Platelet-derived extracellular vesicles play an important role in platelet transfusion therapy

Abstract

Extracellular vesicles (EVs) contain the characteristics of their cell of origin and mediate cell-to-cell communication. Platelet-derived extracellular vesicles (PEVs) not only have procoagulant activity but also contain platelet-derived inflammatory factors (CD40L and mtDNA) that mediate inflammatory responses. Studies have shown that platelets are activated during storage to produce large amounts of PEVs, which may have implications for platelet transfusion therapy. Compared to platelets, PEVs have a longer storage time and greater procoagulant activity, making them an ideal alternative to platelets. This review describes the reasons and mechanisms by which PEVs may have a role in blood transfusion therapy.

Plain Language Summary

What is the context?

Platelet transfusion is a treatment that can be effective in preventing bleeding and reducing the amount of bleeding. However, platelet transfusion may cause some unsatisfactory effects for patients, such as adverse transfusion reactions and poor prognosis in cancer patients. These benefits and harms caused by platelet transfusion may be related to PEVs. With the prolongation of storage time during the shelf life of platelets, PEVs were continuously released and the therapeutic effect of platelet components seems to get worse.

What is new?

This article not only reviews the evidence for PEVs plays a role in blood transfusion therapy but also introduces the mechanism of PEVs in platelet storage lesion and the common methods of isolation and characterization of PEVs.

What is the impact?

It is necessary to improve the method of isolation and purification of PEVs, to increase the purity of PEVs isolation, and to further demonstrate the potential of PEVs to replace platelets. Further research into the mechanisms by which platelets and PEVs affect the prognosis of cancer patients is required.

Keywords:

• Blood transfusion
• extracellular vesicles
• platelet activation
• platelet storage lesions

Introduction

Platelet transfusion is currently an irreplaceable therapy for treating or preventing bleeding and accelerating wound healing in cancer, hematology, surgery, and trauma patients.^1,2 Platelet concentrates (PCs) form nearly ten percent of all blood components (BCs) that are transfused.³ Platelets (PLTs) are usually stored for only 5 days. During storage, the platelets’ morphology and function are gradually damaged, a process known as platelet storage lesion (PSL).^4,5 With the prolongation of storage time, platelet activation increased and platelet extracellular vesicles (PEVs) were continuously released.⁶ EVs (50 nm to over 1 μm in diameter) contain the membrane structure and cytoplasmic components of the originating cells.^7,8 Moreover, EVs are both a means for secretory cells to process intracellular components and an important mediator of intercellular signaling.^6,9 Studies have shown that PEVs are not only rich in tissue factor (TF) and phosphatidylserine (PS) for hemostasis, but also contain significant amounts of growth factors that promote wound healing and inflammatory factors involved in the transfusion response to platelet transfusion.^10,11 Therefore, the efficacy of platelet transfusion and the occurrence of adverse transfusion reactions are likely to be influenced by PEVs.

The immunologic function of platelets

As well as being involved in clotting and mediating inflammatory responses, platelets are now considered to be immune cells (Figure 1).^1,12 Activated platelets not only initiate hemostasis but also perform immune functions.¹³ First of all, activated platelet secrete a large number of biological response regulators (BMRs), including growth factors (kininogen, thrombospondin, etc.), Cytokines/Chemokines (sCD40L/CD40L, CD62P, platelet factor 4 (PF4), interleukin 1β, and factors that regulate the expression of activated normal T cells and secretion (RANTES), etc.).^5,14 Cytokines/chemokines (CKs/CHs) directly or indirectly regulate the innate and adaptive immune system through binding to adhesion receptors expressed on immune cells or other cells such as endothelial cells.^5,14 CD40L is produced during platelet storage and can enhance the interaction of platelets with immune cells, which is associated with serious side effects of platelet transfusion (e.g. transfusion-related acute lung injury) (Figure 1a).^15,16 Moreover, Platelets express immune receptors and Toll-like receptors (TLRs) (Figure 1b). FCR (FcεRII, FcγRIIA, and FcαRI) can recognize immunoglobulins of the IgE, IgG, and IgA subclasses.¹⁷ FcγRIIA on the platelet surface can bind to the Fc parts of anti-PF4/polyanion antibodies leading to heparin-induced thrombocytopenia (HIT).¹⁸ Platelets contain all Toll-like receptors (TLR1 to TLR10), which are key regulators in initiating the innate immune response to foreign organisms.¹⁹ During storage, HMGB1 (a type of DAMPs) can accumulate in platelet components and their association with TLR4 triggers a unique signal transduction response that induces platelet activation and releases immunoregulatory factors.^13,20 In addition, platelets promote the phagocytosis of bacteria by white blood cells (Figure 1c).⁵ Although the innate immune system recognizes the negative charge on the surface of bacteria, the adaptive immune system cannot be activated by the negative charge.²¹ However, platelet-derived PF4, when attached to the negative charge on the bacterial surface, can form a new epitope that can be bound by anti-PF4/polyanionic (IgG-type) antibodies and promote phagocytosis of bacteria by granulocytes.^5,22 IgG-coated bacteria are also engulfed by platelets, which are then activated to release alpha-granules at the site of the bacteria to kill them.^5,22

Figure 1. The immunologic function of platelets. (a) Activated platelets secrete a large number of biological response regulators (BMRs). If patients with previous inflammatory pathological conditions or external factors attracting leukocytes to lung vessels, the BMRs contained in the transfused platelet concentrations will activate neutrophils to produce ROS, which damages the pulmonary endothelium and cause transfusion-related acute lung injury. Moreover, the growth factors are associated with angiogenesis; (b) Platelets express immune receptors, and Toll-like receptors (TLR) are involved in immune regulation. During heparin treatment, if patients with anti-PF4/polyanion antibodies (These antibodies probably result from bacterial infections or heparin treatment), their Fab parts will bind to PF4/heparin complexes, and then FcγRIIA on the platelet surface can bind to the Fc parts of anti-PF4/polyanion antibodies leading to HIT. TLR4 can bind to damage-associated molecular patterns (DAMPs, e.g. HMGB1) to trigger platelet activation and the release of BMRs. (c) Platelets can interact directly or indirectly with bacteria to achieve a bactericidal effect. Lipopolysaccharides on the surface of bacteria activate platelets and promote the production of PF4, which can bind to the negative charge of the bacterial surface, exposing a new epitope. This epitope is bound by anti-PF4/polyanion antibodies, promoting phagocytosis of the bacteria by granulocytes. Platelets can also internalize IgG-encapsulated bacteria to achieve bactericidal effects.

As a multifunctional immune cell, platelet transfusion is a “cell transplantation” that can actively participate in the immune regulation of the host, which may harm the efficacy of transfusion.²³ Numerous mediators produced during platelet storage are involved in a variety of adverse reactions, leading to an increased risk of platelet transfusion and shortened infusion intervals.²⁴ What’s more, PEVs produced during storage are highly homologous to platelets, have similar functions to platelets, and mediate intercellular communication.^7,8 PEVs are believed to be related to enhanced platelet aggregation and regulation of the immune system, may influence the efficacy of platelet transfusion, and participate in the adverse reactions of immune transfusion.^2,7,8

Platelet produce PEVs during storage

Production mechanisms of PEVs

Platelet storage lesions (PSL) began at the time of blood collection and progressed through reprocessing operations and storage. Platelet activation due to the method of platelet concentrate preparation and storage conditions is the main cause of PSL.²⁵ With the prolongation of storage time, the intracellular Ca²⁺ levels were gradually increased, which mediates the activation of gelsolin and the polymerization of actin, ultimately leading to changes in platelet morphology and membrane lipids.²⁶ Under resting conditions, the inner leaflet of the platelet lipid membrane consists mainly of phosphatidylserine (PS) and phosphatidylethanolamine (PE), and the outer leaflet consists mainly of sphingomyelin (SM) and phosphatidylcholine (PC).²⁷ Adenosine triphosphate (ATP)-dependent translocase maintains the stability of the inner leaflet of the platelet membrane (transfers PS and PE to the inner leaflet), and ATP-dependent floppase maintains the stability of the outer leaflet (transfers SM and PC to the outer leaflet).²⁸ The increase of Ca²⁺ level activates Calcium-dependent scramblase and inhibits ATP-dependent transporters and floppase, resulting in asymmetric changes in membrane phospholipid molecules and promoting the expression of PS and PE on the outer leaflet of the platelet membrane (Figure 2).⁸ As the significant alterations of membrane lipids and restructure of the cytoskeleton filaments, the platelet membrane is prone to foaming, and the extracellular domain of the platelet receptor falls off.²⁹ Increased Ca2+ can also lead to increased generation of platelet microvesicles (PMVs) by promoting the activation of calcium-dependent thiol proteases and calpain.³⁰ In addition, the activation of NADPH oxidase (NOX2) during platelet storage causes the production of O2-, which is rapidly converted to H2O2 by superoxide dismutase (SOD), mobilizing intracellular calcium, promoting platelet activation and accelerating PEV production.³¹ Platelet exosome (P-exos) formation begins with membrane bulging into the endosome lumen, which is subsequently converted into a multivesicular body (MVB). Finally, MVBs are either fused to the plasma membrane for release as exosomes or are taken up by lysosomes for degradation.²⁹ Some researchers have proved the process of exosome secretion in several cell types is regulated by Ca²⁺, but whether it applies to P-exos is still unclear.³² Some studies have also proposed that the activation and closure of integrin αIIbβ3, cytoskeletal rearrangement, and its related signaling pathways have a crucial role in the release of EVs.³³

Figure 2. The mechanism of PEVs produced by platelet during storage. With the intracellular Ca²⁺ level was gradually increased during platelet storage, the membrane phospholipid molecules have been asymmetrically changed, which promotes the generation of platelet microvesicles (PMVs). The platelet exosomes (P-exos) generation begins with the membrane bulging into the lumen of endsome, which is converted into a multi-vesicular body (MVB), then the MVBs fuse with the plasma membrane to secrete exosomes. Both PMVs and P-exos belong to PEVs. The biomarkers and contents of platelet-derived extracellular vesicles (PEVs) usually contain CD62P, CD42a, CD9, CD63, CD41b, PF4, and RNA.

PLTs preparation and storage conditions can affect the production and characterization of PEVs

Platelet products are prepared from whole blood by centrifugation.³⁴ The high centrifugal force during preparation leads to increased platelet activation and the production of PEVs. Buffy Coat (BC) prevents direct contact between platelets and the container during centrifugation and reduces platelet activation due to centrifugation. Therefore, there are fewer PEVs than platelet-rich plasma (PRP) PCs in the buffy coat (BC) and apheresis PCs (collected by the Trima device).^34,35 The purified platelets are suspended in plasma, platelet additive solution (PAS), or a mixture of both.^36–38 In addition to saving more plasma products, the use of PAS is also effective in reducing the side effects of plasma on patients; for example, the infusion of PAS-preserved PC results in a significant reduction in allergic reactions in patients.^36,38 In addition, the acetates contained in PAS, by reducing the level of lactic acid and producing bicarbonate, prevent the pH in the PC from falling and reduce the activation of platelets.^36,38 Studies have shown that PCs preserved in PAS contain fewer EVs than those preserved in plasma.³⁵ There are also slight differences in the number and size of PEVs due to the use of different PAS. Compared to PAS-B, the presence of phosphate (improves buffering capacity), potassium, and magnesium (reduces lactate production) in PAS-E further reduces platelet activation and the production of PEVs.³⁸ In addition, the diameter of PEVs in PAS-B increased with storage time, whereas the diameter of PEVs in PAS-E did not change significantly.³⁸

Shaking at room temperature can preserve platelet activity and function well, but also provides a favorable environment for bacterial growth.^39–41 Although platelets are screened before use, window period infections and emerging pathogens limit the accuracy of screening.⁴² Pathogen Inactivation Technology (PRT) inactivates pathogens by interfering with bacterial replication and protein assembly. Common pathogen reduction technology systems available today include Intercept® (amoxicillin and UVA combined), Mirasol® (riboflavin/vitamin B2 and UVA/UVB combined), and Theraflex® (UVC only). Although PRTs are effective in increasing the security of PCs and prolonging storage time, they can exacerbate PSL and promote the release of PEVs.^39,42 Furthermore, PRTs altered the microRNA content of PEVs.^43,44 The main effect of Mirasol® was a slight reduction in the levels of several microRNAs (e.g. hsa-miR-22-3p or hsa-miR-423-5p). Intercept® reduced the levels of some microRNAs (e.g. hsa-miR-185-5p) while increasing the levels of others (e.g. hsa-miR-451a). By transferring microRNAs, PEVs can alter the function of macrophages.²⁹ As hsa-miR-451a can downregulate macrophage migration inhibitory factor (MIF) and promote M1 and M2 polarization in macrophages, the transfer of PEVs containing this MiRNA may affect macrophage function in PC recipients.⁴⁴

It was once thought that cold storage (4°C) and cryopreservation (−80°C) caused dramatic changes in platelet morphology and function, resulting in reduced platelet survival and recovery rates after transfusion.^13,36,39 However, the corrected count increment (CCI) and platelet recovery rates are not fully representative of platelet function.² Recent studies have shown that the hemostatic efficacy of cold-stored PLTs (CCPs) and cryopreserved PLTs (CPPs) is comparable or superior to that of PLTs stored at room temperature.^45,46 This may be related to cold temperatures causing platelets to pre-activate and produce large amounts of PS+ PEVs.^47,48 The concentration of PEVs in CPPs components is 15 times higher than in platelets stored at room temperature and 10 times higher than in CCPs.^47,48 In contrast to PEVs from fresh platelets, CPPs had higher levels of PS+ PEVs and PEVs showed increased expression of several adhesion receptors (e.g. GPIIb, Gpix, and integrin-related proteins).^39,49 CCPs and CPPs are a good alternative to storing platelets at room temperature, not only reducing the risk of bacterial contamination but also preserving the clotting activity of the platelets.^13,36,39 However, due to the high cost, time, complexity, and need for further research, these methods are not yet widely available.³⁶

Studies have shown that PAS can reduce PSLs and platelet activation caused by PRTs and hypothermia.⁵⁰ PAS is also expected to reduce PEVs levels in PRTs and cryogenic-treated PCs, but further experimental analysis is required to prove this.³⁴ Due to several limitations, such as the large number of PEVs required to characterize EVs and the many drawbacks of the methods used to isolate and purify PEVs, most research only compares the number of PEVs in different preparation methods and does not characterize PEVs.^36,51

Separation and characteristic analysis of PEVs

Depending on their release mechanism, PEVs are commonly classified as exosomes, microparticles or microvesicles, and apoptotic bodies (EVs released by apoptotic platelets).⁵² Platelet exosomes (P-exos), with a diameter of about 40–100 nm, are formed by the inward budding of the intracellular granular membrane and released by the exocytosis of secretory granules; Microparticles (MPs), also known as microvesicles (MVs), with a diameter of about 100-1000 nm, are vesicles released by the budding of the cytoplasmic membrane.⁵³ The largest EVs, apoptotic bodies (ABs, 1–5 m in diameter), represented aggregates produced at the late stage of apoptosis, which is cleared by phagocytes immediately after production and regardless of intracellular communication.⁵⁴ When using ultracentrifugation to separate PEVs, 20000 g is used to obtain mainly large PEVs (>200 nm) and 100,000 g to obtain mainly small and medium PEVs (<200 nm).⁵⁵

Separation of PEVs

There is no optimal method to isolate, classify and characterize PEVs.⁵⁶ Common methods for the separation PEVs include ultrafiltration, Polymer precipitation, density gradient centrifugation, ultracentrifugation, asymmetric flow field-flow, size exclusion chromatography (SEC), and immune affinity (Table 1).⁵⁷ SEC has a recovery of up to 90%. Compared to ultracentrifugation, SEC is effective in preventing the aggregation of EVs and is better able to retain the integrity and biological function of PEVs.⁵⁸ Although asymmetric flow-field-flow fractionation is currently the best method for PEVs separation, the yield is poor and the process is very complicated ^[59]. Ultracentrifugation is regarded as the gold standard method for separating EVs populations, allowing for the removal of platelets while obtaining PEVs of moderate purity.⁵⁹ Immune affinity does not efficiently elute the entire EVs from the separation beads, but high purity and exosomes of specific origin are obtained.⁵⁵ It is possible to obtain purer PEVs using a novel method combining multiple separation methods. For example, a combination of size exclusion chromatography and ultracentrifugation has been used to obtain pure PEVs, but the concentrations have not reached the sample concentrations required to test the functional properties of PEVs.⁵⁸

Table I. Commonly used methods and differences in isolation of PEVs.

Strategy	Yield	purity	advantage	limit	references
Ultrafiltration	Medium	Medium	Easy to use Without special equipment and reagents	Possible loss due to clogging and membrane trapping Often used with other centrifugation methods	^Citation60
Polymer precipitation	High	low	Easy to use Avoiding damage to vesicles Suitable for both small and large sample volume	Potential contaminants (co-purifying protein aggregates or residuary polymers)	^Citation61
Density gradient centrifugation	Low	High	Avoiding damage to vesicles Allowing separation of the subpopulation of vesicles	High equipment requirement lengthy duration The potential to damage vesicles by spinning at high speeds Small sample volumes are not suitable	^Citation62
Ultracentrifugation	Low	medium	Current gold standard Suitable for large-volume samples relatively cheap	High equipment requirement lengthy duration The potential to damage vesicles by spinning at high speeds Small sample volumes are not suitable	^Citation59
Size exclusion chromatography (SEC)	High	High	99% of soluble plasma protein and > 95% of HDL can be removed Avoiding the aggregation of vesicles Keep the native state of vesicles [62]	Relatively high device costs lengthy duration Potential lipoprotein contamination	^Citation58
Asymmetric flow field- flow fractionation	Low	High	broad separation range wide variety of eluents	lengthy duration fractionation equipment required Operation is complex Few vesicles in each part	^Citation59,Citation63
Immune affinity	low	High	The high specificity of vesicles subtype isolation	Expensive Eluting EVs may damage vesicles Not suitable for large sample volume	^Citation55

Characteristic analysis of PEVs

Microstructures of PEVs were usually observed under Low Voltage Scanning Electron Microscopy (LVSEM), confocal microscope, atomic force microscope (AFM), transmission electron microscope (TEM), and cryo-electron microscopy (cryo-EM).⁶⁴ Cryo-EM is currently the most advanced method for PEV characterization. Under the observation of cryo-EM, there were three main forms of PEVs released by platelet activation: spherical EVs (50 nm-1 μm in diameter); tubular EVs (1–5 μm in length); and membrane fragments that are neither circular nor tubular.⁶⁵ The quantification and characterization of PEVs were mainly performed using flow cytometry (FC), nanoparticle tracking analysis (NTA), and tunable resistance pulse sensing (TRPs).⁶⁶ Flow cytometry is the gold standard for the detection and characterization of PEVs and is widely used to identify and quantify PS, CD41, and CD62P on the surface of PEVs.⁶⁷ Based on lipid-specific fluorescent probes and light-scattering event detection, the high-sensitivity FC (hs-FC) can detect vesicles down to 60 nm.⁶⁸ In contrast to conventional flow cytometry, NTAs can not only detect EVs smaller than 300 nm in diameter, but also analyze the concentration, refractive index, and size of individual EVs suspended in liquid.⁵⁵ TRPS can accurately measure the size, count, and concentration of EVs and analyze the zeta potential of individual EVs by detecting the resistance pulses of particles passing through the nanopore.⁶⁹ Furthermore, TRPS can distinguish between different types of EVs within a single experiment by tuning the size and conductivity properties of the nanopores.⁷⁰ However, the separated samples need to be pure as NTA and TRP cannot distinguish between PEVs and impurities.⁷¹

In addition to the expression of EVs surface markers (CD9, CD63, TSG101, ALIX), PEVs also express PECAM-1 (CD31), glycoprotein GPIIb/IIIa (CD41/CD61), GPIba (CD42b), GPIaIIa(CD49b/CD29), p-selectin (CD62P), platelet factor 4 (PF4), and phosphatidylserine (PS), which are characteristic of platelets.⁷² According to the recommendations of The Minimum Information for Extracellular Vesicle Studies 2018 (MISEV2018) guidelines, two requirements are needed to characterize EVs: 1. At least three positive protein markers, including ≥ 1 transmembrane/GPI-anchored protein and ≥ 1 cytosolic protein; 2. At least one negative marker.⁷³ As transmembrane proteins, CD9 and CD63 can prove the existence of lipid bilayer of EVs.⁷⁴ CD41b, CD42a, and CD62P can be used as platelet-specific markers.⁷⁵ PF4 is a specific cytoplasmic protein synthesized by platelet alpha-granules and can be present in PEVs⁷⁶. In addition, apolipoproteins A1/2 and B or albumin may be used as a negative control to assess the purity of PEVs.⁷⁵ It is generally believed that P-exos do not expose PS but selectively enrich for the exosome-specific marker CD63, whereas PMVs usually expose PS on its outer surface and contain coagulation-related factors (e.g., FX, prothrombin, etc.).^77,78 In addition, P-exos are rich in protein derived from platelet alpha-granules, while PMVs contain many lipid mediators, mitochondrial proteins, and proteasomes.^29,78,79 High-sensitivity flow cytometry showed a large increase in the number of PEVs during storage, which may have important implications for the transfusion therapy of platelets and is worthy of our attention.⁶⁸

PEVs have an important role in platelet transfusion therapy

The main role of platelet transfusions is to prevent and treat bleeding.⁸⁰ However, platelets also contain a variety of inflammation factors involved in the inflammatory response¹³ and contain molecules related to the regenerative processes promoting angiogenesis as well as tissue regeneration.⁸¹ Therefore, platelet transfusion therapy may have both positive and negative effects on patients, and PEVs may be a major contributor to these effects.⁸²

PEVs as a biomarker of platelet storage lesions

The number of platelets does not change much during PLT storage, but great changes take place in morphology, function, and metabolism.⁸³ The expression of P-selectin (CD62P) is considered to be the gold standard for assessing platelet function and its expression level is negatively correlated with the degree of platelet function loss and the PEVs levels.⁸⁴ Doubling of the number of PEVs in the PC at the end of storage and a significant decrease in platelet surface CD62P expression.^27,83 Therefore, pretransfusion testing for PEVs in platelet products may help prevent patients from ineffective transfusions due to the introduction of severely compromised platelets.

PEVs exert the hemostasis and coagulation function of PCs

In vivo and in vitro experiments, Well-characterized PEVs have strong procoagulant activity.⁸⁵ Platelet-enriched plasma (PEP), derived from platelet-rich plasma (PRP), contains high levels of procoagulant PEVs that retain their function after freeze-thaw cycles.^85,86 PEVs express TF, PS, and GPIIb/IIIa (CD41/CD61), and contain thrombospondin complex and alpha granule-derived factors V and X. Coagulation factor VII binds to TF on the surface of PEVs and activates the coagulation cascade¹¹; PEVs (PS+) provide a negative catalytic surface for coagulation and promote the formation of tenase complexes (FVIIIa and FIXa) and prothrombinase (FVa and FXa).⁸⁷ In addition, PEVs (CD61+) are not only able to attach to fibrin fibers directly, but also enhance the rapid production of thrombin to support the formation of fibrin clots.⁸⁸ Therefore, PEVs (mainly PMVs play the role) can enhance induced hemostasis.⁸²

Even though the surface area of PEVs is approximately 100 times smaller than that of platelets, when thrombin generation and spatial clot growth parameters were measured in ultra-free plasma supplemented with different concentrations of activated platelets or PEVs (activated by the calcium ionophore A23187), the procoagulant activity of individually activated platelets was found to be almost identical to that of individual PEVs.⁸⁹ This suggests that the coagulation capacity of the PEVs activated by calcium ionophore A23187 is around 50–100 times greater than that of activated platelets.⁸⁹ In vitro studies have shown that the procoagulant effect of platelet-free plasma increases with increasing concentration of added PEVs (produced during storage).¹⁰ Furthermore, injection of PEVs isolated from three-day stored de-leukocyte platelet plasma into a mouse bleeding model can significantly reduce hemorrhage.¹⁰ Another study showed that PEVs provide hemostasis by enhancing hemodynamic stability in a mouse model and preventing ischemia from developing.⁸²

PEVs are involved in adverse transfusion reactions

PEVs may play a pro- or anti-inflammatory role in inflammation, according to the expression of surface markers, the release of cargo, and the influence of the microenvironment and receptor cells.⁸² On the one hand, PEVs contain various BRMs and surface receptors (e.g. CD62p and CD61) to promote monocyte adhesion and activation.^8,13 On the other hand, some studies have shown that PEVs can suppress inflammation by inhibiting the activation of macrophages and dendritic cells⁸² and reducing the production of inflammatory cytokines such as TNF-α (produced by macrophages and dendritic cells) and IL-8 (produced by dendritic cells).^90–92

Most studies show that PEVs have pro-inflammatory properties and are involved in adverse platelet transfusion reactions.⁸² PEV in PC express the activation markers CD62P and CD61, which bind to P-selectin glycoprotein ligand-1 (PSGL-1) and Mac-1 expressed on the neutrophil surface, activating neutrophils to form filamentous meshwork structures (neutrophil extracellular traps, NETs) involved in the inflammatory response.^11,13 Many BRMs in PEVs are also associated with inflammation, such as PEVs contain platelet-derived CD40L (CD154), which mediates TRALI (an acute lung injury).^11,15 In addition, PEV transports platelet-derived mitochondria.⁷ Circulating mitochondrial DNA (mtDNA) is an inflammatory factor that is part of the DAMPs and is associated with adverse blood transfusion reactions.⁷ Compared to plasma and RBC products, PCs often contain higher levels of mtDNA, which may explain the much higher incidence of non-hemolytic transfusion reactions with PC transfusions than with red blood cell (RBC) transfusions.^11,93

PEVs lead to poor prognosis of platelet transfusion in cancer patients

Thrombocytopenia is common in cancer patients.⁹⁴ On the one hand, the tumor can infiltrate the body’s hematopoietic organs (bone marrow, spleen), directly leading to thrombocytopenia.⁹⁴ On the other hand, platelet counts are significantly reduced in oncology patients after prolonged chemotherapy.²³ Platelet transfusion significantly reduces the risk of bleeding due to thrombocytopenia in cancer patients and is an irreplaceable means of immediate hemostasis.⁹⁵ However, studies have shown that activated platelets promote the proliferation and metastasis of cancer cells and that cancer cells also activate platelets—a mutually reinforcing vicious circle that may lead to platelet transfusions being highly detrimental to the prognosis of cancer patients.²³ PEVs contain molecules from alpha-granules like platelet-derived growth factor, vascular endothelial growth factor (VEGF), and fibroblast growth factor, which have pro-angiogenic properties.²⁹ MiRNAs in PEVs are involved in angiogenesis (e.g. miRNA Let-7 a) and tumor growth (e.g. miR-126 and miRNA-223).^29,96

Long-term platelet transfusions in cancer patients may be harmful due to the propensity of PEVs to induce thrombosis and support tumors.⁹⁷ Firstly, PEVs suppress immune defense in tumor patients. The miR-183 in PEVs transferred into tumor-associated NK cells inhibits their membrane protein DAP12 activity and suppresses their cytolytic function⁹⁸; The PS receptor (PSR) on immune cells can specifically recognize PS+ PEVs, leading to the innate and adaptive immune systems becoming inhibited in the tumor microenvironment (TME).⁹⁹ Moreover, PEVs promote tumor cell proliferation by activating mitogen-activated protein kinase (MAPK), enhance tumor cell invasion by inducing the secretion of matrix metalloproteinases (MMPs) and activating cyclin D2, and accelerate tumor metastasis by inducing epithelial-mesenchymal transition (ETM).^100,101 In addition, PEVs induce the production of extracellular vesicles (TEVs) in tumor cells, and cancer cells can also stimulate platelet activation to produce PEVs, as TEVs initiate thrombin production.^29,102 Therefore, giving platelet transfusion to oncology patients to reduce the risk of bleeding requires more prudent decision-making, as both platelets and PEVs may increase the progression of cancer.

Discussion

The known mechanisms by which PEVs contribute to platelet transfusions are summarized in Figure 3. Although the procoagulant activity of PEVs was much higher than that of activated platelets, platelet concentrate storage time was negatively associated with clinical efficacy outcomes, which suggests that the effect of platelet transfusion is not better with the increase of PEVs. However, it is interesting to note that in vitro studies have shown that platelet-enhanced plasma, obtained by removing platelets from platelet products, has a better hemostatic effect than platelet-rich plasma, suggesting a role for PEV in promoting coagulation.⁸⁵ We speculate that activated platelets whose morphology and function have changed during platelet storage may interfere with the function of PEVs. PEVs have platelet properties and can retain their function after freeze-thaw cycles.¹⁰ Therefore, in acute situations (e.g. trauma), infusion of PEVs has the potential to be an effective treatment alternative to platelet transfusion. As the difficulty and low purity of PEVs isolation and extraction from stored platelets, it is necessary to further improve the method of isolation and purification of PEVs and to further study their effects on human hemostasis and adverse reactions to blood transfusions.

Figure 3. PEVs contributions in platelet transfusion therapy. (a) PEVs as a biomarker of platelet storage lesions. With the prolongation of storage time, the number of platelets does not change much, but the amount of PEVs in PCs is doubled, and the level of CD62p (the gold standard for the evaluation of PLT function) expression on platelet surface is negatively correlated with PEVs level; (b) PEVs can facilitate their adhesion to leukocytes, then several BRMs produced by PEVs and neutrophil extracellular traps (NETs) formed by activated neutrophils are associated with the inflammatory response; (c) PEVs can inhibit the innate and adaptive immune response in the tumor microenvironment (TME). What’s more, PEVs could promote the proliferation, invasion, and metastasis of tumor cells; (d) PEVs can promote hemostasis and coagulation. Factor VII on the PEVs surface combines with TF to activate the coagulation cascade; PS on the PEVs surface provides a catalytic surface for coagulation and promotes prothrombin and tenase complex formation; and CD61 on the PEVs surface promotes fast thrombin generation and assists in fibrin clotting.

Platelet has been suggested as a multifunctional immune blood cell, adverse progress of cancer patients and adverse transfusion reactions are related to PEVs produced by platelet storage lesion. Transfusion of platelets with short storage time, leucocyte-depleted platelets, and platelet storage in platelet additive solutions (PAS) may reduce the incidence of adverse transfusion reactions.^103–105 Cancer patients’ overall survival could be significantly improved by washing platelets to remove cytokines and PEVs before blood transfusion.²³ Therefore, cancer patients should be advised to transfuse leukocyte-depleted platelets with a short storage time. Further work is needed to demonstrate how platelets and PEVs affect the prognosis of cancer patients.

Authors contributions

ZC wrote the manuscript. JYF and ND drew the figures. PZ, YSH, and HWZ revised the paper. All authors read and approved the final manuscript. Data authentication is not applicable.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the University Sponsored Research Program of Southwest Medical University [Grant Number: 2019ZQL173]