Blood-Borne Microparticles

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.
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 αIIbβ3), CD42b (glycoprotein [GP] Ibα), CD42a (GPIX), CD61 (the β subunit of the integrin αIIbβ3), 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

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Jun 21, 2016 | Posted by in HEMATOLOGY | Comments Off on Blood-Borne Microparticles

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