they demonstrate a novel molecular pathway in which extracellular heme induces the activation of platelet Akt to inhibit mitochondrial complex V, ultimately inducing mtROS. Notably, this study suggests that release of a proportion of the heme-induced secretome is regulated by mtROS production and can be suppressed by mtROS scavengers. Ongoing studies are further characterizing the hemolysis-induced platelet secretome, the downstream effects of secretome products on vascular function, and the extent of regulation of the secretome by mtROS. These studies provide a mechanistic link between hemolysis and platelet degranulation, by which the release of mitogens can lead to the pathogenesis of vascular wall dysfunction. These studies also suggest that platelet mtROS may represent a novel therapeutic target to attenuate vascular dysfunction in hemolytic disorders including SCD. Note: The finding discussed in the above abstract are available as preprint in bioRxiv; doi: https://doi.org/10.1101/2021.08.02.454816 Disclosures No relevant conflicts of interest to declare.

Natural compounds as heme oxygenase-1 inducers to reduce the deleterious consequences following SARS-CoV-2 infection

: The virus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) causes COVID 19 (COronaVIrus Disease 19), a global pandemic with multi-organ failure and resulting in high morbidity and mortality. Some individuals are more vulnerable than others and have deleterious consequences following covid- 19. It has been postulated that Heme oxygenase-1 (HO-1) reduction and free heme may contribute to many of the inflammatory phenomena observed in COVID-19 patients. Therefore, HO-1 inducers could prove to be potential therapeutic or preventive agents for COVID 19. Many of the natural compounds present in fruits and vegetables, such as polyphenols, resulted able to induce HO-1. Aim of this review is to focus on the main foods containing bioactive compounds able to induce HO-1 for an informed choice of foods to use to counteract damage from SARS-CoV-2 infection.

Induced antigen-binding polyspecificity in human serum IgA

Recent studies have shown that polyspecific antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyspecificity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgE and IgG antibodies to acquire polyspecificity following contact with protein-modifying agents has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using Western blotting and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyspecificity.

Effects of Iron Supplements on Heme Scavengers in Pregnancy

In malaria endemic countries, anemia in pregnant women occurs as a result of erythrocyte destruction by Plasmodium infections and other causes including malnutrition. Iron supplementation is recommended as treatment of iron-deficiency anemia. Erythrocyte destruction results in increased release of cytotoxic free heme that is scavenged by haptoglobin (Hp), hemopexin (Hx) and heme oxygenase-1 (HO-1). Paradoxically, iron supplementation in pregnant women has been reported to enhance parasitemia and increase levels of free heme. The relationship between free heme, heme scavengers, and birth outcomes has not been investigated, especially in women who are on iron supplementation. We hypothesized that parasite-infected pregnant women on routine iron supplementation have elevated heme and altered expression of heme scavengers. A cross-sectional study was conducted to determine the association between plasma levels of free heme, HO-1, Hp, Hx, and malaria status in pregnant women who received routine iron supplementation and their birth outcomes. Heme was quantified by colorimetric assay and scavenger protein concentration by ELISA. We demonstrated that iron-supplemented women with asymptomatic parasitemia had increased free heme (mean 75.6 µM; interquartile range [IQR] 38.8–96.5) compared with nonmalaria iron-supplemented women (mean 34.9 µM; IQR 17.4–43.8, P < 0.0001). Women with preterm delivery had lower levels of Hx (mean 656.0 µg/mL; IQR 410.9–861.3) compared with women with full-term delivery (mean: 860.9 µg/mL; IQR 715.2–1055.8, P = 0.0388). Our results indicate that iron supplementation without assessment of circulating levels of free heme and heme scavengers may increase the risk for adverse pregnancy outcomes.

Hemozoin (beta-hematin) formation inhibitors; A promising target for the development of new antimalarials: Current update and A future prospect

Background: Malaria is responsible for a social and an economic burden in most low-income malaria-affected countries. Thus, newer antimalarials are needed to tackle morbidities and mortalities associated with the drug-resistant malarial strains. Haemoglobin digestion inside the food vacuole of malarial parasite would lead to producing redox-active and toxic-free heme. The detoxification process adopted by Plasmodium sp. would give rise to hemozoin (Hz) (beta-hematin) formation. Targeting the pathway of hemozoin formation is considered as a validated target for the discovery of newer antimalarials. Objective: This study aims to collect detailed information about aspects of hemozoin (Hz) (beta-hematin) inhibitors. Methods: A systemic search has been carried out using PubMed, Google Scholar, CNKI, etc., for relevant studies having the keyword, ' hemozoin or beta-hematin' for almost the last 2 decades (2000-2021). Results: This mini-review tries to summarize all the recent advancements made for the developments of synthetic, natural isolated phytoconstituents and plant extracts inhibiting the hemozoin (beta-hematin) formation. Conclusion: thus, would act as promising antimalarial candidates in near future.

Introducing Negatively Charged Residues on the Surface of Fetal Hemoglobin Improves Yields in Escherichia coli

Fetal hemoglobin (HbF) has been developed into an important alternative protein for oxygen therapeutics. Such applications require extensive amounts of proteins, which only can be achieved via recombinant means. However, the expression of vertebrate hemoglobins in heterologous hosts is far from trivial. There are several issues that need to be dealt with. These include, among others, the solubility of the globin chains, equimolar expression of the globin chains, and access to high levels of free heme. In this study, we examined the impact of introducing negative charges on the surface of HbF. Three different HbF mutants were examined, carrying four additional negative charges on the α-subunit (rHbFα4), two additional negative charges on the γ-subunit (rHbFγ2) or a combination of these (rHbFα4/γ2). The increase in negative surface charge in these HbF mutants required the development of an alternate initial capture step in the downstream purification procedures. For the rHbFα4 mutant, we achieved a significantly enhanced yield of purified HbF with no apparent adverse effects on Hb functionality. However, the presence of non-functional Hb portions in the rHbFγ2 and rHbFα4/γ2 samples reduced the yields significantly for those mutants and indicated an imbalanced expression/association of globin chains. Furthermore, the autoxidation studies indicated that the rHbFγ2 and rHbFα4/γ2 mutants also were less oxidatively stable than rHbFα4 and wt rHbF. The study further verified the need for an improved flask culture protocol by optimizing cultivation parameters to enable yield-improving qualities of surface-located mutations.

Excess heme upregulates heme oxygenase 1 and promotes cardiac ferroptosis in mice with sickle cell disease

Sickle cell disease (SCD) is characterized by increased hemolysis which results in plasma heme overload and ultimately cardiovascular complications. Here, we hypothesized that increased heme in SCD causes upregulation of heme oxygenase 1 (Hmox1) which consequently drives cardiomyopathy through ferroptosis, an iron-dependent non-apoptotic form of cell death. First, we demonstrated that the Townes SCD mice had higher levels of hemopexin-free heme in the serum and increased cardiomyopathy, which was corrected by hemopexin supplementation. Cardiomyopathy in SCD mice was associated with upregulation of cardiac Hmox1, and inhibition or induction of Hmox1 improved or worsened cardiac damage, respectively. Since free iron, a product of heme degradation through Hmox1, has been implicated in toxicities including ferroptosis, we evaluated the downstream effects of elevated heme in SCD. Consistent with Hmox1 upregulation and iron overload, levels of lipid peroxidation and ferroptotic markers increased in SCD mice, which were corrected by hemopexin administration. Moreover, ferroptosis inhibitors decreased cardiomyopathy, whereas a ferroptosis inducer erastin exacerbated cardiac damage in SCD and induced cardiac ferroptosis in non-sickling mice. Finally, inhibition or induction of Hmox1 decreased or increased cardiac ferroptosis in SCD mice, respectively. Together, our results identify ferroptosis as a key mechanism of cardiomyopathy in SCD.