key: cord-1018085-b11mhono authors: Puhm, Florian; Flamand, Louis; Boilard, Eric title: Platelet extracellular vesicles in COVID‐19: Potential markers and makers date: 2021-11-03 journal: J Leukoc Biol DOI: 10.1002/jlb.3mir0221-100r sha: 4402f7eb8b3888d3f8f2bdad695430310e9f65ea doc_id: 1018085 cord_uid: b11mhono Platelets and platelet extracellular vesicles (pEV) are at the crossroads of coagulation and immunity. Extracellular vesicles are messengers that not only transmit signals between cells, but also provide information about the status of their cell of origin. Thus, pEVs have potential as both biomarkers of platelet activation and contributors to pathology. Coronavirus Disease‐19 (COVID‐19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), is a complex disease affecting multiple organs and is characterized by a high degree of inflammation and risk of thrombosis in some patients. In this review, we introduce pEVs as valuable biomarkers in disease with a special focus on their potential as predictors of and contributors to COVID‐19. Extracellular vesicles (EV) are small membrane-bound vesicles that contain molecules from their cell of origin. As EVs can be internalized by cellular recipients, they are suggested to mediate cellular signaling. 1, 2 The two most described EV-subtypes are microvesicles and exosomes. 3 Microvesicles are produced by plasma membrane budding and shedding, have a diameter ranging from approximately 100 to 1000 nm 2,4-6 and generally expose phosphatidylserine (PS) although there are exceptions. [7] [8] [9] [10] They bud from cells activated by numerous inflammatory triggers. 11 Exosomes are typically smaller than microvesicles 9 and are released by cells from multivesicular bodies in an exocytosis-dependent mechanism. 6, 12, 13 Platelets are anucleated cell fragments with a diameter of 1 to 3 μm and are produced by megakaryocytes. [14] [15] [16] They prevent bleeding and interact with pathogens and immune cells thereby assisting immune responses. 15, 16 Platelets and megakaryocytes are the major sources of circulating EVs. [17] [18] [19] Similar to platelets, platelet EVs (pEVs) were first recognized as procoagulant entities. [20] [21] [22] However, their roles F I G U R E 1 SARS-CoV-2 infection of lungs and subsequent damage may activate cells in the blood and induce platelet activation, aggregation, and extracellular vesicle release. Extracellular vesicles carry a diverse array of signaling molecules that can influence immune responses and coagulation differential centrifugation. Subsequent studies and electron microscopic analysis identified two main classes of vesicles released from cells: exosomes and microvesicles. 13, [38] [39] [40] During the last 20 years, technological progress has transformed the study of EVs. Advances in flow cytometry-the most commonly used method to detect and quantify EVs-have enabled the analysis of EVs at a higher resolution than ever before, further revealing EV complexity. EVs have historically been categorized into major subclasses such as "microvesicle/microparticle" and "exosomes." However, these narrow definitions have become problematic. Indeed, the vocabulary and methodology describing EVs have expanded at a rapid pace and may lead to confusion upon retrospective examination of EV studies. 3, 41, 42 Therefore, the International Society of Extracellular Vesicles (ISEV) recommends the use of the umbrella term "extracellular-vesicle (EV)" unless specific investigations permit to determine whether EVs were liberated from the plasma membrane by budding or implicated exocytosis, and to include a detailed description of the isolation and detection methods used in the study of EVs. 3, 41, 42 Notably, interpretations of historical studies may have changed with advancements in the technologies and methods used to detect EVs. A common discrepancy is the concentration of EVs per micro-liter in healthy plasma, with a reported concentration of 200 up to 10 9 EVs/μL, which likely depends on the isolation and detection techniques used. 19, 43 The pEV concentration in healthy plasma has been conservatively estimated at around 11,500/μL by cryo-electron microscopy. 19 The most commonly used methods of EV isolation and detection are differential centrifugation and flow cytometry, respectively. However, common pitfalls associated with these techniques are the risk of coisolation and detection of EVs and lipoproteins, the potential of overlooking particularly small EVs due to insufficient resolution, and the risk of damaging EVs during isolation at high centrifugal forces. 43 25, [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] The presence of pEVs is documented in synovial fluid in rheumatoid arthritis, 26 and increased levels of circulating pEVs correlate with disease activity. 56 Moreover, an increase in pEV concentrations was found in lymph in murine models of atherosclerosis and autoimmune arthritis. 24 -27 The number of pEVs is increased in blood in systemic lupus erythematosus (SLE), and higher levels are suggested to associate with declining kidney function. 45 In addition to pEV concentration, pEV content can be used as a biomarker. The protein composition of EVs in disease can be distinguished from that of EVs in healthy controls. For instance, as activated platelets can translate and produce interleukin-1 (IL-1), this cytokine can be packaged into pEVs, which can augment inflammation. 26, 57 Lipid mediators of inflammation, such as prostaglandins and leukotrienes, can also be transported or generated by pEVs given the latter's content of enzymatic machinery and fatty acids. 58 EVs can be released from activated and dying cells, and may therefore, carry self-antigens and damage-associated molecular patterns (DAMPs). Such EVs could have a role as potential biomarkers and may contribute to disease. Indeed, pEVs are associated with autoantibodies in SLE, which suggests that they bear autoantigens and may facilitate cellular activation through activation of Fc receptors. [59] [60] [61] Moreover, platelets and their pEVs can contain DAMPs such as high-mobility group protein 1 (HMGB1), 62 S100A8/9, 63,64 and mitochondrial DAMPs. 59 Another prominent DAMP that can be found on EVs are oxidationspecific epitopes. 65 The latter result from oxidation of polyunsaturated fatty acids and are commonly found on oxidized low-density lipids 46 and in acute coronary syndrome, 48 and as such are likely indicative of tissue damage. pEVs may be useful biomarkers in cancers associated with thrombotic risks and may enhance metastasis. [52] [53] [54] Of particular interest are prostate cancer cells, which are reported to release tissuefactor (TF)-associated EVs (TF + EV). 67 TF is the main initiator of coagulation, 68 and cancer cell TF + EVs may thereby induce platelet activation, 68 leading to the release of pEVs. In the case of coagulation initiated by cancer, TF + EVs are more likely the causative agents in this pathology as opposed to pEVs. However, detection of TFprotein expressed on EVs (TF + EV) is notoriously difficult and generally only achieved through indirect determination of TF-activity. 69, 70 Considering these challenges, pEV-quantification is a potential surrogate marker of TF-activity and platelet activation in cancer and other diseases. Table 1 provides an overview of the different techniques used to identify platelet EV and other blood-borne EV in the literature cited in this section. As indicated in Table 1 , pEVs are most commonly isolated from platelet-poor or platelet-free plasma obtained by differential centrifugation, subsequently labeled for platelet-specific markers (primarily CD41) and detected by flow cytometry. Ideally, it is recommended to perform a two-step centrifugation protocol on whole-blood to obtain platelet-free plasma. 44 Plasma prepared this way can be frozen and stored for long-term. 44 While pEVs have been most extensively studied as biomarkers in noninfectious diseases, pEVs are also found in association with viral infections. [49] [50] [51] 71, 72 Influenza virus H1N1 activates platelets and induces the release of pEVs by a mechanism that implicates thrombin and the activation of FcgRIIa by antibodies directed against this virus. 49 pEVs may contribute to the propagation of HIV, as they can transport C-X-C chemokine receptor type 4 (CXCR4), a coreceptor for HIV, to other cells. 50 In dengue virus infection, pEVs may be released in a Ctype lectin-like receptor 2 (CLEC−2)-dependent manner by platelets 51 and thereby show potential as biomarkers of disease severity 71 by distinguishing between patients who may or may not require platelet transfusion. 72 pEVs in infections and sepsis are discussed in more detail elsewhere. 73 Several studies point to EVs and pEVs as potential biomarkers in COVID-19. 33, 36, 37 Increased levels of circulating pEVs have been observed in patients with SARS-CoV-2 infection. 33, 37 One particular study reported higher numbers of circulating pEVs in patients with nonsevere and severe COVID-19 in comparison with healthy individuals after determining pEV levels in platelet-free plasma. 33 It is intriguing that the increase in pEVs was less pronounced in patients with severe disease relative to those with nonsevere disease. 33 Normalization of pEV numbers to platelet counts still revealed significantly increased levels of pEVs in severe COVID-19, pointing to increased EVbiogenesis during COVID-19. 33 Cappellano et al. 37 It is interesting to note that we and others observed that absolute numbers of pEVs 33, 36 and PS-exposing (PS+) pEVs 33 were significantly lower in severe COVID-19 when compared with nonsevere COVID-19. 33,36 PS + EVs are considered procoagulatory since they provide a negatively charged surface for initiation and maintenance of coagulation. 74 Notably, a decrease in PS+ EVs has also been reported in multiple organ dysfunction syndrome and sepsis, which might point to common mechanisms in these diseases. 75 COVID-19 is indeed a complex disease affecting many organs distant from the lungs and is Refs. Arthritis [25] Lymph (Mouse) Flow-Cyt., Cryo-EM Arthritis [26] Synovial Fluid Arthritis [27] Blood, Synovial Fluid Flow-Cyt., EM, NTA, MassSpec Arthritis [47] Blood, Platelets, Megakaryocytes Arthritis [56] Blood 1550 × g, 20 min, RT Atherosclerosis [24] Lymph (Mouse) Flow-Cyt., Cryo-EM Cancer (Gastric) [52] Blood 1550 × g, 15 min, RT Cancer (Prostate) [53] Blood 2 × 28,00 × g, 15 min, RT Cancer (Breast) [54] Platelet Concentrates Flow-Cyt. Myocardial Infarction [46] Blood Plasma → 2500 × g, 10 Refs. Multiple Sclerosis [55] Blood 160 × g, 10 min, RT Flow-Cyt. SLE [45] Blood characterized by a high degree of thrombosis. Thus, the observed decrease in PS+ pEVs may suggest increased consumption of such EVs in patients with more severe disease as PS is also an "eat-me" signal for cellular removal. 33, 36, 76 In summary, PS+ pEVs may be a biomarker in COVID-19 that distinguishes different stages of disease activity, especially with regard to coagulation and organ damage. [30] [31] [32] by platelet TLR3 86 is also known. TLR3 activation has been associated with EV release by different cell types, 87-89 but has not been described for platelets. Moreover, activation of TLR3 and TLR7 does not induce typical platelet activation, such as granule content release and exposure of activated GPIIbGPIIIa, as seen in response to thrombin stimulation. These interactions may be more similar to the priming or sensitization effect of TLR2 and TLR4. 77, 90 Of note is that SARS-CoV-2 RNA has been found in association with platelets in some COVID-19 patients. 33, 34 Thus, TLR3 and TLR7 may be attractive targets for SARS-CoV-2 platelet interactions and priming of platelets in COVID-19. In a recent study, 91 Detection EV-quantification [33] Blood 200 × g , 15 min → 1000 × g, 10 min → EV in SUP CD41 (AnV: not-platelet specific) Flow-Cyt. EV-quantification and EV-TF-activity [36] Flow-Cyt., Other: TF-activity assay EV-quantification [37] Blood EV were analyzed directly in whole-blood CD41 (CD31, Phalloidin: not-platelet specific) Flow-Cyt. Abbreviations. AnV, Annexin-V; Flow-Cyt., flow cytometry; RT, room temperature; SUP, supernatant; TF, tissue factor. stimulation of TLR9 (recognizes unmethylated CpG DNA) induced oxidative stress in platelets, it also enhanced pEV release when platelets were activated by immune complexes recognized by FcγRIIa. 59 Moreover, antibody-mediated recognition of SARS-CoV-2 via FcγRIIa may play a role in platelet activation and pEV-release, as has been shown for H1N1. 49 Protein in the envelope of viruses are often heavily glycosylated, which may aid in the evasion of adaptive immune responses. 93 Indeed, the SARS-CoV-2 spike (S) protein has multiple glycosylation sites 94 that may be relevant to its function and interactions with target cells. In multiple variants of SARS-CoV-2, mutations to glycosylation sites affect infectiveness. 95 Of note, is that platelets express CLEC-2 (C-type lectin-like type II) and DC-SIGN, 96 which are relevant in HIV-1 and dengue virus infections 51, 96 and platelets may release EVs in a CLEC-2-dependent manner. 51 The primary receptor for SARS-CoV-2 in humans is ACE2 (Angiotensinconverting enzyme 2), 97, 98 which is ubiquitously expressed by type II epithelial cells in the upper and lower respiratory tract. 99 The typical cellular entry route for SARS-CoV-2 is engagement with ACE2 via the Spike protein, subsequent cleavage of the latter by the serine protease TMPRSS2 to enable fusion of the viral and cellular membrane resulting in infection of the target cell. 100 While expression of ACE2 has been shown for vascular endothelial cells and lung macrophages, 99 physiological expression by platelets or megakaryocytes is controversial. 101 Moreover, Koupenova et al. 91 recently reported that SARS-CoV-2 may be taken up by platelets through both ACE2-dependent and independent mechanisms. Cluster of Differentiation 147 (CD147) has been proposed as an alternative receptor for SARS-CoV-2, 102 is commonly expressed in circulating cells, and is associated with risk factors of severe COVID-19 such as obesity, asthma, and chronic obstructive pulmonary disease (COPD). 102,103, CD147 is indeed functionally expressed on platelets, 104,105 but its relevance to SARS-CoV-2 infection has been called into question. 106 Other intriguing targets for direct SARS-CoV-2 platelet interaction are the integrins. Platelet integrin GPIIbGPIIIa binds the three amino acid motif Arg-Gly-Asp (RGD) present in physiological ligands (e.g., fibrinogen, von Willebrand Factor), which is crucial for platelet aggregation. 107, 108 Platelet integrin GPIIbGPIIIa is important in platelet responses and is implicated in pEV-release triggered by FcγRIIA or GPVI receptor activation. 25,26, Furthermore, the Spike protein sequence of SARS-CoV-2 contains an RGD-motif (403-405: Arg-Gly-Asp) within the receptor-binding domain. 109, 110 Thus, platelet integrin GPIIbGPIIIa presents another alternative target receptor for SARS-CoV-2. Platelets may not be stimulated to release pEVs solely upon direct interaction with SARS-CoV-2. Indeed, excessive inflammatory responses and tissue damage, particularly, in the lung and lung microvasculature, are observed in COVID-19 patients. 111, 112 Of interest is that autopsies of 21 patients 113 revealed inflammatory damage and microthrombi in multiple organs (lungs, heart, liver, kindeys, brain), while SARS-CoV-2 infected cells were absent from most of the affected tissues. Notably, SARS-CoV-2 viral RNA copies in a range of 63 to 6,310 copies per milliliter of blood have been detected in a quarter of hospitalized COVID-19 patients. 114 Given that the concentration range of platelets in blood is 150 × 10ˆ8 to 450ˆ8 per mL of blood, platelets would outnumber virus by a factor of 23,771 to 7.14 × 10ˆ6. This would make direct interactions of SARS-CoV-2 with platelets rare events, although the amount of SARS-CoV-2 in the blood may be underestimated viral as RNA may have been degraded. 91 Dissemination of SARS-CoV-2 throughout the circulation is not excluded, but may not be the primary path taken by the virus to affect platelet function and pEV release. As discussed by Chen et al., 115 and nearby vasculature (endothelial cells) causes the production of inflammatory cytokines contributing to an immune response leading to tissue damage. 116, 117 At the same time, damage to tissue may lead to the liberation of DAMPs, including, but not limited to, mitochondria and mitochondrial components and oxidized phospholipids, and TF associated with EVs, which would further amplify the inflammatory and coagulation cascade that stimulates platelet activation and subsequent pEV release. Moreover, the overwhelming inflammation may affect endothelial barrier integrity and thereby lead to increased expression of soluble thrombomodulin, soluble P-selectin, and von Willebrand factor. 117 Indeed, elevated levels of TF activity associated with EVs have been reported in COVID-19, which may directly contribute to excessive coagulation. 36, 70, 118 In addition, ACE2, the primary receptor for SARS-CoV-2 infection in humans, is important in the regulation of the renin-angiotensin-aldosterone system and a deficiency of ACE2 is linked to enhanced risk of inflammation and thrombosis. 98 As COVID-19 is increasingly viewed as a thromboinflammatory disease, 32 it is conceivable that platelet activation in COVID-19 may be a consequence of the inflammation, organ damage, and pathological activation of the coagulation cascade, rather than a consequence of direct virus-platelet interaction. Moreover, at later stages of the disease, secondary effects caused by the tissue damage and inflammatory response, as opposed to viral presence, might become decisive in determining disease outcome. Thus, changes to the presence and activity of pEVs may represent potential risk markers, as in other diseases. 25, [45] [46] [47] [48] [49] [50] [51] 119 The first descriptions of EVs referred to their procoagulant abilities, which are primarily attributed to exposure of the negatively charged phospholipid, PS. 120-122 PS exposure on platelets and pEVs supports propagation of coagulation. [120] [121] [122] In COVID-19, studies report the presence of antiphospholipid antibodies, such as anticardiolipin antibodies, similar to those seen in antiphospholipid syndrome and SLE. [123] [124] [125] [126] These antibodies may target PS-exposing pEVs, thereby, forming immune complexes for cellular activation through Fc receptors. Negatively charged surfaces may also initiate the "intrinsic pathway" of coagulation. 121 However, the main cellular initiator of coagulation is TF. 121 While some procoagulant activity of circulating pEVs has been associated with TF in the past, 127 it has been suggested that platelets may acquire TF from other cells via TF-expressing EVs in a P-selectin glycoprotein ligand-1-dependent manner. 128 However, expression of TF by platelets and pEVs is controversial and direct detection of TF displayed by EVs is challenging. 69 The concentration of TF may be below the detection limit of flow cytometric approaches, but may be sufficiently high to initiate coagulation as concentrations as low as 20 fM suffice to initiate coagulation. 129 Furthermore, TF may be present in an inactive ("encrypted") or active ("decrypted") state. 121 Therefore, it is necessary to determine TF biological activity to confirm its role in a biological process. Given the high prevalence of thrombosis in COVID-19, 30-32 TF activity associated with EVs and its involvement in COVID-19 pathology is of high interest. 36, 70, 118, 130 Several studies 36, 70, 118, 131 report a significant increase in TF activity associated with EVs from COVID-19 patients when compared with healthy controls. Moreover, EV-TF activity was significantly higher in COVID-19 than in sepsis. 36 33 However, the procoagulant activity of EVs was increased in COVID-19, and was thought to depend on the expression of active TF given that the fibrinolytic activity of EVs remained unchanged in these patients. 36 A recent interesting in vitro study by Wang et al. 130 found that infection of human monocytederived macrophages with SARS-CoV-2 spike protein pseudovirus increased TF activity at the cell surface and stimulated the release of EVs with associated TF activity. 130 This study suggests that TF activ-ity depended on "decryption" (activation) of TF by hydrolysis of sphingomyelin via acid sphingomyelinase (ASMase), and not on increased TF protein expression or externalization of PS. 130 This lends further support to a role for SARS-CoV-2 in the "decryption"' of TF and the subsequent release of EVs with associated, active TF. 130 These observations are of clinical importance given the low femtomolar concentrations of EV-associated TF activity reported in COVID-19 patients 36 that are nevertheless sufficient to predict an increased thrombotic risk. The absolute quantification of TF + EVs, or identification of the cellular origin of TF-exposing EVs is challenging and requires careful study. Thus, the distinction of "decrypted" from "encrypted" TF in association with EVs, by the quantification of TF activity, may be a more relevant risk marker in COVID-19. EVs have attracted interest as biomarkers and players in COVID-19 pathology. As COVID-19 is a complex disease affecting multiple organs and is characterized by a high degree of thrombosis, the study of FP wrote the manuscript. EB supervised and reviewed the manuscript. LF reviewed the manuscript. 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FP is recipient of a Postdoctoral fellowship from FRQS. EB is recipient of senior award from the Fonds de Recherche du Québec en Santé (FRQS).