Keywords extracellular vesicles - platelets - coagulation - platelet-derived extracellular
vesicles
Recent developments in extracellular vesicles (EVs) have led to a reimagining of the
idea of a secreted signal.[1 ] Our understanding of communication between cells, tissues, and organs is radically
changing due to the possibility of membrane-bound packages delivering RNA, proteins,
lipids, and organelles.[1 ]
[2 ] A more traditional view of cell–cell communication being upheld by chemical signals
or contact between cells has been extended to include an option of bundled messaging
that come from vesicle-mediated signals.[3 ]
[4 ]
EVs have been of increasing interest to researchers for over 50 years. The story of
their discovery goes back to 1946 when Erwin Chargaff and Randolph West demonstrated
that platelet-free plasma (PFP) was still capable of coagulation.[5 ] A study by Sinauridze and collaborators showed that platelet-derived extracellular
vesicles (PEVs) are 50 to 100 times more potent than thrombin-activated platelets
at promoting coagulation.[6 ] Coagulation parameters of PFP were characterized by standard clotting assays: activated
partial thromboplastin time, prothrombin time and kaolin time, and by determination
of spatial clot growth rate.[6 ]
Workers were even able to isolate the fraction of plasma that gave it this procoagulant
activity by centrifugation, without identifying what was responsible.[5 ] In 1967, Peter Wolf completed this discovery by characterizing the content of this
fraction.[7 ] He showed by electron microscopy that this fraction is made up of particles derived
from activated platelets rich in phospholipids, which he named “platelet dust.”[7 ] Over time this term has been replaced by microparticles (MPs) and then by EVs. The
use of the generic term “extracellular vesicles” is now recommended by the International
Society for Extracellular Vesicles (ISEV) to refer to any particle released from the
cell that is characterized by a lipid bilayer and the absence of a nucleus.[8 ]
EVs are submicron vesicles (size between 10 nm and 1 µm) released into the extracellular
medium by most cell types.[9 ] EVs have a circular shape and are bounded by a lipid bilayer and have varying sizes
depending on their type.[10 ] These EVs can be isolated from different biological fluids (plasma, urine, saliva,
cerebrospinal fluid, cell culture supernatant...).[11 ] For a long time, they were considered to be simple cellular debris, but are now
considered to be true biological information vectors that can modulate the metabolism
of numerous target cells by transferring information and/or modulating cell signaling.
These vesicles carry different nucleic acids (messenger ribonucleic acid [mRNA], microribonucleic
acid [miRNA], transfer ribonucleic acid [tRNA]...), proteins (enzymes, transporters,
and extracellular matrix proteins...) as well as lipids (oleic acid, arachidonic acid...)
derived from their cell of origin, which can induce modifications in the functioning
of the target tissues.[12 ]
Three types of EVs can be distinguished: MPs also called microvesicles, exosomes also
called exovesicles, and apoptotic bodies[13 ] ([Table 1 ]). In blood, the majority of circulating EVs originate from platelets.
Table 1
Release modes and characteristics of EV types[14 ]
[15 ]
Subtypes of EVs
Origin
Size (mm)
Microparticles
Budding of the plasma membrane
100–1,000
Exosomes
Are first formed inside the cells, in structures called multivesicular bodies (MVBs),
and are then released into the extracellular environment following the fusion of these
MVBs with the plasma membrane
40–1,000
Apoptotic bodies
Plasma membrane budding in apoptotic cells
1,000–5,000
Abbreviation: EV, extracellular vesicles.
This review focuses on the classical role PEVs play in hemostasis, as well as recent
studies that demonstrate broader potential therapeutic applications.
Historical Findings on the Role of PEVs
Historical Findings on the Role of PEVs
PEVs account for approximately 80% of circulating EVs in blood.[16 ]
[17 ] They are formed upon activation of platelets by various agonists such as thrombin,
collagen, and complement.[18 ]
Detailed electron microscopic analysis of activated platelets linked data from various
publications and described the release of two different populations of EVs called
microvesicles and exosomes.[19 ] These vesicles play several roles in the regulation of physiological and pathological
functions[20 ] by accelerating the generation of thrombin.[21 ]
[22 ]
[23 ] Also, they have procoagulant activity, the procoagulant properties of these vesicles
are associated with microvesicles but not with exosomes.[19 ]
[24 ]
The procoagulant activity of PEVs arises primarily from the exposure of negatively
charged phospholipids, primarily phosphatidylserine, and tissue factor that provide
a site for the assembly of coagulation factors.[19 ] One early study showed that PEVs have a procoagulant activity that is 50 to 100
times higher than that of activated platelets.[6 ]
Another later study provided the first evidence that PEVs can exert both pro- and
anticoagulant effects. This study showed that PEVs could promote prothrombin activation
but also inactivation of factor Va,[25 ] through binding of protein S to the coagulation inhibitor protein C.[26 ] This shows a possible role of PEVs in maintaining the balance between pro- and anti-thrombotic
states.
The expression of platelet markers by PEVs varies according to the state of the original
cell at the time of release.[27 ]
[28 ] Indeed, all PEVs express the constitutive markers of this cell type (CD41, CD42b,
CD61, or CLEC-2), but only those secreted by activated platelets carry platelet activation
markers (CD62p and CD63) on their surface.[27 ]
[28 ]
PEVs also have other proteins on their surface such as von Willebrand factor and CD31.
However, these proteins are not exclusive to platelets.[29 ] PEVs also have a role in the regulation of immunity (stimulation and suppression),
where they can interact with T cells and regulate their differentiation and regulatory
activity.[30 ]
[31 ] In addition, they can promote the formation of germinal centers and IgG production
by B cells depending on their CD40L content.[32 ]
[33 ] As PEVs can circulate in lymph, they have the potential to transport molecules of
adaptive immunity to lymphoid organs.[34 ]
[35 ]
The cargo carried by PEVs (growth factors, proteins, nucleic acids, organelles, etc.)
also allows them to play a role in intracellular communication.[36 ]
[37 ] Through the exchange of nucleic acids ( mRNA, miRNA, and other types of rubonucleic
acid [RNA]), they can regulate cells at post-transcriptional levels.[38 ]
Therapeutic Potential of PEVs
Therapeutic Potential of PEVs
EVs can be used for cardiac regenerative therapies.[39 ] In a 2005 study, it was shown that PEVs stimulate vascular endothelial growth factor
(VEGF)-dependent revascularization after chronic ischemia, pointing to the potential
regenerative effect of EVs on cardiac regeneration.[40 ] Additionally, a study published in 2015 demonstrated that plasma-derived EVs activate
the toll-like receptor 4 on cardiomyocytes to induce cardioprotective signals following
ischemia–reperfusion.[41 ]
On the other hand, several studies report a significant increase in tissue factor
activity associated with PEVs from coronavirus disease (COVID-19) patients. Indeed,
COVID-19 patients have been shown to have a higher PEV-associated tissue factor activity
associated with thromboembolic events.[42 ]
[43 ]
[44 ]
[45 ]
Researchers are creating new ways to optimize drug efficacy and prevent off-target
effects by developing a better understanding of various diseases and their pathologies.[46 ] Nanoparticles and liposomes, including nanoparticle-based drug delivery technologies,
are gaining considerable attention for delivery of anti-inflammatory drugs or chemotherapy.
Therapeutic EVs may prove to be valuable since they provide efficient cargo delivery
systems for long-distance communication between cells and organs. As a result of rheumatoid
arthritis, which is a chronic inflammatory disease characterized by platelet activation,
the blood is more abundant in PEVs.[47 ] Otherwise, the absence of PEVs has been associated with bleeding and coagulation
disorders, according to several clinical studies. In Scott syndrome, characterized
by mild to moderate bleeding, circulating PEVs are decreased, possibly due to reduced
PS exposure capacity.[48 ]
PEVs are currently being explored as therapeutic tools,[49 ] due to the nature of their molecular cargo, which contains bioactive molecules such
as growth factors, proteins, coagulation factors and non-coding RNAs ([Fig. 1 ]).[50 ]
[51 ]
Fig. 1 The content of platelet-derived extracellular vesicles (PEVs). Different molecules
have been identified at the surface of these extracellular vesicles. CD61, CLEC-2,
and LAMP-1 are constitutively expressed on PEVs surface. Phosphatidylserine as well
as P-selectin are also observed on PEVs. Several studies have shown that PEVs contain
nucleic acids, proteins, and filamin. Moreover, mitochondria can be found in a proportion
of the PEVs.
Microvesicles have been identified as cardio protectors through their association
with Sonic hedgehog factor, known for its muscle tissue repair properties.[52 ] One study showed that procoagulant PEVs were significantly increased in subjects
following cardiac stress induced by dobutamine, a cardiac adrenergic receptor stimulator,
and then decreased after 1 hour.[53 ] This increase was less significant in patients with a history of vascular disease
(coronary heart disease, ischemia, etc.).[53 ] This suggests that the increase in circulating levels of these procoagulant microvesicles,
in the face of this cardiac stress, would be a physiological response which would
nevertheless be diminished in individuals with cardiac pathologies. Therefore, the
possible use of PEVs in the treatment of cardiovascular diseases has been explored.[54 ]
PEVs also participate in the inflammatory process through their influence on cell–cell
interaction and their role in inducing the expression of adhesion molecules and the
release of cytokines by different cell types, also, contain pro-inflammatory cytokines
such as interleukin (IL)-1, IL-6, and tumor necrosis factor α.[55 ]
A recent study has also suggested that PEVs could infiltrate the bone marrow during
an inflammatory state and promote hematopoiesis.[56 ] Other studies have shown that PEVs also possess anti-inflammatory activities and
may suppress inflammation mainly by inhibiting cytokine release.[57 ]
[58 ]
Some populations of PEVs may be particularly suitable for therapy to restore hemostasis
and inhibit vascular permeability.[59 ] The surface of circulating PEVs is 50 to 100 times more pro-coagulant than that
of an activated platelet.[6 ] They express negatively charged phospholipids exposed on their surface, and have
binding sites for coagulation factors such as activated factor V and factor VIII and
thrombin.[6 ]
Results from other studies have shown that PEVs can help treat neurological disorders,
with administration of these vesicles in an animal model of cerebrovascular accident
showing increased neural cell growth at the site of injury, resulting in improved
behavioral outcomes.[60 ] Treatment with PEVs increased the potential for neural stem cells to differentiate
into glia and neurons.[61 ] In addition, growth factors such as platelet-derived growth factor, VEGF, fibroblast
growth factor, and brain-derived neurotrophic factor, which are known to be present
in platelets and their vesicles, may promote neurotrophic effects and neurogenesis.[62 ]
[63 ] PEVs can be used as targeted drug carriers, due to their interaction with other
cells as they express various platelet membrane glycoproteins, such as GPIIbIIIa (CD41/CD61
or integrin αIIb/β3), GPIaIIa (CD49b/CD29), GPIba (CD42b), P-selectin (CD62P), PECAM-1
(CD31), and GP53 (CD63).[64 ]
The ability of cancer cells to internalize PEVs and the known interactions between
tumor and platelets, may make PEVs a suitable cancer treatment.[65 ]
[66 ]
[67 ] Proof of concept that PEVs can be used as vehicles for anti-cancer drugs has been
demonstrated.[68 ] Indeed, PEVs express higher levels of surface proteins like P-selectin and CD41,
as well as lipids such as phosphatidylserine, which contributes to cancer-associated
thrombosis.[6 ] Some native platelet vesicles inhibit tumor growth by transferring miRNA-24.[69 ]
Conclusion
EVs have been shown to have alternative and non-redundant functions. In the past two
decades, a rapidly increasing number of studies and scientific papers have discussed
the functional role of PEVs in many physiological and pathological processes. A wide
range of physiological and pathological processes are regulated by PEVs, including
inflammation, cell communication, coagulation, and metastasis. A further understanding
of PEVs use and manipulation will lead to improved disease diagnosis and associated
morbidity and mortality.
