Cell-derived microparticles : composition and function - Chapter 1: General introduction, aims and outline of this thesis

s

Cell-derived microparticles : composition and function

Biró, É.

Publication date

2008

Link to publication

Citation for published version (APA):

Biró, É. (2008). Cell-derived microparticles : composition and function.

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HAPTER

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General introduction, aims and outline of

this thesis

ell-derived microparticles were observed experimentally as early as 1946, when

Chargaff and West published their findings that the clotting time of plasma,

obtained by centrifugation at 1900 × g, is prolonged by centrifugation at 31000 ×

g, and that “the clotting factor of which the plasma is deprived by high-speed centrifugation

is found in a minute pellet whose sedimentation is brought about by this operation” [1]. In

1967, Wolf published a study “to provide evidence for the occurrence in normal plasma,

serum and fractions derived therefrom of coagulant material in minute particulate form,

sedimentable by high-speed centrifugation and originating from platelets, but

distinguishable from intact platelets”. He called this coagulant material “platelet dust” [2].

During the last 20-30 years extensive research has been done on cell-derived

microparticles. We now know that they can be released not only from platelets, but also

from erythrocytes, leukocytes, endothelial cells, and diverse other cell types, and that such

microparticles are not only present in the circulation but also in other body fluids, such as

synovial and cerebrospinal fluids [3-5].

Microparticles are released by a budding process from the surface membrane of the

cells upon activation or apoptosis, and according to the definition formulated at a 2005

Scientific Subcommittee Meeting of the Working Group of Vascular Biology of the

International Society on Thrombosis and Haemostasis, they are 0.1–1 μm vesicular

structures of diverse cellular origin that lack a nucleus or synthetic capability, but may

contain a membrane skeleton, and varying amounts of surface-exposed phosphatidylserine

(PS). Their composition and function depend upon their cellular origin and the inducer of

the vesiculation.

In vivo, the numbers, cellular origin, composition and function of the microparticles

vary depending on the body fluid analyzed, as well as the pathophysiological state of the

individual. This relationship between microparticles and pathophysiological processes is

complex and two-way, since the formation and composition of microparticles can be

influenced by diverse pathophysiological phenomena as well as medication and, in turn,

microparticles can aggravate or attenuate certain pathophysiological processes.

Processes leading to microparticle release from cells

Microparticles are released from their parent cells upon activation or apoptosis by a

budding process, resulting in right-side out vesicles, as also documented by electron

microscopy [6-10].

Cell activation

Activation of platelets for example with collagen, thrombin, collagen plus thrombin, by

adhesion to von Willebrand factor under high shear stress, or by incubation with sera from

patients with heparin-induced thrombocytopenia in the presence of heparin [11-14] results

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in the formation of platelet-derived microparticles. Similarly, stimulation of leukocytes or

endothelial cells with lipopolysaccharide (LPS) or cytokines such as tumor necrosis

factor-α (TNFfactor-α) or interleukin (IL)-1 [9,15-17] causes the release of microparticles from these

cells. These stimuli act via a membrane receptor. The ligand-receptor interaction induces

changes in the intracellular concentrations of second messengers such as calcium ions and,

in turn, these second messengers influence the activity of protein kinases and phosphatases

and activate calpain, a calcium-dependent cysteine protease. Subsequently, the cytoskeleton

is reorganized / degraded, resulting in membrane blebbing and release of microparticles

[8,12,18-21]. Alternatively, stimulation by the membrane attack complex C5b-9 of the

complement system, which forms lytic pores in cell membranes, or addition of a calcium

ionophore (e.g. A23187) can directly increase the intracellular calcium concentration,

resulting in similar downstream effects as described above, in platelets [18,20-22], as well

as in erythrocytes [23-25], leukocytes [26,27], and endothelial cells [28].

The involvement of the integrin α

β

in platelet microparticle formation has also been

investigated. Integrin α

β

or glycoprotein (GP)IIb-IIIa is the most abundant receptor on

the platelet surface [29] and is involved in inside-out and outside-in signaling processes as

well as platelet aggregation [30]. It has been reported that this complex is required for

microparticle formation upon stimulation of platelets with various agonists [31], but other

studies have found that it has no or only a limited role [32-34]. Evidence has also been

presented that upon storage of platelets, in the absence of agonists and with only minor

platelet activation, release of platelet-derived microparticles is dependent upon α

β

signaling, with a lesser role for calpain activity [35]. Other studies, however, gave

contradictory results, showing a role for platelet activation processes in microparticle

formation during storage [36], or a role for apoptosis-like events mediated either by

caspases or calpain (see below) [37-40]. A role for α

β

in regulating the activation of

calpain has also been described [41]. In summary, the precise role of this integrin in

microparticle generation during activation with different agonists still needs to be clarified.

Apoptosis

Apoptosis, a form of programmed cell death, is characterized by cell shrinkage, loss of

membrane asymmetry and exposure of PS, nuclear condensation, DNA fragmentation, the

release of apoptotic membrane blebs, and the formation of apoptotic bodies [42-45]. A

family of cysteine proteases called caspases plays a central role in the apoptotic machinery

[46]. Membrane blebbing can be induced by the Rho-associated kinase ROCK I, which is

cleaved and thereby activated by caspases. Activated ROCK I subsequently contributes to

phosphorylation of myosin light chains, myosin ATPase activity, and coupling of

actin-myosin filaments to the plasma membrane, thus promoting membrane blebbing and

relocalization of fragmented DNA into blebs and apoptotic bodies [47,48]. Another

possible mechanism is cleavage and thereby activation of the Rho-associated kinase ROCK

II by granzyme B during granule exocytosis-mediated cell death, employed for example by

NK cells and cytotoxic T lymphocytes. Activated ROCK II causes membrane blebbing in a

manner similar to ROCK I [49].

Treatment of endothelial cells with mitomycin C, camptothecin or staurosporin, or

growth factor deprivation of these cells all result in apoptosis and release of microparticles

[50-53].

Although apoptosis is a process pertaining to nucleated cells, some platelet activation

processes such as PS exposure and microparticle formation resemble apoptotic events, and

platelets also contain several caspases [37,54,55]. Whether these caspases play a role in PS

exposure and microparticle formation in platelets upon stimulation with agonists such as

thrombin, collagen plus thrombin or calcium ionophore A23187, is currently a matter of

discussion, and a role for calpain instead of caspases has also been proposed [54-57].

Similarly, apoptosis-like events in platelets during storage have been linked to activation of

caspases in some studies, but others have disputed this and again suggested a role for

calpain in these processes [37-40].

Erythrocytes can also undergo morphological alterations resembling apoptosis,

including PS exposure and microparticle release, and these cells have also been shown to

contain caspases and other components of the apoptotic machinery. However, as is the case

with platelets, the role of caspases in the apoptosis-like events occurring in erythrocytes is

controversial [58,59]. Regarding microparticle release from erythrocytes upon storage, it

has been postulated that under these conditions the normal aging process of these cells is

accelerated, and that this aging process resembles apoptosis, also regarding the mechanism

of microparticle release [60,61].

Composition of cell-derived microparticles

Lipids

Microparticles are surrounded by a lipid bilayer composed mainly of phospholipids and

cholesterol. This lipid bilayer is formed by budding and subsequent release of a part of the

outer membrane of the parent cell (Figure 1). The exact lipid composition of the released

microparticle is dependent upon the type of cell from which it was released and certain

(patho)physiological processes that can modify the lipid composition of membranes.

Cell-derived microparticles isolated from venous blood of healthy individuals,

originating predominantly from platelets but also from erythrocytes, leukocytes and

endothelial cells [4], contain mostly phosphatidylcholine (PC) and sphingomyelin (SM),

with smaller amounts of phosphatidylethanolamine (PE), PS, and phosphatidylinositol (PI),

and minute quantities of the lysophospholipids lyso-PC, lyso-PE and lyso-PS [62].

Platelet-derived microparticles made in vitro by stimulating washed platelets with collagen differ

from this in that they contain less PC and more SM, PE and PS [63]. Quite different is the

phospholipid composition of microparticles isolated from synovial fluid of inflamed joints.

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Resting cell

Activated cell

Microparticles

Figure 1. Schematic representation of the formation and composition of microparticles. Intact resting

cells have an asymmetric phospholipid distribution between the inner and outer leaflet of their plasma membrane, with the aminophospholipids PS and PE (depicted in black) located predominantly in the inner (cytoplasmic) leaflet, and the outer leaflet composed mainly of the choline-containing phospholipids PC and SM (depicted in white). Upon activation of the cell (and upon apoptosis), this phospholipid asymmetry collapses and the aminophospholipids PS and PE become exposed on the outer leaflet as well. Furthermore, depending on the cell type, granule secretion might occur upon activation, via fusion of the granule membranes with the plasma membrane of the cell.

Microparticles are formed by budding and subsequent release of the plasma membrane of activated (or apoptotic) cells. Proteins (depicted in gray) found in or associated with the plasma membrane and proteins found in the cytoplasma can also partition into the microparticles that are being released, either selectively or in a random manner.

PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin

These microparticles are derived mainly from leukocytes and only to a small extent from

erythrocytes and platelets [64], and contain less PC, more PE, and very high quantities of

lysophospholipids compared with microparticles of healthy individuals [3]. The high

quantities of lysophospholipids can be explained by the high concentrations of secretory

phospholipase A

(sPLA

) in the synovial compartment, which can hydrolyze the sn-2 ester

Important to mention is that intact resting cells have an asymmetric phospholipid

distribution between the inner and outer leaflet of their plasma membrane, whereby the

aminophospholipids PS (which is negatively charged) and PE (which is neutral) are located

mainly in the inner leaflet, and the outer leaflet is composed mostly of the

choline-containing (neutral) phospholipids PC and SM. This asymmetric phospholipid distribution

is actively maintained by enzymes such as the ATP-dependent aminophospholipid

translocase and floppase. Inhibition of these enzymes, particularly the aminophospholipid

translocase, and activation of a lipid scramblase results in the collapse of this phospholipid

asymmetry and the exposure of negatively charged phospholipids in the outer leaflet of the

plasma membrane [65]. This is known to occur upon cell activation as well as apoptosis

[15,44,45,66-68], processes that also result in microparticle release from the plasma

membrane of the cells, as described above. Several studies have found that the phospholipid

distribution of the lipid bilayer surrounding the microparticles is also symmetric, with the

microparticles exposing PS on their surface [68-71]. This can be visualized for example by

using fluorescently labeled annexin V (formerly also called, among several other names,

placental anticoagulant protein I), a protein that binds to PS-containing membranes in the

presence of calcium ions [72,73]. However, a few investigators have also reported the

presence of microparticles without negatively charged phospholipids on their surface, i.e.

with an asymmetric phospholipid distribution [52,74,75], questioning the assumption that

PS exposure is a general characteristic of microparticles.

Lipid rafts are cholesterol- and sphingolipid-rich membrane microdomains that play an

important role in membrane trafficking, protein sorting and signal transduction [76-78]. The

fact that certain proteins associated with lipid rafts are selectively enriched in

microparticles (see below) suggests that the lipid rafts themselves, and thus the lipids found

in them, might also be enriched in microparticles compared to their parent cells. The

enrichment of raft lipids, however, has not been directly investigated.

Proteins

Proteins in cells are either found in or associated with the plasma membrane, or are found

within the cell, in the cytoplasma, associated with cell organelles, or in the nucleus. Upon

formation of microparticles, these proteins can partition into the microparticles that are

being released (Figure 1). Some of them are specific for the parent cell type, and thus

enable the determination of the cellular origin of the microparticles.

There is evidence that some proteins are selectively sorted into microparticles, i.e. they

are enriched or depleted in the microparticles compared to their parent cells. For example in

erythrocyte-derived microparticles, depletion of spectrin and enrichment of

acetylcholinesterase, decay accelerating factor (DAF), CD59, stomatin, and complement

receptor 1 has been described [79-84]. Stomatin and the

glycosylphosphatidylinositol-anchored acetylcholinesterase, DAF and CD59 are proteins associated with lipid rafts

[78,85], and their enrichment in the microparticles compared with their parent cells

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suggests that the lipid rafts themselves might be preferentially sorted into microparticles, or

that the rafts are in some other way implicated in the process of microparticle formation.

Enrichment of stomatin has also been shown in platelet-derived microparticles [86]. In

monocyte-derived microparticles, the enrichment of tissue factor (TF) and P-selectin

glycoprotein ligand-1 (PSGL-1), and the depletion of CD45 has been demonstrated, and the

first two proteins have also been shown to localize to lipid rafts [87].

The proteins found in microparticles derived from a certain cell type can also vary

depending upon the stimulus that induces the release of these microparticles. This has been

extensively described for several surface antigens found on endothelial cells, stimulated to

release microparticles by activation or apoptosis [17,52].

Regarding intracellular proteins, very little data are available. The finding that

platelet-derived microparticles contain platelet factor XIII (subunit composition a

) but not plasma

factor XIII (subunit composition a

b

) provides extra evidence that such microparticles are

formed by a budding process [88]. Furthermore, recent studies have shown that caspase 3 is

present in microparticles released from endothelial cells, and that inhibition of

microparticle release results in caspase 3 accumulation in the cells, causing their increased

apoptosis and detachment [53,89]. In platelet-derived microparticles released during storage

of platelets, only procaspase 3 is present during the first few days of storage. Caspase 3 in

the microparticles can only be detected from the 7

_{day onward, probably as a result of}

procaspase 3 processing by caspase 9, which is also present in the microparticles [90]. The

exact significance of these findings still has to be determined.

In recent years large-scale protein identification studies have become feasible using a

proteomic approach. This has also been applied, though to a very limited extent, to the

analysis of microparticles. Microparticles isolated from plasma, platelet-derived

microparticles generated in vitro using adenosine diphosphate (ADP), endothelial

cell-derived microparticles generated using TNFα with concomitant serum-deprivation, and

erythrocyte-derived microparticles generated during blood bank storage have been

analyzed, and hundreds of proteins identified, including those found intracellularly.

Furthermore, the proteome of erythrocyte-derived microparticles has been

semiquantitatively compared to that of erythrocyte membranes, again illustrating the

selective sorting of proteins into the released microparticles [91-95].

Functions of cell-derived microparticles

Thrombosis and hemostasis

Coagulation activation in vivo occurs predominantly via TF and factor VII [96]. Several

ensuing steps in the coagulation activation cascade are dependent upon a negatively

charged phospholipid surface upon which complexes of coagulation factors can assemble.

These complexes bind to the phospholipid surface in the presence of calcium ions via the

negatively charged N-terminal domains of the vitamin K-dependent factors II, VII, IX and

X. These N-terminal domains, also called Gla domains, contain several γ-carboxyglutamic

acid (Gla) residues, which are created by the posttranslational vitamin K-dependent

carboxylation of glutamic acid residues in the liver [97].

Microparticles can expose PS, thereby providing a negatively charged phospholipid

surface [68-71], and may also expose TF, as demonstrated on in vitro [15-17,98-100] as

well as in vivo released microparticles [64,101-104]. This provides the basis for their

procoagulant properties. In addition, they may also have indirect effects on coagulation by

inducing the expression of TF in various cell types. For instance, microparticles released

from leukocytes stimulated by a chemotactic peptide as well as microparticles released

from apoptotic monocytes induce TF expression by endothelial cells in culture [105,106].

Conversely, endothelial cell-derived microparticles generated in vitro bind to monocytic

cells and induce the expression of TF on them. This is partly dependent on the interaction

between intercellular adhesion molecule (ICAM)-1 on the microparticles and β

integrins

on the monocytic cells [107].

Evidence that microparticles are indeed procoagulant is available on several levels.

First, increased concentrations of circulating cell-derived microparticles are present in

clinical conditions associated with a thromboembolic tendency, such as heparin-induced

thrombocytopenia [13], patients with lupus anticoagulant [9], the antiphospholipid

syndrome [108], systemic lupus erythematosus [109], cerebrovascular accidents [110],

acute coronary syndromes [111,112], venous thromboembolism [113], or patients

undergoing cardiac surgery with cardiopulmonary bypass (CPB) [114]. Second, the

decreased capacity of platelets to generate microparticles upon stimulation in Scott

syndrome and a disorder described by Castaman et al. [11,115] is associated with a

bleeding tendency. Third, microparticles isolated from blood or other body fluids of healthy

individuals or patients suffering from various diseases initiate and support coagulation in

vitro [4,64,101,109,116-118]. Microparticles isolated from venous blood of healthy

individuals (originating predominantly from platelets, but also from erythrocytes,

leukocytes and endothelial cells) cause a low but significant coagulation activation in vitro

that is independent of TF/factor VII [4], whereas microparticles from the pericardial blood

of patients undergoing cardiac surgery (originating mainly from erythrocytes, but also from

other cell types such as platelets and leukocytes) and those isolated from synovial fluid of

arthritic patients (derived mainly from leukocytes, and only to a small extent from

erythrocytes and platelets), for example, initiate coagulation in a TF/factor VII-dependent

manner [64,116]. Fourth, microparticles made in vitro by stimulation of platelets with the

calcium ionophore A23187 are thrombogenic in an arterio-venous shunt model in rats

[119].

Studies on the possible mechanisms underlying the procoagulant effect of

microparticles have shown the binding of activated coagulation factors V and X (denoted

factors Va and Xa), which form the prothrombinase complex, and the binding of factors

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VIII and IXa, which, when both activated, comprise the tenase complex. The exact nature

of the binding sites for these coagulation factors still remains to be determined, and the

involvement of specific receptors in addition to the negatively charged phospholipid surface

cannot as yet be excluded [28,120-123]. Further investigations have shown in vitro the

association of procoagulant platelet-derived microparticles with fibrin during thrombus

formation [124] as well as the transfer of TF from leukocytes to platelets via microparticles

released from the leukocytes. This transfer of TF is dependent on the interaction of the

Lewis

_{antigen (CD15) on the leukocyte-derived microparticles with P-selectin on the}

platelets [125]. (P-selectin or CD62P is an adhesion molecule present in the α-granule

membranes of resting platelets and exposed on the platelet surface upon activation and

secretion of the granule contents [126,127].) In addition, using an in vivo laser-induced

endothelial injury model in mice and real-time imaging using intravital high-speed

microscopy, the binding of in vitro generated monocyte-derived microparticles exposing TF

was shown to activated platelets in developing thrombi, mediated by the binding of PSGL-1

on the microparticles to P-selectin on the platelets [128].

Finally, it must be mentioned that microparticles can also have anticoagulant properties

by binding and promoting the activity of protein C. Protein C is a vitamin K-dependent

anticoagulant protein, which in its active form and in the presence of its (also vitamin

K-dependent) cofactor, protein S, cleaves and thereby inactivates factors Va and VIIIa. It is

activated by thrombin bound to thrombomodulin [129]. In a study of factor Va inactivation

by activated protein C on resting and activated platelets, it was found that platelet activation

by thrombin, collagen, or the calcium ionophore A23187 (but not ADP or adrenalin)

resulted in acceleration of factor Va inactivation when compared with non-activated

platelets, and that about 25% of the anticoagulant activity of the platelet suspensions was

associated with the microparticles released from the platelets [130]. Also, it was shown that

protein S binds to platelet-derived microparticles via its Gla domain and stimulates the

binding of protein C as well as activated protein C to these microparticles [131].

Furthermore, the presence of the endothelial protein C receptor (EPCR) has been

demonstrated on microparticles released from cultured endothelial cells or monocytes

isolated from human blood, stimulated with activated protein C or TNFα. EPCR is a

transmembrane protein (located primarily on the endothelium of large blood vessels but

also on monocytes and neutrophil and eosinophil granulocytes) that binds protein C and

enhances its activation by the thrombin/thrombomodulin complex [132-135]. The

microparticle-associated EPCR was full-length, and when using activated protein C as a

stimulus, also contained bound activated protein C, which retained its anticoagulant activity

[136]. Presumably, the relative contribution of the pro- and anticoagulant properties of

microparticles to their overall hemostatic function depends on the specific conditions that

are being investigated.

Inflammation

Cell-derived microparticles play a role in inflammatory processes in various ways. First, by

transporting and transferring bioactive molecules or through direct receptor-ligand

interactions they can activate or alter the function of various cell types, for example by

inducing increased expression of adhesion molecules or increased cytokine production.

Experimental data regarding these functions of platelet-, endothelial cell- and

leukocyte-derived microparticles are summarized in the following paragraphs.

Platelet-derived microparticles contain arachidonic acid [137], which increases the

adhesion of monocytes to endothelial cells by inducing upregulation of leukocyte

function-associated antigen-1 and macrophage antigen-1 on the monocytes and ICAM-1 on the

endothelial cells, and induces chemotaxis of monocytic U937 cells [138]. Furthermore, the

arachidonic acid in the microparticles induces cyclooxygenase (COX)-2 expression as well

as prostaglandin and thromboxane production in monocytic U937 cells [139], and COX-2

expression and prostacyclin production in endothelial cells [137]. Platelet-derived

microparticles also contain and deliver the chemokine RANTES to activated or

atherosclerotic endothelium, thereby promoting monocyte recruitment and arrest [140]. In

addition, platelet-derived microparticles bind neutrophil granulocytes via P-selectin – sialyl

Lewis

_{interactions, resulting in aggregation and activation of these cells, as reflected by}

increased CD11b expression and phagocytic activity [141]. Furthermore, platelet-derived

microparticles can bridge leukocytes via P-selectin – PSGL-1 interactions under flow

conditions [142], and alter the expression of several inflammatory genes in monocytic cells,

as shown using DNA microarray technology [143].

Microparticles released from endothelial cells upon peroxide treatment contain

oxidized phospholipids, which activate neutrophils via their receptor for platelet-activating

factor (PAF), resulting in increased adhesion of these cells as assayed on a gelatin matrix

[144]. Such microparticles also stimulate monocyte adhesion to endothelial cells in a PAF

receptor-mediated process [145].

Microparticles released from activated leukocytes induce endothelial cell activation

and the release of IL-6 and monocyte chemoattractant protein (MCP)-1 from these cells

[105,146]. Also, microparticles released from activated T cells induce the synthesis of TNF

and IL-1β in monocytes [147], and microparticles derived from T cells and monocytes

induce the synthesis of matrix metalloproteinase-1, -3, -9, and -13 as well as IL-6, -8 and

MCP-1 and -2 in fibroblasts [148]. Microparticles from synovial fluid of arthritis patients,

mainly originating from granulocytes and monocytes, increase the synthesis of IL-6, -8,

MCP-1, RANTES, ICAM-1 and vascular endothelial growth factor, and decrease the

synthesis of granulocyte-macrophage colony-stimulating factor in synovial fibroblasts

[149]. Also, microparticles derived from activated monocytic cells contain intravesicular,

bioactive IL-1β, which they deliver to other cells and thereby activate their IL-1 receptors

[150]. Furthermore, transfer of the chemokine receptor CCR5 between cells via cell-derived

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microparticles demonstrates that transfer of receptors may also represent a means by which

microparticles can modulate inflammatory processes [151].

A second type of mechanism by which cell-derived microparticles might possibly

contribute to inflammatory processes is activation of the complement system. In vitro,

microparticles derived from apoptotic Jurkat cells or activated neutrophil granulocytes were

found to bind complement component C1q and activate the classical pathway of

complement, as shown by the deposition of downstream complement components C4 and

C3 [152-154]. However, when comparing ex vivo microparticles isolated from plasma of

healthy individuals and patients suffering from systemic lupus erythematosus, a disease in

which complement activation plays an important pathogenic role, no differences were

found in C1q binding [152]. The binding of other complement components was not

analyzed, leaving the question whether cell-derived microparticles also play a role in

complement activation in vivo unanswered.

Finally, cell-derived microparticles also have anti-inflammatory properties.

Microparticles released from activated neutrophils, for example, do not activate human

macrophages as assessed by their release of IL-8, -10 and TNFα, but increase their release

of the anti-inflammatory cytokine transforming growth factor-β1 (TGFβ1). Furthermore,

these microparticles decrease the production of IL-8, -10 and TNFα in the macrophages in

response to stimuli such as LPS [155]. Additionally, microparticles derived from activated

T cells induce the synthesis of the anti-inflammatory secreted IL-1 receptor antagonist in

monocytes [147].

Other pathophysiological processes

Their have been several reports linking microparticles to other pathophysiological

processes such as endothelium-dependent vasomotor dysfunction [156-160], transmission

of infections [151,161], angiogenesis [162-168], tumor progression and metastasis

[164,169-177], as well as cell survival and apoptosis induction [53,90,174,178]. The role of

microparticles in these processes is partly a consequence of their inflammatory properties

detailed above, but further discussion of these aspects of microparticle function is beyond

the scope of this thesis.

Aims and outline of this thesis

As detailed above, over the past 60 years, and especially during the last 2 decades,

countless studies have been performed to unravel the mechanisms behind the formation of

cell-derived microparticles and to gain detailed knowledge regarding their composition and

function in various disease states. The ultimate goal of these studies was to eventually find

ways to modulate the processes in which microparticles are involved, to ameliorate certain

diseases. However, many issues have still remained unresolved. The studies described in

this thesis therefore aim to further elucidate the composition of cell-derived microparticles

as well as their role in coagulation and inflammation (in particular, complement activation)

in various clinical conditions.

Section I

In Chapter 2, a contribution to a forum on measuring circulating cell-derived

microparticles [179], the methods employed in our laboratory for blood collection and the

initial processing and storage of samples for microparticle analyses are described, as well as

our methods used for isolation and flow cytometric analysis of microparticles.

Section II

In the studies presented in this section, the composition and procoagulant properties of

microparticles of various origin are investigated. In Chapter 3, platelet-derived

microparticles obtained with various stimuli are compared with isolated platelet membrane

fractions (plasma-, granule- and intracellular membranes) regarding their phospholipid

composition and cholesterol content, to determine the origin of microparticle lipids, and to

determine whether these lipids are sorted selectively into microparticles upon their

formation and whether different stimuli result in different lipid compositions of the

microparticles released from the same cell type. Furthermore, the exposure of activation

markers on platelets and platelet-derived microparticles and their relation to the

phospholipid content of the microparticles is determined. In Chapter 4, the

thrombogenicity of cell-derived microparticles isolated from human blood is investigated

for the first time in an in vivo thrombosis model, and the role of TF exposed on the

microparticles is determined herein. In Chapter 5, the phospholipid composition of

endothelial cell-derived microparticles released from resting or IL-1α-stimulated cells is

analyzed. Furthermore, the thrombogenicity of these microparticles and the role of TF

herein is determined in the in vivo thrombosis model also used in Chapter 4. In Chapter 6,

the relationship between the activation status of platelets, assessed by the analysis of their

exposure of the activation markers P-selectin and CD63, and the numbers and P-selectin

and CD63 exposure of the microparticles released from them upon in vitro stimulation is

determined. (CD63 is a member of the tetraspanin superfamily present in the lysosomal-

and dense granule membranes of resting platelets, and exposed on the platelet surface upon

exocytosis of the respective granules [180,181].) Subsequently, the concentrations of

platelet-derived microparticles circulating in vivo and the percentages exposing P-selectin

or CD63 are measured in young and older healthy individuals and patients suffering from

cardiovascular diseases, as a reflection of the platelet activation status in vivo in these

conditions. In Chapter 7, to try and clarify existing inconsistencies in the literature

regarding the role of coagulation, platelet and endothelial cell activation and fibrinolysis in

Fabry disease, markers of these processes as well as their possible association with

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circulating microparticles are analyzed in these patients, also taking into consideration the

severity of the disease and renal function.

Section III

In the studies described in this section, the complement activating properties of

microparticles are investigated in various clinical conditions. First, in Chapter 8, a brief

overview of the complement system is given, and data supporting the pathogenic role of the

complement system in rheumatoid arthritis (RA) are reviewed, as well as therapeutic

advances aimed at influencing the complement system in this disease. Subsequently, in

Chapter 9, microparticles from synovial fluid and plasma of RA patients and plasma of

healthy individuals are analyzed for bound complement components and complement

activator molecules, to investigate whether complement activation occurs on the surface of

these microparticles in vivo. In Chapter 10, microparticles isolated from pericardial wound

blood of patients undergoing cardiac surgery with CPB are analyzed to determine whether

they contribute to complement activation in pericardial blood. Furthermore, the effect of

retransfusion of pericardial blood on systemic complement activation is studied. In

Chapter 11, the possible role of microparticles in complement activation in patients with

myocardial infarction is investigated. Finally, in Chapter 12, the possible role of

microparticles in complement activation in preeclamptic and healthy pregnant women is

analyzed.

References

[1] Chargaff E, West R. The biological significance of the thromboplastic protein of blood. J Biol Chem 1946; 166: 189-197.

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