Blood-Borne Microparticles

JosÉ A. LÓPez

Swaminathan Murugappan

This chapter concerns a blood element that has not been specifically addressed in previous editions of this textbook: bloodborne cell-derived microparticles. Although microparticles have been recognized for many years as important components of the procoagulant function of plasma, it is only in the past decade or so that they have emerged as important actors in many other biologic processes. Their roles are many and diverse. Besides procoagulant activity, they include cellular activation, signaling, endocrine functions, immune modulation, and transfer of biologically important molecules, including proteins, lipids, mRNAs, and microRNAs. The area of microparticle biology is fraught with controversy, owing in large part to the varied methods used to define microparticles, the difficulty in accurately measuring them, and the many artifacts associated with that measurement. Nevertheless, none of these factors justify ignoring them. In this chapter, we attempt to provide a useful way to think about this interesting biologic phenomenon.

DEFINITIONS

Several types of subcellular, membrane-bound particles are found in the blood. The most prevalent are exosomes, apoptotic bodies, and microparticles (FIGURE 36.1).

Exosomes

These particles range in diameter from 50 to 100 nM and are derived from the release of granules containing multivesicular bodies, each multivesicular body producing several exosomes.¹ Multivesicular bodies arise by invagination of granule membranes, producing vesicles whose outer membrane leaflets correspond to the inner membrane leaflet of the granule. Exosomes from immune cells have been the best studied and function in antigen presentation.¹^,²^,³ Conversely, exosomes derived from tumor cells can inhibit the immune response to the tumors by presenting tumor antigens to tolerogenic dendritic cells.⁴^,⁵ Exosomes can also activate the immune response against infectious agents by presenting pathogen-specific antigens.¹

Exosomes can be detected by staining with antibodies against such antigens as CD63, CD81, CD9, and lysosomal-associated membrane protein-1 (LAMP-1), some of which are only found on internal membranes in intact cells.⁶^,⁷ Exosomes are distinguished from microparticles by both their smaller size and their failure to express phosphatidylserine (PS) and are best isolated by a combination of differential centrifugation and sucrose density ultracentrifugation.⁸ They have been studied by a variety of techniques, including transmission electron microscopy (TEM), mass spectrometry, and immunoblotting after isolation by centrifugation.⁸

Apoptotic Bodies

These are membrane-bound cell fragments that arise as cellular blebs from cells undergoing apoptosis and range in size from 1 to 5 µm in diameter, thus overlapping platelets in size.⁹ Like microparticles, they usually express PS on their external membrane leaflets, but unlike microparticles, they often carry DNA derived from the cell of origin.¹⁰

Apoptotic bodies are most commonly identified by flow cytometry, based on both surface antigen expression and annexin V staining (the latter being indicative of surface PS expression) and on their size characteristics. Protocols to isolate apoptotic bodies are lacking, in large part due to the overlap in size and antigen expression they share with other blood components such as microparticles and platelets.

Microparticles

Microparticles, also called microvesicles, are defined as membrane-bound cellular fragments that arise by blebbing from the plasma membranes of cells and range in size from 0.1 to 1.0 µm, with the majority being in the range of 0.2 µm.¹¹^,¹² Blood contains microparticles from a wide variety of cellular sources, including tissue cells and tumors, but the most abundant microparticles arise from blood cells or blood vessel cells in direct contact with the blood, endothelial cells.⁶^,¹³

The most common method for measuring microparticles has been with flow cytometry.¹⁴^,¹⁵ With this method, it is possible to gauge the number and certain phenotypic characteristics of the microparticles, such as their cells of origin (Table 36.1). However, as will be discussed extensively later in this chapter, it is difficult by conventional flow cytometry to accurately count microparticles <500 nm in diameter,¹⁶ unless those particles are stained for phenotype. Most microparticles also express PS on their external membrane leaflet, and this surface characteristic has been used in determining their number.

ORIGINS OF MICROPARTICLES

Blood-borne microparticles are highly heterogeneous with respect to their size and composition, in addition to their cellular sources.¹³ By definition, their sizes span a range of diameters that varies by a factor of 10, from 0.1 to 1 µm. If the shape of a microparticle is assumed to be spherical, this size range translates to a 100-fold difference in surface area and a 1,000-fold difference in volume between the smallest and largest microparticles.

In addition to heterogeneity in size, their cellular sources are extremely varied. In the most common order of abundance
in human blood: platelets, megakaryocytes, endothelial cells, erythrocytes, lymphocytes, monocytes, vascular smooth muscle cells, dendritic cells, and, often, tumor cells.

FIGURE 36.1 Depiction of the different membrane-bound vesicles based on size and comparison to other known biologic particles. As shown, apoptotic bodies, microparticles, and exosomes approximate the sizes of platelets, bacteria, and viruses, respectively.

Platelet and Megakaryocyte Microparticles

The existence of platelet microparticles was first inferred by Chargaff and West¹⁷ in 1946, when they noted that there was a precipitable factor in plasma capable of supporting thrombin generation. In 1967, Wolf¹⁸ described the source of this procoagulant activity as being the platelet and also identified the microparticles morphologically. He termed them “platelet dust.” Wolf also demonstrated that there was a linear relationship between the number of platelets and the quantity of microparticles in the blood, with thrombocytopenic patients having low numbers of microparticles compared to normals and those with polycythemia vera having elevated microparticle numbers.

Microparticles containing platelet markers account for 70% to 90% of circulating microparticles in healthy individuals.¹⁹^,²⁰^,²¹ Markers used to identify these microparticles by antibody staining and flow cytometry include CD41(the α subunit of the integrin α_IIbβ₃), CD42b (glycoprotein [GP] Ibα), CD42a (GPIX), CD61 (the β subunit of the integrin α_IIbβ₃), and CD62P (P-selectin).¹³ Controversy exists over the extent to which platelet microparticles express PS, with some studies using the binding of annexin V as one of the defining characteristics of these microparticles²² and one study finding that up to 80% of microparticles staining with platelet markers did not express PS.²³ This point is an important one, as some methods of microparticle quantification rely on annexin V capture of the microparticles¹⁵ or base their definition of microparticles on the ability to bind annexin V. Despite these concerns, isolation by annexin V capture likely does give a reasonable estimate of the procoagulant capacity of the microparticles.²⁴

Various stimuli are capable of eliciting microparticles from platelets, including thrombin, collagen, complement C5b-9,
high shear stress, and calcium ionophore.²⁵^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³^,³⁴^,³⁵ Microparticle generation is coupled to externalization of PS on the platelet membrane and is a key component of the ability of platelets to support the enzymatic reactions of the coagulation system that eventuate in thrombin production and formation of insoluble fibrin clots.

Table 36.1 Cell-specific surface markers in microparticles

Cell of Origin	Surface Markers
Platelets	CD41 (αIIb) CD61 (beta3) Cd42b (GPIbα) CD62P (P-selectin)
Endothelium	CD31 (PECAM) CD54 (ICAM-1) CD62E (E-selectin) CD105 (endoglin) CD144 (VE-cadherin) CD146 (MUC18)
Red blood cell	CD235a (glycophorin A)
Monocytes	CD14
Lymphocytes	CD4 CD8 CD20 (B-lymphocyte antigen)
Neutrophils	CD66b (carcinoembryonic antigen-related cell-adhesion molecule 8)

Platelets also generate microparticles as they undergo apoptosis, albeit of a different nature than the microparticles produced by agonist stimulation.³⁶^,³⁷^,³⁸^,³⁹

Numerous studies have reported elevated platelet microparticle concentrations in association with a variety of pathologic conditions, including acute coronary syndromes, atherosclerosis, hypertension, diabetes, venous thromboembolism, heparin-induced thrombocytopenia, antiphospholipid syndrome, thrombotic thrombocytopenic purpura, sickle cell disease, human immunodeficiency virus (HIV) infection, sepsis, malignancies, and several other conditions (Table 36.2).

Although it was initially believed that microparticle staining with platelet markers arose entirely from platelets, recent evidence by Flaumenhaft et al.⁴⁰ call this interpretation into question. Using videomicroscopy, these investigators demonstrated the capacity of cultured mouse megakaryocytes to vesiculate, the vesicles arising along the lengths of micropodia. These microparticles expressed CD41, CD42b, and PS. However, they were distinct from platelet microparticles in that they failed to express CD62P and LAMP-1 and they contained uncleaved filamin A, whereas filamin A found in platelet microparticles was uniformly proteolyzed to smaller fragments. In the blood of mice and healthy human volunteers, most of the “platelet” microparticles detected by these investigators did not express CD62P and LAMP-1, leading them to conclude that the microparticles were derived from megakaryocytes. Because both CD62P and LAMP-1 are membrane proteins restricted to granule membranes in unactivated platelets, this finding could also indicate that, in addition to a megakaryocyte source for these particles, they might also arise normally in vivo from platelets that have not been stimulated to release granules.

Endothelium

A second major source of microparticles in the blood is the endothelium. These microparticles arise by blebbing of the endothelial plasma membrane,⁴¹ and they carry a wide variety of endothelial membrane proteins, including CD54 (ICAM-1), CD62E (E-selectin), CD31 (PECAM), CD105 (endoglin), CD144 (VE-cadherin), and CD146 (MUC1).⁶ Because platelet microparticles also express CD31, endothelial cell microparticles are distinguishable by the absence of CD41 staining. A wide variety of stimuli have been reported to induce microparticle production by endothelium, including tumor necrosis factor (TNF)-α, interleukin-1, uremic toxins, oxidized low-density lipoproteins, oxidants such as superoxide and hypochlorous acid, and agents that uncouple nitric oxide synthase.⁴²^,⁴³^,⁴⁴^,⁴⁵^,⁴⁶ Endothelial cells undergoing apoptosis also elaborate microparticles. Microparticles released by cells undergoing apoptosis express more CD31 and less CD62E than those elaborated by cells exposed to activation stimuli.⁴⁷

Endothelial microparticles are elevated in a number of conditions, all of which involve endothelial pathology. These include acute coronary syndromes, hypertension, metabolic syndrome, diabetes, chronic renal failure, systemic lupus erythematosus, sickle cell disease, pulmonary artery hypertension, malaria, and others (Table 36.2).

Erythrocytes

Erythrocyte microparticles make up only a small fraction of microparticles in the blood of normal individuals,¹⁹ but their quantity can be increased considerably in a number of disorders that primarily affect the erythrocyte and other systemic disorders. Hemolytic anemias, both hereditary and acquired, are associated with increased blood microparticle levels, including paroxysmal nocturnal hemoglobinuria,⁴⁸ sickle cell disease,⁴⁹^,⁵⁰^,⁵¹ the thalassemias,⁵²^,⁵³^,⁵⁴ autoimmune hemolytic anemia, malaria,⁵⁵^,⁵⁶ and disorders of the erythrocyte membrane skeleton.⁵⁷^,⁵⁸ Erythrocyte microparticle levels are also often elevated in systemic disorders such as atherosclerosis and chronic renal failure.⁵⁹^,⁶⁰

In addition to conditions that increase erythrocyte microparticles in vivo, microparticles are also produced during extended red cell storage and by conditions that can affect the stored red cells, such as ATP depletion or low pH.⁵⁸^,⁶¹^,⁶²^,⁶³^,⁶⁴ Exposure to agents that elevate intracellular calcium concentrations, such as the calcium ionophore A23187, also induces erythrocytes to produce microparticles. Erythrocyte microparticles are usually identified by staining for glycophorin A (CD235a).⁵⁴^,⁶⁵

Leukocyte Microparticles

Monocytes, neutrophils, and lymphocytes all produce microparticles.⁶⁵ Those derived from monocytes or tissue macrophages are identified by surface expression of CD14 and often are extremely procoagulant by virtue of carrying tissue factor (TF) in addition to expressing anionic phospholipids.⁶⁶ Neutrophils and lymphocytes also shed microparticles. Neutrophil microparticles are elevated in a number of inflammatory states known to be associated with neutrophil activation, including acute vasculitis, preeclampsia, rheumatoid arthritis, and meningitis.⁶⁷^,⁶⁸^,⁶⁹^,⁷⁰^,⁷¹^,⁷² Elevated neutrophil microparticle counts have also been reported in patients with extensive atherosclerosis and in those undergoing hemodialysis.⁶⁰^,⁶⁸

Like monocytes, neutrophils have been reported to elaborate TF-bearing microparticles in response to activation with the anaphylatoxin C5a,⁷³^,⁷⁴^,⁷⁵ although this property might also be a consequence of fusion of TF-bearing microparticles with neutrophils, not from actual TF synthesis by the neutrophils themselves.⁷⁶

MECHANISM OF MICROPARTICLE FORMATION

Several factors have led to platelets being the best studied cell for determining the mechanisms of microvesiculation. First, platelets were the first cell recognized to produce microparticles and for which microvesiculation was noted to be an important part of their physiology.¹⁷^,¹⁸^,⁷⁷ Second, microparticles containing platelet markers are the most prevalent type of microparticle in blood.¹⁹^,²⁰^,²¹^,⁴⁰ Finally, platelets are easy to isolate and produce microparticles rapidly when stimulated.²⁰^,²²

Based on extensive research, it is clear that membrane remodeling and changes in cytoskeletal structure are both involved in the production of microparticles. A simplified illustration of the mechanism of microparticle formation resulting

from cell activation or apoptosis is shown in FIGURES 36.2 and 36.3, respectively.

Table 36.2 Circulating cell-derived microparticles in diseases

Clinical Condition	MP Origin	MP Level in Blood	References
Atherosclerosis	Platelet Endothelium Monocyte	Increased	van der Zee et al.²⁰² Bernal-Mizrachi et al.²⁰³ Koga et al.²⁰⁴ Bulut et al.²⁰⁵ Werner et al.²⁰⁶ Kockx²⁰⁷ Leroyer et al.⁶⁰ Nomura et al.²⁰⁸
Hypertension	Platelet Endothelium	Increased	Preston et al.²⁰⁹
Hyperlipidemia	Endothelium Platelet	Increased	Nomura et al.¹⁴² Boulanger et al.²¹⁰
Acute coronary syndrome	Platelet Endothelium	Increased	Huisse et al.²¹¹ Bernal-Mizrachi et al.²⁰³ van der Zee et al.²⁰² Matsumoto et al.²¹² Singh et al.²¹³ Mallat et al.²¹⁴ Werner et al.²⁰⁶ Heloire et al.²¹⁵
Diabetes	Platelet Endothelium Monocyte	Increased	Feng et al.²¹⁶ Bulut et al.²⁰⁵ Nomura et al.²¹⁷ Nomura et al.²¹⁸ Tan et al.²¹⁹ Ogata et al.²²⁰ Omoto et al.²²¹ Ogata et al.²²²
Deep venous thrombosis and pulmonary embolism	Platelet Endothelium	Increased	Ye and Wang. Throm Res. 2011.²⁸⁶ Barnes et al.²²³ Chirinos et al.²²⁴ Wakefield et al.²²⁵
Heparin-induced thrombocytopenia	Platelet Monocyte	Increased	Kelton et al.²²⁶ Warkentin et al.²²⁷ Hughes et al.¹⁴⁸
Antiphospholipid syndrome	Platelet Endothelium	Increased	Combes et al.⁴³ Dignat-George et al.²²⁸ Morel et al.⁸⁹ Jy et al.²²⁹ Ambrozic et al.²³⁰
Thrombotic thrombocytopenic purpura	Platelet Endothelium	Increased	Kelton et al.²³¹ Jimenez et al.²³² Jimenez et al.²³³
Paroxysmal nocturnal hemoglobinuria	Red blood cell Platelet	Increased	Hugel et al.²³⁴ Kozuma et al.⁴⁸ Wiedmer et al.²³⁵ Sims et al.²³⁶
Sickle cell disease	Platelet Endothelium Red blood cell Monocyte	Increased	Shet et al.⁵⁰ Wun et al.²³⁷ Allan et al.⁴⁹ van Beers et al.⁵¹
Thalassemias	Platelet Red blood cell	Increased	Pattanapanyasat et al.⁵⁴ Pattanapanyasat et al.²³⁸ Habib et al.⁵³ Freikman et al.⁵²
Sepsis	Platelet Endothelium Neutrophil Monocyte Red blood cell	Increased	Meziani et al.²³⁹ Nieuwland et al.⁷¹ Itakura et al.⁷⁰ Soriano et al.²⁴⁰ Fujimi et al.⁶⁹ Joop et al.²¹ Ogura et al.²⁴¹ Walenta et al.²⁴²
HIV	Platelet Monocyte Lymphocyte	Increased	Mayne et al.²⁴³ Corrales-Medina et al.²⁴⁴ Gris et al.²⁴⁵ Mack et al.²⁴⁶ Aupeix et al.¹²⁹ Rozmyslowicz et al.²⁴⁷
Peripheral arterial disease	Platelet	Increased	Tan et al.²⁴⁸ van der Zee et al.²⁰² Nomura et al.²⁰⁸
Strokes	Platelet Endothelium	Increased	Kuriyama et al.²⁴⁹ Simak et al.²⁵⁰ Jung et al.²⁵¹ Lee et al.²⁵²
Chronic renal failure	Platelet Endothelium Red blood cell	Increased	Faure et al.²⁵³ Amabile et al.⁵⁹ Boulanger et al.²⁵⁴ Ando et al.²⁵⁵ Daniel et al.²⁵⁶
Immune thrombocytopenic purpura	Platelet	Increased	Nomura et al.²⁵⁷ Jy et al.²⁵⁸ Nomura et al.²⁵⁹ Ahn and Horstman²⁶⁰ Fontana et al.²⁶¹ Tantawy et al.²⁶²
Malaria	Endothelium Red blood cell Platelet	Increased	Combes et al.²⁶³ Combes et al.²⁶⁴ Coltel et al.⁵⁵ Faille et al.²⁶⁵ Nantakomol et al.⁵⁶
Systemic lupus erythematosus	Platelet Endothelium Leukocyte	Increased	Lazarus et al.²⁶⁶ Dignat-George et al.²²⁸ Nagahama et al.²⁶⁷ Nagahama et al.²⁶⁸ Pereira et al.²⁶⁹ Combes et al.⁴³
Rheumatoid arthritis	Platelet Leukocytes Lymphocyte	Increased	Knijff-Dutmer et al.²⁷⁰ Berckmans et al.⁶⁷ Berckmans et al.¹⁴⁶ Umekita et al.⁷²
Multiple sclerosis	Endothelium Oligodendrocyte (CSF)	Increased	Minagar et al.²⁷¹ Larkin²⁷² Jy et al.²⁷³ Sheremata et al.²⁷⁴ Scolding et al.²⁷⁵
Systemic sclerosis	Platelet Endothelium Leukocyte	Increased	Guiducci et al.²⁷⁶ Nomura et al.²⁷⁷
Vasculitides	Platelet Endothelium Leukocyte	Increased	Brogan and Dillon²⁷⁸ Daniel et al.⁶⁸ Erdbruegger et al.²⁷⁹
Pulmonary arterial hypertension	Endothelium Leukocyte	Increased	Amabile et al.²⁸⁰ Bakouboula et al.²⁸¹
Scott syndrome	Platelet	Decreased	Sims et al.³³ Zwaal et al.⁷⁷ Albrecht et al.⁹⁴
Cancer	Platelet Monocytes Tumors Endothelium	Increased	Toth et al.²⁸² Tilley et al.¹⁹⁷ Campello et al.¹⁹⁸ Khorana et al.²⁰⁰ Uno et al.²⁰¹ Amin et al.²⁸³ Goon et al.²⁸⁴ Kanazawa et al.²⁸⁵ Owens and Mackman⁶⁶