REVIEWNew horizons in the analysis of circulating cell-derived
microparticles
Lawrence L. Horstman, Wenche Jy, Joaquin J. Jimenez, Carlos Bidot and Yeon S. Ahn
The Wallace H Coulter Platelet Laboratory, University of Miami Medical Center, FL, USA(Received for publication on March 24, 2004)

Abstract. Analysis of circulating cell-derived microparticles (MP) is becoming more reﬁned andclinically useful. This review, stemming from lectures given at Tokyo late 2003, does not repeat priorreviews but focusses on new horizons. A major theme is the rising recognition of platelets and their MP(PMP) as key mediators of inﬂammation/immunity. Among the major concepts developed are that (i)many so-called soluble markers of inﬂammation are in reality MP-bound; (ii) PMP and other MP appear to serve important signaling and immune functions including antigen presentation. In conclusion,
MP analysis is poised to enter the mainstream of clinical testing, measuring speciﬁc antigens rather
than gross levels. However, more research is needed to decisively establish their functions, and international standards are needed to allow comparing results from different laboratories. (Keio J Med 53(4): 210–230, December 2004)
Key words: platelet microparticles, cell-derived microparticles, inﬂammation

attempt has been made to exhaustively review all relevant work. The review begins with background to orient readers unfamiliar with MP studies.

Scope and Background
Scope
This review is based on Part I of lectures deliveredin Tokyo, Japan, Nov. 29 (Part I) and Dec 1 (Part II)
2003, graciously sponsored by Otsuka Pharmaceuticals,Ltd. Part I centered on platelet microparticles (PMP),
being an update with new perspectives since our 1999
review1 while Part II focused on endothelial microparticles (EMP). Since our reivew of EMP is now published,2 that topic is not emphasized here. Helpful
reviews by others, each with distinctive perspective, include those by Nomura3 and by Freyssinet.4
As the title indicates, this article features new horizons or challenges in research and applications of
PMP rather than listing established ﬁndings covered inprior reviews. Other cell-derived microparticles (MP),notably leukocyte MP (LMP) and erythrocyte MP(RBCMP), are not a focus of this review but many of
the same principles apply.
This is a critical review in the sense that it highlights
pitfalls in certain methods and assumptions. Because of
the rapidly expanding scope of relevant literature, noWhat are cell-derived MP?
It is now established that all circulating blood cells,
as well as endothelial cells (EC), are capable of releasing membranous fragments (vesicles), of size less than
@1 mm, bearing on their surfaces at least some of theantigenic markers distinctive of the parent cell. Several
circumstances occurring in many disease states are
known to stimulate their release such as (i) activation
or apoptosis induced by numerous agents, (ii) partial orcomplete lysis such as by complement, (iii) oxidative
injury, or (iv) other insults such as high shearing stress.5
Fig. 1 shows schematically these modes of MP generation, as distinct from the detailed mechanisms by which
they are released, discussed later and here labeled‘‘black box’’ to signify that these mechanisms remain
obscure. However, a rise in cytosolic calcium concentration, either from internal stores (dense tubules) orfrom the plasma, appears to be a necessary triggering
event or common pathway for vesicle release.

Presented at the 1337th Meeting of the Keio Medical Society in Tokyo, December 1, 2003.
Reprint requests to: Dr. Lawrence L. Horstman, Wallace H Coulter Platelet Laboratory, University of Miami School of Medicine, 1600 NW
10th Ave, Box R-36A, Miami, FL 33136, telephone: (305) 243-6703, facsmile: (305) 234-5976, e-mail: yahn@med.miami.edu
210Keio J Med 2004; 53 (4): 210–230

211

cytometric and other methods were detailed in ourprevious review,1 and more recently for the case ofendothelial MP.2 A variety of less common but ingenious methods have also been devised, e.g. immunoelectrophoresis.7 Reliable ELISA methods have beendeveloped, particularly by S. Normua et al.,8,9 recently
extended to LMP,10 and by others.11 Flow cytometry isinherantly quantitative but ELISA is not, therefore
several authors quantitate by prothrombinase activityof the particles on the plate;12 however, this method is
dubious for reasons below and results cannot be directly compared to ﬂow cytometry measurements.
Measurement of total phosphate to establish phospholipid (PL) concentration has often been used,1,13 being
reliable but difﬁcult to compare with ﬂow cytometriccounts. A forum on methods by six leading laboratories
is currently in press in Journal of Thrombosis andHaemostasis (October issue).

Fig. 1 Modes and mechanisms of cell-derived microparticle (MP)release. A wide variety of disease states can stimulate release of MP
by mediators known to include antibodies, complement, cytokines,
hypoxia, viscose shear and toxins. The MP phenotype may reﬂect themode of insult, such as activation vs. apoptosis. Detailed mechanismsof vesiculation internal to the cell are incompletely understood.
Abbreviations: ACS acute coronary syndrome; ITP, TTP immune andthrombotic thrombocytopenic purpura; APLS antiphospholipid syndrome; SLE systemic lupus erythematosus; MS multiple slerosis.

In contrast to other circulating lipoprotein particles(HDL/LDL, chylomicrons, various others), electron
micrographic studies from many laboratories revealthat cell-derived MP are quite heterogeneous in density
and size, most of the larger ones being true vesicles(hollow with lamellar membrane) as opposed to onionlike. Many of these shapes and dynamics can be replicated in synthetic liposomes.6 We observed by differential centrifugation and electron microscopy that
EMP are also heterogeneous in density, ranging froma protein-rich fraction (electron-dense in osmium
staining) sedimenting at low-speed to a protein-poorer
fraction sedimenting at high-speed, and all fractions
contained heterogeneous sizes (unpublished). This heterogeneity is conﬁrmed in many published electron
micrographs of PMP.7 As discussed later, there may beoverlap between cell membrane-derived MP and otherMP such as exosomes released by exocytosis from multivesicular bodies (MVB).
How are they measured?
Introduction: PMP and other MP are measured by avariety of methods or combinations of them. Flow

Pitfalls: (1) Annexin V/prothrombinase. In this increasingly popular method,14–21 the primary means ofdetection or capture of MP depends on their binding
annexin V (AnV) in the presence of calcium; secondary
identiﬁcation of MP types (PMP, EMP, LMP) is by
ELISA of cell-speciﬁc antigens; and quantitation isoften by prothrombinase activity (PF3 activity). The
most serious objection to this method is that we havedemonstrated, at least for EMP, that only a fractionof all MP are positive for AnV binding.2 Speciﬁcally,
EMP from activated endothelial cells (EC) were rarely
positive by AnV binding, since 35-fold more werecounted by anti-CD62E in the same samples; and even
EMP from apoptotic cells, though much richer in AnV
binding, gave only half as many positives by AnV as
by CD31. Since AnV binding measures mainly theanionic PL, phosphatidyl serine (PS), which is largely
responsible for prothrombinase activity, that method ofquantitation will also give misleading results. In sum,
therefore, the criterion of AnV binding and quantitation by prothrombinase selectively measures only asubset of MP, potentially giving grossly misleading
results. Similar objections may apply to measurement of
PMP by this method. Likewise, in quantitating RBC
MP by acetyl cholinesterase activity,22 there is no
assurance that all RBC MP have this activity. (2) Centrifugation. Nearly all MP studies rely on preliminary
centrifugations to remove red cells and whole platelets,giving platelet-poor plasma (PPP) in which the MP aremeasured. However, the centrifugation conditions are
not standardized, potentially giving rise to grossly discordant results. For example, one study of MP in preeclampsia21 reported results very different from ﬁndings from our lab,23 probably because the former
authors prepared PPP by centrifuging 2 minutes at
13,000 g, which we ﬁnd sediments @80% of all MP

212detectable in ﬂow cytometry. On the other hand, noamount of centrifugation is sufﬁcient to sediment 100%of MP because, as noted above, some MP are no more
dense than plasma.
The challenge of measuring total MP is therefore
unmet. We have attempted lipophilic ﬂuorescent dyes
but found that they tend to form micelles, giving false
counts even in the absence of MP. More promising
has been the use of lectins such as FITC-labeled Ulex
europaeus since this gives by far the higest counts withpure cell-derived EMP and PMP; however, it may bindto other plasma constituants making it unsuitable forin vivo applications. Other lectins such as Bandeiraea
simplicifolia (a.k.a. isolectin B4, or Griffonia simplicifolia) may offer superior speciﬁcity for EMP but
have not been tried.
A promising alternative would be density gradientcentrifugation as used for separation of HDL/LDL
fractions but that is suitable only for research purposes, being time- and labor-intensive and requiring
expensive equipment. (3) Blood collection. Often
under-appreciated is the importance of blood collectionin MP studies. Platelet activation readily occurs ex vivo
with release of artifactual MP, and some anticoagulants
are known to affect antigens important for measurement, e.g. EDTA extracts calcium required for the
structural integrity of GP IIb/IIIa,24 making it invisible
to ﬂuorescent antibodies often used to identify PMP,
and induces activation as deﬁned by CD62P.25 Even
citrate, commonly preferred for platelet studies, differs
signiﬁcantly in effects on platelets between different
manufacturers and tubes.26 Ahnadi et al. recently compared effects of anticoagulants on platelet activation,ﬁnding signiﬁcant differences.27 Some workers place
the blood on ice in the belief that this will be protective,25 but in fact chilling activates platelets, causing MP
release and loss of GP 1b (or glycocalicin).1,28–30
Furthermore, since many or most platelet membraneglyocoproteins contain disulﬁde links which are functionally important, lessons learned from preservationof homocysteine in sample handling31,32 should apply
to platelet/PMP studies. The -SH/-S-S- redox state isnormally governed mainly by glutathione which, as
might be expected, is associated with homocysteine33
and presumably the thiol redox state of platelets aswell. Redox state of platelet GPIIb/IIIa has been
investigated.34 The thiol protease calpain appears critical to a variety of platelet functions including PMP
production,35,36 more recently concerning conversion
to inﬂammatory phenotype with production of PMP
and PAF.37 Anti-oxidants inhibit P-selectin expression38,39 and platelets normally possess an array ofanti-oxidants,40 but platelet/PMP redox state in relation
to various functions, such as tissue factor (TF) activity
and MP shedding, has not been much investigated.

Horstman LL, et al: Cell-derived microparticles

Need for standardization For reasons below, it islikely that MP analysis will soon enter the mainstreamof clinical testing. Therefore, in view of the profusion of
idiosyncratic methods now in use, few of which can bequantitatively compared to others, there is a pressing
need to hammer out reference standard methods. Flowcytometry is probably the reference method of choice
but agreement must be reached on instrument settings,
sample handling, and standard markers for basic quantitation, e.g. CD144 for EMP, CD42b for PMP, glycophorin for RBC MP, CD45 for LMP, each of deﬁned
target epitope (clone) and ﬂuoresence. There is, however, ample room for debate on preferred markers.
For example, GPIba (CD42b) is shed from platelets
during vesiculation41 but as a marker of PMP in ITP42
gives counts different from CD41 (unpublished). CD61
(GPIIIb, integrin b3) has been suggested as a superiormarker of platelets,43 as might be GP VI44 (CD49, collagen R), or certain lysosomal markers, e.g. CD68positive MP were studied in ITP45 or CD63. Since ﬂow
cytometry is unavailable in many laboratories, ELISA
methods referenced above are often used but improved
quantitation is needed, as on the model of quantitative titers now standard in measuring anticardiolipin
antibodies.46,47
Why measure them? Clinical applications
At the present time, the most solidly establishedfunction of MP is their procoagulant activity (PCA) as
reviewed1 but even this is not yet proven to be a critical
determinant of thrombosis, the evidence being largely
circumstantial (associational), and as noted above, not
all MP are procoagulant. A major purpose of this review is to indicate potentially vital functions of MP not
yet widely appreciated.
To date, the main clinical application of MP analysis
has been the simple correlation of their levels or quantities with various disease states, as amply referenced to19991 and still continuing.48 That is, they serve as general indicators of cell injury, stress, thrombosis, or inﬂammation. More speciﬁcally, dating from about 1990,
PMP levels have been viewed as a marker of plateletactivation.49
However, if this is their only signiﬁcance then one
may doubt the importance of measuring them sinceeasier methods are available, such as CD62P expression
on platelets,50–52 detection of actived GPIIb/IIIa by themAb PAC-1,53 free thromboxane,54–56 or circulating
markers of inﬂammation.57,58
On the other hand, recent developments to bereviewed sugest that MP assay can supply crucial andspeciﬁc information. In the ﬁrst place, it now appears
that many so-called soluble mediators/markers of inﬂammation are in reality MP-bound (next section). In

Keio J Med 2004; 53 (4): 210–230

the second place, we have observed that PMP levels
do not necessarily correlate with CD62P on platelets.
There may exist distinct subtypes (phenotypes) of PMP,
as has been shown for EMP.59,60 For example, we
observed clear correlation of PMP levels with absenceof bleeding in ITP when PMP were measured by
CD42b (GP Ib)42 but not when measured by CD41 (GPIIb) (unpublished).
For these and other reasons below, it is our working
hypothesis that PMP and other MP perform importantsignalling functions not yet appreciated, and that whensuch properties are better understood, assay of speciﬁcmarkers on PMP and other MP, rather than grossquantity alone, will enable improved diagnostics andinsights to underlying pathophysiologies.
Microparticles vs. soluble markers
As a ﬁnal but important introductory topic, it is
now clear that many circulating markers of inﬂammation regarded as soluble are in reality bound to MP,
at least in part. This was defended in some detail in ourrecent review.2 For example, PECAM-1 (CD31) is
widely accepted as a soluble marker61 but we haveroutinely used it to assay PMP and EMP by ﬂow
cytometry.23,62–64 Similarly, E-selectin is widely measured and regarded as a soluble marker of endothelial
stress65–68 but we routinely use it to identify endothelial MP (EMP),59,60 demonstrating that it is actuallyMP-bound, at least in part, because ﬂow cytometry
cannot detect true soluble molecules. ELISA methods
do not distinguish true soluble forms from MP-bound.
Furthermore, ﬁltration of plasma through 0.1 um often
eliminates some or nearly all of such agents from ﬂow
cytometric detection.
Similar considerations apply to many other markers
now regarded as soluble including ICAM-1, VCAM-1,
P-selectin, tissue factor (TF), von Willebrand factor(vWF; partly bound to EMP69 and PMP), thrombomodulin,70 and CD40L.71 It is well established that someof these do exist in true soluble form, usually due to
enzymatic cleavage from the membrane or by posttranslational editing,72 but it is equally well established,by our lab and others, that a signiﬁcant fraction, up to
80–90%, of some of them occur on cell-derived MP,
presumably with their transmembrane domains intact
and normally adjacent proteins present. (Fig. 2)
The practical importance of this lies in the fact that
release of true soluble species occurs by mechanisms
entirely different from membrane vesiculation, hence
reﬂect different pathophysiologies. Secondly, true soluble species often have properties functionally different
from their MP-bound forms (as we have shown forvWF69). In view of these considerations, it is expectedthat when the MP-bound markers are clearly dis-

213

Fig. 2 True soluble proteins arise from mechanisms distinct fromtheir MP-bound form. Immunologic methods such as ELISA will
not distinguish true soluble proteins from their MP-bound forms (suchas CD31 a.k.a. PECAM-1, or CD62E a.k.a. E-selectin), yet they reﬂect different pathophysiologies and may be functionally different.
Soluble forms most often arise from proteolysis such as by matrix
metalloproteases (MMP’s), or by alternative splicing without transmembrane domains; whereas MP-bound forms arise by membrane
vesiculation.

tinguished by independent measurement from the true
soluble species, much clearer relations will emerge between disease states and the marker in question. In
sum, what are now reported as soluble species are inmany cases actually reports of MP. Thus, to the extent
that such so-called soluble markers are recognized as
valuable clinical tools, MP analysis deserves at least
equal recognition.
New Horizons for PMPIntroduction
Two main developments since our 1999 review are
responsible for the new horizons in PMP analysis, (i)increasing recognition of platelets as mediators of inﬂammation and immunity, and (ii) discovery of plateletassociated tissue factor (TF). Table 1 roughly classiﬁes
some platelet-derived agents of interest, many of them
recently discovered, others long-known but with newly
recognized roles. This table is by no means comprehensive; others are listed in other sources4,73 or arecommon knowledge; see any textbook.
Although many of the agents on Table 1 have already been identiﬁed on PMP, others have not. However, insofar as PMP are fragments of the cell membrane, we adopt the working hypothesis that many if
not all platelet membrane-associated agents occur also
on PMP, until shown otherwise. In some cases there isreason to believe that PMP are the sole or main functional vectors of those agents.

214
Table 1

Horstman LL, et al: Cell-derived microparticles
The New Platelet: Some Agents of Recent Interest

I. Mediators of Inﬂammation/Immunity
In general73, 140, 141, 213, 214
RANTES, MIP-1, GRO-a,
164–166, 215
bTG, others
IL-1
160, 161, 163; on LMP 162
CD40, CD40L
175, 176; on PMP 71
VEGF
216
BAFF
184
Fractalkine
178
Fas (on MP)
217
PAF (on PMP)
142
CCR1, 3, 4
179
CXCR4
166, 182; PMP 181
CX3CR1 (& pertussis tox.)
178, 218
Receptor heat-shock protein
208
Thrombospondin
129
Platelet factor 4 (PF4)129b-thromboglobulin
129
sPLA2 (PMP as subtrate)
13, 172
Nitric oxide synthase219
MMP 1, 2, 9
referenced in 186
II. Coagulation/Clotting
Tissue factor (TF)
TF pathway inhibitor (TFPI)
b2GP1 (on PMP)
Thrombomodulin (on LMP)
von Willebrand factor (on EMP)
Factor V/Va
PAI-1

87, 88; on PMP 89–91, 220, 221;
generic or other MP 222, 223
224
94, 95, 98
70
60, 6976
225, 226

III. Pro- and Anti-Angiogenic (see text for more):
VEGF
216
Angiostatin
186
Endostatin
187
IV. Other Receptors
For estrogen, androgen
For leptin?
For shiga toxin?
For erythropoietin (EPO)
For LDL?
For insulin?
insulin R & NO synthase (NOS):
For vasopressin (& DDAVP?)b2 integrin(as in CD11a,b,c, CD18)
V. Miscellaneous
Anti-oxidants
Amyloid precursor protein (APP)
Sialyl Lewis x (CD15s)
JAM proteins
CD59 (C inhibitor)

227
228
229
82
230
219, 231
219, 231
169, 170, 232
233

40
234
235
236–238
239, 240

Where do all these agents come from? On the one
hand, platelets are notorious pack-rats, endocytosing
numerous plasma proteins; for example, IgG/IgM, evenvirus. Much debate has been given to the source ofplatelet tissue factor (TF), possibly from leukocytes;74
and on the source of platelet-derived coagulation factor

V (originally called platelet factor 1),75–78 which also
occurs on PMP.36,79,80
On the other hand, an unexpectedly large variety ofproteins are native to platelets and megakaryocytes,81
as recently underscored by mRNA studies in whichsome 232 proteins additional to those known wereidentiﬁed82 (TF not among them), unexpected because
platelets are so tiny and lack nucleii. Similarly, proteomic approaches to platelet signalling proteins83 andsecreted soluble proteins84 have yielded lengthy newlists. It would be most interesting to see the proteomic
approach applied to PMP and other MP.
PMP in coagulation and thrombosis
As previously reviewed, the best studied role of
PMP has been their procoagulant activity (PCA) by
virtue of providing a suitably anionic phospholipid surface for assembly of the tenase and prothrombinase
complexes, known as platelet factor 3 (PF3) activity.1,85
PMP also carry FV/Va (cited above) from platelets.86
However, these procoagulant properties are passive,
meaning that PMP could support but not themselves
initiate coagulation.
That view changed dramatically with the discoveryof platelet-associated tissue factor (TF).87,88 TF antigen and activity also occur on PMP in signiﬁcantamounts.89–91 It was previously shown that PMP playa special role in engendering TF activity of monocytes92,93 and other cells71 but the recent ﬁnding that
TF is intrinsic to platelets and PMP was a surprise inview of the long history of studies of platelets and TF.
(We observed TF activity of PMP but dismissed it as an
artifact, not suspecting that such a key property could
have been overlooked for so long.) Thus, PMP are
now known to be capable of initiating coagulation as
well as passively supporting it. These two procoagulant
activities of PMP, passive and active, are qualitatively
distinct.
Other modulators of coagulation or clotting havealso been identiﬁed on PMP, for example, b2GP-1,94,95
closely associated with anti-phospholipid antibodies(APLA) in APS.96,97 It exhibits calcium-dependent
membrane binding despite absence of gla domain.98
Theories purporting to explain the correlation between
anti-b2GP-1 with APS have been proposed in terms of
the protein C system;99–101 see next section. We haveshown that EMP carry signiﬁcant vWF,60 that PMP also
carry it, and that EMP-vWF is functionally more active
than free soluble (manuscript submitted).
PMP and the protein C anticoagulant system
As stated in our earlier review,1 Tans et al. proposedthat PMP could perform an anti-coagulant rather than

Keio J Med 2004; 53 (4): 210–230

PS

215

TAFI

also
uPA

PC
inhib.

C4BP

and
/or?
PS
MP

C5a,C3aprotein C
PC

APC

PF4

TM PS

Inactivates

Gla, Ca2+

Kd 0.5nM

thrombinß2GP-1

fibrinaTAFI

inhib.
TF

EPCR
EC (& other?)

FVa
Inact.

FVIIIaPAR-1(Neutrophil elastase can release TM to sTM.)
Fig. 3 Potential sites of interaction of cell-derived MP with the protein C anticoagulant system. This diagram, adapted from several sources,
illustrates the still-growing complexity of the protein C system, critically involved in many thrombotic pathologies, probably including antiphospholipid syndrome as well as normal hemostasis. Although not yet proved, there is evidence that MP may signiﬁcantly modulate several of
the interactions shown, including EMP as site of assembly of the central complex, as discussed in text. Abbreviatons: PC protein C; APC activated protein C; PS protein S; MP cell-derived microparticle; TM thrombomodulin; C4BP complement C4 binding protein; EPCR endothelial
protein C receptor.

procoagulant function in some circumstances by supporting the protein C system.102 More recently, a similar argument has been advanced by Berckmans et al. in
their discussion.103 Conﬁrming other reports, the latterﬁnd that circulating MP in normal controls support lowgrade thrombin generation, but by the contact pathway,
not TF (however, new insights on the contact pathway
raise doubts about its supposed function104). They
argue that low-levels of thrombin are almost entirely
bound to thrombomodulin (TM), leading to activationof protein C and thus inactivation of FVa and FVIIIa;
hence MP-dependent thrombin generation serves ananticoagulant function in normal hemostasis.103
A cartoon of the main players in the protein C/S
system are shown in Fig. 3, based on several sources,105
the intention being to indicate likely roles for MP in thissystem additional to that mentioned above, suspected
because most of the key steps, like those of coagulation,
occur at membrane surfaces. This system is obviously
extremely complex,106 well reviewed elsewhere107–109
and no attempt is made to describe it here. (TAFI inthe ﬁgure, thrombin activable ﬁbrinolysis inhibitor, is
now more properly called carboxypeptidase U.110)
Gris et al. observed that a substantial but variablefraction of protein S is MP-associated and that clinicalassays using PEG precipitation for free vs. C4bp-boundunderestimate it because much of it is precipitated by
PEG with MP.111 Incidentally, this suggests that PEG
might be exploited as a method of MP isolation. The

MP carrying the protein S was not identiﬁed. For recent review of protein S.112 Recently, much interest
has focused on the endothelial protein C receptor(EPCR).113 A true soluble form (sEPCR) can arise by
MMP proteolysis induced by inﬂammation, as citedin;114 but in a recent study,115 a thrombophilic genotype (A3 haplotype) with elevated sEPCR associatedwith thrombosis showed no evidence of lacking thetransmembrane domain, suggesting that the authors
actually measured MP-bound EPCR, probably on
EMP. Similarly, a true soluble form of thrombomodulin(TM) is released by neutrophil elastase,105 yet TM has
been detected on leukocyte MP;70 since endothelium isthe main site of TM, it may be expected also on EMP.
(Neutrophil elastase also acts directly on FV/Va.116)
Circulating TM was elevated in TTP in correlation with
vWF117 but we have recently found that vWF is in partassociated with EMP69 (manuscript submitted).
Another point of involvement of MP with the protein C system is via inhibition of activated protein C
by MP-associated tissue factor (TF),118 likely to also
involve TF pathway inhibitor (TFPI). Platelet factor 4(PF4) is another known participant involved in regulation of clotting through protein C119 and occurs on
PMP. As referenced above, b2GP-1 is rich on PMP and
is known to affect the protein C system, predisposing
to thrombosis and APS. It is difﬁcult, of course, to sortout the many inﬂuences on this system in vivo, owing
to its extreme complexity, only partly shown; for exam-

216

Horstman LL, et al: Cell-derived microparticles

Fig. 4 Potential roles of platelet microparticles (PMP) in angiogenesis. This ﬁgure, adapted from several sources (especially ref’s 186–188),
illustrates another of the ‘‘new horizons’’ of MP studies, since all the promotors and inhibitors of angiogensis shown on the inset table are foundon platelets, and several have been identiﬁed on PMP. It is likely that all membrane-asociated species on platelets also occur also on PMP(working hypothesis). See 3.3 of text. Abbreviations: MMP matrix metalloprotease (numbers 1, 2, 9 identiﬁed on platelets); TIMP tissue
inhibitors of MMP; TF tissue factor (disseminated on microparticles); TFPI is TF pathway inhibitor; tPA tissue-type plasminogen activator; TSP
thrombospondin; PF4 platelet factor 4; VEGF vascular endothelial growth factor; PDGF platelet derived growth factor; bFGF basic ﬁbroblast
growth factor; EGF endothelial growth factor; TGF transforming growth factor.

ple, not shown in Fig. 4 are the actions of PAI-1 andvitronectin.120
Nonetheless, in summary, there are numerous points
at which PMP or other cell-derived MP are known orplausibly suspected to affect the protein C system, either positively or negatively. Further study is clearlyneeded and would certainly be warranted in view of thepivotal importance of this system to hemostasis, thrombosis, and inﬂammation. For example, activated protein
C is the only therapy thus far shown to be effective insepsis;121–124 PMP in sepsis has been studied16,125–127
including by our lab128 (full manuscript submitted).
The New Platelet: Beyond Thrombosis
Introduction
The recent discovery of many additional bioactive
agents on/in platelets and PMP such as those listed
in Table 1 extend the likely role of PMP beyond
thrombosis.
It should be emphasized that not all agents known orsuspected to occur on PMP (or other MP) necessarily
occur on all PMP, as it is now emerging that cellderived MP can occur in multiple phenotypic species,that is, bearing certain restricted sets of proteins; andthat the distribution of phenotypes can vary with thestimulus eliciting their release. For example, Fox noted
different protein compositions on PMP elicted by dibucain vs. calcium ionophore A23187129 and we haveshown different phenotypic sets of EMP depending onwhether the endothelial cells (EC) were activated or
apoptotic.59 Likewise, it was remarked above that PMP
measured by two different markers (CD41, CD42) did
not both correlate with disease activity, implying distinctive subsets.
It is relevant to add that platelets themselvesappear to comprise multiple species. Examples otherthan those discussed in our prior PMP review1,130–133
include differencces in electrophoretic mobility;134 platelet age subpopulations by lectin afﬁnity135 (others
have used bouyancy); uptake of thiazole orange;136 anddistinct subpopulations in chronic myeloid leuekemia(CML)137 and other cancer hemostatic abnormalities.138 We have shown distinctive electrophoretic mobility of platelets from patients with recurring TIA.139
It may be expected that PMP would reﬂect such subpopulations in one or more differentiating markers orfunctional properties.
PMP in inﬂammation
Several roles of platelets in inﬂammation were wellreviewed as of 1994 under editorship of M. Joseph,much of it summarized in Ch. 2 of that book,140 but
were not widely appreciated until recently.73,141 We
suggest that many of those roles may actually beserved by PMP, to greater or lesser extent. Thumbnail
sketches of some of the mediating agents follows.
Platelet activating factor (PAF) is carried mainly on

Keio J Med 2004; 53 (4): 210–230

PMP142 and is known to modulate leukocyte adhesion
to endothelium,142 to contribute to endotoxin-induced
injury,143,144 organ transplant injury,145 and may be
involved in some thrombocytopenias.146 PAF can besigniﬁcant in picomolar amounts147 and may havespecial importance in stroke and brain injury.148 It is
responsible for the release of vWF in response to
DDAVP therapy; see below on vasopressin.
Platelet factor 4 (PF4) has been known since theearly days and has been shown to occur on PMP129 but
has received new attention lately because of a suspected role in atherosclerosis,149 perhaps through its
contribution to priming neutrophils via GM-CSF;150,151
and because it has been identiﬁed as a key antigen inheparin-induced thrombocytopenia (HIT) and HITwith thrombosis (HITT).52,152 For some updated references on PF4 and other inﬂammatory mediators fromplatelets, see Anitua.73 When secreted upon activationof platelets, it localizes to the plasma membrane, atleast in part,153 as do other granule proteins identiﬁed
on PMP (P selectin, thrombospondin,154,155 CD63,156
etc.), supporting our working assumption that many if
not all platelet membrane-associated molecules occur
also on PMP, until proven otherwise. Among many
putative roles, PF4 is involved in regulation of clotting
through protein C119 (Fig. 3).
Thrombospondin (TSP) also has long been known
on PMP129,157 and has diverse activities including
modulating vWF activity,158 complement activity (it copuriﬁes with factor H),159 and angiogenesis. Interleukin
1 (IL-1) has been known for some 15 years to be present in platelets160,161 and has been identiﬁed on monocyte MP,162 hence likely occurs also on PMP. mRNA
shows it is constitituve, not acquired from plasma.163
RANTES, a potent chemotactic factor for eosinophils from platelets,164 has been immuno-histochemically localized to the a-granules, to the open canalicularsystem (OCS), and to the plasma membrane, as was
MIP-1,165 leading to the expectation that they are alsopresent on PMP. Aside from their chemotaxis for eosinophils and basophils, both mediate release of histamine and thus are true inﬂammatory mediators. Threesuch mediators (PF4, RANTES, b-thromboglobulin)were shown to be slowly but signiﬁcantly released fromstored platelet concentrates and may contribute to nonhemolytic transfusion reactions;166 these are probably
on PMP, at least partly.
The platelet receptor(s) for vasopressin is of
special interest to us because we showed that DDAVP(desmopressin, a vasopressin analog), now widely used
as an anti-hemorrhagic drug, can act directly on platelet
to induce a rise in cytosolic calcium and release of
PMP,167 contraray to two prior reports.168,169 This
work was overlooked by two subsequent reviews of
DDAVP and by a recent paper on DDAVP action

217

on platelets,170 possibly because our results were notbelieved in view of the prior negative reports using
supposedly more sensitive calcium reporters. In fact,
the dye we used, CTC, is more sensitive, not less, in thevicinity of membranes such as dense tubules because it
is lipophilic.171 Although the effects shown in vitro
were modest, in vivo DDAVP could act synergisticallywith PAF released from leukocytes in response to
DDAVP, since this is believed to be the mechanism bywhich DDAVP elicits release of vWF from the vascularendothelium.167
Secretory phospholipase A2 (sPLA2), like Creactive protein (CRP), is an acute phase reactant
whose plasma levels increase up to 1000-fold in infections (inﬂammation). It is constitutively present inplatelets and probably on PMP since it has beenshown to act on PMP (but not on intact platelets) to
produce lysophosphatidic acid (LPA), of diverse activities including platelet aggregating, which may be promoted by other products of sPLA2 activity such asarachadonic acid.13 sPLA2 became tightly bound to
added albumin, degraded phosphatidyl serine (PS), andwas suggested to participate in ceramide production
in response to other inﬂammatory mediators. The
observed effects were augmented by sphingomyelinase,the beta toxin of S. aureus.13 More recently, Billy et al.,adopting the view that sPLA2 is mainly anticoagulant,emphasized the role of sPLA2 in degrading phosphatidyl serine (PS) to abolish the prothrombinase activityof MP; this happened unexpectedly rapidly, i.e. only asmall degree of PL membrane hydrolysis abolishedsupport of coagulation.172 In summary, the activities ofhuman sPLA2 (as distinct from that from bee or cobra
venoms), whether anti- or pro-thrombotic, are not yet
entirely clear but it does seem clear that PMP are likely
to be key participants in its actions.
Further stimulating interest in platelets/PMP as inﬂammatory/immune mediators are recent reports on
CD40/CD40L173–177 making it a hot topic; it has been
identiﬁed on PMP in vitro71 and in vivo, being anotherexample of a supposedly soluble molecule which is infact MP-bound, at least in part.
Chemokine receptor CX3 CR1 for fractalkine (andfor pertussis toxin) has been identiﬁed on platelets,178
as have other chemokine receptors.179,180 The mostabundant of these, CXCR4, was readily identiﬁed in
PMP by its bright signal and may play a role in cancer
metastasis181 and juvenile ITP.182 CXCR4 (and CD4)
can be transferred by PMP to other cells, rendering
them susceptible to HIV infection;181 related observations were earlier made for MP-mediated transfer of
CCR5.183
The recent identiﬁcation of the above, and BAFF,184
fractalkine178 and other such mediators (Table 1)
on platelets, underscores the role of platelets as218

Horstman LL, et al: Cell-derived microparticles

major inﬂammatory mediators. PMP are alreadyknown to carry several of these and may be their major
vectors, particularly in the microenvironment of thesite of inﬂammation where platelet activation occurs,with concommitant release of PMP at locally highconcentrations.
PMP in cancer: mediators of angiogenesis & metastasis
Multiple lines of evidence suggest that PMP play aunique and critical role in cancers. A recent ASH abstract reported that PMP (including platelet exosomes)
markedly stimulated metastatic potential of 5 cancer
cell lines, as judged by at least four measures, and related effects were conﬁrmed in vivo.185 Anitua et al.
classiﬁed many platelet-derived substances as pro- andanti-angiogenic.73 Our Fig. 4 summarizes these and related properties, based mainly on Jurasz et al.186 Of
notable interest is the presence of matrix metalloproteases (MMP) 1, 2 and 9 in platelets, which become surface-expressed in response to activation, alongwith their TIMP inhibitors, and angiostatin, releasedin tandem with plasmin by the action of tissue plasminogen activator (tPA) or MMP-9. Platelets also express
and release endostatin,187 said to be a fragment ofplasminogen.188 Platelet activation can be caused bythrombin (which itself possesses angiogenic potential189) or by tumor cells and results in PMP known to

B cell (Raposo 1996)
DC (Zitvogel 1998)

carry at least some of the angiogenic mediators listed,e.g. VEGF and TF;71 TF is also an angiogenic mediator.190 Not shown in the ﬁgure is thrombospondin, another of the many platelet-derived regulators of angiogenesis191 known to be vectored on PMP.
Consequently, if as proposed by Folkman and
Kalluri192 and similarly by Jurasz,186 the difference between common and harmless in situ tumors and theirprogression to lethality hinges on the balance between
pro- and anti-angiogenic factors in circulation, then MP
may well be pivotal in this balance insofar as they are
known vectors of several of these factors. MP from tumor cells have been shown to promote angiogenesis,193
presumably via VEGF and other MP-associated angiogenic factors.194 Oncogenes can be transferred via thatspecies of MP known as apoptotic bodies.195
Exosomes: platelets as antigen-presenting cells (APC)
An exciting recent development in immunology has
been the recognition that certain classes of microparticles, usually collectively called exosomes, perform
crucial antigen-presenting and other cross-talk functions. A cartoon conveying some concepts and key
experiments is shown in Fig. 5, based mainly on Wolfers
et al.196 and sources cited there,197,198 with helpfulcommentary on their intracellular sources, known as
multivesicular bodies (MVB)199 (pg 625). They are

T cellEXOSOMES(40-100nm)(Wolfers, 2001)
tumor
cell

MHCrestricted
responselysosome•CD63(LAMP-1)•CD82•CD11b•lactadherin•CD9... etc.

(multi-vesicularbodies, MVB)

Not released in apoptosis (induced by cisplatin)

specialized
endosome(MHC-I,

anti-tumor
activityDC
CTL

activation

Fig. 5 Exosomes as a variety of cell-derived MP. This ﬁgure sketches some functional activities of exosomes, which may overlap in both detection and function with plasma membrane-derived MP such as PMP. It is adapted from text of ref’s 196–198. Exosomes are expelled frommulti-vesicular bodies associated with a specialized endosome and function to present antigens. They are best characterized from B cells anddendritic cells but are now recognized also in platelets. See 3.4 of text. Abbreviations: DC dendritic cell; MHC major histocompatability complex; CTL cytotoxic lymphocyte; MVB multivesicular bodies.

Keio J Med 2004; 53 (4): 210–230

relevant here because it is now known that exosomes
are released from platelets as well as leukocytes,200,201
and therefore constitute a species of PMP.
Although said to be somewhat smaller at 40–80
nm201 or 60–90 nm196 than PMP normally detected
in ﬂow cytometry (@100 mm), there is some overlap.
For example, in one platelet study, the isolated size
fraction > 100 nm of putative exosomes exhibited thesame key marker CD81 (a.k.a. TAPA-1) as the smaller
high-speed fraction202 but that large size fraction was
not further investigated. Both fractions exhibited lysosomal marker CD63 (LIMP or LAMP-1), one of themarkers often used to identify endosomes but also awell-known surface marker of platelet activation foundon PMP.49,156,203–205 Perhaps relatedly, if lysosomal
markers are signatures of exosomes, Nomura et al.
found MP positive for CD68, a lysosomal marker, in
ITP at levels higher than controls, p < 0.01.95 In view ofthe size overlap, and since they derive from platelet
MVB or alpha-granules, the latter known to mirror theplatelet surface,155 it appears that at least some platelet
exosomes are detected along with PMP in normal ﬂow
cytometry; and likewise for exosomes from other cells
and their respective surface membrane MP since their
granule proteins, too, often mirror the identifying proteins of the plasma membrane.206 Indeed, direct evidence that PMP may function in this way has beenreported.207 If a research-grade ﬂow cytometer isavailable with high-power laser, it should be possible tospeciﬁcally detect and measure exosomes more easily
than by conventional tedious ultracentrifuge methodsof isolation.
The immunologic role of exosomes is probably related to other agents recently identiﬁed on platelets
such as the receptors for heat shock proteins (HSP),
now known to exert pleiotropic effects including activation of dendritic cells. Thus in the report by Hilf
et al.,208 the HSP called Gp96, which activates dendritic
cells (DC) to induce release of pro-inﬂammatory cytokines, is shown to bind to thrombin-activated platelets
10-fold increased relative to resting platelets, attenuating its effects, as in wounds or trauma, and reducing
potential autoimmune responses.
Such work has been widely extended, as in work on
the EAE mouse model of MS employing MBP as antigen,209 of special interest to our lab because of our longinterest in microparticles in MS, initially PMP210 but
more recently EMP.63,11,212
Possibly Related or Unidentiﬁed MP
One often encounters mention of microparticles(MP) in the literature without clear attribution of theirkind or source. For example, we earlier mentioned theﬁnding by Gris et al. of MP associated with protein S

219

but the MP remain to be identiﬁed. In this article wecite other such cases warranting further investigation,
particularly as they imply new functions of MP.
MP bearing mRNA
Circulating mRNA has been of interest in cancer research, but is highly unstable when free in plasma,
thereby raising the question of its persistence, answeredby ﬁnding it is protected against degradation by association with MP.241,242 It had earlier been shown thatmRNA on so-called apoptotic bodies is protected.243
The source and type of MP in question was not fully
addressed. This observation raises the possibility that ageneral function of MP may be to protect agents cariedon them against degradation. We have postulated thatvWF associated with EMP is thereby protected against
proteolytic degradation, maintaining it in active highmultimer form69 (full manuscript submitted).
Filtration affects von Willebrand factor (vWF ) assay
vWF was shown to be depleted in some patients as aresult of ﬁltration of the plasma, causing errors in clinical hematology tests.244 This suggests that vWF (andpossibly other factors affected in that reference) are
MP-associated, to a degree varying with individual
patients. We have directly demonstrated that vWF canoccur bound to EMP and in that form is partly ﬁlterable;69 and that signiﬁcant vWF associates also with
PMP (by electrophoresis and Western blot; unpublished). Of related interest, the same authors (Favaloro
et al.) have shown that storage of blood on ice can also
lead to errors in tests for vWD;245 insofar as cold isknown to induce platelet activation and PMP formation, one may conjecture that cold-generated PMP arebinding platelet vWF in some patients, explaining theerrors through loss of vWF with PMP in centrifugation.
Nucleosomes, etc
Jiang et al. noted an abundance of so-called nucleosomes in patients with SLE, and in that paper246
reports measuring them after administering necrotic or
apoptotic Jurkat cells to mice. This term may be equivalent to the poorly deﬁned MP called apoptotic bodies
released from apoptotic cells, but may be equivalent
to what we term EMP from apoptotic EC, which are
distinct from EMP released from activated EC.59 They
have been shown to be active in the horizontal transfer
of oncogenes.195 Incidentally, it has recently been
demonstrated that exposure of phosphatidyl serine(PS), which is particularly rich on MP from apoptotic
cells, is a true receptor for signalling phagocytosis,247
hence is involved with the clearance of MP exposing

220

Horstman LL, et al: Cell-derived microparticles

this phospholipid. However, as indicated in that survey,
the phagocytosis signal is complex, also involving suchas C1q of complement247 as well as LOX-1, b2GP1-R,
vitronectin R, and Mer receptor tyrosine kinase.247
Other examples abound, such as the transport of
LPS on an MP-like lipid binding protein to its receptor
CD14 and its regulation by TFPI.248 It may be noted,
by the way, that a cytosolic receptor for LPS is now
recognized, through work by G. Nonez and colleagues,
involving NOD1 and NOD2 (now called CARD-4 and
CARD-15), independent of the Toll-like receptors
recognizing extra-cellular ligands, all acting on NF-kB
genes; see perspective with references.249
High- and low-density lipoproteins (HDL, LDL)Best known for their cardiovascular risk (LDL) or
beneﬁt (HDL), these particles occur in a narrow rangeof small sizes (LDL 20–23 nm, HDL 7–13 nm250), alsodeﬁned by characteristic apolipoproteins, hence areunlikely to be confused with cell membrane-derived
MP. Nevertheless, they may be relevant in this context,
because of evidence that they can fuse with or otherwise directly act on platelets.230,251 LDL and HDL are
known to exhibit many activities, some of which arealso found on cell-derived MP, a few examples being
thrombin/AT III complexes,252 procoagulant activity,253 C reactive protein (CRP),254 PAF degrading
enzyme,255 TFPI (formerly called LACI, lipoproteinassociated coagulation inhibitor),256,257 and b2GP1.258
Some of these agents are evidently promiscuous (nonselective) with regard to the phospholipids to which
they bind, hence could associate with more than one
kind of circulating MP. For example, TFPI is heterogeneously distributed in lipoprotein fractions, not necessarily associated with apo-B or apo-A1257 and is foundalso in other plasma fractions and in platelets224 (Byanalogy, the coagulation proteins assemble to active
form with liposomes of many kinds and sources, not justplatelets or PMP or cephalin; e.g. we ﬁnd vegetable
lecithin to be as potent as brain cephalin in coagulation). In sum, HDL/LDL may be relevant to cellderived MP studies via interactions such as fusion andinﬂuence on cell-derived MP sheddding by lipid
exchanges altering the PL composition of lipid rafts
involved in MP shedding.259
Mechanisms of Microparticle Release
Background
Despite many efforts, detailed biophysical knowledge of the mechanisms of MP release remains elusive.
This article surveys some major paradigms now current

and other suggestive clues. Our previous review1 brieﬂysurveyed this topic as of then, in particular, work by
Fox et al., White et al., and especially by Sims, Shattil,
Wiedmer and colleagues in connection with their seriesof studies of PMP release by complement,260 where
they achieved only partial success on the mechanism
question, e.g. use of protein kinase inhibitors.261 However, a few principals have been repeatedly conﬁrmed,
notably the requirement for elevation of cytosolic calcium,36 either from internal stores or through theplasma membrane. Metabolic energy was not required.
Haynes et al. of this institution has well reviewed anddiagrammed calcium handling in platelets,262 althoughknowledge of the relevant calcium channels has since
improved.
The membrane ﬂip/ﬂop paradigm
This concept, due mainly to Zwaal et al. beginning in
the mid-1980s, holds that the process of MP shedding
relies on speciﬁc enzymes (ﬂoppase, scramblase263)
which induce the normally in-facing and more anionicphospholipids of the inner side of the membrane bilayer
to switch, ﬂip, or scramble to the outer (plasma) side;
this causes the platelet (or other cell) to become procoagulant, since anionic phospholipids such as PS are
essential to supporting coagulation, and is accompaniedby the sheddding of procoagulant MP.264,265 (It had
long been known that inside-out erythrocytes could
be prepared,266 and for that matter, mitochondria,267
the chief novel insight of Zwaal et al. being to draw aconnection between procoagulant activity and membrane sidedness; and the hypothesis that this was controlled by one or more enzymes.)
This was surveyed in our prior PMP review but
is reiterated here because of what we believe to be
unwarranted generalizations now commonly drawn
from that concept, speciﬁcally, that a deﬁning feature of
MP is exposure of PS. For example, as remarked earlier, many laboratories now deﬁne, measure, capture, orotherwise isolate MP on the basis of annexin V binding,which is quite speciﬁc for PS; but we ﬁnd that only afraction of MP are PS-positive, depending on theirmode of production, e.g. apoptosis vs. activation.2 The
review by J.M. Freyssinet is properly skeptical of claims
about certain enzymes (scramblase, ﬂoppase) inducing
the ﬂip-ﬂop and associated MP release,4 yet makes that
hypothesis a central organizing principle, as we havecriticized in a letter-to-the-editor.268 Other evidence
that platelet activation is not necessarily coupled tomembrane ﬂip-ﬂop is seen, for example, in work by Leo
et al., who found that knock-out mice lacking SLP-76
responded to agonists with 10-fold increased CD62P,
yet showed no increase in surface PS expression;44
unfortunately, they did not measure MP release.

Keio J Med 2004; 53 (4): 210–230

The lipid raft concept
It had been known for a long time that many if not
all proteins of the plasma membrane exhibit a high degree of lateral mobility,269 manifested in such phenomena as receptor clustering, dimerization, and capping ofleukocytes.270–272 Recently, the concept of lipid rafts
has emerged, in part to account for aspects of thismobility, being ﬂoating islands of distinctive lipid composition bearing restricted sets of proteins (such as
GPI-anchored), and are studied by their differential
solubility in detergents.273–280 Erythrocytes (RBC)
have been good models for study281 and their rafts help
us to understand earlier ﬁndings such as the selective
shedding of RBC MP enriched in acetyl cholinesterase.282 The latest ASH meeting included new ﬁndings
on rafts related to TF activity283 and platelet cytoskeleton.284 Some authors speaks of just one kind of raftbut others imply several. Sun et al. has sought to relatescramblase activity to lipid rafts.285
We have observed by ﬂuorescence microscopy theclustering (capping) of PECAM-1 as a prelude to theshedding of MP enriched in it,286 suggesting that speciﬁc kinds of rafts are preferentially shed as MP. This
would explain our ﬁnding of multiple phenotypes of
EMP,59 i.e. on the assumption that they reﬂect distinctive raft species. Millan et al. has studied rafts in theuropods of leukocytes,287 further hinting at a link between rafts and blebbing, vesiculation. Relatedly, we
have observed that the shedding of endothelial MP(EMP) in vivo is highly sensitive to the lipid proﬁle ofthe volunteer before and after a high-fat meal259 (inpress), consistent with the fact that the composition andstability of rafts are very sensitive to levels and compositions of particular plasma lipid particles and transfer
proteins. For example, Frenkel et al., following earlier
studies investigating release of erythrocyte microparticles (RBC MP) in response to incubation with liposomes of various compositions, which induce various
shape changes (such as to echinocytic), demonstrated
that concentration of cholesterol in the membrane is
key to vesicle shedding, and does not depend on
ATP.22 Thus, the now emerging elucidation of lipid
rafts promises better understanding of the shedding of
MP with distinctive antigen composition.
Liposome models and complement (C)Using artiﬁcal liposomes as models of MP, it has
been shown that those bearing negative or positive
charge bind and activate complement (C) leading tolysis by the classical and alternative pathways, respectively, while neutral liposomes have no effect.288 They
readily identiﬁed activated C on the liposomes. Suchstudies are important for application of liposomes as

221drug delivery systems but may have limited relevanceto natural MP since physiologic MP often or usually
bear agents which modulate the action of C, such as
sialyl groups known to bind the C inhibitor, factor H,288
and probably other more speciﬁc membrane-associatedinhibitors of C-mediated autologous lysis such as DAF
and CD59, which we brieﬂy studied;240 for authoritative review.289 On the other hand, C-mediated lysis of
opsonized platelets, whose study was pioneered by Sims
et al.1 and which we have also investigated,290 results inthe membrane attack complex (MAC) being largely
carried away with the resulting PMP so that the cellrecovers. C was readily detected on the resulting PMP(also EMP291), suggesting, by the way, that C-mediatedplatelet or endothelial injury, as may occur in somepatients with ITP, TTP, APS, etc., could be identiﬁedby measuing C-associated MP. Perhaps relatedly, Taylor et al., using a baboon model of thrombosis inducedby TNF and anti-protein C in the setting of partial stasisof the superﬁcial femoral vein, showed that infusionof phospholipid vesicles greatly ampliﬁed the thrombotic response,292 although for reasons above, thephysiologic relevance of artiﬁcial liposome infusion is
doubtful. We trust, however, that current liposomebased drug delivery systems are at last aware of thesepitfalls.293
Parting remarks
Although a detailed understanding of MP shedding
is not yet at hand, it is likely to be furthered by recent
advances in understanding cytoplasmic vesicle trafﬁc,
uropod formation,294 inter-cellular membrane fusions,
granule secretions and related events, including oftdrawn parallels between neurons and platelets; severalof these topics are reviewed by Reed et al.295 As longappreciated,129 all these events seem to involve thecytoskeleton (although the striking models of Baumgart
et al. show such properties without any protein6).
Summary & Conclusions
The central message of this review has been to indicate new horizons for research and clinical applicationsof PMP and other MP. Speciﬁcally, we envision a second generation of MP assays in which speciﬁc functional agents on MP rather than gross quantities of MP
constitute the analyte. This expected new generation of
assays will most likely focus on the putative immune
and inﬂammatory roles of MP, and will beneﬁt from therealization that many so-called soluble markers are in
reality MP-bound, at least in part.
However, the word putative should be emphasized
because it remains to be proved that MP are indeed
crucial carriers of such agents, whose quantitation could

222

Horstman LL, et al: Cell-derived microparticles

be of real clinical value. In other words, realization ofthis vision entails the considerable preliminary challenge of demonstrating exactly which of the many likely
functions outlined above are in fact important and are
directly reﬂected by quantitation of the MP carrying theagents in question.

16.

Acknowledgements: This work was supported by the Wallace H
Coulter Foundation and Funds from Mary Beth Weiss, Jane andCharles Bosco, and gifts from Frank Smather.

17.

15.

References18.
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. Nomura S: Function and clinical signiﬁcance of platelet-derived
microparticles. Int J Hematol 2001; 74: 397–404
4. Freyssinet JM: Cellular microparticles: what are they bad orgood for? J Thromb Haemost 2003; 1: 1655–1662
5. Miyazaki Y, Nomura S, Miyake T, Kagawa H, Kitada C, Taniguchi H, Komiyama Y, Fujimura Y, Ikeda Y, Fukuhara S: High
shear stress can initiate both platelet aggregation and sheddingof procoagulant containing microparticles. Blood 1996; 88:3456–3464
6. Baumgart T, Hess ST, Webb WW: Imaging coexisting ﬂuid
domains in biomembrane models coupling curvature and line
tension. Nature 2003; 425: 821–824
7. George JN, Thoi LL, McManus LM, Reimann TA: Isolation ofhuman platelet membrane microparticles from plasma and serum. Blood 1982; 60: 834–840
8. Kokawa T, Nomura S, Yanabu M, Yasunaga K: Detectionof platelet antigen for antiplatelet antibodies in idiopathic
thrombocytopenic purpura by ﬂow cytometry, antigen-capture
ELISA, and immunoblotting: a comparative study. Eur J
Haematol 1993; 50: 74–80
9. Osumi K, Ozeki Y, Saito S, Nagamura Y, Ito H, Kimura Y,
Ogura H, Nomura S: Development and assessment of enzyme
immunoassay for platelet-derived microparticles. Thromb Haemost 2001; 85: 326–330
10. Matsumoto N, Nomura S, Kamihata H, Kimura Y, Iwasaka T:
Increased level of oxidized LDL-dependent monocyte-derived
microparticles in acute coronary syndrome. Thromb Haemost
2004; 91: 146–154
11. Miyamoto S, Marcinkiewicz C, Edmunds LH Jr, Niewiarowski
S: Measurement of platelet microparticles during cardiopulmonary bypass by means of captured ELISA for GPIIb/IIIa.
Thromb Haemost 1998; 80: 225–230
12. 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 plaquethrombogenicity. Circulation 1999; 99: 348–353
13. Fourcade O, Simon MF, Viode C, Rugani N, Leballe F, Ragab
A, Fournie B, Sarda L, Chap H: Secretory phospholipase A2
generates the novel lipid mediator lysophosphatidic acid inmembrane microvesicles shed from activated cells. Cell 1995;
80: 919–927
14. 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

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

circulating blood of patients with acute coronary syndromes.
Circulation 2000; 101: 841–843
Aupeix K, Hugel B, Martin T, Bischoff P, Lill H, Pasquali JL,
Freyssinet JM: The signiﬁcance of shed membrane particles
during programmed cell death in vitro, and in vivo, in HIV-1
infection. J Clin Invest 1997; 99: 1546–1554
Joop K, Berckmans RJ, Nieuwland R, Berkhout J, Romijn FP,
Hack CE, Sturk A: Microparticles from patients with multiple
organ dysfunction syndrome and sepsis support coagulation
through multiple mechanisms. Thromb Haemost 2001; 85: 810–
820
VanWijk MJ, Boer K, Berckmans RJ, Meijers JC, van der Post
JA, Sturk A, VanBavel E, Nieuwland R: Enhanced coagulation
activation in preeclampsia: the role of APC resistance, microparticles and other plasma constituents. Thromb Haemost 2002;
88: 415–420
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: 450–456
Vanwijk MJ, Svedas E, Boer K, Nieuwland R, Vanbavel E,
Kublickiene KR: Isolated microparticles, but not whole plasma,from women with preeclampsia impair endothelium-dependent
relaxation in isolated myometrial arteries from healthy pregnant women. Am J Obstet Gynecol 2002; 187: 1686–1693
Sabatier F, Darmon P, Hugel B, Combes V, Sanmarco M, Velut
JG, Arnoux D, Charpiot P, Freyssinet JM, Oliver C, et al: Type
1 and type 2 diabetic patients display different patterns of cellular microparticles. Diabetes 2002; 51: 2840–2845
Bretelle F, Sabatier F, Desprez D, Camoin L, Grunebaum L,
Combes V, D’Ercole C, Dignat-George F: Circulating microparticles: a marker of procoagulant state in normal pregnancy
and pregnancy complicated by preeclampsia or intrauterine
growth restriction. Thromb Haemost 2003; 89: 486–492
Frenkel EJ, Kuypers FA, Op den Kamp JA, Roelofsen
B, Ott P: Effect of membrane cholesterol on dimyristoylphosphatidylcholine-induced vesiculation of human red
blood cells. Biochim Biophys Acta 1986; 855: 293–301
Gonzalez-Quintero VH, Jimenez JJ, Jy W, Mauro LM, Hortman L, O’Sullivan MJ, Ahn Y: Elevated plasma endothelial
microparticles in preeclampsia. Am J Obstet Gynecol 2003; 189:
589–593
Charo IF, Kieffer N, Phillips DR: Platelet membrane glycoproteins. In: Robert W. Colman et al. eds, Hemostasis andThrombosis: Basic Principles and Clinical Practice, 4th Ed, PA,
Philadelphia, Lippincott Williams & Wilkins, 2001; Ch. 22
Macey M, Azam U, McCarthy D, Webb L, Chapman ES,
Okrongly D, Zelmanovic D, Newland A: Evaluation of the
anticoagulants EDTA and citrate, theophylline, adenosine, anddipyridamole (CTAD) for assessing platelet activation on the
ADVIA 120 hematology system. Clin Chem 2002; 48: 891–899
Philippe J, De Logi E, Baele G: Comparison of ﬁve different
citrated tubes and their in vitro effects on platelet activation.
Clin Chem 2004; 50: 656–658
Ahnadi CE, Sabrinah Chapman E, Lepine M, Okrongly D,
Pujol-Moix N, Hernandez A, Boughrassa F, Grant AM:
Assessment of platelet activation in several different anticoagulants by the Advia 120 Hematology System, ﬂuorescence
ﬂow cytometry, and electron microscopy. Thromb Haemost
2003; 90: 940–948
Hoffmeister KM, Josefsson EC, Isaac NA, Clausen H, Hartwig
JH, Stossel TP: Glycosylation restores survival of chilled blood
platelets. Science 2003; 301: 1531–1534
Tablin F, Gousset K, Field CL, Crowe JH: Cold-induced platelet raft aggregation requires actin cytoskeletal reorganization,and is calcium independent. Blood 2002; 100(11): 63b #3723

Keio J Med 2004; 53 (4): 210–230
30. Gousset K, Tsvetkova NM, Crowe JH, Tablin F: Important roleof raft aggregation in the signaling events of cold-induced platelet activation. Biochim Biophys Acta 2004; 1660: 7–15
31. Bowron A, Stansbie D: Addition of 3-deazaadenosine to vacutainer tubes stabilizes whole-blood homocysteine for at least 6
hours at ambient temperature. Clin Chem 2003; 49: 835
32. Clark S, Youngman LD, Sullivan J, Peto R, Collins R: Stabilization of homocysteine in unseparated blood over several days:a solution for epidemiological studies. Clin Chem 2003; 49: 518–
520
33. Parodi O, Baudo F, Devoto E, Campolo J, Parodi G,
Parolini M, Caruso R, Brunelli C, Accinni R: Blood glutathione detects prooxidant patterns in patients with mild
hyperhomocysteinemia at risk for atherosclerotic vascular
events. Blood 2002; 100(11): 112b #3926
34. Walsh GM, Sheehan D, Kinsella A, Moran N, O’Neill S: Redox
modulation of integrin [correction of integin] alpha IIb beta 3
involves a novel allosteric regulation of its thiol isomerase
activity. Biochemistry 2004; 43: 473–480
35. Pasquet JM, Toti F, Nurden AT, Dachary-Prigent J: Procoagulant activity and active calpain in platelet-derived microparticles. Thromb Res 1996; 82: 509–522
36. Wiedmer T, Shattil SJ, Cunningham M, Sims PJ: Role of calcium and calpain in complement-induced vesiculation of theplatelet plasma membrane and in the exposure of the platelet
factor Va receptor. Biochemistry 1990; 29: 623–632
37. Jackson SP, Kanagasundaram V, Kulkarni S: Phenotypic conversion of platelets from a prothrombotic to a proinﬂammatory
adhesive state: involvement of the calcium-dependent thiol
protease, calpain. Blood 2003; 102(11): 66b #3968
38. Lindenblatt N, Schareck W, Belusa L, Maria Nickels R, Dieter
Menger M, Vollmar B: Anti-oxidant ebselen delays microvascular thrombus formation in the rat cremaster muscle byinhibiting platelet P-selectin expression. Thromb Haemost
2003; 90: 882–892
39. Xia L, Pan J, Yao L, McEver RP: A proteasome inhibitor, anantioxidant, or a salicylate, but not a glucocorticoid, blocks
constitutive and cytokine-inducible expression of P-selectin inhuman endothelial cells. Blood 1998; 91: 1625–1632
40. Del Principe D, Frega G, Palumbo G: Platelet-leukocyte interactions: platelets possess an antioxidant armamentarium.
Thromb Haemost 2003; 90: 759–760
41. Bode AP, Knupp CL: Platelet vesiculation and loss of GP Ib
during storage at 4 C. Thromb Haemost 1991; 65(6): 1072 #356
42. Jy W, Horstman LL, Arce M, Ahn YS: Clinical signiﬁcance ofplatelet microparticles in autoimmune thrombocytopenias. J
Lab Clin Med 1992; 119: 334–345
43. von Ahsen N, Ehrlich B, Scott CS, Riggert J, Oellerich M:
Cryoglobulins interfere with platelet counts by optical and impedance methods but not with the CD61 immunoplatelet count.
Clin Chem 2001; 47: 1858–1860
44. Leo L, Di Paola J, Judd BA, Koretzky GA, Lentz SR: Role of
the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen. Blood 2002; 100: 2839–2844
45. Nomura S, Yanabu M, Kido H, Lan XG, Ichiyoshi H, Katsura
K, Miyake T, Miyazaki Y, Kagawa H, Fukuhara S: Signiﬁcanceof cytokines and CD68-positive microparticles in immune
thrombocytopenic purpura. Eur J Haematol 1995; 55: 49–56
46. Harris EN, Pierangeli S, Birch D: Anticardiolipin wet workshop
report. Fifth International Symposium on antiphospholipid
antibodies. Am J Clin Pathol 1994; 101: 616–624
47. Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW,
Piette JC, Brey R, Derksen R, Harris EN, Hughes GR, et al:International consensus statement on preliminary classiﬁcation
criteria for deﬁnite antiphospholipid syndrome: report of aninternational workshop. Arthritis Rheum 1999; 42: 1309–1311

223
48.

49.

50.

51.
52.

53.

54.

55.

56.

57.
58.

59.

60.

61.

62.

63.

64.

Morel O, Jesel L, Chauvin M, Freyssinet JM, Toti F: Eptiﬁbatide-induced thrombocytopenia and circulating procoagulant
platelet-derived microparticles in a patient with acute coronary
syndrome. J Thromb Haemost 2003; 1: 2685–2687
Abrams CS, Ellison N, Budzynski AZ, Shattil SJ: Direct detection of activated platelets and platelet-derived microparticles inhumans. Blood 1990; 75: 128–138
Shattil SJ, Cunningham M, Hoxie JA: Detection of activatedplatelets in whole blood using activation-dependent monoclonal
antibodies and ﬂow cytometry. Blood 1987; 70: 307–315
Michelson AD: Flow cytometry: a clinical test of platelet function. Blood 1996; 87: 4925–4936
Jy W, Mao WW, Horstman LL, Valant PA, Ahn YS: A
ﬂow cytometric assay of platelet activation marker P-selectin(CD62P) distinguishes heparin-induced thrombocytopenia(HIT) from HIT with thrombosis (HITT). Thromb Haemost
1999; 82: 1255–1259
Schwarz M, Katagiri Y, Kotani M, Bassler N, Loefﬂer C,
Bode C, Peter K: Reversibility versus persistence of GPIIb/
IIIa blocker-induced conformational change of GPIIb/IIIa(alphaIIbbeta3, CD41/CD61). J Pharmacol Exp Ther 2004; 308:
1002–1011
Forastiero R, Martinuzzo M, Carreras LO, Maclouf J: Antibeta2 glycoprotein I antibodies and platelet activation inpatients with antiphospholipid antibodies: association withincreased excretion of platelet-derived thromboxane urinary
metabolites. Thromb Haemost 1998; 79: 42–45
Barry MC, Kelly CJ, Stokes K, Abdih H, Sheehan S, Burke P,
Bouchier Hayes D: Glyceryl trinitrate prevents neutrophil
activation but not thromboxane release following ischaemiareperfusion injury. Br J Surg 1996; 83: 1095–1100
Martinuzzo ME, Maclouf J, Carreras LO, Levy-Toledano S:
Antiphospholipid antibodies enhance thrombin-induced platelet activation and thromboxane formation. Thromb Haemost
1993; 70: 667–671
Van Lente F: Markers of inﬂammation as predictors in cardiovascular disease. Clin Chim Acta 2000; 293: 31–52
Schmitz G, Rothe G, Ruf A, Barlage S, Tschope D, Clemetson
KJ, Goodall AH, Michelson AD, Nurden AT, Shankey TV:
European Working Group on Clinical Cell Analysis: Consensus
protocol for the ﬂow cytometric characterisation of platelet
function. Thromb Haemost 1998; 79: 885–896
Jimenez JJ, Jy W, Mauro LM, Soderland C, Horstman LL, Ahn
YS: Endothelial cells release phenotypically and quantitatively
distinct microparticles in activation and apoptosis. Thromb Res
2003; 109: 175–180
Jimenez JJ, Jy W, Mauro LM, Horstman LL, Soderland C,
Ahn YS: Endothelial microparticles released in thrombotic
thrombocytopenic purpura express von Willebrand factor andmarkers of endothelial activation. Br J Haematol 2003; 123:
896–902
Losy J, Niezgoda A, Wender M: Increased serum levels ofsoluble PECAM-1 in multiple sclerosis patients with brain
gadolinium-enhancing lesions. J Neuroimmunol 1999; 99: 169–
172
Jimenez JJ, Jy W, Mauro LM, Horstman LL, Ahn YS: Elevatedendothelial microparticles in thrombotic thrombocytopenic
purpura: ﬁndings from brain and renal microvascular cell culture and patients with active disease. Br J Haematol 2001; 112:
81–90
Minagar A, Jy W, Jimenez JJ, Sheremata WA, Mauro LM, Mao
WW, Horstman LL, Ahn YS: Elevated plasma endothelial
microparticles in multiple sclerosis. Neurology 2001; 56: 1319–
1324
Bernal-Mizrachi L, Jy W, Jimenez JJ, Pastor J, Mauro LM,
Horstman LL, de Marchena E, Ahn YS: High levels of circu-

224

65.

66.

67.

68.

69.

70.

71.

72.
73.

74.

75.

76.
77.

78.

79.

80.

81.

Horstman LL, et al: Cell-derived microparticles
lating endothelial microparticles in patients with acute coronary
syndromes. Am Heart J 2003; 145: 962–970
Roldan V, Marin F, Lip GY, Blann AD: Soluble E-selectin in
cardiovascular disease and its risk factors. A review of the literature. Thromb Haemost 2003; 90: 1007–1020
Semaan HB, Gurbel PA, Anderson JL, Muhlestein JB, Carlquist JF, Horne BD, Serebruany VL: Soluble VCAM-1 andE-selectin, but not ICAM-1 discriminate endothelial injury inpatients with documented coronary artery disease. Cardiology
2000; 93: 7–10
Hwang SJ, Ballantyne CM, Sharrett AR, Smith LC, Davis CE,
Gotto AM Jr, Boerwinkle E: Circulating adhesion molecules
VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis andincident coronary heart disease cases: the Atherosclerosis Risk
In Communities (ARIC) study. Circulation 1997; 96: 4219–4225
Lieuw-a-Fa M, Schalkwijk C, van Hinsbergh VW: Distinct accumulation patterns of soluble forms of E-selectin, VCAM-1
and ICAM-1 upon infusion of TNFalpha in tumor patients.
Thromb Haemost 2003; 89: 1052–1057
Jy W, Jimenez JJ, Mauro LM, Horstman LL, Bidot CJ, Tiede
MP, Ahn E, Ahn YS: Endothelial microparticles (EMP) interact with platelets via a vWF dependent pathway to form platelet aggregates, more resistent to dissociation than those induced
by soluble vWF. Blood 2003; 102(11): 783a #2896
Satta N, Freyssinet JM, Toti F: The signiﬁcance of humanmonocyte thrombomodulin during membrane vesiculation andafter stimulation by lipopolysaccharide. Br J Haematol 1997;
96: 534–542
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 liganddependent manner: Possible role in angiogenesis and metastasis. Blood 2002; 100(11): 63b #3721
Heaney ML, Golde DW: Soluble cytokine receptors. Blood
1996; 87: 847–857
Anitua E, Andia I, Ardanza B, Nurden P, Nurden AT: Autologous platelets as a source of proteins for healing and tissue
regeneration. Thromb Haemost 2004; 91: 4–15
Rauch U, Bonderman D, Bohrmann B, Badimon JJ, Himber J,
Riederer MA, Nemerson Y: Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor.
Blood 2000; 96: 170–175
Camire RM, Pollak ES, Kaushansky K, Tracy PB: Secretable
human platelet-derived factor V originates from the plasmapool. Blood 1998; 92: 3035–3041
Colman RW: Where does platelet factor V originate? Blood
1999; 93: 3152–3153
Sun H, Yang TL, Yang A, Wang X, Ginsburg D: The murine
platelet and plasma factor V pools are biosynthetically distinct
and sufﬁcient for minimal hemostasis. Blood 2003; 102: 2856–
2861
Yang TL, Pipe SW, Yang A, Ginsburg D: Biosynthetic origin
and functional signiﬁcance of murine platelet factor V. Blood
2003; 102: 2851–2855
Bode AP, Sandberg H, Dombrose FA, Lentz BR: Associationof factor V activity with membranous vesicles released fromhuman platelets: requirement for platelet stimulation. Thromb
Res 1985; 39: 49–61
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
Veljkovic DK, Cramer EM, Alimardani G, Fichelson S, Masse
JM, Hayward CP: Studies of alpha-granule proteins in cultured
human megakaryocytes. Thromb Haemost 2003; 90: 844–852

82. Fink L, Holschermann H, Kwapiszewska G, Muyal JP, Lengemann B, Bohle RM, Santoso S: Characterization of plateletspeciﬁc mRNA by real-time PCR after laser-assisted microdissection. Thromb Haemost 2003; 90: 749–756
83. Garcia A, Prabhakar S, Hughan S, Anderson TW, Brock CJ,
Pearce AC, Dwek RA, Watson SP, Hebestreit HF, Zitzmann
N: Differential proteome analysis of TRAP-activated platelets:
involvement of DOK-2 and phosphorylation of RGS proteins.
Blood 2004; 103: 2088–2095
84. Coppinger JA, Cagney G, Toomey S, Kislinger T, Belton O,
McRedmond JP, Cahill DJ, Emili A, Fitzgerald DJ, Maguire
PB: Characterization of the proteins released from activatedplatelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004; 103: 2096–2104
85. Jy W, Horstman LL, Wang F, Duncan RC, Ahn YS: Platelet
factor 3 in plasma fractions: its relation to microparticle size andthromboses. Thromb Res 1995; 80: 471–482
86. Rand MD, Kalafatis M, Mann KG: Platelet coagulation factor
Va: the major secretory platelet phosphoprotein. Blood 1994;
83: 2180–2190
87. Zillmann A, Luther T, Muller I, Kotzsch M, Spannagl M,
Kauke T, Oelschlagel U, Zahler S, Engelmann B: Plateletassociated tissue factor contributes to the collagen-triggered
activation of blood coagulation. Biochem Biophys Res Commun 2001; 281: 603–609
88. Goodall AH, Jones CI, Lamb DJ, Tull SP, Payne DA, Appleby
JA: Blood borne tissue factor is present on platelets in the absence of leukocyte microparticles. Blood 2002; 100(11): 56a#200
89. Siddiqui FA, Desai H, Amirkhosravi A, Siddiqui TF, Meyer T,
Amaya M, Francis JL: Platelet microparticles from healthy
donors contain tissue factor. Blood 2002; 100(11): 497a #1936
90. Muller I, Klocke A, Alex M, Kotzsch M, Luther T, Morgenstern E, Zieseniss S, Zahler S, Preissner K, Engelmann B:
Intravascular tissue factor initiates coagulation via circulating
microvesicles and platelets. FASEB J 2003; 17: 476–478
91. Scholz T, Temmler U, Krause S, Heptinstall S, Losche W:
Transfer of tissue factor from platelets to monocytes: role ofplatelet-derived microvesicles and CD62P. Thromb Haemost
2002; 88: 1033–1038
92. Amirkhosravi A, Alexander M, May K, Francis DA, Warnes G,
Biggerstaff J, Francis JL: The importance of platelets in the expression of monocyte tissue factor antigen measured by a newwhole blood ﬂow cytometric assay. Thromb Haemost 1996; 75:
87–95
93. Osterud B: The role of platelets in decrypting monocyte tissue
factor. Semin Hematol 2001; 38: 2–5
94. Nomura S, Komiyama Y, Matsuura E, Kokawa T, Takahashi H,
Koike T: Binding of beta 2-glycoprotein I to platelet-derived
microparticles. Br J Haematol 1993; 85: 639–640
95. Nomura S, Yanabu M, Miyake T, Miyazaki Y, Kido H, Kagawa
H, Fukuhara S, Komiyama Y, Matsuura E, Koike T: Relationship of microparticles with beta 2-glycoprotein I and P-selectin
positivity to anticardiolipin antibodies in immune thrombocytopenic purpura. Ann Hematol 1995; 70: 25–30
96. Roubey RA, Pratt CW, Buyon JP, Winﬁeld JB: Lupus anticoagulant activity of autoimmune antiphospholipid antibodies
is dependent upon beta 2-glycoprotein I. J Clin Invest 1992; 90:
1100–1104
97. Navarro M, Cervera R, Teixido M, Reverter JC, Font J, LopezSoto A, Monteagudo J, Escolar G, Ingelmo M: Antibodies toendothelial cells and to beta 2-glycoprotein I in the antiphospholipid syndrome: prevalence and isotype distribution. Br
J Rheumatol 1996; 35: 523–528
98. Vallar L, Regnault V, Latger-Cannard V, Lecompte T: Beta2glycoprotein I binding to platelet microparticle membrane spe-

Keio J Med 2004; 53 (4): 210–230

99.

100.

101.

102.

103.

104.

105.
106.
107.
108.

109.

110.

111.

112.

113.
114.

115.

116.

ciﬁcally reduces immunoreactivity of glycoproteins IIb/IIIa.
Thromb Haemost 2001; 85: 314–319
Salemink I, Blezer R, Willems GM, Galli M, Bevers E, Lindhout T: Antibodies to beta2-glycoprotein I associated with
antiphospholipid syndrome suppress the inhibitory activity oftissue factor pathway inhibitor. Thromb Haemost 2000; 84:
653–656
Merrill JT, Zhang HW, Shen C, Butman BT, Jeffries EP, Lahita
RG, Myones BL: Enhancement of protein S anticoagulant
function by beta2-glycoprotein I, a major target antigen of
antiphospholipid antibodies: beta2-glycoprotein I interfereswith binding of protein S to its plasma inhibitor, C4b-binding
protein. Thromb Haemost 1999; 81: 748–757
Mori T, Takeya H, Nishioka J, Gabazza EC, Suzuki K: beta 2Glycoprotein I modulates the anticoagulant activity of activatedprotein C on the phospholipid surface. Thromb Haemost 1996;
75: 49–55
Tans G, Rosing J, Thomassen MC, Heeb MJ, Zwaal RF, Grifﬁn
JH: Comparison of anticoagulant and procoagulant activities ofstimulated platelets and platelet-derived microparticles. Blood
1991; 77: 2641–2648
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
Schmaier AH: The physiologic basis of assembly and activationof the plasma kallikrein/kinin system. Thromb Haemost 2004;
91: 1–3
Weiler H, Isermann BH: Thrombomodulin. J Thromb Haemost
2003; 1: 1515–1524
Marlar RA: The protein C system – how complex is it? Thromb
Haemost 2001; 85: 756–757
Esmon CT: The protein C anticoagulant pathway. Arterioscler
Thromb 1992; 12: 135–145
Esmon CT: Protein C anticoagulant pathway and its role incontrolling microvascular thrombosis and inﬂammation. Crit
Care Med 2001; 29: S48–51
Dahlback B, Villoutreix BO: Molecular recognition in the protein C anticoagulant pathway. J Thromb Haemost 2003; 1:
1525–1534
Leurs J, Nerme V, Sim Y, Hendriks D: Carboxypeptidase U(TAFIa) prevents lysis from proceeding into the propagation
phase through a threshold-dependent mechanism. J Thromb
Haemost 2004; 2: 416–423
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 humanimmunodeﬁciency virus infection. Thromb Haemost 1996; 76:
38–45
Rezende SM, Simmonds RE, Lane DA: Coagulation, inﬂammation, and apoptosis: different roles for protein S and the protein
S-C4b binding protein complex. Blood 2004; 103: 1192–1201
Esmon CT: The endothelial cell protein C receptor. Thromb
Haemost 2000; 83: 639–643
Stearns-Kurosawa DJ, Swindle K, D’Angelo A, Della Valle P,
Fattorini A, Caron N, Grimaux M, Woodhams B, Kurosawa S:
Plasma levels of endothelial protein C receptor respond to
anticoagulant treatment. Blood 2002; 99: 526–530
Saposnik B, Reny JL, Gaussem P, Emmerich J, Aiach M, Gandrille S: A haplotype of the EPCR gene is associated withincreased plasma levels of sEPCR and is a candidate risk factor
for thrombosis. Blood 2004; 103: 1311–1318
Samis JA, Garrett M, Manuel RP, Nesheim ME, Giles AR:
Human neutrophil elastase activates human factor V but inactivates thrombin-activated human factor V. Blood 1997; 90:1065–1074

225
117.

118.

119.

120.

121.

122.
123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

Takahashi H, Hanano M, Wada K, Tatewaki W, Niwano H,
Tsubouchi J, Nakano M, Nakamura T, Shibata A: Circulating
thrombomodulin in thrombotic thrombocytopenic purpura. Am
J Hematol 1991; 38: 174–177
Varadi K, Siekmann J, Turecek PL, Schwarz HP, Marder VJ:
Phospholipid-bound tissue factor modulates both thrombin
generation and APC-mediated factor Va inactivation. Thromb
Haemost 1999; 82: 1673–1679
Dudek AZ, Pennell CA, Key NS, Slungaard A: Platelet factor 4(PF4) binds to both the GAG domain of thrombomodulin and
the GLA domain of protein C: potential role in the regulationof clotting (abstract). J Invest Med 1996; 44(3): 213A
Rezaie AR: Vitronectin functions as a cofactor for rapid inhibition of activated protein C by plasminogen activator inhibitor-1. Implications for the mechanism of proﬁbrinolytic actionof activated protein C. J Biol Chem 2001; 276: 15567–15570
Joyce DE, Grinnell BW: Recombinant human activated protein
C attenuates the inﬂammatory response in endothelium andmonocytes by modulating nuclear factor-kappaB. Crit Care
Med 2002; 30: S288–293
Hotchkiss RS, Karl IE: The pathophysiology and treatment ofsepsis. N Engl J Med 2003; 348: 138–150
Taylor FB Jr, Stearns-Kurosawa DJ, Kurosawa S, Ferrell G,
Chang AC, Laszik Z, Kosanke S, Peer G, Esmon CT: The
endothelial cell protein C receptor aids in host defense against
Escherichia coli sepsis. Blood 2000; 95: 1680–1686
Dhainaut JF, Yan SB, Margolis BD, Lorente JA, Russell JA,
Freebairn RC, Spapen HD, Riess H, Basson B, Johnson G 3rd,et al; PROWESS Sepsis Study Group: Drotrecogin alfa (activated) (recombinant human activated protein C) reduces hostcoagulopathy response in patients with severe sepsis. Thromb
Haemost 2003; 90: 642–653
Ogura H, Kawasaki T, Tanaka H, Koh T, Tanaka R, Ozeki
Y, Hosotsubo H, Kuwagata Y, Shimazu T, Sugimoto H: Activated platelets enhance microparticle formation and plateletleukocyte interaction in severe trauma and sepsis. J Trauma
2001; 50: 801–809
Ogura H, Kawasaki T, Tanaka H, Koh T, Tanaka R, Ozeki
Y, Hosotsubo H, Kuwagata Y, Shimazu T, Sugimoto H: Activated platelets enhance microparticle formation and plateletleukocyte interaction in severe trauma and sepsis. J Trauma
2001; 50: 801–809
Nieuwland R, Berckmans RJ, McGregor S, Boing AN, Romijn
FP, Westendorp RG, Hack CE, Sturk A: Cellular origin andprocoagulant properties of microparticles in meningococcal
sepsis. Blood 2000; 95: 930–935
Soriano AO, Jy W, Valdivia MA, Velasquez HS, Chirinos J,
Tiede MP, Ahn E, Kett D, Horstman LL, Jimenez JJ, Ahn YS:
Reduced endothelial and platelet microparticles and their interaction with leukocytes correlate with organ dysfunction andpredict mortality in severe sepsis. Blood 2003; 102: 58b #3934
Fox JE, Austin CD, Boyles JK, Steffen PK: Role of the membrane skeleton in preventing the shedding of procoagulant-richmicrovesicles from the platelet plasma membrane. J Cell Biol
1990; 111: 483–493
Behnke O, Forer A: Blood platelet heterogeneity: evidence fortwo classes of platelets in man and rat. Br J Haematol 1993; 84:
686–693
Kunicki TJ, Orchekowski R, Annis D, Honda Y: Variability ofintegrin alpha 2 beta 1 activity on human platelets. Blood 1993;
82: 2693–2703
Johnston GI, Pickett EB, McEver RP, George JN: Heterogeneity of platelet secretion in response to thrombin demonstrated by ﬂuorescence ﬂow cytometry. Blood 1987; 69: 1401–
1403
Frojmovic M, Wong T: Dynamic measurements of the platelet

226

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.
149.

150.

Horstman LL, et al: Cell-derived microparticles
membrane glycoprotein IIb-IIIa receptor for ﬁbrinogen by ﬂow
cytometry. II. Platelet size-dependent subpopulations. Biophys
J 1991; 59: 828–837
Jung SM, Tanoue K, Yamazaki H: The electrophoretic mobility
heterogeneity of human platelet subpopulations of different
buoyant densities. Thromb Haemost 1985; 53: 188–194
Dhar A, Ganguly P: Lectin fractionation of rabbit blood platelets: isolation of subpopulations of different ages. Prog Clin Biol
Res 1988; 283: 77–81
Kienast J, Schmitz G: Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation ofthrombocytopenic disorders. Blood 1990; 75: 116–121
Michelson AD: Flow cytometric detection of distinct subpopulations of platelets in individual patients. Prog Clin Biol
Res 1988; 283: 131–136
Holmes CE, Bouchard BA, Barbick RR, Safdar H, Tracy PB:
The existence of platelet subpopulations in patients withmyeloproliferative disease: preliminary correlation with clinically abnormal hemostasis. Blood 2003; 102(11): 787a #2909
Jy W, Horstman LL, Homolak D, Ahn YS: Electrophoretic
properties of platelets from normal, thrombotic and ITP
patients by doppler electrophoretic light scattering analysis.
Platelets 1995; 6: 354–58
Herd CM, Page CP: Do platelets have a role as inﬂammatory
cells? In: Joseph M, ed, Immunopharmacology of Platelets,
London/New York, Academic Press, 1995: 1–12
Weyrich AS, Lindemann S, Zimmerman GA: The evolving roleof platelets in inﬂammation. J Thromb Haemost 2003; 1: 1897–
1905
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
Murakami K, Okajima K, Uchiba M, Johno M, Okabe
H, Takatsuki K: A novel platelet activating factor antagonist,
SM-12502, attenuates endotoxin-induced disseminated intravascular coagulation and acute pulmonary vascular injury byinhibiting TNF production in rats. Thromb Haemost 1996; 75:
965–970
Siebeck M, Weipert J, Keser C, Kohl J, Spannagl M, Machleidt
W, Schweiberer L: A triazolodiazepine platelet activating factor
receptor antagonist (WEB 2086) reduces pulmonary dysfunction during endotoxin shock in swine. J Trauma 1991; 31: 942–
949
Qayumi AK, Jamieson WR, Poostizadeh A: Effects of plateletactivating factor antagonist CV-3988 in preservation of heart
and lung for transplantation. Ann Thorac Surg 1991; 52: 1026–
1032
Meade CJ, Heuer H, Kempe R: Biochemical pharmacology ofplatelet-activating factor (and PAF antagonists) in relation toclinical and experimental thrombocytopenia. Biochem Pharmacol 1991; 41: 657–668
James-Kracke MR, Sexe RB, Shukla SD: Picomolar plateletactivating factor mobilizes Ca to change platelet shape without
activating phospholipase C or protein kinase C; simultaneous
ﬂuorometric measurement of intracellular free Ca concentration and aggregation. J Pharmacol Exp Ther 1994; 271: 824–831
Lindsberg PJ, Hallenbeck JM, Feuerstein G: Platelet-activating
factor in stroke and brain injury. Ann Neurol 1991; 30: 117–129
Pitsilos S, Hunt J, Mohler ER, Prabhakar AM, Poncz M,
Dawicki J, Khalapyan TZ, Wolfe ML, Fairman R, Mitchell M,
Carpenter J, Golden MA, Cines DB, Sachais BS: Platelet factor
4 localization in carotid atherosclerotic plaques: correlation
with clinical parameters. Thromb Haemost 2003; 90: 1112–1120
Aziz KA, Cawley JC, Zuzel M: Platelets prime PMN via
released PF4: mechanism of priming and synergy with GMCSF. Br J Haematol 1995; 91: 846–853

151. Pabst MJ: Priming of neutrophils, In: Hellwell PG, ed, Immunopharmacology of neutrophils, Williams TJ, London/New
York, Academic Press, 1994: 195–221
152. Visentin GP, Ford SE, Scott JP, Aster RH: Antibodies frompatients with heparin-induced thrombocytopenia/thrombosis
are speciﬁc for platelet factor 4 complexed with heparin orbound to endothelial cells. J Clin Invest 1994; 93: 81–88
153. Capitanio AM, Niewiarowski S, Rucinski B, Tuszynski GP,
Cierniewski CS, Hershock D, Kornecki E: Interaction of platelet factor 4 with human platelets. Biochim Biophys Acta 1985;
839: 161–173
154. McGregor JL: The role of human platelet membrane receptors
in inﬂamation [Ch 4]. In: Joseph M, ed. Immunopharmacologyof Platelets. London/New York: Academic Press, 1995: 66–82
155. Berger G, Masse JM, Cramer EM: Alpha-granule membrane
mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX, and V. Blood 1996; 87: 1385–1395
156. Israels SJ, McMillan-Ward EM, Easton J, Robertson C, McNicol A: CD63 associates with the alphaIIb beta3 integrin-CD9complex on the surface of activated platelets. Thromb Haemost
2001; 85: 134–141
157. White JG: Platelet membrane ultrastructure and its changes
during platelet activation. In: Jamieson GA, ed. Platelet Membrane Receptors: Molecular Biology, Immunology, Biochemistry and Pathology. NY: Alan R. Liss Inc, 1988: 1–32
158. Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman
CN, Hogg PJ: The von Willebrand factor-reducing activity ofthrombospondin-1 is located in the calcium-binding/C-terminal
sequence and requires a free thiol at position 974. Blood 2002;
100: 2832–2838
159. Carron JA, Bates RC, Smith AI, Tetoz T, Arellano A, Gordon
DL, Burns GF: Factor H co-puriﬁes with thrombospondin isolated from platelet secretate. Biochim Biophys Acta 1996; 1289:
305–311160. Hawrylowicz CM, Santoro SA, Platt FM, Unanue ER: Activated platelets express IL-1 activity. J Immunol 1989; 143:
4015–4018
161. Hawrylowicz CM, Howells GL, Feldmann M: Platelet-derived
interleukin 1 induces human endothelial adhesion molecule expression and cytokine production. J Exp Med 1991; 174: 785–790
162. MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA,
Surprenant A: Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 2001; 15: 825–835
163. Loppnow H, Bil R, Hirt S, Schonbeck U, Herzberg M, Werdan
K, Rietschel ET, Brandt E, Flad HD: Platelet-derived interleukin-1 induces cytokine production, but not proliferation ofhuman vascular smooth muscle cells. Blood 1998; 91: 134–141
164. Kameyoshi Y, Schroder JM, Christophers E, Yamamoto S:
Identiﬁcation of the cytokine RANTES released from platelets
as an eosinophil chemotactic factor. Int Arch Allergy Immunol
1994; 104: 49–51
165. Klinger MH, Wilhelm D, Bubel S, Sticherling M, Schroder JM,
Kuhnel W: Immunocytochemical localization of the chemokines RANTES and MIP-1 alpha within human platelets andtheir release during storage. Int Arch Allergy Immunol 1995;
107: 541–546
166. Bubel S, Wilhelm D, Entelmann M, Kirchner H, Kluter H:
Chemokines in stored platelet concentrates. Transfusion 1996;
36: 445–449
167. Horstman LL, Valle-Riestra BJ, Jy W, Wang F, Mao W, Ahn
YS: Desmopressin (DDAVP) acts on platelets to generate
platelet microparticles and enhanced procoagulant activity.
Thromb Res 1995; 79: 163–174
168. Ghosh S, Rao AK: Lack of effect of 1-desamino-8-D-arginine
vasopressin on direct adhesion of platelets to collagen. Thromb
Res 1993; 70: 417–422

Keio J Med 2004; 53 (4): 210–230
169. Yang X, Disa J, Rao AK: Effect of 1-desamino-8-D-arginine
vasopressin (DDAVP) on human platelets. Thromb Res 1990;
59: 809–818
170. Reiter RA, Mayr F, Blazicek H, Galehr E, Jilma-Stohlawetz P,
Domanovits H, Jilma B: Desmopressin antagonizes the in vitro
platelet dysfunction induced by GPIIb/IIIa inhibitors and aspirin. Blood 2003; 102: 4594–4599
171. Jy W, Haynes DH: Intracellular calcium storage and release in
the human platelet. Chlorotetracycline as a continuous monitor.
Circ Res 1984; 55: 595–608
172. Billy D, Speijer H, Zwaal RF, Hack EC, Hermens WT: Anticoagulant and membrane-degrading effects of secretory (nonpancreatic) phospholipase A2 are inhibited in plasma. Thromb
Haemost 2002; 87: 978–984
173. Anand SX, Viles-Gonzalez JF, Badimon JJ, Cavusoglu E,
Marmur JD: Membrane-associated CD40L and sCD40L in
atherothrombotic disease. Thromb Haemost 2003; 90: 377–
384
174. Henn V, Steinbach S, Buchner K, Presek P, Kroczek RA: The
inﬂammatory action of CD40 ligand (CD154) expressed on
activated human platelets is temporally limited by coexpressed
CD40. Blood 2001; 98: 1047–1054
175. Hermann A, Rauch BH, Braun M, Schror K, Weber AA: Platelet CD40 ligand (CD40L)-subcellular localization, regulationof expression, and inhibition by clopidogrel. Platelets 2001; 12:74–82176. Inwald DP, McDowall A, Peters MJ, Callard RE, Klein NJ:
CD40 is constitutively expressed on platelets and provides anovel mechanism for platelet activation. Circ Res 2003; 92:
1041–1048
177. Krueger LA, Furman MI, Barnard MR, Frelinger AL, Michelson AD: Release of soluble CD40L from platelets is regulated
by ﬁbrinogen binding to GPIIb-IIIa, actin polymerizatin, and amatrix matalloproteinase. Blood 2002; 100(11): 251a #952
178. Schafer A, Schulz C, Eigenthaler M, Fraccarollo D, Kobsar A,
Gawaz M, Ertl G, Walter U, Bauersachs J: Novel role of themembrane-bound chemokine fractalkine in platelet activation
and adhesion. Blood 2004; 103: 407–412
179. Clemetson KJ, Clemetson JM, Proudfoot AE, Power CA, Baggiolini M, Wells TN: Functional expression of CCR1, CCR3,
CCR4, and CXCR4 chemokine receptors on human platelets.
Blood 2000; 96: 4046–4054
180. Wang JF, Liu ZY, Groopman JE: The alpha-chemokine receptor CXCR4 is expressed on the megakaryocytic lineage fromprogenitor to platelets and modulates migration and adhesion.
Blood 1998; 92: 756–764
181. Rozmyslowicz T, Murphy SL, Conover DO, Wroblewski K,
Ratajczak MZ, Gaulton GN: The role of microparticles in HIV1 infection. Blood 2003; 102(11): 278a #987
182. Sheng GY, Huang XL, Bai ST: Study on CXCR4 receptor
on megakaryocytes and its ligand in bone marrow in children
with acute idiopathic thrombocytopenic purpura. Blood 2003;
102(11): 65b #3962
183. Mack M, Kleinschmidt A, Bruhl H, Klier C, Nelson PJ, Cihak J,
Plachy J, Stangassinger M, Erﬂe V, Schlondorff D: Transfer ofthe chemokine receptor CCR5 between cells by membranederived microparticles: a mechanism for cellular human immunodeﬁciency virus 1 infection. Nat Med 2000; 6: 769–775
184. Momoi A, Tsukada N, Nishio M, Aoki S, Fuse I, et al: Platelets
express functional BAFF, which can support B cell survival.
Blood 2003; 102(11): 57b #3930
185. Janowska-Wieczorek A, Kijowski J, Marquez LA, Ratajczak J,
Ratajczak MZ: Microvesicles derived from activated platelets:
an under-appreciated modulator of the metastatic potential oftumor cells. Blood 2003; 102(11): 73b #3998
186. Jurasz P, Alonso D, Castro-Blanco S, Murad F, Radomski MW:

227

187.

188.
189.

190.

191.

192.
193.

194.

195.

196.

197.

198.

199.

200.

201.

202.

203.

204.

Generation and role of angiostatin in human platelets. Blood
2003; 102: 3217–3223
Ma L, Hollenberg MD, Wallace JL: Thrombin-induced platelet endostatin release is blocked by a proteinase activatedreceptor-4 (PAR4) antagonist. Br J Pharmacol 2001; 134: 701–
704
Carrell R: And a ﬁnal message from antithrombin (Editorial).
Blood 2004; 103: 1179
Bahou WF: Thrombin’s faces revealed: cellular effects include
induction of neoangiogenesis. J Thromb Haemost 2003; 1:
2078–2080
Belting M, Dorrell M, Sandgren S, Ahamad J, Dorﬂeutner A,
Carmeliet P, Mueller B, Friedlander M, Ruf W: Tissue factor
signalling in angiogenesis. Blood 2003; 102(11): 13a #33
Taraboletti G, Roberts D, Liotta LA, Giavazzi R: Platelet
thrombospondin modulates endothelial cell adhesion, motility,and growth: a potential angiogenesis regulatory factor. J Cell
Biol 1990; 111: 765–772
Folkman J, Kalluri R: Cancer without disease. Nature 2004;
427: 787
Kim CW, Lee HM, Lee TH, Kang C, Kleinman HK, Gho YS:
Extracellular membrane vesicles from tumor cells promoteangiogenesis via sphingomyelin. Cancer Res 2002; 62: 6312–
6317
Kim HK, Song KS, Park YS, Kang YH, Lee YJ, Lee KR, Kim
HK, Ryu KW, Bae JM, Kim S: Elevated levels of circulatingplatelet microparticles, VEGF, IL-6 and RANTES in patientswith gastric cancer: possible role of a metastasis predictor. Eur J
Cancer 2003; 39: 184–191
Bergsmedh A, Szeles A, Henriksson M, Bratt A, Folkman MJ,
Spetz AL, Holmgren L: Horizontal transfer of oncogenes by
uptake of apoptotic bodies. Proc Natl Acad Sci USA 2001; 98:
6407–6411
Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier
C, Flament C, Pouzieux S, Faure F, Tursz T, et al: Tumorderived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 2001; 7: 297–303
Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ: B lymphocytes secrete antigenpresenting vesicles. J Exp Med 1996; 183: 1161–1172
Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza
D, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 1998; 4: 594–
600
Manoury B, Hewitt EW, Morrice N, Dando PM, Barrett AJ,
Watts C: An asparaginyl endopeptidase processes a microbial
antigen for class II MHC presentation. Nature 1998; 396: 695–
699
Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ: Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 1999;
94: 3791–3799
Denzer K, Kleijmeer MJ, Heijnen HF, Stoorvogel W, Geuze
HJ: Exosome: from internal vesicle of the multivesicular bodyto intercellular signaling device. J Cell Sci 2000; 113: 3365–3374
Fritzsching B, Schwer B, Kartenbeck J, Pedal A, Horejsi V, Ott
M: Release and intercellular transfer of cell surface CD81 via
microparticles. J Immunol 2002; 169: 5531–5537
Abrams C, Shattil SJ: Immunological detection of activatedplatelets in clinical disorders. Thromb Haemost 1991; 65: 467–
473
Kannan K, Divers SG, Lurie AA, Chervenak R, Fukuda M,
Holcombe RF: Cell surface expression of lysosome-associatedmembrane protein-2 (lamp2) and CD63 as markers of in vivo

228

205.

206.

207.

208.

209.

210.

211.

212.

213.

214.

215.

216.

217.

218.

219.

220.

Horstman LL, et al: Cell-derived microparticles
platelet activation in malignancy. Eur J Haematol 1995; 55:
145–151
Ciferri S, Emiliani C, Guglielmini G, Orlacchio A, Nenci GG,
Gresele P: Platelets release their lysosomal content in vivo inhumans upon activation. Thromb Haemost 2000; 83: 157–164
Borregaard N, Kjeldsen L, Lollike K, Sengelov H: Granules
and vesicles of human neutrophils. The role of endomembranes
as source of plasma membrane proteins. Eur J Haematol 1993;
51: 318–322
Majka M, Zupanska B, Ratajczak MZ: Evidence that plateletderived microvesicles transfer HPA1a antigen to the surface ofboth endothelial cells and CD34+ hematopoietic stem/progenitor cells: a new perspective on the pathogenesis of immune
thrombocytopenia. Blood 2003; 102(11): 294a #1051
Hilf N, Singh-Jasuja H, Schwarzmaier P, Gouttefangeas C,
Rammensee HG, Schild H: Human platelets express heat shockprotein receptors and regulate dendritic cell maturation. Blood
2002; 99: 3676–3682
Patel DM, Arnold PY, White GA, Nardella JP, Mannie MD:Class II MHC/peptide complexes are released from APC and
are acquired by T cell responders during speciﬁc antigen recognition. J Immunol 1999; 163: 5201–5210
Kolodny L, Ahn YS, Jy W, Horstman LL, Sheremata WA:
Evidence of platelet activation in multiple sclerosis. Ann Neurol 1996; 40: 520 #M117
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 microparticle (L-EMP)
complexes. Blood 2003; 102(11): 72b #3994
Jy W, Jimenez JJ, Minagar A, Mauro L, Horstman LL,
Bidot CJ, Sheremata WA, Ahn YS: Endothelial microparticles(EMP) enhance adhesion and transmigration of monocytes:
EMP-monocyte conjugates as a marker of disease activity inmultiple sclerosis (MS). Blood 2002; 100(11): 460a #1783
McEver RP: Adhesive interactions of leukocytes, platelets, andthe vessel wall during hemostasis and inﬂammation. Thromb
Haemost 2001; 86: 746–756
Elzey BD, Tian J, Jensen RJ, Swanson AK, Lees JR, Lentz SR,
Stein CS, Nieswandt B, Wang Y, Davidson BL, et al: Plateletmediated modulation of adaptive immunity. A communication
link between innate and adaptive immune compartments. Immunity 2003; 19: 9–19
Kameyoshi Y, Dorschner A, Mallet AI, Christophers E,
Schroder JM: Cytokine RANTES released by thrombin-stimulated platelets is a potent attractant for human eosinophils. J
Exp Med 1992; 176: 587–592
Wartiovaara U, Salven P, Mikkola H, Lassila R, Kaukonen J,
Joukov V, Orpana A, Ristimaki A, Heikinheimo M, Joensuu H,
et al: Peripheral blood platelets express VEGF-C and VEGF
which are released during platelet activation. Thromb Haemost
1998; 80: 171–175
Albanese J, Meterissian S, Kontogiannea M, Dubreuil C, Hand
A, Sorba S, Dainiak N: Biologically active Fas antigen and its
cognate ligand are expressed on plasma membrane-derived
extracellular vesicles. Blood 1998; 91: 3862–3874
Banga HS, Walker RK, Winberry LK, Rittenhouse SE: Pertussis toxin can activate human platelets. Comparative effects ofholotoxin and its ADP-ribosylating S1 subunit. J Biol Chem
1987; 262: 14871–14874
Fleming I, Schulz C, Fichtlscherer B, Kemp BE, Fisslthaler B,
Busse R: AMP-activated protein kinase (AMPK) regulates theinsulin-induced activation of the nitric oxide synthase in humanplatelets. Thromb Haemost 2003; 90: 863–871
Kagawa H, Komiyama Y, Nakamura S, Miyake T, Miyazaki Y,
Hamamoto K, Masuda M, Takahashi H, Nomura S, Fukuhara

221.

222.

223.

224.

225.

226.

227.

228.

229.

230.

231.

232.

233.

234.

235.

236.

S: Expression of functional tissue factor on small vesicles oflipopolysaccharide-stimulated human vascular endothelial cells.
Thromb Res 1998; 91: 297–304
Chou J, Mackman N, Skoloff GM, Pedersen B, Furie BC, Furie
B: Initiation and propagation of the arterial thrombus in vivo:
contribution of vessel wall and blood-borne microparticle tissue
factor. Blood 2003; 102(11): 6a #6Bonderman D, Teml A, Jakowitsch J, Adlbrecht C, Gyongyosi
M, Sperker W, Lass H, Mosgoeller W, Glogar DH, Probst P,
Maurer G, Nemerson Y, Lang IM: Coronary no-reﬂow iscaused by shedding of active tissue factor from dissected atherosclerotic plaque. Blood 2002; 99: 2794–2800
Shet AS, Aras O, Gupta K, Hass M, Solovey A, Key NS, Hebbel RP: Sickle blood contains tissue factor positive microparticles derived from endothelial cells and monocytes. Blood
2002; 100(11): 45a #157
Novotny WF, Girard TJ, Miletich JP, Broze GJ Jr: Platelets
secrete a coagulation inhibitor functionally and antigenicallysimilar to the lipoprotein associated coagulation inhibitor.
Blood 1988; 72: 2020–2025
Braaten JV, Handt S, Jerome WG, Kirkpatrick J, Lewis JC,
Hantgan RR: Regulation of ﬁbrinolysis by platelet-released
plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-ﬁbrin thrombus. Blood 1993; 81: 1290–1299
Stringer HA, van Swieten P, Heijnen HF, Sixma JJ, Pannekoek
H: Plasminogen activator inhibitor-1 released from activatedplatelets plays a key role in thrombolysis resistance. Studies
with thrombi generated in the Chandler loop. Arterioscler
Thromb 1994; 14: 1452–1458
Khetawat G, Faraday N, Nealen ML, Vijayan KV, Bolton E,
Noga SJ, Bray PF: Human megakaryocytes and platelets contain the estrogen receptor beta and androgen receptor (AR):testosterone regulates AR expression. Blood 2000; 95: 2289–
2296
Giandomenico G, Czekay RP, Koschnick S, Loskutoff DJ:
Inﬂuence of leptin on platelet function. Blood 2003; 102(11):69b #3981
Ghosh SA, Polanowska-Grabowska RK, Fujii J, Obrig T, Gear
AR: Shiga toxin binds to activated platelets. J Thromb Haemost 2004; 2: 499–506
Hackeng CM, Relou IA, Pladet MW, Gorter G, van Rijn HJ,
Akkerman JW: Early platelet activation by low density lipoprotein via p38MAP kinase. Thromb Haemost 1999; 82: 1749–
1756
Akkerman JWN, Eybrechts KL, Kroner C, Ferreira IA: IRS-1
mediates platelet inhibition by insulin via the inhibitory G protein Gi. Blood 2003; 102(11): 775a #2861
Hirasawa A, Shibata K, Kotosai K, Tsujimoto G: Cloning,
functional expression and tissue distribution of human cDNAfor the vascular-type vasopressin receptor. Biochem Biophys
Res Commun 1994; 203: 72–79
Philippeaux MM, Vesin C, Tacchini-Cottier F, Piguet PF: Activated human platelets express beta2 integrin. Eur J Haematol
1996; 56: 130–137
Nomura S, Komiyama Y, Miyake T, Miyazaki Y, Kido H,
Suzuki M, Kagawa H, Yanabu M, Takahashi H, Fukuhara S:
Amyloid beta-protein precursor-rich platelet microparticles inthrombotic disease. Thromb Haemost 1994; 72: 519–522
Cooling LL, Zhang DS, Walker KE, Koerner TA: Detection inhuman blood platelets of sialyl Lewis X gangliosides, potential
ligands for CD62 and other selectins. Glycobiology 1995; 5:
571–581
Santoso S, Sachs UJH, Kroll H, Linder M, Ruf A, Preissner
KT: JAM-3, a new platelet receptor for the Mac-1 integrin.
Blood 2002; 100(11): 259a #988

Keio J Med 2004; 53 (4): 210–230
237. Babinsky A, Kedees MH, Arthar H, Ahmed T, Batuman O,
Erlich YH, Hussain MM, Kornecki E: F11 receptor (F11R/
JAM) mediates platelet adhesion to inﬂamed endothelial cells.
Blood 2002; 100(11): 258a #984
238. Kornecki E, Babinska A, Kedees MH, Athar H, Sobocka MB,
Sobacki T, Erlich YH, Hussain MM, Jiang XC: F11R, a platelet
adhesion protein critical for the development of thrombosis andatherosclerosis. Blood 2002; 100(11): 75b #3772
239. Morgan BP: Isolation and characterization of the complementinhibiting protein CD59 antigen from platelet membranes.
Biochem J 1992; 282: 409–413
240. Horstman LL, Jy W, Morgan BP, Ahn YS: CD59 expression onplatelets in ITP and PNH. La Revista de Investigacion Clinica(Suppl) 1994; 110: 212 #a109
241. Anker P, Stroun M: Progress in the knowledge of circulating
nucleic acids: plasma RNA is particle-associated. Can it become
a general detection marker for a cancer blood test? Clin Chem
2002; 48: 1210–1211
242. Ng EK, Tsui NB, Lam NY, Chiu RW, Yu SC, Wong SC, Lo
ES, Rainer TH, Johnson PJ, Lo YM: Presence of ﬁlterable andnonﬁlterable mRNA in the plasma of cancer patients andhealthy individuals. Clin Chem 2002; 48: 1212–1217
243. Hasselmann DO, Rappl G, Tilgen W, Reinhold U: Extracellular tyrosinase mRNA within apoptotic bodies is protectedfrom degradation in human serum. Clin Chem 2001; 47: 1488–
1489
244. Favaloro EJ, Mohammed A, Coombs R, Mehrabani PA: Filtered plasma as a potential cause of clinical misdiagnosis: inappropriate testing in a haematology laboratory. Br J Biomed Sci
1995; 52: 243–248
245. Favaloro EJ, Nair SC, Forsyth CJ: Collection and transport ofsamples for laboratory testing in von Willebrand’s disease(VWD): time for a reappraisal? Thromb Haemost 2001; 86:
1589–1590
246. Jiang N, Reich CF 3rd, Pisetsky DS: Role of macrophages in
the generation of circulating blood nucleosomes from dead anddying cells. Blood 2003; 102: 2243–2250
247. Savill J, Gregory C, Haslett C: Cell biology. Eat me or die.
Science 2003; 302: 1516–1517
248. Park CT, Creasey AA, Wright SD: Tissue factor pathway inhibitor blocks cellular effects of endotoxin by binding to endotoxin and interfering with transfer to CD14. Blood 1997; 89:4268–4274
249. Roberts JP: An immunological role in the CARDS. The Scientist 2003; 17: 29–30
250. Barzilai N, Atzmon G, Schechter C, Schaefer EJ, Cupples AL,
Lipton R, Cheng S, Shuldiner AR: Unique lipoprotein phenotype and genotype associated with exceptional longevity.
JAMA 2003; 290: 2030–2040
251. Lerch PG, Spycher MO, Doran JE: Reconstituted high density
lipoprotein (rHDL) modulates platelet activity in vitro and ex
vivo. Thromb Haemost 1998; 80: 316–320
252. Murai K, Kowata S, Sako M, Ito T, Inoue H, Kaneko Y, Nakai
K, Ito E, Ishida Y: Thrombin-antithrombin III/HDL (highdensity lipoprotein) is one of the regulators of platelet production in chronic immune thrombocytopenia. Blood 2003;
102(11): 299a #1069
253. Dennis MW, Downey C, Brufatto N, Nesheim ME, Stevenson
K, Hock Toh C: Prothrombinase enhancement through quantitative and qualitative changes affecting very low density lipoprotein in complex with C-reactive protein. Thromb Haemost
2004; 91: 522–530
254. Toh CH, Dennis MW, Colin D: Procoagulant enhancement
through quantitative and qualitative changes affecting very low
density lipoprotein in complex with C-reactive protein. Blood
2003; 102(11): 90b #4067

229
255.

256.

257.

258.

259.

260.

261.

262.

263.

264.

265.

266.

267.

268.
269.
270.

271.

272.

Tselepis AD, Dentan C, Karabina SA, Chapman MJ, Ninio E:
PAF-degrading acetylhydrolase is preferentially associated with
dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme. Arterioscler Thromb Vasc Biol 1995; 15: 1764–1773
Hansen JB, Huseby NE, Sandset PM, Svensson B, Lyngmo V,
Nordoy A: Tissue-factor pathway inhibitor and lipoproteins.
Evidence for association with and regulation by LDL in humanplasma. Arterioscler Thromb 1994; 14: 223–229
Lesnik P, Vonica A, Guerin M, Moreau M, Chapman MJ:
Anticoagulant activity of tissue factor pathway inhibitor in human plasma is preferentially associated with dense subspeciesof LDL and HDL and with Lp(a). Arterioscler Thromb 1993;
13: 1066–1075
Polz E, Wurm H, Kostner GM: Investigations on beta 2glycoprotein-I in the rat: isolation from serum and demonstration in lipoprotein density fractions. Int J Biochem 1980; 11:
265–270
Jimenez JJ, Ferreira A, Peter A, Jy W, Mauro LM, Mendez AJ,
deMarchena E, Soderland C, Horstman LL, Ahn YS: Elevatedendothelial microparticles (EMP) and decreased endothelial
progenitors are associated with increased serum cholesterollevels. Blood 2003; 102(11): 73b #3997
Sims PJ, Wiedmer T: The response of human platelets to activated components of the complement system. Immunol Today
1991; 12: 338–342
Wiedmer T, Sims PJ: Participation of protein kinases in complement C5b-9-induced shedding of platelet plasma membrane
vesicles. Blood 1991; 78: 2880–2886
Haynes DH: Effects of cyclic nucleotides and protein kinases onplatelet calcium homeostasis and mobilization. Platelets 1993; 4:
231–43
Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ:
Molecular cloning of human plasma membrane phospholipid
scramblase. A protein mediating transbilayer movement ofplasma membrane phospholipids. J Biol Chem 1997; 272:
18240–18244
Zwaal RF, Comfurius P, Bevers EM: Platelet procoagulant
activity and microvesicle formation. Its putative role in hemostasis and thrombosis. Biochim Biophys Acta 1992; 1180: 1–8
Zwaal RF, Schroit AJ: Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89:1121–1132
Seymour CA, Muallem S, Lew VL: Preparation of inside-out
vesicles from red blood cells in one step, In: Ellory JC, Young
JD, eds, Red cell Membranes, a Methodological Approach,London/New York, Academic Press, 1982; 219–222
Racker E, Horstman LL: Partial resolution of the enzymes catalyzing oxidative phosphorylation. 13. Structure and function ofsubmitochondrial particles completely resolved with respect tocoupling factor. J Biol Chem 1967; 242: 2547–2551
Ahn YS, Jy W, Horstman LL, Jimenez JJ: Letter on review by
JM Freyssinet. J Thromb Haemost 2004; (in press)
White JG: Functional signiﬁcance of mobile receptors on
human platelets. Arterioscler Thromb 1993; 13: 1236–1243
Nurden P: Bidirectional trafﬁcking of membrane glycoproteins
following platelet activation in suspension. Thromb Haemost
1997; 78: 1305–1315
Coller BS, Kutok JL, Scudder LE, Galanakis DK, West SM,
Rudomen GS, Springer KT: Studies of activated GPIIb/IIIa
receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to highdensity ﬁbrinogen. J Clin Invest 1993; 92: 2796–2806
Simmons SR, Sims PA, Albrecht RM: alpha IIb beta 3 redistribution triggered by receptor cross-linking. Arterioscler Thromb
Vasc Biol 1997; 17: 3311–3320

230
273. Bodin S, Viala C, Ragab A, Payrastre B: A critical role of lipid
rafts in the organization of a key FcgammaRIIa-mediated signaling pathway in human platelets. Thromb Haemost 2003; 89:
318–330
274. Dietzen DJ, Jack GG, Page KL, Tetzloff TA, Hall CL, Mast
AE: Localization of tissue factor pathway inhibitor to lipid rafts
is not required for inhibition of factor VIIa/tissue factor activity.
Thromb Haemost 2003; 89: 65–73
275. Simons K, Toomre D: Lipid rafts and signal transduction. Nat
Rev Mol Cell Biol 2000; 1: 31–39
276. Anderson RG, Jacobson K: A role for lipid shells in targeting
proteins to caveolae, rafts, and other lipid domains. Science
2002; 296: 1821–1825
277. Czech MP: Lipid rafts and insulin action. Nature 2000; 407:
147–148278. Simons K, Ikonen E: Functional rafts in cell membranes.
Nature 1997; 387: 569–572
279. Freed EO: Virology. Rafting with Ebola. Science 2002; 296: 279
280. Frank PG, Woodman SE, Park DS, Lisanti MP: Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc
Biol 2003; 23: 1161–1618
281. Salzer U, Prohaska R: Stomatin, ﬂotillin-1, and ﬂotillin-2 aremajor integral proteins of erythrocyte lipid rafts. Blood 2001;
97: 1141–1143
282. Butikofer P, Kuypers FA, Xu CM, Chiu DT, Lubin B: Enrichment of two glycosyl-phosphatidylinositol-anchored proteins,acetylcholinesterase and decay accelerating factor, in vesicles
released from human red blood cells. Blood 1989; 74: 1481–
1485
283. Dietzen DJ, Page KL, Tetzloff TA: Lipid rafts are necessary fortonic inhibition of cellular tissue factor procoagulant activity.
Blood 2003; 102(11): 791a #2926
284. Munday AD, Shrimpton CN, Lopez JA: Relationship of lipid
rafts and the platelet cytoskeleton. Blood 2003; 102(11): 772a
#2849
285. Sun J, Nanjundan M, Pike LJ, Wiedmer T, Sims PJ: Plasma
membrane phospholipid scramblase 1 is enriched in lipid rafts
and interacts with the epidermal growth factor receptor. Biochemistry 2002; 41: 6338–6345

Horstman LL, et al: Cell-derived microparticles
286. Jy W, Jimenez JJ, Mauro LM, Ahn YS, Newton KR, Mendez
AJ, Arnold PI, Schultz DR: Agonist-induced capping of adhesion proteins and microparticle shedding in cultures of humanrenal microvascular endothelial cells. Endothelium 2002; 9:
179–189287. Millan J, Montoya MC, Sancho D, Sanchez-Madrid F, Alonso
MA: Lipid rafts mediate biosynthetic transport to the T lymphocyte uropod subdomain and are necessary for uropod integrity and function. Blood 2002; 99: 978–984
288. Chonn A, Cullis PR, Devine DV: The role of surface charge in
the activation of the classical and alternative pathways of complement by liposomes. J Immunol 1991; 146: 4234–4241
289. Morgan BP, Meri S: Membrane proteins that protect againstcomplement lysis. Springer Semin Immunopathol 1994; 15:
369–396290. Horstman LL, Jy W, Schultz DR, Mao WW, Ahn YS: Complement-mediated fragmentation and lysis of opsonized platelets:ender differences in sensitivity. J Lab Clin Med 1994; 123: 515–
525
291. Hamilton KK, Hattori R, Esmon CT, Sims PJ: Complement
proteins C5b-9 induce vesiculation of the endothelial plasma
membrane and expose catalytic surface for assembly of theprothrombinase enzyme complex. J Biol Chem 1990; 265: 3809–
3814
292. Taylor FB Jr, He SE, Chang AC, Box J, Ferrell G, Lee D,
Lockhart M, Peer G, Esmon CT: Infusion of phospholipid
vesicles ampliﬁes the local thrombotic response to TNF andanti-protein C into a consumptive response. Thromb Haemost
1996; 75: 578–584
293. Jamil H, Sheikh S, Ahmad I: Liposomes: The next generation.
Modern Drug Discovery 2004; Jan: 36–39
294. Sanchez-Mateos P, Campanero MR, del Pozo MA, SanchezMadrid F: Regulatory role of CD43 leukosialin on integrinmediated T-cell adhesion to endothelial and extracellular matrix ligands and its polar redistribution to a cellular uropod.
Blood 1995; 86: 2228–2239
295. Reed GL, Fitzgerald ML, Polgar J: Molecular mechanisms ofplatelet exocytosis: insights into the ‘‘secrete’’ life of thrombocytes. Blood 2000; 96: 3334–3342