Hemolysis and cell-free drive an intrinsic mechanism for human disease

Mark T. Gladwin, … , Tamir Kanias, Daniel B. Kim-Shapiro

J Clin Invest. 2012;122(4):1205-1208. https://doi.org/10.1172/JCI62972.

Commentary

Blood transfusion represents the first and most prescribed cell-based therapy; however, clinical safety and efficacy trials are lacking. Clinical cohort studies have suggested that massive transfusion and/or transfusion of aged stored may contribute to multiorgan dysfunction in susceptible patients. In this issue of the JCI, Baek and colleagues report that aged stored blood hemolyzes after massive transfusion in a guinea pig model. led to vascular and kidney injury that was mediated by cell-free plasma hemoglobin and prevented by coinfusion of the specific hemoglobin scavenger protein, . These studies support an expanding body of research indicating that intravascular hemolysis is a pathological mechanism in several human diseases, including multiorgan dysfunction after either massive transfusion or hemoglobin-based therapy, the hemoglobinopathies, , and other acquired and genetic hemolytic conditions.

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Hemolysis and cell-free hemoglobin drive an intrinsic mechanism for human disease Mark T. Gladwin,1 Tamir Kanias,1 and Daniel B. Kim-Shapiro2

1Vascular Medicine Institute and Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 2Department of Physics and Translational Science Center, Wake Forest University, Winston-Salem, North Carolina, USA.

Blood transfusion represents the first and most prescribed cell-based ther- membrane integrity (e.g., reducing deform- apy; however, clinical safety and efficacy trials are lacking. Clinical cohort ability and increasing rigidity), alter rheo- studies have suggested that massive transfusion and/or transfusion of logical properties, and cause hemolysis aged stored blood may contribute to multiorgan dysfunction in suscepti- and have been proposed to contribute in ble patients. In this issue of the JCI, Baek and colleagues report that aged some way to the risk of transfusion of aged stored blood hemolyzes after massive transfusion in a guinea pig model. blood. However, the epidemiologically Hemolysis led to vascular and kidney injury that was mediated by cell-free established relationship between age of plasma hemoglobin and prevented by coinfusion of the specific hemoglo- transfused blood and risk of adverse clini- bin scavenger protein, haptoglobin. These studies support an expanding cal outcomes is confounded by a number body of research indicating that intravascular hemolysis is a pathological of important variables. First, transfusion mechanism in several human diseases, including multiorgan dysfunction of multiple units of blood increases the after either massive red blood cell transfusion or hemoglobin-based blood probability that an older unit is given, cre- substitute therapy, the hemoglobinopathies, malaria, and other acquired ating uncertainty about the role of massive and genetic hemolytic conditions. transfusion as opposed to that from a stor- age lesion mechanism (2). Second, sicker Blood transfusion and ischemic heart disease; it is also clearly patients receive more units (3). Finally, the storage lesion beneficial as a supportive and exchange O blood group units are more rare and are Blood transfusion is one of the first and therapy for hemoglobinopathies. However, more rapidly depleted from the inventory, most prescribed cell-based therapies. an increasing number of studies suggest so that patients who have type O blood are Despite the frequency with which blood that massive transfusion — defined as the more likely to receive fresh blood, leading transfusion is prescribed, the timing, dose, transfusion of approximately one complete to fundamental differences among groups and established placebo-adjusted benefits blood volume within the first 24 hours in published case-controlled cohort studies of this “drug” have not been established. of resuscitation — may increase the risk of that may influence outcomes (4). Blood transfusion is clearly beneficial in multiorgan dysfunction, respiratory and In order to address the major variables a multitude of clinical conditions, such as renal insufficiency, and death (1–5). confounding the assessment of risk of massive traumatic and surgical hemorrhage, Studies in patients who have undergone transfusing blood stored for a long time, critical , and anemia associated with cardiac surgery or experienced trauma or NIH-funded well-controlled transfusion critical illness have suggested that the age studies in preclinical animal models and Conflict of interest: Mark T. Gladwin and Daniel B. of the transfused blood may relate to the human placebo-controlled clinical trials Kim-Shapiro are named as co-inventors on an NIH risk of adverse clinical outcomes (1). The are being performed. While the clinical government patent for the use of nitrite salts for cardio- vascular conditions. combined effects of storage on red blood trials are underway, because of safety and Citation for this article: J Clin Invest. 2012; cells have been termed the “red blood cell ethical considerations, these trials evalu- 122(4):1205–1208. doi:10.1172/JCI62972. storage lesion.” These effects modulate ate only modest ranges of storage time as

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Figure 1 Mechanisms by which hemolysis and cell-free hemoglobin can medi- ate vascular injury. Intravascular hemolysis releases cell-free hemo- globin (Hb) into the plasma. The hemoglobin is maintained in the fer- rous redox state, which will react with NO in a near-diffusion limited reaction to inhibit NO signaling. Extravasation of hemoglobin in the subendothelial space and reductive recycling back to the ferrous state may enhance this pathological effect. Oxidation of hemoglobin, particularly in the kidney, from the ferric state to the ferryl state may further drive fenton and peroxidase oxidative cellular injury (involving the lipid peroxyl species, LOO•), leading to acute kidney injury. Free and iron promote inflammatory injury via activation of innate immune responses in and monocytes. Compensatory pathways that normally control these NO dioxygenation and oxidation reactions include haptoglobin- and -mediated sequestra- tion of hemoglobin dimer and heme, respectively. Downstream Nrf-2, hemoxygenase, and biliverdin reductase signaling detoxify heme and iron and provide catalytic antioxidant, antiproliferative, and antiinflam- matory protective signaling.

well as limited transfusion volumes and Intravascular hemolysis and associated with iron deposition within the patients with lower severity of illness. It the storage lesion supporting vasa vasorum. Major effects is possible that a deleterious effect of red Using red cell ektacytometry and shear- on renal function were also observed, with cell storage may only be observed with the stress analysis, Baek and colleagues report upregulation of Nrf-2–dependent antioxi- longest storage time, with massive transfu- that red blood rigidity dant gene expression signatures; dilation sion, and/or in susceptible recipients. In increased at differing rates for human and of proximal and distal tubules, which is contrast, preclinical animal model studies guinea pig red blood cells as a function of consistent with tubular degeneration; and uniformly evaluate massive transfusion at storage duration (5), with human blood elevation of levels of creatinine. the extremes of storage duration. stored for 28 days comparable to guinea Importantly, washing the stored red blood In this issue of the JCI, Baek and col- pig blood stored for 14 days and human cells did not prevent these effects, as the leagues evaluate a preclinical animal model blood stored for 42 days (which is the limit aged cells continued to hemolyze intravas- of massive transfusion (5). They report the of storage in the US) comparable to guinea cularly after transfusion. effects in guinea pigs of massive transfusion pig blood stored for 28 days. After massive Given the association between the nega- of either fresh blood stored for 2 days or of 80% of the blood tive effects of transfusion of old blood blood stored for 21 or 28 days. They found volume with leukocyte-depleted stored and substantial intravascular hemolysis, that the older blood caused , blood, substantial intravascular hemoly- resulting in accumulation of reduced fer- vascular injury, and renal insufficiency sis was observed, together with accumula- rous oxyhemoglobin, Baek and colleagues and that this was directly related to the tion of reduced ferrous oxyhemoglobin in infused the high-affinity hemoglobin scav- intensity of posttransfusion intravascular the plasma of animals receiving old blood. enger haptoglobin along with the stored hemolysis and the levels of cell-free, non- While the transfusion of new blood was not blood (5). Upon binding to hemoglobin, haptoglobin–bound, plasma hemoglobin. associated with changes in blood pressure, haptoglobin is cleared from the circula- These findings are consistent with recent old blood was observed to have marked tion via the monocyte/ hap- observations that blood transfusion can acute hypertensive effects, which were toglobin-hemoglobin scavenger receptor mediate endothelial injury and impaired coupled to increases in the NO-consuming CD163. Based on observations that the vascular function as a result of the most properties of plasma and in vivo reduction vasoactive effects, vascular injury, and fundamental of storage lesions, hemolysis of levels of NO metabolites (nitrate and renal Nrf-2–dependent gene expression (6–8). The beneficial effects they observe nitrite). Interestingly, old blood but not changes and dysfunction were ameliorat- with coinfusion of haptoglobin (5) confirm new blood caused coagulative necrosis of ed by coinfusion of haptoglobin with the a mechanism intrinsic to hemoglobin and the aortic arch, from the luminal tunica aged blood, the authors propose that the identify a new potential therapy. intima to the tunica media, and this was pathophysiology of transfusion of aged

1206 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 4 April 2012 commentaries stored blood is intrinsically mediated by layer around the red blood cell, and by an the molecular size of the hemoglobin, the cell-free plasma hemoglobin. These find- intrinsic NO diffusional barrier in the red lesser the hypertensive effect. It has been ings are consistent with recent observa- blood cell submembrane (20). Accordingly, proposed that the large molecular mass tions that the storage lesion may relate, at both the red blood cell and haptoglobin limits extravasation. It is therefore likely least in part, to the fundamental effects of prevent hemoglobin extravasation into the that high mass hemoglobin-haptoglobin hemolysis and cell-free plasma hemoglo- subendothelial compartments in which multimers will not extravasate and will bin on vascular function (6–8). NO scavenging can directly interrupt NO exhibit reduced NO scavenging, and this is diffusion to smooth muscle. consistent with the reduced renal clearance Rust reactions — pleiotropic toxicity The toxicity of intravascular cell-free of free hemoglobin observed by Baek and of cell-free plasma hemoglobin, hemoglobin does not stop at NO scav- colleagues after haptoglobin infusion (5). heme, iron, and oxygen enging. Additional oxidation reactions Future studies to image labeled hemoglo- The new studies of red blood cell storage are catalyzed by hemoglobin, heme, and bin extravasation coupled to studies of in (5–7) support a growing appreciation that iron. Reactions of hydrogen peroxide vivo endothelial NO signaling will be nec- intravascular hemolysis represents a fun- with ferrous and ferric hemoglobin drive essary to test the effects of haptoglobin on damental mechanism for human disease the fenton and peroxidase reactions to hemoglobin extravasation and NO inhibi- (9–11). This concept evolved from clinical generate hydroxyl and ferryl heme radi- tion in the subendothelial compartment. trial observations that trauma patients cals. These oxidation reactions can redox receiving hemoglobin-based oxygen car- cycle, leading to lipid peroxidation and Conclusion — potentially novel riers developed hypertension and multi- cellular injury. Recent studies have high- therapy to mitigate hemolysis- organ injury caused by hemoglobin-NO lighted the importance of this reaction mediated vasculopathy scavenging reactions (12–14) and clinical in the renal injury that is associated with The work of Baek and colleagues, in this observations of vasculopathic complica- myoglobinemia from issue of the JCI (5), supports the hypothesis tions in patients with hemoglobinopathies (21), which appears pathologically similar that intravascular hemolysis represents a and (15, 16). Intravas- to the kidney injury observed by Baek and fundamental mechanism for human disease cular hemolysis releases hemoglobin and colleagues (5). Downstream reactions may (9, 10). The hemoglobin-mediated vasculop- enzymes, such as arginase 1, into plasma. relate to heme and iron release from the athy and end-organ toxicity is likely relevant An efficient hemoglobin and heme scav- hemoglobin protein. Heme can amplify to hemolysis in malaria, cardiopulmonary enging system, consistenting of haptoglo- innate immune responses in (22), bypass, and genetic red cell enzymopathies, bin, CD163, and hemopexin, sequesters while free iron can amplify inflammatory membranopathies, and hemoglobinopa- these redox-active molecules. When these responses after the intracellular uptake thies. A potent role for haptoglobin in limit- scavenging systems are saturated by excess and catabolism of a subset of damaged, ing end-organ injury opens the door to new hemoglobin, cell free plasma hemoglobin stored red blood cells by the monocyte/ therapies targeting hemoglobin and heme accumulates and extravasates within the macrophage system (23). These studies, in sequestration and catabolism. Infusions vasculature and organs (10). sum, highlight the remarkable toxicity of of haptoglobin and hemopexin have now A number of NO depletion and ROS hemoglobin, iron, heme, and oxygen when clearly been shown to limit hemoglobin- generation pathological pathways medi- liberated from the reductive intracellular and heme-mediated toxicity in preclinical ated by cell-free plasma hemoglobin have red blood cell environment. models of blood transfusion and sepsis (22). been defined (Figure 1). A primary effector These findings raise interesting questions is oxyhemoglobin, which is largely main- Hemoglobin complex size and surrounding potential protective effects tained in the reduced state in plasma and extravasation matter of plasma or transfusion to can react in a rapid and irreversible dioxy- An interesting and yet to be explained obser- increase the concentration of haptoglobin genation reaction with NO to form methe- vation of Baek and colleagues is that hap- and hemopexin and provide a rationale for moglobin and nitrate (7, 17). This reaction toglobin binding to hemoglobin reduces the development of haptoglobin-enriched of NO with oxyhemoglobin accounts for the vasoconstrictive effects of hemoglobin, and hemopexin-enriched blood products or the acute vasoactive responses seen with yet paradoxically increases the half-life of recombinant protein preparations. While infusions of hemoglobin-based oxygen hemoglobin in the circulation and does not the high-molecular weight of these mol- carriers and cell-free plasma hemoglobin inhibit the intrinsic reactivity of hemoglo- ecules provides challenges to recombinant (14). It also causes a NO resistance state of bin with NO (5). While the mechanism(s) protein generation and delivery, alternative in diseases such for this was not explored, the observations approaches using aptamer and small-mol- as sickle cell and malaria (9, 18, 19). The are consistent with extravasation of hemo- ecule technology may be successful. The hypertensive effects of cell-free plasma globin and subcellular compartmental NO design and testing of hemoglobin-binding hemoglobin relate to the disruption of inhibition. The mixtures of haptoglobin and hemoglobin-metabolism therapeu- important NO diffusional barriers between used by Baek and colleagues include all tics should be considered for transfusion the endothelium and the normally com- three of the haptoglobin isoforms (1-1, of stored blood, , partmentalized intracellular hemoglobin. 1-2, and 2-2), which form disulfide bonds malaria, and hemoglobinopathies. NO normally is able to signal because the and high-molecular weight multimers. The hemoglobin reactions are effectively shield- vasopressor effect of hemoglobin-based Acknowledgments ed by a cell-free zone between the endothe- oxygen carriers has been shown to cor- M.T. Gladwin receives research sup- lium and red blood cells caused by laminar relate with NO scavenging reaction rates port from NIH grants R01HL098032, flow and the glycocalyx, by an unstirred and with molecular size (14, 17); the higher RO1HL096973, and PO1HL103455; the

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