The Role of Microparticles in Inflammation and Thrombosis
S. P. Ardoin MD, DUMC 3212, Durham, NC 27710, USA. E-mail: firstname.lastname@example.org
Microparticles (MP) are small membrane-bound vesicles that circulate in the peripheral blood and play active roles in thrombosis, inflammation and vascular reactivity. While MP can be released from nearly every cell type, most investigation has focused on MP of platelet, leucocyte and endothelial cell origin. Cells can release MP during activation or death. Flow cytometry is the usual method to quantify MP; the small size of these structures and lack of standardization in methodology complicate measurement. As MP contain surface and cytoplasmic contents of the parent cells and bear phosphatidylserine, antibodies to specific cell surface markers and annexin V can be used for identification. Through various mechanisms, MP participate in haemostasis and have procoagulant potential in disease. MP contribute to inflammation via their influence on cell–cell interactions and cytokine release, and MP also function in mediating vascular tone. In several disease states characterized by inflammation and vascular dysfunction, MP subpopulations are elevated, correlate with clinical events, and may have important roles in pathogenesis. In the rheumatic conditions such as rheumatoid arthritis and systemic lupus erythematosus, MP are potentially important markers of disease activity and have an increasingly recognized role in immunopathogenesis. It is clear that MP play an important role in atherosclerosis, and study of these structures may provide insight into the link between chronic inflammatory conditions and accelerated atherosclerosis. As biomarkers, MP allow access to usually inaccessible tissues such as the endothelium. Further research will hopefully lead to interventions targeting MP release and function.
Originally described as inert ‘platelet dust’, microparticles (MP) are small (0.1–1 μm) membrane-bound vesicles that circulate in the blood and mediate inflammation and thrombosis . The most abundant MP in the blood form from platelets, although MP in the periphery can also arise from lymphocytes, monocytes and endothelial cells among other cell types. MP contain membrane, cytoplasmic and nuclear constituents characteristic of their precursor cells and differ in size and composition from other subcellular structures such as apoptotic bodies and exosomes . While all subcellular structures may have physiological activities, they likely vary depending upon the array of molecules present as well as access to the blood where they can act distal to their site of origin.
As shown in in vitro experiments, MP release from cells occurs with both activation and cell death, either apoptosis or necrosis. While MP are present in peripheral blood of healthy individuals, marked elevations occur in many disease states. These conditions include inflammatory and autoimmune diseases, atherosclerosis, malignancy and infection among others. In general, these conditions are characterized by high cell turnover or cell death, settings associated with the presumed mechanism for MP generation; in addition, these diseases have an immune component, suggesting a link between MP and inflammation.
In view of their origin, MP may represent novel markers of disease activity, serving as phenotypically defined remnants of cells critically involved in pathogenesis. Furthermore, MP can function as disease effectors, playing a role in local as well long-range signalling in cellular processes that underlie inflammation and thrombosis.
Cellular release of microparticles
While activation and cell death are settings for MP release, the exact mechanisms of these processes remain unknown. During cell activation, a rise in intracellular calcium is followed by remodelling of the plasma membrane. This membrane modification can cause phosphatidylserine exposure and bleb formation, leading to the extrusion of MP which incorporate surface proteins and other contents of the originating cell . For platelets, in vitro studies have shown that MP release can occur with activation by a variety of stimuli including adrenaline, adenosine diphosphate, thrombin, collagen, Ca2+ ionophore A23187, complement membrane attack complex and shear stress [4, 5].
In apoptosis, MP release is associated with membrane blebbing, a characteristic feature of programmed cell death. Blebbing involves a dynamic redistribution of cellular contents, perhaps related to volume stress that occurs as cells die; in this process, the activation of Rho-associated kinase 1 (ROCK-1) is critical . ROCK-1, an effector of Rho GTPases, is essential for apoptotic membrane blebbing. Not all cells bleb, however, and the blebbing process can differ during the stage of apoptosis. MP release appears to occur late in the death process and may occur concurrently with cell fragmentation and the formation of apoptotic bodies; apoptotic bodies represent shrunken and collapsed cells with nuclear fragmentation.
As these considerations suggest, the mechanisms for MP formation likely differ in the settings of cell activation and apoptosis. These differences could lead to variations in MP size as well as macromolecular composition, both surface and internal. As an example of these differences, the expression of cell membrane molecules on endothelial MP varies depending upon whether their formation results from cell activation or apoptosis . Whether these structural difference influence function is not yet known.
While any eukaryotic cell undergoing activation or death should theoretically release MP, in the investigation of the physiology of these structures, attention has focused primarily on circulating cell types (e.g. platelets, leucocytes and erythrocytes) as well as vascular cells including endothelial cells. Other cells, including tumours, smooth muscle cell and synovial cells can also release MP, although these MP may be more readily detected in the tissue or origin or at sites of inflammation (e.g. in the synovium or synovial fluid), rather than the blood.
Given the generation of MP during exocytosis or blebbing of membranes, their origin can be tracked by cell-specific protein markers. Thus, the presence of CD4, CD3 or CD8 on the MP surface indicates lymphoid origin while platelet MP are marked by the expression of glycoproteins IIb–IIIa, P-selectin/CD42a. Similarly, endothelial MP display surface CD31 or CD146 [5, 8]. MP can express a different set of surface markers than the precursor cells; however, as has been observed with erythrocyte MP . The rules for the incorporation of different proteins into MP are not known.
The measurement of microparticles
Conventional flow cytometry (FACS) is the most commonly used method for analysing the number, size, and properties of MP. By definition, MP are small structures (0.1–1 μm) which display characteristic forward and side scatter patterns. As in the case of FACS analysis of cells, antibodies to cell surface molecules allow the identification of specific MP subpopulations. Because of MP size, however, the amount of any surface marker is drastically reduced in comparison to intact cells, thereby limiting detection. In addition to proteins, the presence of phosphatidylserine on the outer surface of the MP membrane allows binding of annexin V which can also be used to identify and enumerate MP.
An important limitation of flow cytometry concerns detection of small MP (e.g. <300 nm in diameter). With structures of these dimensions, detection is limited by ‘noise’ in these instruments; conventional FACS instruments are well designed to measure cells which are 10- to 100-fold greater in diameter than MP. An alternate method for identifying MP involves binding assays in a solid-phase or microtiter plate format. In this approach, antibodies to cell surface molecules can capture particles for subsequent detection by another antibody or a functional assay for thrombosis, for example. While such assays can assess the total amount of MP-related material in a specimen, they cannot provide information on the number or size of particles. MP analysis can be impacted not only by type of assay, but also by the manner in which blood is collected and processed, including sampling site, needle diameter, centrifugation and freeze-thaw methods. At present, MP analysis is constrained by lack of standardized MP assays, a challenge being addressed by the Working Group on Vascular Biology of the Scientific and Standardization Committee of the International Society on Thrombosis and Hemostasis .
In contrast to their initial description as dormant cell debris, MP can serve as physiologically active effectors, playing important roles in inflammation, haemostasis, thrombosis, angiogenesis and vascular reactivity. Because of their small size, MP readily circulate in the vasculature allowing participation in both local and long-range signalling. In their interaction with cells, MP bind via surface ligands, representing a kind of direct long distance cell–cell communication for cells typically remote from each other. Furthermore, in a novel mechanism for the intercellular interaction, MP can transfer surface molecules, including receptors, to other cells as well exchange membrane and cytoplasmic proteins [10, 11]. Receptor transfer can enlarge the responses that can be mounted by a given cell type; such transfer can also confer sensitivity to infection by an organism (e.g. HIV) requiring a particular cell surface molecules to allow entry .
Bleeding and thrombosis
Since platelet MP were the first species identified, investigation on the role of these structures in health and disease has long focused on the clotting system. Along with platelets themselves, platelet-derived MP may contribute to haemostasis . Supporting this possibility is the observation that platelet MP numbers rise during acute bleeding episodes associated with haemophilia, for example . Furthermore, MP circulating in healthy individuals may stimulate low-grade thrombin generation necessary for normal protein C activation . In the setting of idiopathic thrombocytopenia, elevated platelet MP may play a role in preventing bleeding complications .
Microparticles have diverse properties that could promote the pathogenesis of thrombotic disorders. Phosphatidylserine and tissue factor are both exposed on MP outer membranes and are central players in the coagulation cascade. Additionally, MP interact with factors Va, VIII and IXa, thereby facilitating assembly of prothrombinase complex [17–20]. Among other activities, platelet MP bind β2-glycoprotein-1 antibodies, suggesting a possible role in antiphospholipid antibody syndrome (APS) . Of note, endothelial MP bear large von Willebrand factor multimers, which promote and stabilize platelet aggregates .
As shown in in vitro experiments, isolated MP from patients in various clinical settings (including sepsis, thrombotic thrombocytopenic purpura, sickle cell disease and cardiopulmonary bypass grafting) have procoagulant activity [23–26]. This activity may be relevant clinically as MP concentrations are elevated in prothrombotic disorders including acute coronary syndromes, venous thromboembolism, heparin-induced thrombocytopenia, vasculitis, paroxysmal nocturnal haemoglobinuria and thrombotic thrombocytopenic purpura [27–32].
Investigation into the properties of MP has revealed a significant impact of these structures on vascular tone. Platelet MP induce cyclo-oxygenase-2 expression in human umbilical vein endothelial cells and in U-937 monocytoid cells, thereby leading to the release of prostacyclin [33, 34].
Additionally, platelet MP can serve as a reservoir of thromboxane A2 and, in a rabbit model, can promote arterial vasoconstriction . As shown in in vitro experiments, the endothelium is an important target of MP action. Thus, endothelial MP can modulate nitric oxide-induced endothelial relaxation in rat models , while MP derived from T lymphocytes can promote endothelial dysfunction through effects on nitric oxide- and prostacyclin-dependent vasodilation . These activities indicate a broad potential for MP for modulating vascular cell function.
Coupled with their effects on thrombosis, MP have potent proinflammatory activities and are potentially important mediators of rheumatologic and other inflammatory diseases. Among these activities, platelet MP can induce binding of monocytes to endothelial cells and promote survival of haematopoietic cells . Furthermore, platelet MP can promote leucocyte–leucocyte aggregation, likely due to interactions between P-selectin expressed on platelet MP and its ligand on leucocytes . The mechanisms by which MP mediate these effects may include the transfer arachidonic acid to other cells, leading to increased adhesion of monocytes to endothelium [34, 40]. Other proinflammatory roles of MP include the secretion of IL-1β .
In contrast to actions promoting inflammation, T cell-derived MP can induce macrophage apoptosis. This killing action can potentially impair a key element of the immune system and trigger in turn MP release from the dying macrophage to augment other immunological and vascular events .
Microparticles in disease
Like other consequences of cell activation or death, elevated MP levels occur in the blood of patients with many different diseases. As a group, these diseases are characterized by immune system as well as vascular abnormalities. In autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), cardiovascular disease is markedly accentuated. As these conditions have increased MP, a pathogenic link is possible. These clinical associations will be reviewed briefly.
A growing body of literature demonstrates a clear association between MP and atherosclerosis. Endothelial MP are elevated in acute coronary syndromes and correlate with the angiographic evidence of high-risk lesions [27, 42]. Interestingly, subclinical atherosclerosis, as assessed by ultrasound, is also associated with increased levels of leucocyte-derived MP, suggesting a predictive value for MP measurement . In this condition, at least some MP may arise in atherosclerotic lesions themselves, since, in human plaque specimens, macrophage and lymphocyte MP are abundant and account for a large amount of tissue factor activity . These findings implicate MP in plaque thrombogenicity. MP may also contribute to atherosclerosis by functional effects as intracoronary endothelial-dependent vasodilation during coronary catheterization in human subjects may be impaired in the presence of high levels of circulating endothelial MP .
In view of the effects of MP in atherosclerosis, it is of interest that the HMG-CoA reductase inhibitor, fluvastatin, can decrease the in vitro release of endothelial MP from human coronary artery endothelial cells . Thus, one of the beneficial effects of statins, widely used for primary and secondary prevention of coronary artery disease, may result from a decrease in the number MP impinging on atherosclerotic vessels.
Diabetes, metabolic syndrome
Levels of platelet- and monocyte-derived MP are elevated in the blood of patients with type 2 diabetes mellitus. In cross-sectional studies of patients, elevated levels of monocyte MP correlate with nephropathy, retinopathy and neuropathy [47, 48], while elevated platelet MP number correlate with symptomatic atherosclerosis in type 2 diabetes mellitus [49, 50]. Of note, in patients with the metabolic syndrome, endothelial MP are also elevated .
Platelet-derived MP have been implicated in the metastatic and angiogenic capability of lung cancer and breast cancer cells [52–54]. Additionally, MP may increase the risk of thrombosis associated with tumours, as increased levels of tissue factor-positive MP occur in colorectal cancer and are associated with elevated D-dimer levels .
Pregnancy is a complex physiological setting whose elements – cell growth, modulation of immune reactivity, new blood vessel formation – have all been associated with MP generation and action. Not surprisingly, therefore, total MP concentrations are elevated in normal pregnancy , while in women with recurrent pregnancy loss, total and endothelial MP numbers are greater than parous controls [57, 58]. The situation with respect to pre-eclampsia is less certain since studies have shown that platelet MP numbers in pre-eclampsia may be reduced or equivalent to those of controls with comparable platelet counts [56, 59]. Other studies, however, have shown that granulocyte and endothelial MP subpopulations are increased in the blood of pre-eclamptic patients, and correlate with hypertension [60–62]. While the relationship between MP number and clinical event is not clear, a direct effector role of MP in the vascular dysregulation of pre-eclampsia is suggested by the finding that prolonged exposure of healthy myometrial arteries (removed from healthy women at the time of Caesarean section) to isolated MP from pre-eclamptic women impairs endothelium-dependent relaxation .
In multiple sclerosis (MS), a disease characterized by inflammation and demyelination of the central nervous system, endothelial MP level rise during disease exacerbation, normalize in remission, and correlate with MRI evidence of active disease . A role of MP in disrupting the blood–brain barrier in MS is suggested by findings that endothelial MP from blood of MS patients enhance binding of monocytes to human brain microvascular endothelial cells (BMVEC) and promote monocyte migration through BMVEC monolayers . Of interest, interferon-β1b, an effective therapy for MS, inhibits release of endothelial MP .
In the pathogenesis of RA, inflammation, angiogenesis and thrombosis occur prominently and the course of disease is marked by accelerated atherosclerosis. MP may play a role in both articular and extra-articular manifestation and therefore could serve as biomarkers. In a study of patients with RA, platelet MP levels in the blood were elevated and correlated with disease activity as measured by Disease Activity Score (DAS)-28 . Another study did not confirm these findings, however, there were significant differences in the study populations .
While platelet MP predominate in the plasma of RA subjects, in the synovial fluid, MP derived from granulocytes and monocytes dominate. Also, present in synovial fluid are T cell, B cell, platelet and erythrocyte MP . Synovial MP from both RA and some non-RA patients stimulate tissue factor/factor VII-dependent thrombin generation. This local hypercoaguability could play a role in intra-articular inflammation and the formation of fibrin clots, known as ‘rice bodies’ .
Microparticles, in addition to promoting inflammation, could also promote cartilage and bone erosion via effects on synovial fibroblast activity. Thus, as shown in in vitro experiments using cells obtained from patients with RA and other arthritides, incubation of fibroblast-like synoviocytes with autologous MP induces expression of MCP-1, IL-6, IL-8, VEGF, ICAM-1 and RANTES and a decrease in GMCSF . Furthermore, in in vitro studies, MP derived from T cells and monocytes can induce synovial fibroblast production of matrix metalloproteinases (MMP), including MMP-1, MMP-3, MMP-9 and MMP-13 . Together, these findings suggest that MP can mediate cellular crosstalk responsible for synovial activation and articular destruction.
Given the endothelial injury associated with vasculitis, it is not surprising that endothelial MP are elevated in children with various vasculitides and correlate with disease activity . Among patients with a variety of nephropathies, levels of granulocyte and platelet MP are also significantly elevated in acute vasculitis .
Systemic lupus erythematosus and antiphospholipid antibody syndrome
Systemic lupus erythematosus is a prototypic autoimmune disease characterized by systemic inflammation, thrombosis and accelerated atherosclerosis. In addition to immune hyperactivity, the pathogenesis of lupus involves either aberrant levels of apoptosis or a failure to clear apoptotic cells or debris appropriately. Such debris, which could include MP, could contribute to disease pathogenesis because of its immune properties, including stimulation of cytokines alone or in the context of immune complexes. In view of the multiple abnormalities operative in SLE, the presence of MP in patient blood could provide a novel marker for functional disturbance in the major cell populations underlying disease.
In a study looking at patients with primary APS, SLE and RA, median levels of platelet-derived MP did not differ significantly between the groups, although several SLE patients had elevated MP concentrations . In contrast, levels of endothelial-derived MP were elevated in subjects with APS compared to healthy controls and controls with non-APS thrombosis. Additionally, in vitro, APS plasma induced a fourfold increase in release of endothelial MP from human umbilical endothelial cells . In one study, elevated MP, primarily platelet-derived, were elevated in SLE and correlated with thrombin generation, although levels did not correlate with disease activity or presence of antiphospholipid antibody .
As this review indicates, MP are neither inert nor dust. Rather, they are potentially important players in immune-mediated diseases that can participate in local and long-range cell–cell communication. Furthermore, MP may represent unique markers of disease activity for typically inaccessible tissues such as the endothelium. In a link that has been yet been well explored, there is a potentially important connection between actions of MP and the accelerated atherosclerosis associated with chronic inflammatory diseases such as RA and SLE. Future research will hopefully define the precise roles of MP subpopulations in rheumatic and other diseases and allow the development of new therapeutic strategies that target either MP release or action.