Influence of red blood cell-derived microparticles upon vasoregulation

Abstract

Here we review recent data and the evolving understanding of the role of red blood cell-derived microparticles (RMPs) in normal physiology and in disease progression. Microparticles (MPs) are small membrane vesicles derived from various parent cell types. MPs are produced in response to a variety of stimuli through several cytoskeletal and membrane phospholipid changes. MPs have been investigated as potential biomarkers for multiple disease processes and are thought to have biological effects, most notably in: promotion of coagulation, production and handling of reactive oxygen species, immune modulation, angiogenesis, and in apoptosis. Specifically, RMPs are produced normally during RBC maturation and their production is accelerated during processing and storage for transfusion. Several factors during RBC storage are known to trigger RMP production, including: increased intracellular calcium, increased potassium leakage, and energy failure with ATP depletion. Of note, RMP composition differs from that of intact RBCs, and the nature and composition of RMP components are affected by both storage duration and the character of storage solutions. Recognised RMP bioactivities include: promotion of coagulation, immune modulation, and promotion of endothelial adhesion, as well as influence upon vasoregulation via nitric oxide (NO) scavenging. Of particular relevance, RMPs are more avid NO scavengers than intact RBCs and this feature has been proposed as a mechanism for the impaired oxygen delivery homeostasis that has been observed following transfusion. Preliminary human studies demonstrate that circulating RMP abundance increases with RBC transfusion and is associated with altered plasma vasoactivity and abnormal vasoregulation. In summary, RMPs are submicron particles released from stored RBCs, with demonstrated vasoactive properties that appear to disturb oxygen delivery homeostasis. The clinical impact of RMPs in transfusion recipients is an area of continued investigation.

Microparticles

Release of small membrane vesicles (termed microparticles [MPs]) by activated and/or apoptotic cells was first described over 40 years ago¹. MPs are formally defined as cell-derived vesicles that are 0.1–1.0 μm in size and are categorised by membrane proteins and cytosolic material specific to various parent cell populations² (Figure 1). MPs are distinguished from exosomes and apoptotic bodies by size, composition and mechanism of formation³ (Figure 2). Exosomes are generally smaller (40–100 nm) and are formed by a multistep process that involves intracellular generation and subsequent extrusion of vesicles; apoptotic bodies are much larger (1–5 μm) and are formed by membrane shedding during apoptosis⁴.

Figure 1.

Cellular microparticles: a mobile storage pool of bioactive effectors.

Membrane microparticles are shed from the plasma membrane of stimulated cells, harbouring cytoplasmic proteins as well as bioactive lipids implicated in a variety of fundamental processes. MHC: major histocompatibility complex; GPI: glycosylphosphatidylinositol. (Adapted with permission from Hugel et al., 2005¹¹³.)

Figure 2.

Size classes of extracellular vesicles.

Exosomes are formed through inward membrane budding, leading to formation of 40–100 nm intracellular vesicles which accumulate within multivesicular bodies that are subsequently released to the extracellular milieu. Apoptotic bodies may contain DNA and/or organelles and are formed during the late stages of apoptosis, after cell shrinkage. Microparticles are formed from the outward blebbing of membrane and released into the extracellular space. (Adapted with permission from Burger et al., 2013³).

Microparticle-cell interaction

Microparticles serve as vehicles for inter-cellular exchange of biological material and information for which two principle mechanisms have been proposed: 1) MPs act similarly to circulating signaling modules, activating receptors on target cells by presenting organised clusters of membrane-associated bioactive molecules⁵; and/or 2) direct transfer of MP content, including proteins, bioactive lipids or RNA to recipient cell. These mechanisms result in phenotypic modification and reprogramming of cell functions⁶. As such, MP-based cell-cell communication enables a unique form of remote signalling from parental to target cells by presenting a complex arrangement of receptor ligands for membrane receptors that is paired with concentrated payloads of bioactive molecules and substrate for intracellular delivery⁷.

Microparticles as biomarkers of disease processes

Microparticle formation is enhanced under conditions of stress and injury and so MPs have been investigated as potential disease biomarkers. As such, it is important to recognise that circulating MP levels are determined by the balance between MP formation and clearance. MP levels, in particular platelet, leucocyte and endothelium-related MPs, are known to increase in states of vascular injury, pro-thrombotic and proinflammatory states. These include diabetes⁸, pulmonary hypertension⁹, chronic kidney disease¹⁰, preeclampsia¹¹, atherosclerosis¹², and heart failure¹³, amongst others.

Formation of microparticles

Microparticles arise from diverse cell types, including those related to vessels (endothelial cells, and vascular smooth muscle cells)¹⁴, blood (erythrocytes¹⁵, platelets and leucocytes), cardiomyocytes¹⁶ and podocytes³, as well as various cancers¹⁷ and progenitor cell populations¹⁸. MP formation occurs by outward blebbing and shedding of the plasma membrane¹⁹. This poorly understood process appears to involve two main steps: 1) initial cytoskeletal re-organisation that precedes outward membrane budding²⁰ (actin filament rearrangements are essential to this process and are influenced by a number of intracellular enzymes including calpain²¹, rho kinase²² and transglutaminase²³) (Figure 3); and 2) externalisation of phosphatidylserine (PS), a negatively charged aminophospholipid found almost exclusively on the plasma membrane inner leaflet in healthy cells²⁴. PS “sidedness” is controlled by an ATP/calcium-dependent system involving 3 distinct enzymes: flippase, floppase and scramblase²⁵. Of note, defective PS externalisation underlies Scott syndrome, a bleeding disorder associated with diminished platelet MP shedding²⁶.

Figure 3.

Mechanisms proposed for cytoskeleton remodelling leading to microparticle (MP) formation.

Under normal conditions, aminophospholipids (phosphatidylserine and phosphatidylethanolamine) are found exclusively on the inner leaflet of the plasma membrane. During MP formation, membrane asymmetry is lost as aminophospholipids redistribute to the outer leaflet of the plasma membrane. Cytoskeletal re-organisation results in the outward blebbing of the plasma membrane and may be dependent upon actin polymerisation, caspase 2/Rho kinase, calpain and/or transglutaminase 2. Such processes may vary between different cell types. MP formation appears to occur selectively in lipid-rich microdomains (lipid rafts/caveolae) within the plasma membrane. (Adapted with permission from Burger et al., 2013³). MP: microparticles.

Studies of cultured cells have identified several stimuli for MP formation, including: hormones, fatty acids, reactive oxygen species (e.g. hydrogen peroxide)²⁷ and increased intracellular calcium levels²⁸. Activation of several surface receptors has also been shown to drive MP production, such as by tumour necrosis factor (TNF)-α²⁹ on monocytes, leucocytes and neutrophils, as well as by pro-inflammatory (lipopolysaccharide³⁰, shiga toxin³⁰, and cytokines³¹) and pro-coagulant ligands (thrombin³², collagen³³, and norepinephrine³⁴) on platelets and Toll-like receptor 4 on dendritic cells³⁵ (Figure 4).

Figure 4.

Stimuli for microparticle (MP) formation from platelets, endothelial cells and leucocytes.

A summary of the stimuli which promote MP formation from platelets, endothelial cells and leucocytes. A23187: calcium ionophore A23187; TRAP: thrombin receptor activating peptide; NE: norepinephrine; ROS: reactive oxygen species; EPO: erythropoietin; IL-6: interleukin 6; TNFα: tumour necrosis factor α; LPS: lipopolysaccharide; sCD40I: soluble CD40 ligand; PMA: phorbol 12-myristate 13-acetate; FMLP: formyl-methionyl-leucyl-phenylalanine; ANCA: anti-neutrophil cytoplasmic antibodies; CRP: c-reactive protein; Ang II: angiotensin II; IL-1α: interleukin 1α; PAI-1: plasminogen activator inhibitor-1. (Adapted with permission from Burger et al., 2013³).

Microparticle clearance

Microparticle elimination via the mononuclear phagocyte system (MPS) is believed to regulate circulating MP availability for target-cell fusion; however, less is known about this process than about MP formation³. Macrophages ingest co-cultured MPs and externalised PS is thought to signal scavenger receptors promoting MP endocytosis³². IgM expressed on the MP surface has also been shown to stimulate MP clearance by macrophages³⁶.

Microparticle-mediated biological effects

Although cell activation enhances MP shedding, exocytosis is a constitutive process for the majority of cell types³⁷. Depending on the stimulus, however, protein content of both “cytoplasm” and membrane for MPs derived from the same cell lineage can vary³⁸. Moreover, the enzymes that govern plasma membrane remodelling during MP shedding can be selective, depending on the activating agonist and/or the microenvironments of parental cells^39,40. Such tight regulation of MP production suggests that MPs may be important mediators of cell-cell communication. Of note, MPs are internalised by a variety of cells (macrophages and endothelial cells amongst others) in a dose-dependent manner, enabling “MP cargo” transfer between cells in a fashion that influences both functional and phenotypic characteristics of the target cells⁴¹.

The most well characterised processes influenced by MPs include the following items.

1) Coagulation

Coagulation is perhaps the most clearly established example of MP biology. Platelet-derived MPs have effects similar to activated platelets in initiation of thrombin generation and clot propagation, despite having at least 2-orders-of-magnitude difference in surface area⁴². Moreover, externalised phospholipids (mainly PS) create a negatively charged surface that anchors cationic domains of proteins involved in assembly of the multi-component complex involved in the thrombin burst⁴³.

2) Oxidative stress

Microparticles of different derivation, produced under various stimuli, have been shown to affect the enzymatic systems controlling reactive oxygen species generation. Both endothelial and monocyte-derived MPs are known to increase superoxide⁴⁴ and hydrogen peroxide production⁴⁵, as well as to uncouple nitric oxide synthase (NOS)⁴⁶. However, activated T-cell-related MPs have been shown to decrease reactive oxygen species production and to increase nitric oxide (NO) production⁴⁷.

3) Inflammation

Pro-inflammatory stimuli provoke MP shedding; MPs may also directly contribute to the inflammatory response in an amplifying signalling loop (e.g. PMN-derived MPs promote endothelial IL-6 and monocyte chemotactic protein release)⁴⁸. MPs are also thought to promote inter-cellular inflammatory cell interaction and adhesion; specifically, endothelial MPs increase expression of endothelial adhesion molecules and facilitate monocyte-endothelial cell interactions⁴⁹, in addition to binding to monocytes and promoting transendothelial migration⁵⁰.

4) Angiogenesis

Platelet-derived MPs have been implicated in regulation of angiogenesis. In rats, following myocardial ischemia, platelet MPs increase both post-ischaemic capillary density and proliferation⁵¹, and are reported to influence survival and tube formation of human umbilical vein endothelial cells⁵². This is not surprising, as platelets are known to contain at least 20 angiogenesis-regulating factors. Moreover, certain stimulated T-cell-related MPs have been shown to inhibit angiogenesis both in vivo and in vitro⁵³.

5) Apoptosis

Endothelial and monocyte MPs appear to promote cellular senescence and apoptosis in circulating angiogenic and endothelial progenitor cells, respectively^54,55. This process is thought to be dependent upon phagocytosis of MPs that contain high amounts of membrane arachidonic acid, leading to caspase activation and apoptosis initiation⁵⁶.

Red blood cell microparticles

Microparticle formation from red blood cells (RBCs) occurs during maturation and is accelerated during RBC storage⁵⁷. RBC MPs are generally smaller than MPs of other origin, are more homogenous in size (approximately 0.15 μm in diameter), and are often accompanied by smaller vesicles, termed nanovesicles⁵⁸. Of note, during their 120-day lifespan, RBCs lose approximately 20% of their volume through vesicle emission, increasing intraerythrocytic haemoglobin (Hb) concentration by approximately 14%. Metrics for production rates, circulating number and volume are presented in Table I⁵⁹. It was originally thought that vesiculation served to rid RBCs (which lack lysosomes) of damaged or harmful components that may accumulate over time, such as denatured Hb, C5b-9 complement attack complexes or Band 3 neoantigens⁶⁰. It has also been suggested that RMP shedding promotes recognition and clearance of senescent and/or damaged RBCs by removing integral self-marker membrane proteins (e.g. CD47)⁶¹.

Table I.

Estimated total circulating number, volume and rate of production of RMP, intact RBCs and their respective ratios in a healthy adult male (Willekens et al., 2008⁵⁹).

	Total circulating number			Volume per RMP/RBC			Rate of production
	RMP		RBC	RMP		RBC	RMP		RBC
Absolute number	8.5×108		2.5×1013	0.065 μm3		88 μm3	5.8×108/sec		1.4×106/sec
RMP:RBC ratio		3.4×10−5:1			7.38×10−4:1			8,120:1

RMP: red blood cell-derived microparticles; RBC: red blood cell.

RMP production

Red blood cells spontaneously shed PS-positive MPs⁶²; each RBC is estimated to generate approximately 230 vesicles during its lifespan⁶³. Similar to other cell types, membrane phospholipid rearrangement is thought to be an integral step in RMP formation. The normal asymmetric distribution of the lipid bilayer is controlled by 3 different elements; flippase (ATP-dependent enzyme that promotes inward orientation of negatively charged lipids), floppase (responsible for maintaining outward orientation of phosphatidylcholine), and scramblase (facilitating bidirectional movement of all phospholipids)⁶⁴. During RBC storage, ATP depletion and potassium leakage reduce flippase activity, while elevated intracellular calcium increases scramblase activity; consequently membrane asymmetry is lost, PS is exposed on the RBC surface, and vesicle shedding is promoted⁶². There is some controversy with regard to the RBC sub-population most responsible for RMP production. Some suggest that senescent RBCs are responsible for the majority of RMP production in vivo⁶⁵, while others have shown that during storage younger RBC populations produce the majority of RMPs⁵⁷. Of note, RMP composition/content may vary with specific storage conditions as well as with storage duration⁶⁶. For example, RMPs generated in vitro by stimulation with Ca ionophores differ in size and cytoskeletal protein structure than RMPs produced during RBC storage^58,67. RMPs isolated during storage seem to have less variation in size and shape than those isolated from circulation⁵⁷. Moreover, hypotonic, alkaline storage solutions are associated with increased RMP production and with an RMP population that has significantly less cholesterol, phospholipids, as well as band 3 and protein 4.1⁵⁷. Leucoreduction appears to diminish RMP production by up to 40–50%, particularly under anaerobic conditions⁶⁸. More generally, RBC storage is characterised by progressive depletion of energy resources and antioxidant defences, enabling accumulation of oxidative lesions, particularly involving the cytoskeleton and Band 3⁶⁹. Vesiculation may allow RBCs to eliminate such markers, as well as other harmful components that accumulate during storage⁷⁰.

Triggers for RMP production

Little is known about the specific signalling pathways that regulate RBC vesiculation, both during RBC aging in vivo and during ex vivo storage. Furthermore, it is also important to recognise that unique changes may occur to stored RBCs in vivo following transfusion. Several triggers for RBC MP production have been identified, mostly pertaining to the changes RBCs undergo during storage. These include the following:

1. Increased cytosolic calcium (Ca²⁺) is perhaps the most well characterised trigger for activation of Ca²⁺-dependent proteases, leading to cytoskeletal damage and activation of Ca²⁺-dependent scramblase; both processes are integral to PS exposure and to MP shedding²⁴.

2. ATP depletion impairs performance of the major ATP-dependent transporter proteins (flippase and floppase) responsible for maintaining cell membrane asymmetry; loss of asymmetry promotes MP budding and shedding⁶².

3. Increased potassium (K⁺) leakage has also been linked to disturbed erythrocytic membrane transporter activity, disturbing maintenance of membrane structure and is thought to lead to MP formation⁶².

4. Other cascades arising from energy failure in RBCs have been shown to increase RBC vesiculation and MP formation. These include G protein-coupled receptor signalling, the phosphoinositide 3-kinase (PI3K-Akt protein kinase B) pathway, the Jak-STAT (Janus kinase-signal transducer and activator of transcription) pathway, and the Raf-MEK (mitogen-activated protein kinase)-ERK (extracellular signal-regulated kinase) pathway²⁵.

RMPs differ from intact RBCs

Proteomic analysis has demonstrated that RMPs contain many RBC proteins including carbonic anhydrase, peroxiredoxins and 14-3-3 proteins (regulators of a number of processes, such as modulation of protein kinase activity and signal transduction)⁷¹; however, RMPs are structurally and functionally different from intact RBCs (e.g. RMPs are not “just small RBCs”). As discussed earlier, the process of RMP shedding involves loss of normal RBC membrane asymmetry, and as a consequence there is a greater density of negatively charged phospholipids (e.g. PS) on the outer RMP membrane. RMP membranes are also enriched with specific proteins (Band 3 and Band 3 dimers) in comparison to parent RBCs⁶⁰. Moreover, in comparison to intact RBCs, storage-related RMPs appear enriched with stomatin, relatively depleted in actin, and to have more stable glycophorin A⁷¹. Disruption of normal RBC cytoskeletal protein structure⁷² is also integral to RMP shedding⁷³ and distinguishes daughter from parent cells. These structural characteristics, in addition to the discrepancy in size, result in differential “streaming characteristics” between RBCs and RMPs, with preferential RMP circulation in proximity to endothelial cells in the “cell-free” zone of the micro-circulation⁷⁴. (It is to be noted that this feature has significant physiological implications; see below). Finally, RMPs encapsulate a significant amount of Hb⁷⁵, which confers physiological characteristics closer to cell-free Hb than to intact RBCs, particularly with regard to interactions with nitric oxide.

Proposed RMP biological effects

There is increasing recognition of the biological effects of RMP, particularly in the context of transfusion. Proposed effects include the following items.

1) Promotion of coagulation by RMPs

It has been well established that negatively charged surfaces activate zymogen components of the coagulation cascade and it appears that RMPs act as pro-coagulant factors in this fashion. RMPs increase thrombin generation in the presence of low exogenous tissue factor and, in fact, are capable of initiating and propagating thrombin generation even in the absence of tissue factor⁷⁶. There is some evidence that this pro-coagulant activity is dependent on Factor XII⁷⁷. Some authors have suggested that tissue factor may be expressed on the RMP surface, contributing to their pro-coagulant effect⁷⁸. Of note, RMP abundance is known to increase in certain pathological states, including sickle cell crises, which are associated with a hypercoagulable state⁷⁹. Interestingly, RMPs also bind protein S, a co-factor for activated protein C, enhancing the degradation of Factors VIIIa and Va, inhibiting tenase and prothrombinase, and thus increasing clot breakdown⁸⁰. As such, depending on context, balance amongst this and the other above mentioned effects may determine the RMP coagulation phenotype⁸⁰.

2) Nitric oxide (NO) scavenging by RMPs

Nitric oxide (NO) is a vasodilator subserving physiological reflexes that maintain dynamic matching between regional blood flow and tissue respiration⁸¹. Of note, Hb reacts with NO in a diffusion-limited oxidation reaction that quenches NO bioactivity, disrupting vasoregulation and oxygen delivery homeostasis. Under normal conditions, this effect is limited by Hb compartmentalisation within RBCs⁸². Moreover, constraints upon Hb:NO interaction are substantially influenced by RBC size and membrane architecture⁸³; however, reaction between de-compartmentalised, cell-free Hb and NO lacks such constraints⁸⁴. Notably RMP:NO interactions more closely mirror those of cell-free Hb than that of intact RBCs, and as such, RMPs act as potent NO scavengers (RMP reaction with NO is approximately 1,000-fold faster than with RBC-encapsulated Hb⁸⁵ and is only 2.5-3-fold slower than with cell-free Hb⁸⁵). The potential impact of NO quenching by RMPs (following transfusion) dramatically increases with the increase in RMP abundance observed during storage. The degree by which RMPs influence NO bioavailability in vivo is dependent on several factors, most importantly, the degree to which RMPs enter the cell-free zone in the microcirculation (e.g. stream in proximity to endothelium)⁷⁴.

3) Immune modulation by RMPs

Transfusion related immune modulation (TRIM) is a recognised, but poorly characterised, complication of transfusion. Given the known RMP increase with storage duration, a role for RMPs in TRIM pathobiology has been postulated⁸⁶. RMPs influence the impact of antigen presenting cells (APC) and boost mitogen driven T-cell responses⁸⁷. Specifically, RMPs amplify APC-based induction of pro-inflammatory cytokines and chemokines from peripheral blood mononuclear cells (PBMC) and promote PBMC survival⁸⁷. Alternatively, RMPs may exert an immunosuppressive effect, by decreasing the release of various cytokines such as TNF-α, IL-8, or IL-10⁸⁸. Of note, production of sickle cell-derived RMP (SS RMPs) is enhanced by inflammatory conditions. When engulfed by myeloid cells, SS RMPs promote pro-inflammatory cytokine secretion and endothelial cell adhesion, suggesting crosstalk between circulating inflammatory cells, and SS RMPs contributes to sickle cell disease pathogenesis⁸⁹. In addition, RMPs have also been demonstrated to bear blood group antigens⁹⁰; transfusion-related RMPs may, therefore, represent a significant immunogenic load⁵⁹ and contribute to the severity of alloimmunisation in chronically transfused patients⁹⁰.

4) Promotion of endothelial adhesion by RMPs

As noted above, RMPs appear to play a significant role in the pathophysiology of sickle cell disease (SCD), during which RMP production is enhanced and promotes pro-inflammatory cytokine secretion⁷⁹. SS RMPs have also been shown to enhance adhesion of intact RBCs to endothelial cells⁸⁹. Of note, up to one-third of cell-free haeme in SCD may be carried by circulating RMPs⁹¹. Moreover, externalised membrane PS on SS RMPs retains haeme on the external RMP surface. Such haeme-laden SS RMPs transfer haeme directly to vascular endothelium, generating oxidative stress and endothelial apoptosis⁹¹. Such linkage of haemolysis to endothelial injury has been proposed as a trigger for SCD vaso-occlusive crises.

RMP clearance

Similar to other MP populations, RMPs are also likely to be cleared via the MPS, in particular by hepatic Kupffer cells⁹². Such removal appears to occur very rapidly and is thought to be mediated by PS-binding scavenger receptors and senescent cell antigen-specific autoantibodies⁹². Immunological analysis of RMPs elaborated by senescent RBCS demonstrates Band 3 clustering; this RMP subpopulation may, therefore, be cleared in a similar fashion to that for intact senescent RBCs⁵⁹.

RMPs and RBC storage

The RBC storage lesion is compromised by altered metabolism and biomechanics. In particular, energy failure (with impaired ATP and reduced equivalent production) is a common characteristic and is associated with a significant acidosis, decrease in 2,3-diphosphoglycerate levels, and failure of the membrane Na⁺/K⁺ ATPase pump with continuous potassium leakage^93,94 (e.g. conditions known to promote RMP genesis^95,96). Morphologically, stored RBCs slowly morph from smooth biconcave discs, to spiculated echinocytes, to dense spheroechinocytes. These changes are related to membrane loss consequent to RMP formation, as well as loss of cell volume regulation and diminished deformability⁵⁷, thus greatly impacting post-transfusion survival and rheology⁹⁷. In fact, RMP genesis appears to be directly proportional to storage duration, with a 20-fold increase after 50 days⁷¹ (Figure 5). The doubling time for RMP concentration during storage is estimated to be nine days (95% CI: 7.7–10.7 days)⁸⁵. The specific reasons for such significant RMP production remain unknown although it has been suggested that storage activates a physiological process that serves (in vivo) as a means to prevent premature RBC clearance by shedding membrane proteins that would otherwise signal RBC senecence^59,98.

Figure 5.

Microparticle (MP) count in red blood cell units during storage (without centrifugation).

Data are expressed as the mean±SD (n=7). At day 5: 3,371±1,188 MPs/μL were counted, whereas at day 50: MPs had approximately 20-fold (64,858±37,846 MPs/μL). MPs were stained with anti-human CD47. (Adapted with permission from Rubin et al.,⁷¹).

Physiological impact of storage-generated RMPs during RBC transfusion

Oxygen delivery homeostasis and vasoregulation

Tissue oxygen delivery is a function of blood O₂ content and regional blood flow, with the latter being the principle determinant. The volume and distribution of regional blood flow is actively regulated to maintain dynamic coupling between O₂ delivery and tissue respiration⁹⁹. It is now commonly appreciated that RBCs act as both sensors and transducers in this physiology by regulating bioavailability of vasoactive effectors in plasma (and thereby, regulating resistance vessel caliber)⁸¹. This key physiological reflex is termed hypoxic vasodilation (HVD) and is primarily mediated by RBC-transported NO^100–102. As such, by serving as HVD effector elements, RBCs function has a key role in maintaining O₂ delivery homeostasis (Figure 6). This paracrine RBC function is governed by O₂-linked transitions in Hb conformation which, because of differing reactions of deoxy- and oxy-Hb with NO, transduce regional pO₂ gradients into NO bioactivity, thereby effecting vasodilation to resolve perfusion insufficiency (e.g. HVD)⁸². This physiology is disrupted when RBCs release Hb into plasma. Specifically, although Hb packaging in RBCs blunts NO consumption approximately 1,000-fold, once released, free Hb (and Hb containing RMP) readily inactivates NO, preventing facile NO-based traffic between RBCs and endothelium^74,103.

Figure 6.

Red blood cells (RBC) transduce regional O₂ gradients in tissue to control nitric oxide (NO) bioactivity in plasma by trapping or delivering NO groups as a function of haemoglobin (Hb) O₂ saturation (Hb SO₂).

(A) Circulating NO groups are processed by Hb into the highly vasoactive (thiol-based) NO congener, S-nitrosothiol (SNO). By exporting SNOs as a function of Hb deoxygenation, RBCs precisely dispense vasodilator bioactivity in direct proportion to the lack of regional blood flow. (B) O₂ delivery homeostasis requires biochemical coupling of vessel tone to environmental cues that match perfusion sufficiency to metabolic demand. Because oxygenated Hb (oxy Hb) and deoxygenated Hb (deoxy Hb) process NO differently, allosteric transitions in Hb conformation afford context-responsive (O₂-coupled) control of NO bioavailability, thereby linking the sensor and effector arms of this system. Specifically, Hb conformation governs the equilibria among (A) deoxygenated Hb FeNO (NO sink), (B) oxygenated SNO-Hb (NO store), and (C) acceptor thiols including the membrane protein SNO-AE-1 (bioactive NO source). Direct SNO export from RBCs or S-transnitrosylation from RBCs to plasma thiols (D) yields vasoactive SNOs, which influence resistance vessel caliber and close this signalling loop. Thus, RBCs either trap (A) or export (D) NO groups to optimise blood flow. (C) NO processing in RBCs (A and B) couples vessel tone to tissue PO₂; this system subserves hypoxic vasodilation in the arterial periphery and thereby calibrates blood flow to regional tissue hypoxia. (Adapted with permission from Doctor and Stamler, 2011⁸¹. ^©American Physiological Society). RSH: peptide or protein containing a thiol group.

Transfusion and vasoregulation

There is substantial evidence to suggest that RBC transfusion impairs HVD, although the mechanism has not been fully elucidated^93,104,105. In addition to direct effects of released free Hb upon NO bioavailability in plasma, haemolysis may also impair endothelial NO production, possibly via release of arginase (which, through substrate depletion, constrains eNOS activity)^85,106. Moreover, intact stored RBCs, with increasing storage duration, are 2–4 fold more avid NO scavengers than fresh RBCs¹⁰⁷ and exhibit more pronounced inhibition of NO-mediated vasodilation. Furthermore, there is growing evidence to suggest stored RBC-derived free-Hb and Hb-rich RMPs dampens normal NO bioactivity in the microcirculation, leading to physiologically significant HVD impairment^85,108–110. This latter observation is supported by in vivo data demonstrating RMP contributes to the initiation of vaso-occlusive crises in sickle cell disease⁹¹.

RMP impact upon vasoregulation

As discussed above, HVD is the principle physiological reflex that maintains dynamic coupling between regional oxygen delivery and tissue respiration, particularly during physiological stress. Evidence suggests that RMPs impair RBC-based HVD support of oxygen delivery homeostasis, by: 1) preferentially streaming in the cell-free zone of the microcirculation; and 2) acting as an NO sink. Specifically, animal studies demonstrate a significantly greater increase in mean arterial pressure (MAP) upon infusion of cell-free supernatants of longer stored RBC units (39 days) vs fresher units (4 days)⁸⁵ (Figure 7). This increase correlates with the amount of extra-erythrocytic Hb (both free and in RMPs) in supernatants. Moreover, RMP half-life in this model was approximately 15–20 minutes, consistent with the time course for changes in blood pressure that occur with infusion of stored RBC supernatants⁸⁵. This is particularly important since, unlike cell-free Hb, RMPs are not bound by haptoglobin and may, therefore, contribute significantly to this phenomenon⁸⁵. Early results from the study of RMPs upon vasoregulation in critically-ill humans suggest an increase in circulating RMPs concurrent with transfusion, but not in MPs of other origin (Figure 8A). Moreover, these RMPs appear to increase NO scavenging (Figure 8B) and alter plasma vasoactivity (Figure 8C). In addition, in these subjects, a transfusion-associated increase in RMPs was associated with a significant increase in systemic vascular resistance and a corresponding decrease in cardiac output. Concurrently, tissue re-oxygenation following experimental vaso-occlusion (e.g. dynamic near infrared spectroscopy, a measure of HVD capacity¹¹¹) was significantly dampened post transfusion as compared to pre- and intra-transfusion phases (Figure 8D)¹¹².

Figure 7.

Vasoactivity of infused packed red cell supernatant/plasma.

(A) Experimental time line for packed red cell supernatant infusions. Rats were stabilised for 30 minutes (min) after surgery, and blood gasses were drawn as indicated (BG 1 and BG 2). Supernatant (1.6 mL) of packed red blood cells stored for either 4 or 39 days was infused for 40 min, after which the rats were followed for 1 hour (n=5). (B) Change in mean arterial pressure (MAP) over time after packed red blood cell (RBC) supernatant infusion and 60-min follow up. (C) Average percentage peak increase in MAP after infusion of packed RBC supernatants (RBC sup) (p=0.003). (D) Correlation (solid line) between packed red blood cell supernatant haeme concentration and percentage increase in MAP after 40-min infusion of packed red blood cell supernatant stored for either four days (black solid circle) or 39 days (black solid square; r²=0.65). Each data point was obtained from a separate rat infusion experiment, in a different rat (2 groups of n=5). All values are displayed as mean standard error of mean (SEM). Student t-test was used to compare the 2 groups of rats. Avg.: average; conc.: concentration. (Adapted with permission from Donadee et al., 2011⁸⁵).

Figure 8.

Pilot data of the vasoregulatory effects of transfusion related RMPs in humans.

(A) Circulating RMP abundance during RBC transfusion. Box and whisker plot of RMP concentration in plasma (flow cytometry, CD235a antibody) demonstrates an increasing trend during RBC transfusion (RM ANOVA). (B) NO scavenging relation to RBC transfusions. Using a validated NO consumption assay (Wang et al., 2004), we observe a ~1.3 fold increase in plasma NO scavenging following RBC transfusions (p=0.63, RM ANOVA). (C. Plasma vasoactivity is altered by RBC transfusions. Using a validated in vitro rabbit aortic ring array to assess vasoactivity, there was a 1.3 0.35 and 1.36 0.24 fold increase in plasma vasoconstrictive capacity during and after RBC transfusions compared to the pre-transfusion state, respectively. D. Change in hypoxic vasodilatory capacity in relation to RBC transfusion. A validated Dynamic Near Infrared Spectroscopy (NIRS) vascular occlusion test before, during and after RBC transfusion. Linear regression analysis showed a significantly slower rate of tissue resaturation post-transfusion as compared to the pre-and intra-transfusion states (p=0.0092). The rate of tissue resaturation is thought to correlate with capacity for capillary bed recruitment and hypoxic vasodilation (Said et al., 2016¹¹²).

Conclusions

Microparticles are submicron particles of various cell origins, the composition of which is dependent on parent cell identity, that form in response to a multitude of stimuli through cell membrane re-organisation, blebbing and shedding. MPs are thought to have several biological effects and may serve as vehicles for inter-cellular communication. RMPs form spontaneously during the RBC lifespan, with content and a cytoskeletal structure distinct from intact RBCs. RMP production accelerates during RBC storage due to associated biochemical changes: increased cytosolic calcium, ATP depletion, and increased potassium leakage. Moreover, RMP composition is affected by the trigger for their formation and by different storage conditions.

Proposed RMP biological effects include promotion of coagulation, immune modulation, and enhanced endothelial adhesion. Of particular importance, RMPs demonstrate significant NO trapping/consumption, disrupting regional matching between blood flow and tissue respiration that is essential to oxygen delivery homeostasis physiology. These effects have been demonstrated in animal models evaluating storage-related RMPs, which appear to provoke an increase in systemic vascular tone and blood pressure following infusion of cell-free RBC unit supernatants. This effect progresses with storage duration. Pilot data in transfused humans demonstrate similar findings, with an increase in circulating RMPs, in plasma NO scavenging, in systemic vascular resistance, and a reduction in tissue capacity for re-oxygenation following experimental vaso-occlusion.