91
From: Current Clinical Neurology: Inflammatory Disorders of the Nervous System:
Pathogenesis, Immunology, and Clinical Management
Edited by: A. Minagar and J. S. Alexander © Humana Press Inc., Totowa, NJ
Circulating Cell-Derived Microparticles in Thrombotic and Inflammatory Disorders
Wenche Jy, Lawrence L. Horstman, Joaquin J. Jimenez, Alireza Minagar, and Yeon S. Ahn
1. BACKGROUND 1.1. Introduction
It is now recognized that all circulating blood cells, as well as endothelial cells (EC), continuously shed small membranous vesicles (microparticles [MPs]), which are approximately less than 1 μm, and that levels of circulating MPs are sensitive indicators of disease activity. The first type exten- sively studied in patients was platelet MP (PMPs) (1). Currently, endothelial-derived MP (EMPs) have risen to the fore as sensitive markers of EC perturbation, recently reviewed (2) and further considered in this article. Although other reviews may differ in viewpoint and emphasis (3,4), it is generally agreed that circulating MPs comprise different subspecies of membrane vesicles released from endothelium and blood cells, such as platelets, leukocytes, and red blood cells (RBCs). MPs containing negatively charged phospatidylserine (PS) and/or tissue factor are highly procoagulant.
MPs that express specific adhesion molecules are capable of interacting with leukocytes and endothelia to initiate inflammatory responses.
Leukocyte MPs (LMPs) are less commonly studied, even though they are potentially of great interest. Because erythrocyte MPs (RBCMPs) are rarest in blood and are studied mainly in relation to disorders involving RBCs, such as hemolytic anemias, sickle-cell disease, and thallasemias, they are not considered in this chapter.
The focus of this review is on MPs as markers of inflammation and thrombosis. Additionally, their potential role in pathophysiology is also a major topic. Accordingly, we develop some novel hypothe- ses implied by experimental findings concerning the pathophysiology of certain disease conditions.
1.2. Methodologies
At present, results from different laboratories on the same type of patient may differ radically
because of differences in methodological procedures. This situation led us to write a recent forum
article in which principals of six laboratories active in MP studies presented their methods side by
side (5). Although most employ flow-cytometric techniques based on addition of fluorescent-labeled
antihuman monoclonal antibodies to the MPs in plasma (or resuspended after centrifugation), it is
evident (5) that different results will be found by different laboratories from the same sample
owing to differences in methods. We will not attempt to discuss methodological details, but this
situation must be noted when examining findings from different laboratories. Future workshops
are planned to improve agreement on measurement methods.
1.3. Soluble Inflammatory Markers
Several studies of inflammatory disorders have relied on measurement of so-called soluble markers of endothelial perturbation. For example, CD31 (platelet-endothelial cell adhesion molecule-1 [PECAM-1]) has been used to assess disease activity in multiple sclerosis (MS) (6).
However, we have demonstrated that the majority of CD31 can be removed by filtration through 0.1 μm filter and that it is clearly associated with PMP and EMP (7–10); thus, it is not a true soluble species. (A minimum of several hundred fluorescent molecules is needed to trig- ger a signal in clinical flow cytometers; therefore, they cannot detect true soluble molecules.) Similarly, E-selectin (CD62E) is widely measured as a soluble marker of endothelial stress (11–14), but we routinely use it to identify EMP (15–17), demonstrating that it is actually at least partially MP-bound.
Similar considerations apply to many other markers now regarded as soluble, including intercel- lular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, tissue factor (TF), von Willebrand factor (vWF; partly bound to EMP [18] and PMP), thrombo- modulin (19), and CD40L (20). As our recent review further details (2), these observations lead to the conclusion that many or most so-called soluble markers of EC disturbance are in reality not soluble, but are at least partially bound to MPs.
It is well established that some of these markers do exist in true soluble form, usually owing to enzymatic cleavage from the membrane or by posttranslational editing (21), but it is equally well established by our lab and others that a significant fraction, up to 80 to 90%, of these markers occur on cell-derived MPs, presumably with their transmembrane domains intact and normally adjacent proteins present.
The practical importance of this lies in the fact that release of true soluble species occurs by mech- anisms entirely different from membrane vesiculation and hence reflect different pathophysiologies.
Additionally, true soluble species often have properties functionally different from their MP-bound forms, as we have shown for vWF (18). In view of these considerations, it is expected that when the MP-bound markers are clearly distinguished by independent measurement from the true soluble species, better-defined relations will emerge between disease states and the marker in question.
In summary, many so-called soluble species are in reality MPs. Accordingly, to the extent that they are recognized as valuable clinical and research tools, MP analysis deserves at least equal recognition.
1.4. Generation of MPs
MPs can be released under many different conditions, such as (a) activation or apoptosis induced by numerous agents; (b) partial or complete lysis, such as by complement; (c) oxida- tive injury; or (d) other insults, such as high-shearing stress (22). The detailed mechanisms for MP release remain obscure. However, a rise in cytosolic calcium concentration, either from internal stores or from plasma membrane, appears to be a necessary triggering event or com- mon pathway for vesicle release. Elevated cytoplasmic calcium has been shown to activate sev- eral cytoplasmic enzymes involving MP shedding. First, elevated calcium can induce cytoskeletal contraction, which is thought to be the driving force for the formation of mem- brane blebs (23). To allow the membrane blebs to be shed from plasma membrane, the mem- brane cytoskeleton must be broken down. It has been demonstrated that calcium-dependent proteases, such as calpain (24) and caspases (25) are capable of breaking down cytoskeleton and facilitating MP releases.
1.5. Composition of MPs
MPs consist mainly of phospholipids and proteins. Their composition depends on the cell origin
and the cellular processes inducing their release. Inside the vesicles, MPs may carry some cytoplas-
mic materials. The redistributed lipid bilayers of plasma membrane have been shown to be disturbed
prior to the release of MPs (26), leading to the expression of negatively charged PS on the MP. The
expression of PS on MP has been shown to play an important role in blood coagulation (26).
Weerheim et al. (27) have analyzed the phospholipid composition of cell-derived microparticles in normal blood and found that the composition comprises 60% phosphatidylcholine (PC), with remainder in sphingomyelin, phosphatidylethanolamine (PE), and phosphatidylserine (PS). In contrast, Fourcade et al. have reported that MPs from synovial fluids from inflamed joints of arthritis patients contained evenly distributed PC, PE, sphingomyelin, and lysophospholipids (28). MPs in the synovial fluids are mainly derived from leukocytes. However, MPs in blood are mainly released from platelets (29,30). These studies indicate that the difference in MP lipid composition may be caused by differences in cell origin and types of stimulation.
The surface antigens on MPs are a unique indicator of parent cell status. We have found that EMPs derived from activated ECs are enriched with CD62E and CD54 antigens. In contrast, EMPs derived from apoptotic ECs are enriched with CD31 antigen (15). However, not all the surface antigens on the parent cells are expressed on MPs. For example, T cell-derived MPs lack CD28 and CD45 antigens, which are among the most abundant antigens on the parent T cells (31). We have also observed that CD51 antigens are highly expressed on ECs but seldom on EMPs.
Together, it is very likely that MP shedding is a well-controlled process. It may involve membrane antigen clustering/capping and the formation of lipid raft (32,33).
2. CIRCULATING MPS IN THROMBOTIC AND INFLAMMATORY DISEASES
Berckmans et al. have assayed the number, cellular origin, and thrombin-generation properties of MPs in healthy individuals (29). They found that normal blood contains the highest number of PMPs (237 × 10
6/L), as compared to EMP (64 × 10
6/L), granulocyte MPs (46 × 10
6/L), or red cell MPs (28 × 10
6/L). In this chapter, we review only MPs originating from platelets, endothelia, and leukocytes.
2.1. Platelet Microparticles
Among all cell-derived MPs, PMPs were the first discovered (34) and are the most widely stud- ied. Glycoprotein (GP) IIb/IIIa (CD41), GP Ib/IX (CD42), and CD62P are the most frequently used markers to label and quantitate PMPs. In addition, they also carry other factors including vascular endothelial growth factor (VEGF) (35), thrombospondin (36), platelet-activating factor (PAF) (37), β-amyloid protein precursor (38), anticoagulant protein C/S (39), and complement components (40).
These factors carried by PMP can further amplify PMP’s role in thrombosis and inflammation.
Abnormal PMP have been reported in many thrombotic and inflammatory disorders including immune thrombocytopenic purpura (ITP), transient ischemic attacks (TIAs), acute coronary syndrome (ACS), thrombosis, antiphospholipid antibodies, lupus anticoagulant, thrombotic thrombocytopenic purpura (TTP), heparin-induced thrombocytopenia/heparin-induced thrombocytopenia with throm- bosis (HIT/HITT), paroxysmal nocturnal hemoglobinuria (PNH), and multiple sclerosis (MS). For detailed information, please refer to our previous review of this subject (1). In contrast, low-PMP generation was reported in Scott’s syndrome, a rare bleeding disorder (41). Most of these studies focus quantitatively on the EMP levels and do not follow up longitudinally on the studied patients.
The value of PMP as a predictive marker in prethrombotic state remained mostly unknown.
In addition to assaying the PMP counts, some studies tried to identify the functionality of PMP by examining special antigens or proteins on PMP. For example, Tans et al. found that some PMPs contain anticoagulant factor protein C/S; therefore, they concluded that some PMP are anticoagulant (39).
2.2. Endothelial Microparticles
EMPs have been investigated intensively in the past several years. Cumulative studies have
shown that EMPs comprise subsets of membrane vesicles with different surface antigens. There
is no consensus on which markers or methodologies are best for evaluating EMPs in disease
activities. Sometimes, conflicting results arise from different research groups owing to their different
methodologies.
2.2.1. Lupus Anticoagulant
The first report of clinical application of EMP assay was by Combes et al. in lupus anticoagulant (42). Using CD31/CD51 markers, they found that patients with lupus anticoagulant had significantly higher EMPs (by twofold) than normal controls. Interestingly, the levels of EMPs were not reduced in those treated for thrombosis with anticoagulant.
2.2.2. Thrombotic Thrombocytopenic Purpura
Jimenez et al. reported elevated EMPs in patients with TTP by measuring CD31 +/CD42b- EMP species and observed that EMP levels rose in acute stages, returned to normal in remission, and correlated with disease activity (7). In a more recent study, CD62E rather CD31 was used as the main marker, yielding higher EMP counts and better correlation with other markers (16).
2.2.3. Multiple Sclerosis
Minagar et al. reported an elevation of CD31
+EMPs during MS exacerbations in contrast to remission. However, CD51
+EMPs do not distinguish exacerbation from remission (8). Elevated CD31
+EMPs were well correlated with the presence of gadolinium-enhancing lesions in brain mag- netic resonance imagings. Jy et al. used a simple in vitro model of transendothelial migration (TEM) to study the role of EMPs in the transmigration of the monocytic cell U937 (5). The most recent work demonstrated that (a) plasma from MS patients sharply increase TEM; (b) pretreatment of the leuko- cytes with EMPs further facilitated TEM; (c) leukocytes exhibited bound EMPs after passing through the monolayer; and (d) drugs, such as interferon- β-1b and danazol, inhibited TEM (17).
2.2.4. Acute Coronary Syndrome
Mallat et al. (43) measured MPs in ACS by first capturing MP with immobilized Annexin V (ANV), quantitating them by prothrombinase activity, then identifying cell origins using an ELISA method with anti-CD3, -CD11a, -CD31, -CD146, or -GPIb. Their main finding was that EMP, but not other MP, were elevated by 2.5-fold in acute myocardial infarction (MI). In this study, neither EMP nor other MP levels differentiated between stable angina (SA) and normal controls, between unstable angina (UA) and MI, or between UA and controls.
At the same time, Bernal-Mizrachi et al. (9) similarly found elevated EMP in ACS when they studied a larger number of patients (N = 84) using flow cytometry. A number of Bernal-Mizrachi’s findings are notable. First, they found distinctly different EMP results when they used two markers, CD31
+and CD51
+. For example, CD51
+EMP, although clearly elevated in ACS relative to con- trols, did not distinguish new ACS from recurring disease, as CD31
+EMP did. The authors con- cluded that CD31
+EMP mainly acts as a marker of acute events, whereas CD51
+EMP reflects chronic endothelial stress, which has also been observed in MS (8).
2.2.5. Hypertension
Preston et al. (44) investigated a possible relationship between hypertension (HTN) and endothelial injury, as measured primarily by EMPs. They studied patients with untreated severe (diastolic blood pressure [BP] ≥120) or mild (BP > 95 <100) HTN compared to normal controls.
They observed that EMP was highest in severe HTN (p = 0.002) and showed a significant correla- tion with systolic and diastolic BP. Interestingly, they found no correlation between BP and soluble markers of endothelial activation, such as sVCAM-1, and thus concluded that EMP assay appears to be the most sensitive method for assessing BP-induced effects on the endothelium and subsequent risk of impending hypertensive vascular and organ damage.
2.2.6. Preeclampsia
Gonzalez-Qintero et al. (10) applied EMP analysis to a prospective, case-controlled study of
20 patients with preeclampsia (PE) and 20 healthly pregnant controls. They demonstrated that a
significant elevation of CD31 +/CD42- EMP in PE (10). In a more recent follow-up that also
employed CD62E, results were clearer and more dramatic. Additionally, EMP correlated with proteinuria in patients with PE. They also observed a correlation between EMP and mean arterial BP, as in the work by Preston et al. cited earlier (see Section 2.2.5.). VanWijk et al. (4) also investigated MP in patients with PE. They failed to demonstrated significant differences in EMP levels between patients and controls. We suggest that the discrepancy may have been caused by the use of different methodologies.
2.2.7. Diabetes Mellitus
Sabatier et al. (45) compared MP numbers in controls vs patients with diabetes mellitus types 1 and 2, finding that patients with type 1 had elevated PMP, EMP, and total MP. Patients with diabetes mellitus type 2 showed elevation only in total MP, not in PMP or EMP. The authors suggested that these findings may be related to vascular complications in diabetes mellitus.
2.2.8. Paroxysmal Nocturnal Hemoglobinuria and Sickle Cell Crisis
Simak et al. (46) studied circulating EMP in PNH, aplastic anaemia (AA), and sickle cell dis- ease (SCD). They found that both CD54
+and CD144
+EMP were significantly elevated in PNH and SCD but not in AA or healthy controls. Their findings indicate an involvement of endothelial injury in the acute phase of PNH and SC crisis.
2.3. Leukocyte Microparticles
The role of LMP as a maker of disease activities recently has received increasing attention. Sev- eral laboratories have reported that LMPs were elevated in thrombotic or inflammatory disorders.
Biro et al. (47) have shown that MP originated from granulocyte-expressed TF, indicating that they may promote thrombus formation in a tissue factor-dependent manner.
2.3.1. Preeclampsia
VanWijk et al. (4) have investigated the cellular origin and numbers of circulating MP in normal pregnancy and PE. They found that the number of circulating MPs was unaltered in pregnancy and PE; however, numbers of T-cell and granulocyte MPs are increased in PE. Whether these altered MP levels cause vascular dysfunction in PE or are a consequence of the disease remains to be established.
2.3.2. Sepsis
Nieuwland et al. (48) have studied circulating MPs in meningococcal sepsis. They found that on admission, all patients had increased levels of MPs originating from platelets or granu- locytes when compared to controls. In addition, they reported that these MPs supported thrombin generation more strongly in vitro than controls did. Plasma from the patient with the most fulminant disease course and severe disseminated intravascular coagulation contained MPs that expressed both CD14 and TF, and these microparticles demonstrated extreme thrombin generation in vitro.
2.3.3. Antiphospholipid Syndrome
Nagahama et al. (49) have reported that the concentration of monocyte-derived and platelet-derived MP in patients with antiphospholipid syndrome (APS) was significantly higher than that in normal subjects and patients with systemic lupus erythematosus. Twenty one of the 37 APS patients (56.8%) had elevated levels of anti-oxLDL antibody. In addition, the patients with elevated monocyte-derived MPs were frequently associated with positive anti-oxLDL antibody (49).
2.3.4. Sickle Cell Disease
Shet et al. (50) found total MPs were elevated in crisis and steady state in subjects with SCD,
compared to those without it. These MP were derived from erythrocytes, platelets, monocytes,
and EC. Total TF-positive MPs were elevated in SCD crisis vs steady-state and control subjects
and were derived from both monocytes and EC. Their data support the concept that SCD is an inflammatory state with monocyte and endothelial activation and abnormal TF activity.
2.3.5. Trauma
Fujimi et al. (51) have evaluated the production of polymorphonuclear leukocyte (PMNL)- derived MPs in severely injured patients at three time points: days 0 to 1, days 2 to 5, and days 6 to 12 after the trauma event. They found that production of PMNL-derived MPs increased along with adhesion-molecule expression on days 2 to 5 after severe trauma. CD62L expression was enhanced on MPs at all three time points, and CD11b expression was enhanced on MPs less than 1.0 μm in diameter at all three time points. However, soluble E2-selectin and thrombomodulin in blood did not change significantly between time points, indicating no significant endothelial injury.
2.3.6. Venous Thrombosis
Myers et al. (52) have studied P-selectin and leukocyte MPs in thrombogenesis in mice. The evaluation of MP revealed that mice with the highest thrombus mass showed a high amount of mean channel fluorescence for MAC-1 (phycoerythrin) antibody, indicative of leukocyte MPs. An antibody directed against PSGL-1 was more effective than rPSGL-Ig in decreasing TM and limiting leukocyte-derived MP fluorescence. These data suggest that leukocyte MPs are associated with venous thrombus formation.
3. FUNCTIONS OF MICROPARTICLES 3.1. Coagulation
Exposing TF usually triggersblood coagulation. Completing the coagulation cascade, not only requires coagulation factors and calcium ions but also a membrane surface exposing negatively charged phospholipids, such as PS, to facilitate the formation of tenase and prothrombinase com- plex. MPs have been shown to carry anionic phospholipids, especially PS and TF. Both of these procoagulant activities are known to occur on EMP, LMP, and PMP (7,43,47,53,54); however, it is not yet possible to ascertain the relative importance of these activities on EMP vs PMP and LMP or on whole cell surfaces (such as PF3 activity of activated platelets or TF expression on leukocytes or activated EC).
TF activity in particular has been difficult to quantify; conflicting reports may be caused by variable expression of “cryptic” TF and masking by tissue factor pathway inhibitor (55,56) and partly to sensitivity to sulfhydryl redox state (57). Although more work is needed, much evi- dence suggests that EMP likely have important roles in coagulation, especially at local sites of injury, not only by virtue of PF3 and TF activities but also by possibly modulating the protein C/S-thrombomodulin anticoagulant system.
Although the majority of MP are procoagulant, some reports also demonstrate that MP may be anticoagulant under certain conditions. Tans et al. suggested that PMP could serve an anti- coagulant function by supporting the protein C/S/TM pathway (39). Gris et al. (58) demon- strated that a large part of protein S is MP-associated and that clinical assays using PEG precipitation cause underestimation of protein S because much of it is precipitated with MP.
More recently but in a similar spirit, Berckmans et al. have shown that low levels of thrombin generation by MP in normal controls occurs via the contact pathway (independent of TF) and may serve an anticoagulant function because protein C could be activated by the trace of thrombin (29).
3.2. Inflammation
Jy et al. (59) first demonstrated the interaction of PMPs and leukocytes. They showed that
PMPs can bind to leukocytes via a P-selectin-dependent pathway. The binding of PMPs to leuko-
cytes leads to leukocyte activation and aggregation. Barry et al. (60) showed that PMPs interact
with ECs, which results in upregulation of CD54 and the subsequent adhesion of monocytes.
Mesri and his coworker also showed that leukocyte microparticles can interact with endothelium to induce interleukin-6 release (61).
Recently, Sabattier et al. (62) demonstrated that EMPs can bind to and activate cultured monocytes, as judged by induced expression of TF antigen and that this effect could be largely inhibited by anti-CD54. Building on previous work on PMP–leukocyte interactions, Jy et al. (59) had independently found similar results (including selective inhibition of the interaction by anti-CD54) but used a different approach: studying leukocytes in whole blood and measuring leukocyte activation in response to added EMP by expression of CD11b (5). Their findings also indicated that EMPs interacted only weakly with PMNs relative to monocytes and hardly at all with lymphocytes.
Of particular interest was the finding that EMPs generated by apoptosis in ECs were weak compared to equal concentration of EMPs from ECs activated by tumor necrosis factor (TNF)- α (5).
This may be the result of higher levels of CD54
+EMP induced by TNF- α compared to apoptotic ECs. In a binding study using U937 cells exposed to EMP labeled with various markers, the EMPs labeled with ICAM-1 (CD54) exhibited the greatest apparent binding, followed by EMPs labeled with PECAM-1 (CD31), E-selectin (CD62E), and vitronectin receptor (CD51). Accord- ingly, the CD54- labeled EMP was largely depleted in the cell-free supernatant, consistent with the majority binding to the U937 cells (5). Those authors also proposed that the relatively low concentration of CD54
+EMP found in blood in various disease states is explained by the finding that these EMPs preferentially and strongly bind to leukocytes, reducing their free concentration.
Finally, they demonstrated that monocytes with adhering EMPs were facilitated in their passage through an endothelial monolayer (5). Taken together, these findings suggest that at least one function of EMPs may be to modulate inflammation via leukocyte activation and transendothelial migration. The complete interaction between MPs and platelets, leukocytes and endothelium was summarized in Fig. 1.
Fig. 1. Current findings on interactions among MPs species and cells.
3.3. Vascular Function
It has been reported that MPs released from platelets or leukocytes can influence endothelial function. Boulanger et al. (63) reported that MPs from patients with acute MI impaired endothelium- dependent relaxation in isolated arteries. In contrast, MPs isolated from patients with non-ischemic chest pain had no such effect. High concentrations of microparticles from patients with MI affected neither endothelium-independent relaxation to sodium nitroprusside nor expression of the endothe- lial nitric oxide (NO) synthase. The origin of the MP isolated from patients with MI has not been identified. Their data indicate that circulating MPs from patients with MI selectively impair the endothelial NO transduction pathway. VanWijk et al. also demonstrated that MPs isolated from patients with PE impaired endothelium-dependent relaxation in isolated myometrial arteries (64). It is suggested that these MPs may contain oxidized phospholipids, which are a potent inhibitor of endothelial function (65). Some MPs, however, may have a beneficial effect on endothelium. Barry et al. showed that PMPs can transfer arachidonic acid to ECs, which results in prostacyclin produc- tion and will induce vascular relaxation (66). It has been reported recently that PMPs promote pro- liferation, survival, migration, and tube formation in human umbilical vein endothelial cells (67).
In addition, PMPs were also shown to augment endothelial progenitor cell differentiation in peripheral blood mononuclear cells. Their results suggest that lipid components of the PMP may be major active factors and that protein components may be minor contributors.
4. CONCLUSION
Cell-derived MPs have received increasing attention in recent years, both as a diagnostic aid and investigative tool. Because they carry markers of the parent cell, including those induced by activation or apoptosis, EMPs can provide valuable information on the status of the parent cell, which can be obtained no other way. In addition, there is a growing belief that MPs can function as important diffusible vectors of specific adhesins and cytokines promoting cellular interactions and signal transmission.
Thus, MP analysis constitutes a new avenue for investigation of pathologies in various dis- eases. Although still considered investigational, recent results from several laboratories suggest that MP analysis may be poised to enter the mainstream of clinical testing. In summary, it appears that MP analysis is emerging as the method of choice for assessing cell involvement in disease states. Although few studies have yet undertaken direct comparison of results using EMP com- pared to soluble markers, our EMP results for TTP, coronary artery disease, and MS appear to offer superior discrimination of clinical states as compared to other published studies employing soluble markers of endothelial disturbance.
REFERENCES
1. Horstman LL, Ahn YS. Platelet microparticles: a wide-angle perspective. Crit Rev Oncol Hematol 1999;30:111–142.
2. Horstman LL, Jy W, Jimenez JJ, Ahn YS. Endothelial microparticles as markers of endothelial dysfunction. Front Biosci 2004;9:1118–1135.
3. Freyssinet JM. Cellular microparticles: what are they bad or good for? J Thromb Haemost 2003;1:1655–1662.
4. VanWijk MJ, Nieuwland R, Boer K, van der Post JA, VanBavel E, Sturk A. Microparticle subpopulations are increased in preeclampsia: possible involvement in vascular dysfunction? Am J Obstet Gynecol 2002;187:4506.
5. Jy W, Minagar A, Jimenez JJ, et al. Endothelial microparticles (EMP) bind and activate monocytes: elevated EMP-monocyte conjugates in multiple sclerosis. Front Biosci 2004;9:3137–3144.
6. Losy J, Niezgoda A, Wender M. Increased serum levels of soluble PECAM-1 in multiple sclerosis patients with brain gadolinum-enhancing lesions. J Neuroimmunol 1999;99:169–172.
7. Jimenez J, Jy W, Mauro L, Horstman L, Ahn YS. Elevated endothelial microparticles in thrombotic thrombocy- topenic purpura (TTP): findings from brain and renal microvascular cell culture and patients with active disease.
Br J Haematol 2001;112:81–90.
8. Minagar A, Jy W, Jimenez JJ, Mauro LM, Horstman LL, Ahn YS, Sheremata WA. Elevated plasma endothelial microparticles in multiple sclerosis. Neurology 2001;56:1319–1324.
9. Bernal-Mizrachi L, Jy W, Jimenez JJ, et al. High levels of circulating endothelial microparticles in patients with acute coronary syndromes. Am Heart J 2003;145:962–970.
10. Gonzalez-Quintero V, Jimenez JJ, Jy W, Mauro LM, Horstman L, O’Sullivan M, Ahn YS. Elevated plasma endothelial microparticles in preeclampsia. Am J Obstet Gynecol 2003;189:589–593.
11. Roldan V, Marin F, Lip GYH, Blann AD. Soluble E-selectin in cardiovascular disease and its risk factors. Thromb Haemost 2003;90:1007–1020.
12. Seeman HB, Gurbel PA, Anderson JL, Muhlestein JB, Carlquist JF, Horne BD, Serebruany VL. Soluble VCAM-1 and E-selectin, but not ICAM-1, discriminate endothelial injury in patients with documented coronary artery disease.
Cardiology 2000;93:7–10.
13. Hwang SJ, Ballantyne CM, Sharrett R, Smith LC, Davis CE, Gotto AM, Boerwinkle E. Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases.
Circulation 1997;96:4219–4225.
14. Lieuw-a-Fa M, Schalkwijk C, vanHinsbergh VWM. Distinct accumulation patterns of soluble forms of E-selectin, VCAM-1 and ICAM-1 upon infusion of TNF-alpha in tumor patients. Thromb Haemost 2003;89:1052–1057.
15. Jimenez JJ, Jy W, Mauro L, Soderland C, Horstman LL, Ahn YS. Endothelial cells release phenotypically and quan- titatively distinct microparticles in activation and apoptosis. Thromb Res 2003;109:175–180.
16. Jimenez JJ, Jy W, Mauro LM, Horstman LL, Soderland C, Ahn YS. Endothelial microparticles released in throm- botic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation. Br J Haematol 2003;12:896–902.
17. Jimenez JJ, Jy W, Mauro LM, Minagar A, Solderland C, Horstman LL, Ahn YS. Transendothelial migration (TEM) in multiple sclerosis (MS): induction by patient plasma and its augmentation by leukocyte endothelial microparticles (L-EMP) complexes. Blood 2003;102:72b Ab 9.
18. Jy W, Jimenez JJ, Mauro LM, et al. Endothelial microparticles (EMP) interact with platelets via a vWF dependent pathway to form platelet aggregates, more resistant to dissociation than those induced by soluble vWF. Blood 2003;102:783a Ab 2896.
19. Satta N, Freyssinet JM, Toti F. The significance of human monocyte thrombomodulin during membrane vesiculation and after stimulation by lipopolysaccharide. Brit J Haematol 1997;96:534–542.
20. Amirkhosravi A, Meyer T, Sackel D, Desai H, Biddinger R, Amaya M, Francis JL. Platelet microparticles upregulate TF and VEGF in endothelial and melanoma cells in a CD40 ligand-dependent manner: Possible role in angiogenesis and metastasis. Blood 2002;100(11, Part II):63b Ab 3721.
21. Heaney ML, Golde DW. Soluble cytokine receptors. Blood 1996;87:847–857.
22. Miyazaki Y, Nomura S, Miyake T, et al. High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles. Blood 1996;88:3456–3464.
23. Mills JC, Stone NL, Erhardt J, Pittman RN. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol 1998;140:627–636.
24. Fox JE, Austin CD, Boyles JK, Steffen PK. Role of the membrane skeleton in preventing the shedding of procoagulant- rich microvesicles from the platelet plasma membrane. J Cell Biol 1990;111:483–493.
25. Rohn TT, Cusack SM, Kessinger SR, Oxford JT. Caspase activation independent of cell death is required for proper cell dispersal and correct morphology in PC12 cells. Exp Cell Res 2004;295:215–225.
26. Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997;89:1121–1132.
27. Weerheim AM, Kolb AM, Sturk A, Nieuwland R. Phospholipid composition of cell-derived microparticles deter- mined by one-dimensional high-performance thin-layer chromatography. Anal Biochem 2002;302:191–198.
28. Fourcade O, Simon MF, Viode C, et al. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell 1995;80:919–927.
29. Berckmans RJ, Neiuwland R, Boing AN, Romijn FP, Hack CE, Sturk A. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 2001;85:639–646.
30. Berckmans RJ, Nieuwland R, Tak PP, et al. Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism. Arthritis Rheum 2002;
46:2857–2866.
31. Blanchard N, Lankar D, Faure F, Regnault A, Dumont C, Raposo G, Hivroz C. TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex. J Immunol 2002;168:3235–3241.
32. Jy W, Jimenez JJ, Mauro LM, et al. Agonist-induced capping of adhesion proteins and microparticle shedding in cultures of human renal microvascular endothelial cells. Endothelium 2002;9:179–189.
33. Salzer U, Hinterdorfer P, Hunger U, Borken C, Prohaska R. Ca(++)-dependent vesicle release from erythrocytes involves stomatin-specific lipid rafts, synexin (annexin VII), and sorcin. Blood 2002;99:2569–2577.
34. Wolf P. The nature and significanc of platelet product in human plasma Br J Haematol 1967;13:269–288.
35. Wartiovaara U, Salven P, Mikkola H, et al. Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation. Thromb Haemost 1998;80:171–175.
36. George JN, Pickett EB, Saucerman S, McEver RP, Kunicki TJ, Kieffer N, Newman PJ. Platelet surface glycoproteins.
Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 1986;78:340–348.
37. Iwamoto S, Kawasaki T, Kambayashi J, Ariyoshi H, Monden M. Platelet microparticles: a carrier of platelet-activating factor? Biochem Biophys Res Commun 1996;218:940–944.
38. Nomura S, Komiyama Y, Miyake T, et al. Amyloid beta-protein precursor-rich platelet microparticles in thrombotic disease. Thromb Haemost 1994;72:519–522.
39. Tans G, Rosing J, Thomassen MC, Heeb MJ, Zwaal RF, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991;77:2641–2648.
40. Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity. J Biol Chem 1988;263:18205–18212.
41. Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ. Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity. J Biol Chem 1989;264:17,049–17,057.
42. Combes V, Simon AC, Grau GE, et al. In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest 1999;104:93–102.
43. Mallat Z, Hugel B, Ohan J, Leseche G, Freyssinet JM, Tedgui A. Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity. Circulation 1999;99:348–353.
44. Preston RA, Jy W, Jimenez JJ, et al. Effects of severe hypertension on endothelial and platelet microparticles. Hyper- tension 2003;41:211–217.
45. Sabatier F, Darmon P, Hugel B, et al. Type 1 and type 2 diabetic patients display different patterns of cellular microparticles. Diabetes 2002;51:2840–2845.
46. Simak J, Holada K, Risitano AM, Zivny JH, Young NS, Vostal JG. Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria. Br J Haematol 2004;125:804–813.
47. Biro E, Sturk-Maquelin KN, Vogel GM, et al. Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner. J Thromb Haemost 2003;1:2561–2568.
48. Nieuwland R, Berckmans RJ, McGregor S, et al. Cellular origin and procoagulant properties of microparticles in meningococcal sepsis. Blood 2000;95:930–935.
49. Nagahama M, Nomura S, Kanazawa S, Ozaki Y, Kagawa H, Fukuhara S. Significance of anti-oxidized LDL antibody and monocyte-derived microparticles in anti-phospholipid antibody syndrome. Autoimmunity 2003;36:125–131.
50. Shet AS, Aras O, Gupta K, et al. Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 2003;102:2678–2683.
51. Fujimi S, Ogura H, Tanaka H, et al. Increased production of leukocyte microparticles with enhanced expression of adhe- sion molecules from activated polymorphonuclear leukocytes in severely injured patients. J Trauma 2003;54:114–119.
52. Myers DD, Hawley AE, Farris DM, et al. P-selectin and leukocyte microparticles are associated with venous throm- bogenesis. J Vasc Surg 2003;38:1075–1089.
53. Kagawa H, Komiyama Y, Nakamura S, et al. Expression of functional tissue factor on small vesicles of lipopolysaccharide-stimulated human vascular endothelial cells. Thromb Res 1998;91:297–304.
54. Mallat Z, Benamer H, Hugel B, Benessiano J, Steg PG, Freyssinet JM, Tedgui A. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes.
Circulation 2000;101:841–843.
55. Bajaj MS, Birktoft JJ, Steer SA, Bajaj SP. Structure and biology of tissue factor pathway inhibitor. Thromb Haemost 2001;86:959–972.
56. Camerer E, Kolsto AB, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation. Thromb Res 1996;81:1–41.
57. Horstman LL, Cast L, Jy W, Jimenez JJ, Ahn YS. Tissue factor activity is controlled by its inhibitor and redox state:
findings in endothelial microparticles, monocytes and a porcine trauma model. Presented at the 19th Annual ISTH Congress, Birmingham, U.K. July 12–18, 2003.
58. Gris JC, Toulon P, Brun S, Maugard C, Sarlat C, Schved JF, Berlan J. The relationship between plasma microparticles, protein S and anticardiolipin antibodies in patients with human immunodeficiency virus infection. Thromb Haemost 1996;76:38–45.
59. Jy W, Mao WW, Horstman L, Tao J, Ahn YS. Platelet microparticles bind, activate and aggregate neutrophils in vitro. Blood Cells Mol Dis 1995;21:217–231.
60. Barry OP, Pratico D, Savani RC, FitzGerald GA. Modulation of monocyte-endothelial cell interactions by platelet microparticles. J Clin Invest 1998;102:136–144.
61. Mesri M, Altieri DC. Endothelial cell activation by leukocyte microparticles. J Immunol 1998;161:4382–4387.
62. Sabatier F, Roux V, Anfosso F, Camoin L, Sampol J, Dignat-George F. Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor-dependent procoagulant activity. Blood 2002;99:3962–3970.
63. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, Mallat Z. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 2001;104:2649–2652.
64. VanWijk MJ, Svedas E, Boer K, Nieuwland R, Vanbavel E, Kublickiene KR. Isolated microparticles, but not whole plasma, from women with preeclampsia impaired endothelium-dependent relaxation in isolated myometrial arteries from healthy pregnant women. Am J Obstet Gynecol 2002;187:1686–1693.
65. Rikitake Y, Hirata K, Kawashima S, et al. Inhibition of endothelium-dependent arterial relaxation by oxidized phos- phatidylcholine. Atherosclerosis 2000;152:79–87.
66. Barry OP, Pratico D, Lawson JA, FitzGerald GA. Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles. J Clin Invest 1997;99:2118–2127.
67. Kim HK, Song KS, Chung JH, Lee KR, Lee SN. Platelet microparticles induce angiogenesis in vitro. Br J Haematol 2004;124:376–384.
CME QUESTIONS
1. Which statement is correct?
A. It is now recognized that all circulating blood cells, as well as endothelial cells, continuously shed small membranous vesicles (microparticles), which are approximately less than 1.0 μm.
B. Plasma levels of circulating microparticles may be sensitive indicators of disease activity.
C. Endothelial microparticles have been reported as markers of endothelial disturbance.
D. All of the above are correct.
2. Which statement about microparticles is correct?
A. Microparticles consist mainly of phospholipids and proteins.
B. Composition of microparticles depends on the cell origin and the cellular processes inducing their release. Inside the vesicles, microparticles may carry some cytoplasmic materials.
C. Microparticles carry one or multiple adhesion molecules from their parent cells.
D. All of the above are correct.
3. Which statement is incorrect?
A. Elevated endothelial microparticles plasma levels have been reported in multiple sclerosis.
B. Microparticles probably have a role in coagulation.
C. Microparticles interact with leukocytes and participate in the cascade of inflammation.
D. Microparticles have no known function in endothelial biology.
