439 �� 2009 Schattauer GmbH, Stuttgart Cell-derived microparticles in haemostasis and vascular medicine Laurent Burnier1 Pierre Fontana2 Brenda R. Kwak3 Anne Angelillo-Scherrer1 1Service and Central Laboratory of Hematology, Centre Hospitalier Universitaire Vaudais and University of Lausanne, Lausanne, Switzerland 2Division of Angiology and Haemostasis, Geneva University Hospitals and University of Geneva, Geneva, Switzerland 3Division of Cardiology, Geneva University Hospitals and University of Geneva, Geneva, Switzerland Summary Considerable interest for cell-derived microparticles has emerged, pointing out their essential role in haemostatic re- sponse and their potential as disease markers, but also their im- plication in a wide range of physiological and pathological pro- cesses. They derive from different cell types including platelets ��� the main source of microparticles ��� but also from red blood cells, leukocytes and endothelial cells, and they circulate in blood. Despite difficulties encountered in analyzing them and Keywords Microparticle, ectosome, haemostasis, vascular pathologies disparities of results obtained with a wide range of methods, microparticle generation processes are now better understood. However, a generally admitted definition of microparticles is currently lacking. For all these reasons we decided to review the literature regarding microparticles in their widest definition, in- cluding ectosomes and exosomes, and to focus mainly on their role in haemostasis and vascular medicine. Thromb Haemost 2009 101: 439���451 Review Article Correspondence to: Anne Angelillo-Scherrer Service and Central Laboratory of Hematology Centre Hospitalier Universitaire Vaudois Rue du Bugnon 46, CH-1011 Lausanne, Switzerland Tel.: +41 21 314 42 22, Fax: +41 21 314 41 80 E-mail: anne.angelillo-scherrer@chuv.ch Financial support: This work was supported by the Swiss National Foundation for Scientific Research Grant PP00B���106690/1, The Swiss League for Cancer Research OCS 01775���08���2005 and the Leenaards��� Foundation. Received: August 14, 2008 Accepted after major revision: October 29, 2008 Prepublished online: February 9, 2009 doi:10.1160/TH08-08-0521 Introduction Blood contains microparticles (MPs) derived from different cell types, including mainly platelets, but also red blood cells, granu- locytes, monocytes, lymphocytes and endothelial cells (ECs). Overproduction of MPs has been related to various physiological and pathophysiological conditions such as cell adhesion, apopto- sis, immune response, vascular function, vascular remodeling and angiogenesis, haemostasis and thrombosis, cardiovascular diseases, cancer, infections, as well as normal and pathological pregnancy. About 60 years ago, prolongation of plasma clotting time was observed after high-speed centrifugation, suggesting that sedi- mentable procoagulant particles are present in plasma and can be removed by centrifugation (1). In 1967, Wolf (2) showed that pla- telet shedding after activation results in ���platelet dust���. A population of platelet-derived MPs (PMPs) is generated during platelet activation, whereas other PMPs populations are derived from megakaryocytes during megakaryopoiesis (3���5) or quiescent circulating platelets, or might result from platelet apoptosis (6). MPs from other cells may be released during cell activation, cell injury or following cell activation-independent processes, including senescence and apoptosis. Carrying markers from their parental cells (Table 1), MPs are used as in- vestigation and diagnostic tools (7). After a general overview of MPs and the processes of their formation, a brief review of the methods to detect MPs will be presented. We will then systematically outline the role of MPs in haemostasis and vascular medicine (Table 2). Search strategy Studies whose title and/or abstract contained the terms ���micro- particles���, ���microvesicles���, ���ectosomes��� combined with ���hae- mostasis��� or ���vascular medicine��� were searched in the PubMed database until September 23, 2008. Relevant articles were se- lected according to abstract content. To supplement the search, citations in pertinent review articles were examined (6���9). One- hundred-seventy-seven publications were cited in this review. Microparticles (MPs): definition MPs are vesicles that bud off from cells, lack a nucleus, contain a membrane skeleton and are defined by their size and ex- pression on their surface of antigens specific of parental cells (Table 1) (10���12). These phospholipid vesicles are less than

440 Burnier et al. Microparticles in haemostasis and vascular medicine 1 ��m of diameter. Recently, tissue factor (TF)-bearing MPs have been shown to vary in size from 332 to 501 nm, using imped- ance-based flow cytometry (13). The minimal size of MPs was defined as 0.1 ��m because commonly used flow cytometers are unable to distinguish between smaller particles and the elec- tronic noise. The upper size of MPs was fixed just below 1 ��m because a single bigger MP might be difficult to distinguish from MPs aggregates, platelets, or MPs-platelet aggregates. Together with the usage of impedance-based flow cytometry, the develop- ment of digital-acquiring flow cytometers and more thinner laser beam has improved the discrimination and characterization of MPs (14). To reliably define MPs, the terms exosomes and ectosomes need to be introduced. Exosomes originate from multivesicular bodies and exocytosis of endocytic bodies, and ectosomes di- rectly originate from the membrane surface (Fig. 1) (15, 16). In this review, we will mainly use the commonly used term ���micro- particles���, keeping in mind their definition as ectosomes. Some authors use the term ���microparticles��� without a clear definition or a clear analysis of the origin of the vesicles described in their article. It is therefore possible that in this review the correct defi- nition of MPs is not strictly applied everywhere. A clear dis- cussion about problems and need for a correct definition of all microvesicles has been recently published (17). The vascular bi- ology group within the Scientific Subcommittees of the Scien- tific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) has proposed recently to create an international workshop by using calibrated microbeads to clarify confusing data regarding MPs numeration, and the first step will be to establish a normal range for MPs (18, 19). Another point risen during this recent meeting was the dis- crepancy between the protein or lipid content of MPs and their numbers reported in the literature. It was therefore suggested to express MP concentrations in protein and/or lipid equivalents. MP formation The current knowledge on MP formation is mainly issued from experiments performed in vitro on isolated or cultured cells. However, mechanisms involved in MP formation in vivo remain essentially unknown. In steady-state, the cell membrane is asymmetric regarding the composition and the distribution of phospholipids in its inner and outer layers: phosphatidylcholine and sphingomyelin are lo- cated in the outer layer, while phosphatidylserine (PS) and phos- phatidyl-ethanolamine (PE) are present in the inner layer. This asymmetric distribution of phospholipids in the membrane is maintained by a three piece enzyme system: flippase, floppase and scramblase (Fig. 2). Flippase is an aminophospholipid trans- locase that specifically translocates PS and PE from the outside to the inside of the bilayer membrane. Floppase transports phos- pholipids from the inner to the outer leaflet. Floppase does not specifically act on transport of aminophospholipids and prob- ably works together with flippase. Scramblase, whose role is thought to be the transportation of phospholipids between the two monolayers of the cell membrane, is inactive in steady-state. Successive mechanisms initiate MP (ectosome) formation during cell activation or other cell processes including apoptosis and senescence (Fig. 3): 1. Calcium is released by the endoplasmic reticulum. 2. Calcium inactivates flippase and activates floppase and scramblase, inducing the loss of phospholipids asymmetry between the inner and the outer leaflets. Contacts between aminophospholipids and cytoskeleton are then disrupted. 3. In addition, calcium release leads to activation of two enzymes: calpain and gelsolin. Calpain hydrolyzes actin- binding proteins that decreases association of actin with membranes glycoproteins (20, 21), while gelsolin (only in platelets) is involved in the cleavage of the actin capping pro- teins (22). Protein anchorage to the cytoskeleton is therefore disrupted, re- sulting in membrane budding and microparticles shedding (23). The main mechanism of PMPs formation is therefore calpain dependant. Nevertheless, calpain-independent microparticles formation can occur, such as when platelets are stimulated in vitro with collagen and thrombin in absence of stirring (21) or when cells are stimulated with the complement proteins C5b-9 (24). MPs enriched in PE and PS exposed on their outer surface are released. MPs isolated from blood (mainly PMPs) comprise 60% phosphatidylcholine, 20% sphingomyelin, 9% PE, the re- mainder being minor quantities of other phospholipids (25). However, not all MPs exhibit PE and PS on their surface. For example, EC MPs originating from activated ECs are different in their lipid composition than those derived from apoptotic ECs. Indeed, annexin V binding sites are modestly but significantly Cellular origin of microparticles Marker References Red blood cell CD235a (141, 174) Leukocyte CD45 (159, 174) Granulocyte CD66b (141, 159, 174) Monocyte CD14 (141, 159, 174) Lymphocyte CD4 CD8 CD20 (141, 159, 174) (159, 174) (159, 174) Platelet CD31* CD41 CD41a CD42a CD42b CD61 CD62P (10, 111) (141, 174) (174) (174) (174) (174) (141) Endothelial cell** CD31 CD34 CD54 CD62E CD51 CD105 CD106 CD144 CD146 (26, 141, 175) (174) (26, 141) (26, 174���176) (41, 174) (174) (26, 175) (38, 141) (146) * in association with CD42, ** CD42 negative (114, 141), MPs harbouring a CD62E/CD144/CD146 phenotype are the more likely to originate from endothelial cells (19). Table 1: Markers for cell-derived microparticles.