• Open Access

Microparticles in Health and Disease

Authors


Corresponding author: M.A. McMichael, Department of Veterinary Clinical Sciences, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Dr., Urbana, IL 61802; e-mail: mmcm@illinois.edu.

Abstract

Microparticles (MPs), small membrane-derived vesicles, are derived from many cell types and released into the circulation. Microparticles can express antigens, and contain cell surface proteins, cytoplasmic contents, and nuclear components from their cell of origin that determines their composition, characterization, and transfer of biologic information. Certain prompts for this release include shear stress, complement activation, proapoptotic stimulation, cellular damage, or agonist interaction with cell surface receptors. Release can be physiologic or pathologic and is associated with proinflammatory and procoagulant effects and has been implicated in thrombotic states. Microparticles also contribute to systemic inflammation and cardiovascular, hematologic, and oncologic disease states. The study of MPs in human medicine is rapidly advancing and extends into the physiology of health, the pathophysiology of disease, and the role of MPs in transfusion medicine. In veterinary medicine, published work on MPs has been limited to the area of inherited disorders, blood storage, and leukoreduction (LR). Microparticle research is still in its infancy, and this review should be seen as a snapshot of what is currently known. As research continues important limitations, including variations in preanalytic variables such as collection, storage, or centrifugation, and limitations of quantitation are coming to the forefront. Correlation of quantitation of MPs with assays of activity will hopefully shed light on the true nature of MPs in health and disease. This review will focus on the role of cellular exocytic vesiculation in health, disease, and transfusion medicine.

Abbreviations
LR

leukoreduction

MPs

microparticles

PE

phycoerythrin

Microparticles (MPs) are submicron (<1 μm diameter) membrane-derived exocytic vesicles that are released into the circulation in vivo and generated in stored blood products ex vivo. Platelets, endothelial cells, erythrocytes, polymorphonuclear leukocytes, lymphocytes, and monocytes all produce MPs in a tightly controlled process prompted by stimuli such as shear stress, complement activation, proapoptotic stimulation, or cellular damage.[1-3] Physiologic and pathologic processes are responsible for microvesicle formation in both healthy and diseased individuals[4] and the presence of these vesicles has proven detrimental for recipients of blood transfusions.[1, 5-10] Microparticles typically express antigens, and contain cell surface proteins, cytoplasmic contents, and nuclear components from their cell of origin. These biomolecules determine their composition, characterization, and transfer of biologic information.[11-14] Microparticles have been associated with profound proinflammatory and procoagulant effects and have been implicated as an essential part of both normal and abnormal coagulation (Table 1). They also contribute to systemic inflammation and cardiovascular, hematologic, and oncologic disease states.[15, 16] The study of MPs in human medicine is rapidly advancing, and extends into the physiology of health, the pathophysiology of disease, and the role of MPs in transfusion medicine. In veterinary medicine, published work on MPs has been limited to the area of inherited disorders, blood storage, and leukoreduction (LR).1 This review will focus on the role of cellular exocytic vesiculation in health, disease, and transfusion medicine.

Table 1. Possible roles of microparticles in hemostasis
 Target, Ligand, or Substrate
  1. Microparticles can participate in hemostasis by providing membrane-anchored proteins that act as regulatory units, receptors for binding, or both. The proteins expressed depend on the cell of origin. Microparticles also provide the activated (phosphatidylserine containing) membrane surface necessary to support enzymatic reactions required for the generation of various participants in hemostatic processes.

  2. a

    Note that tissue factor is membrane anchored, and does not participate in hemostasis unless it binds factor VII or factor VIIa.

  3. b

    Factor VIIa, an enzyme, has low activity unless bound to tissue factor.

Procoagulant
Membrane anchored
Tissue factoraFactor VII, factor VIIa
Membrane binding
Factor VIIabFactor IXa, factor Xa, tissue factor
Factor Xa–Factor VaProthrombin
Factor IXa–Factor VIIIaFactor X
Anticoagulant
Membrane anchored
Tissue factor pathway inhibitorFactor Xa, tissue factor-factor VIIa
ThrombomodulinProtein C
Endothelial protein C receptorProtein C
Heparan sulfate proteoglycansAntithrombin
Membrane binding
Activated protein C-activated protein SFactor Va, factor VIIIa
Profibrinolytic
Membrane anchored
α-EnolasePlasminogen
Urokinase plasminogen activator receptorUrokinase plasminogen activator
Antifibrinolytic
Membrane anchored
ThrombomodulinThrombin-activatable fibrinolysis inhibitor

Microparticle Formation and Properties

Microparticles are generated from their cell of origin by a budding of the cell membrane secondary to disruption of the normal phospholipid asymmetry. The distribution of phospholipids across the membrane bilayer of eukaryotic cells is highly organized and asymmetric. Under normal physiologic conditions, the aminophospholipids, phosphatidylserine (PS,) and phosphatidylethanolamine (PE) are sequestered to the inner membrane leaflet, whereas the choline-containing phospholipids, phosphatidylcholine, and sphingomyelin are arranged on the outer membrane.[16] The maintenance of this asymmetry is critical and regulated by the balance of various substrate and direction-specific adenosine triphosphate (ATP)-dependent transporters, referred to as flippases and floppases.[16, 17] Flippases govern inward movement of phospholipids to the cytosolic membrane leaflet and floppases control outward luminal translocation. A 3rd group of lipid transporters, scramblases, result in rapid reorganization of the lipid asymmetry in a bidirectional calcium-dependent and energy-independent manner.[17] The maintenance of the normal phospholipid asymmetry is dependent on an ATP-dependent transporter, the aminophospholipid translocase, responsible for rapid directional movement of PS and PE from the outer to the inner leaflet.[18] The activities of flippases and floppases are thought to generate and maintain lipid asymmetry, whereas the bidirectional scramblases are involved in its reorganization. Thus, when cellular activation occurs, there is redistribution of the transbilayer phospholipid membrane symmetry and in platelets, a dramatic change in membrane-associated proteins, resulting in the production of MPs (Fig 1).

Figure 1.

(A) Schematic representation of the resting cytoskeleton. Calcium is stored in the endoplasmic reticulum (ER). Scramblase is inactive, while translocase is active. Translocase transports phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outside layer of the cell membrane to the inside, maintaining PS and PE inside. One molecule of ATP is necessary for each PS molecule transported. Floppase is an ATP-dependent protein that contributes to maintaining the physiological membrane asymmetry. (For the sake of clarity, proteins expressed on the surfaces of the cell and the microparticle have not been included in the figure.) (B) Cellular activation (incomplete schematic). Intracellular calcium rises as a consequence of its release from internal stores (ER) and its entry across the plasma membrane from the extracellular compartment. Recently, a transmembrane protein, TMEM16F, was identified as important in calcium-dependent scramblase activity. In platelets, TMEM16F forms a calcium-activated cation channel required for lipid redistribution during blood coagulation. Increased cytoplasmic calcium leads to activation of calpain and gelsolin. Calpain cleaves long actin filaments. Gelsolin cleaves the actin-capping proteins. The raised cytoplasmic calcium also activates scramblase and inactivates translocase. Phospholipid asymmetry begins to be compromised. Phosphatidylserine externalization and microparticle generation are dependent on high levels of sustained calcium entry and intracellular ionized calcium mobilization. (C) Cytoskeletal disruption after cellular activation. Spectrin and actin are cleaved. At this point, protein anchorage to the cytoskeleton is disrupted allowing membrane budding. (D) Generated microparticle exposing increased phosphatidylserine on the external surface. Modified with permission from Piccin et al.[16]

Phospholipid Flip-Flop

In the resting state, spontaneous phospholipid translocation in the absence of a transporter is slow, thus maintaining the stability of lipid asymmetry in quiescent cells.[19] This is postulated, although unproven, to result from resistance in the translocation of polar head groups through the hydrophobic core as well as increased tension created when new phospholipids insert themselves into the receiving monolayer.[19] During cell stimulation, aminophospholipids are translocated to the outer leaflet, mediated through a reduction in flippase activity and increases in both floppase and scramblase activation. This scrambling of phospholipids occurs between the 2 leaflets irrespective of the chemical nature of the polar head groups.[18] A transmembrane protein, TMEM16F, is important in calcium-dependent scramblase activity.[20] In platelets, TMEM16F forms a calcium-activated cation channel required for lipid redistribution during blood coagulation.[17] Interestingly, in patients with Scott syndrome, there is a deficiency in phospholipid scramblase activity found to be associated with a mutation in the gene encoding TMEM16F, causing early transmembrane protein termination.[20] A bleeding disorder ensues wherein both PS exposure and MP release are deficient.[18, 20] A hereditary bleeding disorder with characteristic features of Scott syndrome and caused by a deficiency in platelet procoagulant activity occurs in German Shepherd dogs.[21]

Cytosolic calcium concentration is responsible for initiating the cascade of enzymatic events that rapidly changes the expression of the phospholipids in the membrane bilayer, with a concomitant rise in procoagulant activity.[22, 23] Changes in cell shape, membrane blebbing, and release of microvesicles occur secondary to the increase in intracellular cytosolic calcium.[22] Calcium homeostasis is tightly regulated and is essential for causing phospholipid migration. Important calcium-dependent mechanisms include translocase inhibition, flippase transporter inactivation, and disruption of the cytoskeleton.[18] The rise in intracellular calcium is a result of its release from internal stores (endoplasmic reticulum), but also influx through the plasma membrane.[18] In platelets, agonist-induced stimulation of different receptors culminates in the activation of phospholipase C isoforms, leading to the production of inositol-1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 binds to its receptors and initiates calcium release from cytosolic stores.[18] Depletion of calcium from intracellular stores is sensed by the stromal interacting molecules 1 (STIM-1), which triggers the influx of calcium from the extracellular compartment. This latter movement is mediated by DAG and the mechanism is referred to as store-operated calcium entry.[18]

An increase in intracellular calcium activates gelsolin in platelets. Gelsolin cleaves capping proteins at the end of actin filaments, allowing for platelet cytoskeletal reorganization.[16, 24] Calpain, also stimulated by the increase in cytosolic calcium, cleaves long cytoskeletal filaments, facilitates MP shedding, and activates apoptosis.[16] In contrast to other cell types, erythrocytes do not contain cytoplasmic actin filaments. Their cytoskeleton is instead composed of short actin filaments that are closely adherent to the cellular membrane and cross-linked by spectrin.[25]

Microparticle Generation and Composition

Cytoskeletal disruption follows cellular activation. This disruption of the phospholipid asymmetry is modulated in vivo by such stimuli as shear stress, oxidative stress, cytokine release (tumor necrosis factor, IL-6), complement activation, hypoxia, apoptosis, cell damage, or can be agonist-induced in platelets and other cell types.[2] Membrane contracture and budding occur, leading to the generation of MPs that are proinflammatory and procoagulant in nature because of the expression of active phospholipids on their outer leaflet.[22]

The stimulus that triggers the production of MPs regulates the composition of these vesicles and, thus, the transfer of biologic information.[14] Microparticle composition is a reflection of the original cell's redistributed plasma membrane. Microparticles contain membrane, cytoplasmic, and nuclear constituents specific to their cell of origin.[14] For example, proteins from endothelial-derived MPs are mostly metabolic enzymes, proteins involved in adhesions and fusion, and cytoskeletal-associated proteins,[26] whereas those from platelet-derived MPs are surface glycoproteins or chemokines.[27] Furthermore, the lipid environment can modify the activity of proteins carried by MPs. For instance, cholesterol enrichment of human monocytes can induce production of procoagulant MPs expressing surface tissue factor and PS.[28] Microparticles also contain nucleic acids allowing for transfer of genetic material to target cells.[14] Microparticles consequently directly interact with ligands on target cells, activate cascade signaling, and transfer proteins, mRNA, and bioactive lipids.[14] Target cells are able to acquire new surface antigens and biologic activities via these mechanisms. Interestingly, transfer of information is dependent on the origin and composition of the MPs, rather than their concentration in circulation.[29, 30]

Pathophysiology of Microparticles

Role of Microparticles in vivo

Membrane vesiculation occurs in vivo as a result of various stimuli and signaling mechanisms (Table 2). Under normal physiologic conditions, MPs are involved in tightly controlled biologic functions,[3] including inflammation and hemostasis,[31] transfer of surface proteins,[32] and angiogenesis.[33] Phosphatidylserine on the surface of MPs provides binding sites for coagulation enzyme assembly,[34] enhances platelet adhesion to the endothelium,[35] and is important for tissue factor function[36] and thrombin production.[31]

Table 2. Stimuli for microparticle production from various cell types
PlateletsT-CellsMonocytesNeutrophilsEndothelial Cells
  1. Stimuli (y-axis) resulting in microparticle generation from various cell types, including platelets, endothelial cells, and leukocytes (x-axis). A23187, calcium ionophore; ANCA, antineutrophil cytoplasmic antibodies; AngII, angiotensin II; CRP, C-reactive protein; EPO, erythropoietin; FMLP, N-formylmethionyl-leucyl-phenylalanine; IL, interleukin; LPS, lipopolysaccharide; NE, noradrenaline; PAI-1, plasminogen activator inhibitor-1; PMA, phorbol myristyl acetate; ROS, reactive oxygen species; sCD40l, soluble CD40 ligand; TNF, tumor necrosis factor; TRAP, thrombin receptor-activating peptide.

NEPhytohemagglutinLPSFMLPCamptothecin
EPOStaurosporinA23187PMAUremic toxins
TNFαTNFαTNFαANCACRP
TRAPPMAEtoposideBacteremiaTNFα
ROSEtoposideFas ligand Ang II
IL-6Actinomycin D  ROS
LPS   LPS
Collagen   Hyperglycemia
Shiga toxin   Thrombin
Thrombin   IL-1α
sCD40l   PAI-1
A23187    

Current information suggests that erythrocyte-derived MPs are generated as a mechanism to prevent premature erythrocyte removal by Kupffer cells in the liver.[1, 37] These MPs might help protect erythrocyte viability via removal of the C5-9 complement attack complex, band 3 neoantigen, IgG, nonfunctional methemoglobin, and other harmful membrane-associated agents.[3, 38-40]

The production of platelet-derived MPs is triggered by thrombin, collagen, and exposure to high shear. As a consequence, platelet-derived MPs are present both in normal circulation and during pathologic conditions.[41-44] The interaction of platelet-derived MPs with endothelial cells,[45] polymorphonuclear cells,[46] or monocytic cells[46, 47] results in activation of the target cell. Platelet-derived MPs also modulate monocyte-endothelial interactions[48] and leukocyte-leukocyte interactions,[49] inhibit apoptosis of polymorphonuclear leukocytes,[50] and induce chemotaxis.[47, 51] Platelet-derived MPs additionally stimulate proliferation of endothelial cells, smooth muscle cells, and hematopoietic cells.[52-54] Furthermore, platelet-derived MPs express both pro- and anticoagulant proteins[43, 55] and interact with fibrin.[41, 56]

Leukocyte-derived MPs initiate signal transduction and stimulate inflammatory and procoagulant endothelial cell responses through expression of endothelial cell adhesion molecules and functional tissue factor, up-regulation of inflammatory and chemotactic chemokines, and increased monocyte adhesiveness.[32, 45, 48, 57] Platelet- and monocyte-derived MPs form functional prothrombinase complexes and increase procoagulant activity and aberrant fibrin deposition in vivo.[58, 59]

Endothelial cell-derived MPs increase vascular procoagulant and proinflammatory activity and can induce thromboembolic complications.[60] They cause endothelial dysfunction and contribute to the pathophysiology of diseases such as chronic renal failure and coronary artery disease.[61, 62] Increased numbers of circulating endothelial cell-derived MPs are a marker of endothelial injury and systemic vascular remodeling.

Apoptosis

Microparticles are generated in a tightly controlled process on the surface of endothelial and circulating blood cells under physiologic conditions during apoptosis. Phosphatidylserine on the surface of apoptotic cells and MPs is a recognition signal for removal of these cells and fragments from the circulation by activated macrophages.[63] Appropriate apoptosis is critical to the clearance of aged cells, necrotic remnants, and proinflammatory debris.[64] Microparticle formation is an important defense mechanism against the complement cascade, allowing membrane shedding of complement components from the cell surface.[65] Shedding of MPs from endothelial cells is likely beneficial, as it prevents premature endothelial cell apoptosis and detachment.[66] Without this strict regulation, accumulation of autoantigens in tissues can occur, resulting in inflammatory and immunologic responses with detrimental effects.[64] In particular, inappropriate clearance of apoptotic remnants may result in systemic autoimmune diseases, such as systemic lupus erythematosus in humans.[67]

Coagulation, Thrombosis, and Inflammation

Microparticles play a vital role in normal hemostasis and pathologic thrombosis. The expression of PS on the outer leaflet is of critical importance in supporting membrane-dependent enzymatic reactions.[31] In fact, procoagulant activity is lost and thrombin production is delayed with the removal of PS-bearing MPs.[68] Tissue factor expression on the surface of some MPs (particularly of monocyte origin) could be an important reservoir for blood-borne tissue factor,[69] and might contribute to thrombin generation within forming clots. Circulating tissue factor could be “encrypted” such that it is inactive until signals associated with vascular injury cause decryption.[1, 70, 71] Expression of ligands on the MP surface allows them to mediate binding to other MPs, and to monocytes and endothelial cells via selectins, capturing the circulating tissue factor into the developing clot.[1, 71] Microparticles additionally can express down-regulators of thrombin such as thrombomodulin, tissue factor pathway inhibitor,[72] and endothelial protein C receptor.[68, 73]

The expression of negatively charged phospholipids on the membrane surface is vital for activity of many of the coagulation enzyme complexes (Table 1).[1] Platelet- and endothelial cell-derived MPs provide the required binding sites for both zymogen and activated factors X, IX, V, VIII, and zymogen prothrombin.[16, 34, 74-76] Platelet-derived MPs have 50–100 times more procoagulant activity than activated platelets,[1, 77] and erythrocyte-derived MP membranes are also procoagulant in nature.[2, 78]

Microparticles are normally formed in low numbers in vivo under tightly regulated conditions[4] and have controlled hemostatic properties. In contrast, pathologic MPs circulate at high concentrations and could predispose to thrombosis.[79] Microparticles bearing active tissue factor accumulate and allow for unregulated thrombus formation and fibrin generation, predisposing to thrombotic complications in human diseases such as neoplasia, pre-eclampsia, rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria, and atherosclerosis.[16, 71]

Microparticles also play a key role in inflammation and vascular function; in particular, endothelial cell-derived MPs might be directly involved in endothelial dysfunction.[13] Regulation of vascular tone is accomplished by endothelial MPs through nitric oxide (NO) and prostacyclin production.[80] In addition, generation of endothelial cell-derived MPs during oxidative stress results in biologically active oxidized phospholipids that promote neutrophil activation and monocyte adherence to the endothelial cells.[13, 81] Leukocyte-derived MPs also stimulate cytokine release and induce tissue factor expression by endothelial cells, leading to increased proinflammatory and procoagulant activity.[32, 57]

Disease States

Circulating MPs play roles in a variety of human diseases, and are biomarkers of vascular injury and inflammation in atherosclerosis, coronary endothelial dysfunction, acute myocardial infarction, type 2 diabetes, hypertension, acute ischemic stroke, hypertriglyceridemia, and metabolic syndrome.[13, 82] It is not clear whether MPs are the cause or the consequence of these vascular disease states.[13] The severity of disease is often correlated with both concentration and relative activity of MPs in circulation.[2] The nature of the circulating MP population varies depending on the etiology of the disorder. As a consequence, specific types of MPs could represent useful therapeutic targets.[83-86] An example of this involves human patients with rheumatoid arthritis, wherein platelet MPs likely contribute to the pathophysiology of this disease and resultant joint inflammation via stimulation of the collagen receptor glycoprotein VI (GPVI).[87] Antagonism of this receptor is a novel therapeutic approach in human medicine, as humans and mice that lack GPVI fail to develop this disease.

Studies involving human patients with myocardial infarction, sepsis, diabetes, pre-eclampsia, metabolic syndrome, and obstructive sleep apnea have documented that circulating MPs induce endothelial dysfunction.[29, 83, 86, 88-91] Proposed mechanisms include inactivity of endothelial NO reductase and reduction in NO bioavailability resulting in a decrease in NO signaling,[29, 83, 88, 89] increase in protein nitration on endothelial cells,[90, 92] and enhancement of oxidative stress.[90]

In contrast, in a mouse model of endothelial dysfunction, there are leukocyte-derived MPs that stimulated the production of NO, caused coronary vasodilation, and reversed endothelial dysfunction.[93] Beneficial MPs might also play a role in development, hemostasis, angiogenesis, wound healing, and tissue remodeling.[94] For example, in patients with hemophilia, MP levels are persistently increased as compared with normal individuals, and can elevate even further with acute bleeding.[94] Thus, MPs could exert either beneficial or deleterious effects in disease states depending on cellular origin, stimulus for production, and the clinical setting.[13, 94, 95]

Cell-Specific Microparticles

Studies of the cellular origin of MPs in disease population have elucidated their roles and pathophysiologic mechanisms in some diseases. Although MP formation likely occurs in tissues, there is currently no accepted method of characterizing MPs from specific tissues. Our knowledge of MP formation is, therefore, quite limited to a small subset of cells.

Leukocytes

Increased leukocyte-derived MPs, in particular monocyte-derived MPs, occur in patients with multiple organ failure with severe disseminated intravascular coagulation[96, 97] and in those with acute myocardial infarction.[98] Circulating leukocyte-derived MPs are also elevated in humans with pulmonary hypertension and are thought to be a marker for monitoring of vascular inflammation in these patients.[60] Atherosclerotic plaques contain leukocyte-derived MPs. These MPs stimulate cytokine release, bear tissue factor, and expose PS, leading to proinflammatory and procoagulant activity.[32, 57, 99-101]

Endothelial Cells

The presence of endothelial cell-derived MPs is closely related to endothelial damage.[102] They are predictors of death from cardiovascular disease and major adverse cardiovascular events in patients with pulmonary hypertension and coronary artery disease.[61, 103-105] Endothelial cell-derived MPs might be associated with high-risk coronary lesions and could be a useful marker for the risk of acute coronary events.[106] Increased circulating endothelial cell-derived MPs are also associated with acute ischemic stroke,[107-109] severe systemic and pulmonary hypertension,[110, 111] and vascular dysfunction in diseases such as end-stage renal failure.[13, 112] Consequent induction of inflammatory responses contributes to acute lung injury, as a function of increased pulmonary and systemic IL-1β and TNF-α levels and neutrophil migration to the lungs.[18, 113] In vitro experiments suggest that endothelial cell-derived MPs modulate vascular tone by influencing NO and prostacyclin.[80] They are associated with endothelial dysfunction in diabetes,[88, 102] metabolic syndrome,[86, 90] vasculitis,[114] myocardial infarction,[83] malaria,[12, 115] and pre-eclampsia in humans.[89, 116] In animal models, endothelial-derived MPs cause endothelial dysfunction in pulmonary arterial hypertension[30] and endothelial progenitor dysfunction in stroke and diabetes.[117] Endothelial-derived MP levels are elevated in elderly postsurgical patients,[118] and are thought to be a measure of early lung destruction in smokers.[119] In meningococcal sepsis, endothelial-derived MP levels are increased[96] and injection of endothelial-derived MPs into mouse and rat lung demonstrated features of acute lung injury.[120]

Platelets

Platelet-derived MPs interact with target cells to induce various biologic responses, including activation of endothelial cells[45] and polymorphonuclear leukocytes,[121] stimulation of cytokine secretion and tissue factor expression in endothelial cells,[32] and inhibition of apoptosis of polymorphonuclear leukocytes.[50, 54] Two distinct pathways can control platelet PS exposure and procoagulant activity in vitro, namely a calcium-dependent, apoptotic regulator (caspase)-independent pathway induced by physiologic agonists and an apoptosis-mediated (Bak and Bax) and -regulated (caspase) pathway independent of platelet activation.[122]

In addition, platelet-derived MPs are involved in blood coagulation, participate in the pathogenesis of atherosclerosis and vascular injury in inflammation,[123] and promote bone cell proliferation.[33, 124] Interestingly, as the ability to form MPs is essential for physiologic coagulation, a defect in this pathway leads to the clinical bleeding disorder Scott syndrome in humans and its veterinary equivalent, a hereditary bleeding disorder in an inbred colony of German Shepherd dogs characterized by a lack of platelet procoagulant activity.[16, 21, 33] Conversely, increased levels of platelet-derived MPs occur in humans with pulmonary hypertension.[60] These MPs could contribute to the risk for thromboembolic complications via enhanced platelet reactivity. High numbers of platelet-derived MPs have also been reported with cerebral vaso-occlusive events[125] and deep vein thrombosis with or without pulmonary embolism in humans.[16, 126] There is strong evidence that platelet-derived MPs also directly affect pulmonary pressure[127] because of induction of smooth muscle cell proliferation and intima thickening by MP-derived thromboxane A2.[41] Platelet-derived MP concentrations are also elevated in sepsis,[96] heparin-induced thrombocytopenia,[128] idiopathic thrombocytopenia purpura,[129] cerebrovascular events,[130] unstable angina,[131] acute myocardial infarction,[132] and cardiopulmonary bypass.[133]

A subset of platelets, termed coated-platelets, retain high levels of α-granule-derived procoagulant proteins after dual stimulation by both collagen and thrombin.[134] Using a novel methodology for quantitating MPs, coated-platelets cause release of MPs in greater amounts than platelets that are stimulated by collagen or thrombin alone.[134] Bodipy-labeling allows observation of MP generation by prelabeling platelets with low levels of a membrane-permeable fluorescent dye. Visualization of MP generation after several different agonists are added allows quantitation of MPs release from platelets.[134]

Erythrocytes

Erythrocyte-derived MPs are elevated in humans with malaria and are highest in patients with severe falciparum malaria, with parasitized red cells producing >10 times more MPs than nonparasitized red cells.[135] Erythrocyte-derived MP production increases as parasites mature, and is inhibited by N-acetylcysteine, suggesting that erythrocyte-derived MP production is mediated by oxidative stress.[135] Erythrocyte-derived MPs scavenge NO 1000 times faster than intact red blood cells, leading to severe vasoconstriction.[136] These MPs likely contribute to the prothrombotic state associated with hemolytic disorders, including sickle cell anemia, paroxysmal nocturnal hemoglobinuria, thalassemia, hemoglobin H, hereditary spherocytosis, and elliptocytosis.[1, 7, 137, 138] Prevention of vesiculation may reduce the hypercoagulability associated with these pathologies.[35]

Role of Microparticles ex vivo

Erythrocyte concentrate transfusions can have deleterious clinical effects in patients, associated with the immunomodulating and proinflammatory properties of the product.[139, 140] These transfusion complications include febrile nonhemolytic reactions, immunosuppression, thrombocytopenia, urticaria, transfusion-related acute lung injury (TRALI), and transfusion-associated immunomodulation (TRIM).[141, 142] Contaminating leukocytes in erythrocyte concentrate units are a major contributor to adverse events causing a marked inflammatory response, indicated by rise in leukocyte, fibrinogen, and C-reactive protein concentrations in dogs.[143] The number and type of MPs in erythrocyte concentrates might correlate with the documented increase in posttransfusion inflammation in vivo. Vesicle concentrations increase with blood unit storage, but this increase is attenuated by prestorage leukodepletion of either canine or human blood products.[38, 144, 145] Transfusion of older stored erythrocyte concentrates causes more profound inflammatory responses and more in vivo hemolysis than does transfusion of fresher units in both dogs2 and humans.[146]

The cellular origin of MPs formed in the supernatants of stored erythrocyte concentrates is an area of active investigation. In stored human blood, produced MPs are derived from platelets, leukocytes, and erythrocytes, with the latter predominating. Generation of MP subtypes varies considerably over storage time, with all subtypes increasing significantly but at differing time points during the duration of storage.[6] These MPs are able to both activate neutrophils and enhance thrombin generation,[6] supporting the concept that aged blood products are proinflammatory and procoagulant in nature. Leukodepletion before storage decreased production of MPs derived from all 3 cell types.[6] Studies of MP profiles in stored animal blood have been hampered by the lack of availability of suitable reagents that cross-react with animal cells.

Red Blood Cell Storage Lesion

The purpose of erythrocyte transfusions is to improve oxygen delivery to the tissues by increasing the hemoglobin concentration and promoting efficient oxygen-hemoglobin dissociation. The deformability of the erythrocyte is critical in maintaining adequate tissue oxygenation as the cell must appropriately navigate the microcirculation for oxygen delivery.[147] Factors impacting deformability include surface area to volume ratio, membrane elasticity, and intracellular viscosity.[147, 148] Changes to shape and deformability lead to the production of erythrocyte-derived MPs in the microvasculature[147] and in stored products. In vivo, abnormal cells are removed by the spleen,[137] but they accumulate ex vivo over time in stored erythrocyte concentrates.

Erythrocytes develop storage lesions as a function of ex vivo conditions such as temperature and nature of the storage medium.[1] Initially, erythrocytes reversibly change shape from biconcave disks to echinocytes because of ATP depletion.[149] Later, irreversible spheroechinocytes are formed, likely from complete depletion of the adenine nucleotides, ATP, ADP, and AMP.[150, 151] Thus, erythrocytes develop rigid membranes and MPs are generated. Vesiculation and loss of membrane,[138, 152] protein oxidation,[153, 154] and lipid peroxidation occur[155] leading to a loss of deformability[147, 156, 157] and decreased surface area.[137] Further increased rigidity adversely impacts the survival of transfused cells. Erythrocyte-derived MPs scavenge NO, inhibiting in vitro NO availability and signaling.[136] Other accompanying alterations include an increase in the concentrations of supernatant lipids, potassium, lactate, and free hemoglobin, and reduction in pH, glucose, 2,3-diphosphoglycerate, sodium, and ATP.[1, 158]

Adverse Effects of Transfusion

The role of MPs is a growing area of investigation in human transfusion medicine. Erythrocyte- and platelet-derived MPs have recently been implicated as mediators for TRALI, where neutrophils are excessively activated and sequester into the pulmonary tissue.[159] Both platelet-derived MPs (via interactions between P-selectin and its ligand)[160, 161] and erythrocyte-derived MPs (via enriched concentrations of complement and IgG) can activate neutrophils.[6, 162] In addition, the slow leakage or generation of platelet agonists can contribute to microparticle generation in stored blood units in addition to ongoing apoptotic events.

It is likely that MPs are involved in other transfusion-mediated effects, including febrile nonhemolytic transfusion reactions, immunosuppression, thrombocytopenia, urticaria, and TRIM. Recent evidence indicates that an inflammatory response follows autologous transfusion of stored erythrocytes to normal dogs[143] or humans.[5, 146] The importance of this inflammatory reaction is not clear, but may be underappreciated in critically ill patients attributable to pre-existing inflammatory processes. Leukocytes, platelets, and MPs all seem to be involved in the development of inflammation, with leukodepletion attenuating the response. Laboratory biomarkers of inflammation, oxidation, and procoagulant activity are being studied to identify patients at risk for serious adverse transfusion effects.[6]

Detection of Microparticles

Microparticle concentrations are a net reflection of their rate of formation minus their rate of elimination. Plasma concentrations, therefore, reflect the dynamic balance between formation and elimination. Multiple methodologies are currently available for detection and characterization of MPs with regard to size, morphology, concentration, biochemical composition, and cellular origin[163] (Table 3). The gold standard for evaluation of human cell-derived MPs currently is flow cytometry. Protein-conjugated fluorophores are generally implemented as markers for specific detection of the particles of interest.[164, 165] Flow cytometry allows for reproducible enumeration of MPs with interassay and intra-assay variability in ~7–12% and 2–6%, respectively.[109, 166] Some of the limitations of flow cytometry include insensitive detection limits for particles 100–400 nm leading to underestimation of MP numbers because of smaller ones being missed. In addition, antibody binding might not occur attributable to the small fraction of surface antigens available.

Table 3. Advantages and disadvantages of assays utilized in the detection of microparticles
AssayQuantitation MethodAdvantagesDisadvantages

Flow cytometry

Currently the Gold Standard for testing

Protein-conjugated fluorophores detect light scattering properties of MPsAvailable at most research institutions, rapid analysis, single sample can analyze multiple antigensCostly, not readily available, Range of 100–400 nm may be insensitive, antibody-dependent cell-specific origin detection

Immunoassays

Acceptable for testing with limitations

ELISA detects surface antigen on MPsAvailable at most research institutions, less costly than flow cytometry, not restricted by sizeAnalysis done in batches, no size determination possible

Functional assays

Acceptable for testing with limitations

Procoagulant activity detectionReadily available assays, procoagulant activity recordedAnalysis done in batches, no size determination possible, measures procoagulant activity—not specific MP detection

Nanoparticle analysis

Experimental testing technology

Light-scattering properties of MPs detected, video capture of MP motionMP size detection, quantitation of MPsTechnology not readily available, long analysis times, costly

Atomic force microscopy

Experimental testing technology

Scanning of MPs utilizing specialized microscopeMP size detection very accurate, allows for 3D structure of MP and quantitation of MPsTechnology not readily available, cell origin detection requires antibody coating, limited to small sample size

Other scattering and fluorescence methods are also available for MP isolation, but are less commonly employed.[163] These include optical and fluorescence microscopy, fluorescence-based antibody array system, ELISA, solid-phase capture, immunoelectrophoresis, and a chronometric assay.[163, 167-169] ELISA uses either annexin V or antibodies to surface antigens and has the advantage of being readily available and easy to use. Functional assays can be used to determine the procoagulant activity of the sample and our laboratory has reported increased MP procoagulant activity associated with storage time in packed red blood cell units by means of a new functional assay.3 Disadvantages of this approach include inability to quantitate the MPs and the detection of a specific biologic variable with no information on other MP functions. Nanoparticle analysis allows for MP enumeration and visualization of MPs of 25–1500 nm in size and is an emerging technology that has great potential.[170]

Lack of method standardization, inconsistent recovery, and ex vivo microvesiculation all complicate the interpretation and direct comparison of findings from studies of MPs. Characteristics and components of the collected sample (high blood viscosity, small particles such as lipoproteins and platelets, proteins such as fibrinogen and albumin) might influence results.[15, 163, 171] Methodological factors also impact the ultimate results. These include the nature of venipuncture, the anticoagulant used, the time between blood collection and processing, and the centrifugation and washing methods. With the exception of murine-derived MPs, there are very few reagents that have the established sensitivity and specificity to be used for microparticle detection and characterization. Future development and validation of antibodies for use in species other than humans and rodents is needed as this remains a critical challenge in the development of microparticle research in veterinary medicine.

To reduce ex vivo microvesiculation of leukocytes and erythrocytes by shear forces, blood sample collection should be performed by slow-pull syringe venipuncture with 21-gauge (or larger) needles.[15, 172] Use of ACD or sodium citrate as the anticoagulant is recommended.[15] The majority of current protocols for detection of MPs involve washing and centrifugation techniques that minimize background signals contributed by plasma proteins.[15] The methodological approaches for washing and centrifugation are highly variable. Concentration of MPs can help to reduce the problematic signal to noise ratio,[15, 169] but centrifugation force should be conservative to minimize shear force. Centrifugation separates particles based on the size and density of the particle. Lower centrifugal forces will pellet larger and denser particles, whereas small cell fragments require more force and longer times to pellet. Analysis of fresh samples minimizes the potential for fragmentation of residual cells during the freeze/thaw cycle.[15]

Diagnostic, Protective, and Possible Therapeutic Effects of Microparticles

Microparticles have complex roles in a variety of processes, including inflammation, thrombosis, and alterations of vascular reactivity.[18]

Measurement of MP number and type may be used as a screening measure for diagnostic purposes.[15] Risk of hemorrhage or thrombosis in hematologic and oncologic disease may be indicated in part by circulating MPs, in particular tissue factor-bearing MPs. Currently, there are multiple clinical trials in human medicine using MPs as biomarkers of drug treatment efficacy, spanning across the fields of surgery, medicine, oncology, and hematology.[14]

The possible protective potential of MPs is currently under active investigation. Anti-inflammatory and anticoagulant properties and roles in cytoprotection, vascular hyporeactivity, and immunity are being explored.[18] Neutrophil-derived MPs release potent anti-inflammatory effectors (such as annexin A1) at the onset of an inflammatory response, thereby favoring earlier resolution.[173] Annexin A1 inhibits adhesion of stimulated neutrophils to the endothelium via binding to PS.[18, 174] Anticoagulant activities of MPs are a function of expression of thrombomodulin, tissue factor pathway inhibitor, and endothelial protein C receptor.[31] Microparticles may also be cytoprotective via modulation of cytokine and antiapoptotic activity and endothelial barrier stabilization.[175] Circulating MPs from patients with septic shock exert a protective role in vascular function from vascular hyporeactivity[18, 92] and patients with decreased concentrations of endothelial cell-, platelet-, and leukocyte-derived MPs had higher mortality rates and organ dysfunction.[18, 176] Microparticles also contain major histocompatibility complex molecules and induce antigen-specific T-cell activation, and can be immunoprotective in acute allograft rejection after heart transplantation.[18, 177]

In the future, MPs could serve as therapeutic targets in disease. They may someday be suitable targets for thromboprophylaxis, further advancing the strategies to combat conditions with high risk for thrombus formation. Some medications are known to reduce the numbers of circulating MPs, thus minimizing their detrimental effects. Both cilostazol, a selective cyclic AMP phosphodiesterase inhibitor and antiplatelet agent, and ticlopidine, an ADP antagonist, decrease platelet-derived MPs.[178, 179] The latter also impacts monocyte-derived MPs.[179] Losartan and eposartan, antihypertensive agents, lowered or normalized numbers of monocyte- and platelet-derived MPs. Short-term megadose vitamin C showed decreased circulating apoptotic MPs and hindering endothelial cell apoptosis in humans with congestive heart failure, resulting in improvements in endothelial function.[180] Other drugs used in hyperlipidemic diabetic patients and those with the metabolic syndrome[13] have similar effects.

One future target is the generation of in vitro engineered MPs that could be used in vivo. These particles could be produced on demand with specific unique properties and characteristics. Engineered MPs could be designed to overexpress proteins from the cell of origin.[14] For instance, MPs with specific properties related to angiogenesis could be useful in ischemia-reperfusion injury where angiogenesis and new vessel formation is advantageous, or conversely, in the reduction in tumor growth through angiogenesis inhibition.[14] Furthermore, the ability of MPs to transfer proteins and mRNA messages from their cell of origin into target cells results in modification in the cell's phenotype. Microparticles, in a manner analogous to use of plasmids in bacteria, could someday be used to treat genetic or acquired diseases.

Conclusion and Future Directions

Microparticle generation occurs under physiologic and pathologic conditions in vivo as well as ex vivo in stored blood. The role of MPs in hemostasis,[31] thrombosis,[2] transfer of surface proteins,[32] angiogenesis,[33] and apoptosis[65] is tightly controlled and well recognized. There is much less information about MP elimination, and research into this area will shed light on the dynamic equilibrium between formation and elimination. Most recently, the study of MPs has shifted to highlight their roles in disease, investigate their detrimental and beneficial effects, understand their usefulness as biomarkers, and evaluate them as therapeutic targets. The understanding of MPs is evolving at a rapid pace. Their importance in the pathophysiology of many human diseases is likely of relevance for veterinary patients. Research on MPs currently faces several challenges in the area of standardization of methods of detection as well as in standardization of preanalytical variables. Validation of techniques of MP enumeration is essential to allow multicenter comparison of MP generation. Future studies are needed to further our understanding of MPs in animals, and their function in health, disease, and transfusion medicine.

Acknowledgment

Conflict of interest: Authors disclose no conflict of interest.

Footnotes

  1. 1

    Throughout this review, all statements that do not denote a specific species refer to the human medical literature

  2. 2

    Smith SA, McMichael M, Herring JM, et al. Serum biomarkers indicated hemolysis and an inflammatory response to transfusion of autologous stored erythrocyte concentrates. J Vet Intern Med 2012;26(3):778-9

  3. 3

    Ngwenyama TR, McMichael M, Smith SA, et al. Quantitation of procoagulant phospholipid in erythrocyte concentrates stored with and without leukoreduction. J Vet Emerg Crit Care 2011;21(S1):S1-26

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