Bench-to-bedside review: Circulating microparticles - a new player in sepsis?

In sepsis, inflammation and thrombosis are both the cause and the result of interactions between circulating (for example, leukocytes and platelets), endothelial and smooth muscle cells. Microparticles are proinflammatory and procoagulant fragments originating from plasma membrane generated after cellular activation and released in body fluids. In the vessel, they constitute a pool of bioactive effectors pulled from diverse cellular origins and may act as intercellular messengers. Microparticles expose phosphatidylserine, a procoagulant phospholipid made accessible after membrane remodelling, and tissue factor, the initiator of blood coagulation at the endothelial and leukocyte surface. They constitute a secretion pathway for IL-1β and up-regulate the proinflammatory response of target cells. Microparticles circulate at low levels in healthy individuals, but undergo phenotypic and quantitative changes that could play a pathophysiological role in inflammatory diseases. Microparticles may participate in the pathogenesis of sepsis through multiple ways. They are able to regulate vascular tone and are potent vascular proinflammatory and procoagulant mediators. Microparticles' abilities are of increasing interest in deciphering the mechanisms underlying the multiple organ dysfunction of septic shock.


Introduction
In the 1960s and 70s Wolf [1] was the fi rst to describe platelet derivatives of less than 0.1 μm as procoagulant vesicles. Later, having been given the name of 'microparticles' (MPs), these vesicles were described as membrane-derived nano-fragments (0.05 to 1 μm) that are active in coagulation and infl ammation. MPs are released in the extracellular environment through a membrane reorganization and blebbing process following cell activation or apoptosis. Th ey constitute a storage pool of bioactive eff ectors with varied cellular origins and are able to act as intercellular messengers [2]. Th ey are present in body fl uids where they refl ect normal tissue homeostasis, but undergo phenotypic and quantitative changes to play a pathophysiological role in several diseases, most of them associated with thrombotic disorders [3,4] (Figure 1).
MPs often convey tissue factor (TF) that may contribute to the dissemination of coagulopathy in sepsis [5,6] and cytokines up-regulating deleterious infl ammatory responses [7]. Circulating MPs can also provoke vascular dysfunction, and they reduce available nitric oxide (NO) and increase levels of reactive oxygen species, thereby promoting oxidative stress [8].
Th is review will focus on the role of MPs during sepsis, with a special emphasis on coagulation and infl ammation disturbances.

Microparticles are potential intercellular messengers during sepsis
Sepsis is a syndrome characterized by excessive cellular activation involving a systemic infl ammatory response to severe infection. Its most severe form may lead to septic shock. Th e ongoing circulatory failure is characterized by vasoplegia-related arterial hypotension and may include vasopressor resistance, and myocardial and local blood fl ow impairments. Infl ammation plays a key role in the acute activation of the vascular wall and is associated with local thrombosis and changes in vasomotricity [9]. Th us, the endothelium-derived TF initiates the coagulation process and a proteolytic cascade [10]. Th e endothelial damage furthermore leads to the expression of adhesion molecules and other vasoactive factors involved in infl ammation and coagulation.

Biogenesis and general features of microparticles
MPs are produced following cellular activation or apoptosis. Th e increase in intracellular calcium activates various cytosolic enzymes, including calpains, that cleave

Abstract
In sepsis, infl ammation and thrombosis are both the cause and the result of interactions between circulating (for example, leukocytes and platelets), endothelial and smooth muscle cells. Microparticles are proinfl ammatory and procoagulant fragments originating from plasma membrane generated after cellular activation and released in body fl uids. In the vessel, they constitute a pool of bioactive eff ectors pulled from diverse cellular origins and may act as intercellular messengers. Microparticles expose phosphatidylserine, a procoagulant phospholipid made accessible after membrane remodelling, and tissue factor, the initiator of blood coagulation at the endothelial and leukocyte surface. They constitute a secretion pathway for IL-1β and upregulate the proinfl ammatory response of target cells. Microparticles circulate at low levels in healthy individuals, but undergo phenotypic and quantitative changes that could play a pathophysiological role in infl ammatory diseases. Microparticles may participate in the pathogenesis of sepsis through multiple ways. They are able to regulate vascular tone and are potent vascular proinfl ammatory and procoagulant mediators. Microparticles' abilities are of increasing interest in deciphering the mechanisms underlying the multiple organ dysfunction of septic shock. the cytoskeleton and facilitate the role of procaspase-3 in apoptosis [11]. As a response to stimulus, the cytoskeleton is reorganized and the asymmetric distribution of the phospholipid membrane modifi ed with exposure of phosphatidylserine at the cell surface. Cellular blebbing then occurs, ultimately leading to the release of MPs. In addition to phosphatidylserine exposure, protein-lipid raft domains are formed and furnish the MP with its specifi cities and biological roles [12] (Figure 1).
Th e cellular origin of MPs can be determined by assessment of the antigens that they expose at their surface. However, the complete protein content of MPs remains diffi cult to establish. More than 300 proteins have been reported by proteomics, some of which are cytosolic and some membranous [13]. Th e MP phenotype is, however, known to vary according to cellular origin and parental cell response to stimulus [7,14].

Microparticle survival and clearance
Although bearing phosphatidylserine, which is a signal for phagocytosis, MPs seem to survive longer than their parental apoptotic cell, probably because of their size, which does not allow optimal exposure of a cluster of senescence signals. Dasgupta and colleagues [15] recently described the major role of lactadherin in the removal of phosphatidylserine-expressing platelet MPs from human The plasma membrane is reorganised with active externalisation of phosphatidylserine (PhtdSer; a negatively charged phospholipid) and internalisation of phosphatidylcholine (insert). MPs bear intracytoplasmic and membrane-bound eff ectors from the originating cells, such as tissue factor (TF) and endothelial protein C receptor (EPCR) (endothelial cells and monocytes), CD-14 (monocytes) or glycoprotein (GP) Ibα-IX-V , P-selectin or integrins (platelets).
plasma. Lactadherin is a macrophage opsonin that mediates the clearance of apoptotic lymphocytes and knockout lactadherin (-/-) mice have increased levels of circulating platelet MPs and a hypercoagulable state; lactadherin supplementation restores the normal clearance of MPs. To date, there are no data on the eff ect of MP clearance on the haemostatic balance under physiological or pathological settings.

Microparticles as messengers in blood fl ow
As mediators of cellular communication, MPs are actors and possible mediators in the interplay between thrombosis and infl ammation, a process previously described for vascular injury in infl ammatory diseases [5]. Th ey can transfer receptors, organelles, mRNA and other proteins to target cells [16] and also comprise a secretion pathway for several cytokines, such as mature IL-1β [17]. Th e multiple properties of MPs and the variety of their possible cellular targets support them having a key role in cell reprogramming and tissue remodeling with physiological or pathological consequences [4]. Th us, MPs could play a major role in propagating proinfl ammatory and procoagulant states in sepsis. In the vascular compartment, including the arterial wall, the particular settings of sepsis and the tuning abilities of MPs point to the endothelium as a pivotal target [18,19].

How to detect and measure microparticles?
Th e International Society for Th rombosis and Haemostasis (ISTH) provides information on technical procedures and recommendations for the detection and measurement of MPs. Although no standardized procedures for MP measurement are available yet, a consensus is forming on blood sampling and MP isolation by centrifugation steps that avoid exosome contamination of MP samples [20]. Several assays and phenotyping methods coexist, but these are not necessarily comparable, thus making the interpretation of results across studies diffi cult. MPs can be analyzed through capture techniques (using immobilized annexin V -a high affi nity probe for phosphatidylserine -quantitative assessment, or insolubilized antibodies for phenotyping) combined with a functional prothrombinase assay. Flow cytometry is another method for the study of MPs. Th is method allows quantifi cation and determination of cellular origin via the use of specifi c fl uorescent antibodies and calibration beads. Th e protein content of MPs can also be assayed and expressed in molecular mass units [21]. Caution should be taken in the interpretation of MP analyses, taking into account the pitfalls of each method and the purpose of the experiment or clinical investigation. Furthermore, control cohorts are of prime importance in clinical investigations of MP pattern variations.

Microparticles as a player in coagulation disorders of sepsis
In the defence against pathogens, haemostasis is as fundamentally important as innate immunity and complement-mediated cell lysis. Haemostasis is activated during sepsis and septic shock, leading to thrombin and fi brin generation with dual eff ects: limitation of pathogen diff usion and invasion; and fi brin deposition in vessels, resulting in thrombotic microangiopathy or disseminated intravascular coagulopathy. As detailed above, MPs are effi cient eff ectors in the haemostatic response and pathogenic markers of thrombotic disorders (Figure 2).

Microparticles and thrombin generation
Th rombin generation requires activation of coagulation factors, which is made possible after their assembly on a catalytic surface constituted of anionic phospholipids. Cell activation constitutes the fi rst step by furnishing exposed phosphatidylserine with a negative charge. Th e required remodelling of plasma membrane, resulting in phosphatidylserine translocation to the outer leafl et of the plasma membrane, occurs in platelets, endothelial cells and monocytes at sites of vascular damage or injury. Calcium ion-mediated interactions between gammacarboxyl groups of vitamin-K-dependent factors and phosphatidylserine comprise the key step in this assembly, explaining the effi cacy of anti-vitamin K treatments in hypercoagulable states [22]. At the monocyte surface a possible encrypted pre formed TF would be de-encrypted by plasma membrane remodelling, thereby allowing the (auto-)activation of factor VII. Indeed, TF expression at the surface of monocyte-derived MPs has been demonstrated during in vitro endotoxin stimulation [23]. Although TF is the primary initiator of blood coagulation, whether there is a blood-borne TF (activity) is still debated, but there is growing evidence that this activity is directly tied to MPs [5,24]. TF-bearing MPs can interact with neutrophil granulocytes by 'paracrine transfer' , as demonstrated in vitro [25][26][27]. Circulating MPs bearing active TF have been associated with a thrombotic status in human meningo coccal sepsis [28] and a primate Ebola fever model [29], pointing to their possible role in the dissemination of a procoagulant potential.

Microparticles and amplifi cation loops in thrombin generation
TF-driven coagulation is under the control of Tissue factor pathway inhibitor (TFPI), an inhibitory complex, with factor Xa and protein S as cofactors. Although this inhibits TF-induced thrombin generation, thrombin is still generated during the propagation phase via the Josso loop: platelet-exposed factor XI (of megakaryocytic origin) is activated by the GP Ibα -thrombin complex present on the surface of activated platelets. Activated platelets, and released GP Ibα -FXIa bearing MPs, may, in turn, be responsible for increased thrombin generation [30][31][32]. In addition to blood-borne TF conveyed by MPs, polymorphonuclear (PMN)-derived MPs likely contribute to an additional amplifi cation loop in the generation of thrombin mediated by MPs (Figure 2).
MPs could contribute to such amplifi cation loops in sepsis. Indeed, ex vivo activation of human neutrophils by endotoxin, platelet activating factor or phorbol myris tate acetate can generate MPs bearing active integrin α M β 2 (CD11b/CD18), which is able to activate GP Ibα [33,34].

Microparticles in the control of thrombin generation, cytoprotection and tissue remodelling
Interestingly, several cellular models showed that α M β 2 exposed at the MP surface can interact with other ligands, such as urokinase plasminogen activator, plas mino gen and metalloproteases MMP-2 and -5, suggesting a role in fi brinolysis and in local tissue remodelling [30,34,35]. MPs may also display antithrombotic activities, which would be overwhelmed by procoagulant activities when MPs are released under highly thrombotic conditions, as observed during sepsis or myocardial infarction. Indeed, in purifi ed monocyte suspensions, thrombomodulin anti coagulant activity and TF coexist at the MP surface, but when released by lipopolysaccharide treatment, the TF activity is predominant on MPs [31]. Th e presence of the anticoagulant endothelial protein C receptor (EPCR) at the surface of endothelial-derived MPs (mpEPCR) is another example of a cytoprotective element attached to MPs [32]; EPCR is involved in the activation of anticoagulant protein C by the thrombin-thrombomodulin complex. mpEPCR,

Figure 2. Microparticles and blood coagulation. (A)
The plasma membrane of endothelial cells and monocytes is reorganised, with externalisation of phosphatidylserine -a negatively charged phospholipid -and encrypted tissue factor (TF) expression, allowing factor VII (FVIIa) activation and thrombin (FIIa) generation at the cell surface. Blebbing occurs, with release of microparticles (MPs) bearing TF, resulting in an increased surface for procoagulant reactions. Platelet adhesion and aggregation also occur with the release of MPs; platelets and MPs bear GP Ibα , a cofactor for factor XI activation by thrombin, leading to the propagation phase with high levels of thrombin generation and fi brin formation. Endothelial TF-bearing MPs allow transfer of TF to PMNs, increasing TF dissemination and thrombotic microangiopathy or disseminated intravascular coagulopathy. (B) TF initiation of blood coagulation is quickly down-regulated by tissue factor pathway inhibitor (TFPI) on endothelial and monocytic cell surfaces, as on MPs. Endothelial protein C receptor (EPCR)-bound protein C is activated by the thrombin-thrombomodulin complex and activated protein C (APC) inhibits factor Va and factor VIIIa, limiting the propagation phase of thrombin generation. EPCR-bound APC also regulates NF-ΚB, with cytoprotective eff ects on endothelial cells and monocytes. APC induces blebbing, with emission of EPCR-bearing MPs able to activate protein C, resulting in the dissemination of anticoagulant and antiapoptotic activities. LPS, lipopolysaccharide; PhtdSer, phosphatidylserine; PMN, polymorphonuclear. released in response to activated protein C (APC), may switch the procoagu lant properties of endothelial MPs to anti coagulant and anti-apoptotic properties. On the surface of MPs bearing mpEPCR, APC inactivates procoagulant cofactors factor Va and factor VIIIa, thereby down-regulating thrombin generation. Because a circulating soluble form of EPCR (sEPCR) has been described in sepsis, and its concentration possibly correlates with the severity of the illness, the respective contributions of mpEPCR and sEPCR is a matter of clinical relevance. sEPCR binds protein C and APC, thereby blunting their actions. Th e effi cacy of therapeutic activated protein C (rhAPC; drotrecogin alfa (activated)) may depend on the balance between circulating sEPCR and mpEPCR [32]. Recent investigations in human endothelial cells reported that free rhAPC and rhAPC bound to mpEPCR have similar eff ects. rhAPC cleaves protease activated receptor-1 and induces signifi cant changes in the activation/inhibition of genes with direct anti-apoptotic eff ects or indirect cell barrier protective eff ects, the latter requiring transactivation of KDR (vascular endothelial growth factor receptor 2/kinase insert domain receptor) via the phosphoinositide 3-kinase-Akt pathway and S1P 1 (sphingosine 1-phosphate receptor) [36]. In sepsis, procoagulant MPs of endothelial, platelet, erythroid, and leukocyte origins have been reported [28,37].

Microparticles as potential eff ectors in the infl ammatory response of sepsis
Circulating MPs have been reported to be present in various infl ammatory diseases, including sepsis [7]. MPs are a source of phospholipids, a substrate for phospholipase A2, which facilitates platelet aggregation [38,39]; they may also provoke vascular infl ammation during sepsis via lysophosphatidic acid and facilitate chemotactic migration of platelets and/or leukocytes to the endothelium, thus playing the role of trigger in the production of monocyte cytokines (IL-1β, IL-8 and tumour necrosis factor-α) [8,40,41] (Figure 3).

Microparticles targeting the endothelial function
During sepsis, the endothelial function is altered and the endothelial surface becomes proadhesive, procoagulant and antifi brinolytic [42]. Th e endothelium is one of the primary targets of circulating MPs, as demonstrated by Barry and colleagues [43] in vitro. Indeed, they showed that arachidonic acid exposed by platelet MPs promotes the up-regulation of cyclooxygenase-2 (COX-2) and inter cellular adhesion molecules in endothelial cells. Platelet-derived MPs have been shown to modulate interactions between monocytes and endothelial cells. Released proinfl ammatory endothelial cytokines may themselves also contribute to the production of MPs [44], thereby amplifying the infl ammatory response and the consecu tive alteration of the vascular function [45]. Platelet activat ing factor present in endothelial cells and leuko cytes is also involved in the proinfl ammatory eff ect of MPs [46].

Endothelial microparticles and infl ammatory status
Circulating MPs of endothelial origin may thus vary with respect to quantity and phenotype according to the endothelial response and have been reported in infl ammatory diseases and disorders [47]; the endothelial response to infl ammation stimuli may be immediate, delayed or refl ect a chronic endothelial activation. Th ey were reported to participate in the regulation of arterial tone in several diseases in which oxidative stress is involved, such as human acute coronary syndromes [48] or preeclampsia [49] associated with altered NO bioavailability [50].
Sepsis induces a phenotypic change of the endothelium and the endothelial surface becomes proinfl ammatory, expresses cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1) [51] and becomes prothrombotic through the increased expression of membrane TF and the inhibition of thrombomodulin and EPCR synthesis. In parallel, endothelial cells become capable of recruiting and activating platelets [52].

Microparticles contribute to the spreading of infl ammatory and prothrombotic vascular status
MPs may be considered as both the cause and the consequence of infl ammatory diseases through multiple amplifi cation and regulatory loops aff ecting vascular cell functions. In vitro incubation of leukocyte-derived MPs with endothelial cells allowed Mesri and Altieri [53] to demonstrate their role in the secretion of infl ammatory IL-6 and in the production of TF [54]. Furthermore, platelet MPs are able to deliver RANTES at the infl amed endothelium, thus promoting leukocyte recruitment and diapedesis [55]. MPs may aff ect the smooth muscle tissue through the activation of the transcription factor NF-κB and favour the expression of inducible NO-synthase and COX-2, resulting in an increase in NO and vasodilator prostanoids, leading to arterial hyporeactivity [45].
Th e interactions between platelets, leukocytes and endothelium clearly contribute to the vascular dysfunction observed in sepsis and various MPs were reported to alter the arterial wall directly or indirectly [56,57].
Endothelial MPs may play a role in the spread of sepsis infl ammatory responses leading to multiple organ dysfunc tion [18,58]. Th ey may participate in the potentialisation of the procoagulant state associated with sepsis by providing renewed lipid surfaces of human endothelial cells for the generation of thrombin and by up-regulating monocyte TF expression, as demonstrated in vitro [59]. In sepsis, blockade of the human TF pathway by TFPI is very quickly overridden, clearing the way for a detrimental procoagulant state [28]. Indeed, in humans, a single endotoxin administration provokes a signifi cant increase in endothelial-cell-or monocyte-derived MPs displaying potentiated TF [60]. Th is state is worsened by the exhaustion and/or faulty activation of the two other regulatory molecules, antithrombin and APC.
Several reports illustrate well the cascade of interwoven events that link cellular activation, TF up-regulation, the release of MPs presenting active TF and the triggering of disseminated intravascular coagulopathy and shock [28,29]. With regard to vascular tone, MPs could promote the signifi cant vasoplegia observed in septic shock [8]. Arachidonic acid transfer may up-regulate COX-2 expression and the production of prostacyclin, which is implicated in vasodilation and the inhibition of platelet activation [32].

Microparticles and oxidative stress
During sepsis, generated MPs are involved in the modi fication of the oxidative status; they form micro aggregates with circulating neutrophil granulocytes and markedly increase oxidative activity [8]. Subunits of NADPH oxidase have been identifi ed in endothelial-and plateletderived MPs associated with increased produc tion of reactive oxygen species [18,61] (Figure 3).

Conclusion
Th e systemic infl ammatory response that is a characteristic feature of sepsis is a major cause of cellular dysfunction that may lead to the exaggerated generation of MPs. Th ese plasma membrane fragments are circulating markers of vascular infl ammatory diseases. Th ey also MPs may be considered as both the cause and the consequence of infl ammation through multiple amplifi cation and regulatory loops aff ecting vascular cells and functions. Thus, MPs contribute to the spread of infl ammatory and prothrombotic vascular status and they may aff ect the smooth muscle tissue through adhesion molecules, activation of NF-κB and the expression of inducible nitric oxide synthase and cyclooxygenase-2, with an increase in nitric oxide and vasodilator prostanoids, leading to arterial hyporeactivity. MPs form microaggregates with circulating neutrophil granulocytes and platelets and are involved in the modifi cation of the oxidative status, markedly increasing oxidative activity. Subunits of NADPH oxidase have been identifi ed in MPs associated with increased production of reactive oxygen species. AA, arachidonic acid; COX = cyclooxygenase; ICAM, intercellular cell adhesion molecule; iNOS, inducible NO-synthase; LPS, lipopolysaccharide; NO, nitric oxide; PAF, platelet activating factor; R, Rantes; ROS, reactive oxygen species; TF, tissue factor; VCAM, vascular cell adhesion molecule. behave as pathogenic shuttles able to disseminate their deleterious proinfl amatory and procoagulant potential in the systemic circulation and may be implicated in the multiple organ dysfunction characterizing sepsis and septic shock. To date, however, we have insuffi cient evidence to determine whether MPs are major players or bystanders in the development of the sepsis syndrome.

Competing interests
The authors declare that they have no competing interests.