Thrombosis Detection and role of circulating microparticles in thrombosis M. J. Mooberry ABSTRACT N.S. Key Microparticles (MPs) are submicron vesicles released from the plasma membrane of eukaryotic cells Department of Medicine, Division of in response to apoptosis or certain activators. MPs have been associated with the processes of coag - Hematology and Oncology, University ulation, inflammation, the immune response, cancer metastasis, and angiogenesis, amongst others. of North Carolina, Chapel Hill, NC, Both phosphatidylserine exposure and the presence of tissue factor in the MP membrane may account USA for their procoagulant nature. Elevated numbers of MPs in plasma have been reported in numerous prothrombotic conditions; however, to date there are few data on true causality linking MPs to the genesis of thrombosis. Numerous methodologies have been employed to characterize and enumerate Hematology Education: MPs, although detection is challenging due to their submicron size. Flow cytometry remains the most the education program for the frequently utilized strategy for MP detection; however, it is associated with significant technological annual congress of the European limitations. Pre-analytical and analytical variables can influence the detection of MPs, rendering data Hematology Association interpretation difficult. Additionally, lack of methodologic standardization confounds the issue further, although efforts are currently underway to address this limitation. Together, these issues make com - 2012;6:381-388 parison of study results difficult and remain one of the main obstacles preventing MP analysis from being adopted by clinical laboratories. Moving forward, it will be important for the scientific commu - nity to agree upon standardized procedures for the isolation, detection, characterization, and enumer - Acknowledgements: MJM was ation of MPs. supported by grant NIH grant T32 HL007149, and NSK was supported by RO1 HL095096. Introduction supports the notion that not all MPs carry PS on their surface 6-10 and that the PS content may Historical perspective vary depending on the cell of origin and stim - ulus or mechanism by which they are Microparticles (MPs) are thought to have formed. 11 Whether this is due to a true lack of been first described by Chargaff and West 1 in PS exposure or whether the expression is the mid-20 th century as a “precipitable factor” below the detection threshold of conventional present in platelet poor plasma that was techniques is unclear. 12 To complicate matters, endowed with the ability to generate throm - it has also been theorized that the presence of bin. Wolf 2 in 1967 described “platelet dust” a cell-specific antigen on the surface of a MP that was formed as a result of platelet shed - does not necessarily identify its cell of origin. ding. This “platelet dust”, which exhibited Soluble antigens from a different cell type procoagulant activity, was detectable in the may adhere to MPs, or fusion may occur 0.1 to 0.3 mm size range by transmission elec - between MPs from one cell type with the cel - tron micrography after ultracentrifugation of lular membrane of a different cell type, there - diluted plasma. Now understood to be platelet by theoretically setting the stage for release of MPs (PMPs), this observation has led to an a second MP expressing an “adopted” anti - exponential growth in the study of MPs, and gen. 13,14 with it a greater understanding of their biolog - MPs must also be distinguished from two ic relevance. other bioactive vesicles released from cells, namely exosomes and apoptotic bodies (AptB). 15 Exosomes are preformed vesicles Definition less than 100 nm that are generated in endo - cytic multivesicular bodies and released via MPs are defined as heterogeneous, submi - exocytosis upon fusion with the cell mem - cron (0.1 to 1 mm) vesicles released from the brane. They are more homogeneous in size membrane of eukaryotic cells in response to than MPs, carry different membrane antigens, specific stimuli or apoptotic cell death. MPs and play an important role in the immune have an intact phospholipid membrane and response and antigen presentation. 16-18 AptB lack a nucleus but express membrane antigens are produced during the latter stages of cell specific to their cell of origin. 3 The working apoptosis, as their name implies. 19 They are definition of an MP has previously included typically larger than MPs and exosomes (in both the size discrimination, as well as the the range of 1-3 mm), although a few may be presence of phosphatidylserine (PS) on the smaller (0.5 mm). 20 Similar to MPs, they outer membrane. 4,5 Newer evidence, however, express PS on their surface; however, in con - Hematology Education: the education programme for the annual congress of the European Hematology Association | 2012; 6(1) | 381 | 17 th Congress of the European Hematology Association trast to MPs, AptB carry DNA and histones, which is one Functions of their hallmarks. 20,21 Once thought to be involved only in coagulation, MPs are now known to be involved in numerous other biologic Sources of circulating MPs functions, including inflammation, modulation of the MPs are most commonly derived from circulating immune response, angiogenesis, vascular function, can - blood cells and endothelial cells, although other cells, cer metastasis/invasiveness, and a growing list of oth - such as tumor cells 22 and smooth muscle cells 23 are capa - ers. 36,37 As a result of the presence of multiple bioactive ble of producing MPs as well. They can be detected in molecules, including ligands, receptors, counter-recep - healthy individuals, and PMPs have generally been tors, mRNA, and microRNA, MPs serve as a storage pool accepted as the most abundant subtype of MP in this set - of effectors that can modulate the biologic properties of ting. 24,25 More recent data suggest that a significant portion other cells. Potential (patho)physiologic mechanisms of PMPs may instead be derived from megakaryocytes include the transfer of membrane receptors, release of and not circulating platelets. 26 In addition, other groups proteins or biolipids, and exchange of genetic information have challenged the dogma that PMPs are the most abun - by transfer of mRNA or microRNA. 28,38 Given the expand - dant subpopulation of circulating MPs in healthy individ - ing list of functions linked to MPs, it is not surprising that uals and have reported that endothelial MPs (EMPs) are the list of disease states in which elevated numbers of more abundant than PMPs. 27 MPs have been reported continues to grow, and now includes multiple inflammatory disorders, cardiovascular Formation and clearance diseases, infectious diseases, such as malaria and HIV, The formation and release of MPs from parent cells and autoimmune disorders. 9,39 typically occurs upon stimulation or induction of apopto - sis. It may be considered a broad primitive response to Microparticles and coagulation stress that is shared by all eukaryotic cells, 28 and is With respect to the studied association(s) with coagula - thought to reflect a dynamic balance between cell prolif - tion processes, the potential hemostatic and/or thrombotic eration, stimulation, and death. 29 Specific stimuli known function of MPs may be related to the presence of PS on to induce formation of MPs include activation by sub - the outer membrane, as well as the possible presence of stances, such as endotoxin or cytokines, partial or com - tissue factor (TF). MP-associated PS provides a catalytic plete lysis, such as by complement, oxidative injury, and surface for the assembly of the enzymatic tenase and pro - high shear stress, 30,31 although this list is not exhaustive. thrombinase complexes that initiate and maintain coagula - Mechanistically, new evidence continues to emerge tion. 40 This function may underlie the contribution of MPs regarding the cellular processes that lead to formation and to both the physiologic process of hemostasis, as well as release of MPs. This topic has recently been extensively the pathologic process of thrombosis. 41 Interestingly, the reviewed, 32 but in brief, loss of cellular membrane phos - surface area of a PMP generated ex vivo has approximately pholipid asymmetry with resultant PS exposure appears 50- to 100-fold higher procoagulant activity than the same to be a critical component of MP formation. This process area on an activated platelet, 42 which may help account for is governed by several phospholipid transporters (“flip - the potential pathogenicity/thrombogenicity of MPs. pase”, “floppase” and “scramblase”), which under basal Tissue factor (TF) is the principal physiological initiator of conditions preserve the normal phospholipid asymmetry coagulation in vivo through its interactions with of the cellular membrane, with PS and phos - FVII/FVIIa and is constitutively expressed by most non - phatidylethanolamine confined primarily to the inner vascular cells. 43 Its presence on monocyte-derived MPs leaflet. Calcium influx also appears to be a necessary pre - and tumor-derived MPs is well established; however, requisite for MP formation, as it contributes to both PS whether PMPs or EMPs truly express TF remains a matter externalization, as well as membrane cytoskeleton of debate. 44,45 Although likely only a small fraction of total remodeling through activation of calpains and caspases TF in the blood, MP-borne TF is likely to be functionally necessary for cleavage of certain cytoskeletal proteins active and may thus contribute to the procoagulant nature such as filamin. Upon stimulation, the loss of phospho - of MPs. Indeed, MPs have been studied in numerous lipid asymmetry along with cytoskeletal disruption even - thrombotic conditions, where elevated numbers have been tually leads to membrane blebbing and MP formation and described, 46 but primarily in cross sectional studies. In the release. Newer evidence points to a potential role for flux remainder of this review, we will examine the challenges of other ions, such as Na +, Cl –, and K –, in PS externaliza - associated with MP detection, and then evaluate the poten - tion, as well as a potential role for mitochondria-associat - tial role of MPs in thrombosis.