Platelet-derived Microparticles Research Articles

Differences in coagulation-relevant parameters: Comparing cryoprecipitate and a human fibrinogen concentrate.

Variable fibrinogen content within cryoprecipitate makes accurate dosing challenging in patients with coagulopathic bleeding, in addition to pathogen transmission risks associated with its administration. Purified and standardized human fibrinogen concentrates (HFCs) represent reliable alternatives. Full cryoprecipitate characterization is required to inform selection of an appropriate fibrinogen source for supplementation therapy. Extended biochemical comparison of pooled cryoprecipitate and HFC (Fibryga, Octapharma) was performed using commercially available assays to determine levels of variability in cryoprecipitate and HFC. In addition to standard procoagulant factors, measurements included activities of platelet-derived microparticles (PMPs) and plasminogen, and levels of fibrin degradation products. Cryoprecipitate contains lower fibrinogen levels than HFC (4.83 vs.19.73 g/L; p<0.001), translating to approximately half the amount of fibrinogen per standard cryoprecipitate dose (two pools, pre-pooled from five donations each) vs. HFC (2.14 vs. 3.95 g; p<0.001). Factor XIII (FXIII) levels were also lower in cryoprecipitate vs. HFC (192.17 vs. 328.33 IU/dL; p = 0.002). Levels of procoagulants in cryoprecipitate, such as von Willebrand Factor (VWF) and factor VIII (FVIII), were highly variable, as was PMP activity. A standard cryoprecipitate dose contains significantly higher levels of measured plasminogen and D-dimer fragments than a standard HFC dose. The tested HFC is a more reliable fibrinogen and FXIII source for accurate dosing compared with cryoprecipitate. Cryoprecipitate appears considerably less predictable for bleeding management due to wide variation in pro- and anticoagulation factors, the presence of PMPs, and the potential to elevate VWF and FVIII to prothrombotic levels.

Pro-Tumorigenic and Thrombotic Activities of Platelets in Lung Cancer.

Aside from their key protective roles in hemostasis and innate immunity, platelets are now recognized as having multifaceted, adverse roles in the pathogenesis, progression and outcome of many types of human malignancy. The most consistent and compelling evidence in this context has been derived from the notable association of elevated circulating platelet counts with the onset and prognosis of various human malignancies, particularly lung cancer, which represents the primary focus of the current review. Key topics include an overview of the association of lung cancer with the circulating platelet count, as well as the mechanisms of platelet-mediated, pro-tumorigenic immunosuppression, particularly the role of transforming growth factor beta 1. These issues are followed by a discussion regarding the pro-tumorigenic role of platelet-derived microparticles (PMPs), the most abundant type of microparticles (MPs) in human blood. In this context, the presence of increased levels of PMPs in the blood of lung cancer patients has been associated with tumor growth, invasion, angiogenesis and metastasis, which correlate with disease progression and decreased survival times. The final section of the review addresses, firstly, the role of cancer-related platelet activation and thrombosis in the pathogenesis of secondary cardiovascular disorders and the associated mortality, particularly in lung cancer, which is second only to disease progression; secondly, the review addresses the potential role of antiplatelet agents in the adjunctive therapy of cancer.

In-vivo functions and regulation of polyphosphate in the vascular system.

Polyphosphate, an inorganic polymer consisting of linearly linked phosphate subunits, is ubiquitously found in living organisms. Functions and regulation of the polymer have been analyzed in plants, bacteria and yeast; however, the roles of polyphosphate in mammals are still emerging. In contrast to synthetic polyphosphate that has been extensively utilized in ex-vivo studies, natural polyphosphate is complexed with bivalent cations (mostly Ca2+) and regardless of chain length, forms microparticles that are retained on the surface of procoagulant platelets, platelet-derived microparticles and cancer extracellular vesicles. On cell surfaces, these Ca2+/polyphosphate aggregates initiate the factor XII-driven contact system, triggering proinflammatory and procoagulant reactions through the kallikrein kinin system and intrinsic pathway of coagulation, respectively. Polyphosphate inhibitors interfere with thrombosis while sparing hemostasis, replicating the effect of factor XII neutralizing agents. Furthermore, polyphosphate binds to platelet factor 4, which has implications for autoimmune thrombotic diseases, such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT), potentially contributing to their pathogenesis. The metabolism and organ-specific distribution of the polymer remain incompletely defined and is the topic of ongoing research. Polyphosphate acts as a procoagulant and proinflammatory mediator. Neutralizing polyphosphate provides well tolerated thromboprotection, mimicking the effects of factor XII deficiency.

Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

It is well established that inflammation and platelets promote multiple processes of cancer malignancy. Recently, platelets have received attention for their role in carcinogenesis through the production of microvesicles or platelet-derived microparticles (PMPs), which transfer their biological content to cancer cells. We have previously characterized a new subpopulation of these microparticles (termed mito-microparticles), which package functional mitochondria. The potential of mitochondria transfer to cancer cells is particularly impactful as many aspects of mitochondrial biology (i.e., cell growth, apoptosis inhibition, and drug resistance) coincide with cancer hallmarks and disease progression. These metabolic aspects are particularly notable in chronic lymphocytic leukemia (CLL), which is characterized by a relentless accumulation of proliferating, immunologically dysfunctional, mature B-lymphocytes that fail to undergo apoptosis. The present study aimed to investigate the role of PMPs on CLL metabolic plasticity leading to cancer cell phenotypic changes. CLL cell lines were co-incubated with different concentrations of human PMPs, and their impact on cell proliferation, mitochondrial DNA copy number, OCR level, ATP production, and ROS content was evaluated. Essential genes involved in metabolic-reprogramming were identified using the bioinformatics tools, examined between patients with early and advanced CLL stages, and then validated in PMP-recipient CLLs. Finally, the impact of the induced metabolic reprogramming on CLLs' growth, survival, mobility, and invasiveness was tested against anti-cancer drugs Cytarabine, Venetoclax, and Plumbagin. The data demonstrated the potency of PMPs in inducing tumoral growth and invasiveness in CLLs through mitochondrial internalization and OXPHOS stimulation which was in line with metabolic shift reported in CLL patients from early to advanced stages. This metabolic rewiring also improved CLL cells' resistance to Cytarabine, Venetoclax, and Plumbagin chemo drugs. Altogether, these findings depict a new platelet-mediated pathway of cancer pathogenesis. We also highlight the impact of PMPs in CLL metabolic reprogramming and disease progression.

Platelet-Derived Microparticles and Autoimmune Diseases.

Extracellular microparticles provide a means of cell-to-cell communication and can promote information exchanges between adjacent or distant cells. Platelets are cell fragments that are derived from megakaryocytes. Their main functions are to stop bleeding, regulate inflammation, and maintain the integrity of blood vessels. When platelets are activated, they can perform related tasks by secreting platelet-derived microparticles that contain lipids, proteins, nucleic acids, and even organelles. There are differences in the circulating platelet levels in many autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid antibody syndrome, and Sjogren's syndrome. In this paper, the latest findings in the research field of platelet-derived microparticles are reviewed, including the potential pathogenesis of platelet-derived microparticles in various types of immune diseases, their potential as related markers, and for monitoring the progress and prognosis of disease treatment are expounded.

CARD12: Shear Stress Renders Redistribution of P2Y12 and PAR-1 Receptors from Platelets to Microparticles: A Cue to Limited Effectiveness of Antiplatelet Therapy in MCS

Background: Platelet exposure to hypershear stress generated by mechanical circulatory support (MCS) results in platelet dysfunction, thrombosis, and bleeding coagulopathy. Antiplatelet agents, targeting ADP-evoked P2Y12 and thrombin-evoked PAR1 platelet receptors, have limited effectiveness reducing MCS thrombosis, rather favoring bleeding. Shear-mediated platelet dysfunction is associated with the downregulation of platelet adhesion receptors, decreased aggregation response, and generation of platelet-derived microparticles (PDMPs). We tested the hypothesis that MCS shear stress alters the surface expression of agonist receptors P2Y12 and PAR1 on platelets and PDMPs, thus affecting platelet aggregation response induced by ADP, collagen, and TRAP-6. Methods: Human platelets were exposed to shear stress (30-70 dynes/cm2, 10’) or sonication; PDMPs were isolated via differential centrifugation. P2Y12, PAR1, & αIIbβ3 expression on platelets and PDMPs was visualized by immunostaining and multi-colored flow cytometry. Platelets were distinguished from PDMPs by forward/side scatter vs. standard fluorescent nanobeads SPHEROTM. Platelet aggregation in plasma induced by ADP, collagen, and TRAP-6 was assessed by optical aggregometry. Results: Platelet exposure to increasing shear stress and sonication resulted in a decrease in P2Y12+ and PAR1+ platelet populations (Fig. 1A&B). The baseline surface density of P2Y12 on platelets was very low and slightly increased after 70 dyne/cm2 shear (Fig. 1C). While also low, PAR1 density on platelets tended to decrease with increasing shear stress (Fig. 1D). Shear stress induced generation of P2Y12+ and PAR1+ PDMPs, accounting for 3.5±0.6% and 4.3±0.5% after 70 dyne/cm2 shear, respectively. Remarkably, P2Y12 and PAR1 surface density on PDMPs was between 7 and 10-fold higher than on platelets (Fig. 1C&D). When added to intact platelets, PDMPs significantly decreased platelet aggregation in plasma induced by ADP, collagen, and TRAP-6. Conclusions: Elevated shear stress promotes generation of PDMPs carrying increased amounts of P2Y12 and PAR1 on their surface; the surface density of these receptors on sheared platelets was also affected. Sheared PDMPs reduced platelet aggregation likely acting as competitive inhibitors imposing steric difficulties for platelets to aggregate and thus favoring MCS bleeding phenotype and limiting the effectiveness of antiplatelet agents.

P83: Circulating Platelet-Derived Microparticles Modulate Platelet Adhesion Under Flow: Modeling Primary Thrombus Formation in a Microfluidic System

Background: Elevated levels of circulating platelet-derived microparticles (PDMPs) are associated with a range of pathologies, thought to increase thrombotic risk. We hypothesized that addition of PDMPs to whole blood would alter platelet adhesion and thrombogenicity, with its effect further modulated by shear stress magnitude. We tested the effect of addition of PDMPs to whole blood on platelet adhesion and primary thrombus formation on collagen under a range of shear flows using a microfluidic system. Methods: PDMPs were generated via sonication of gel-filtered human platelets and separated via differential centrifugation. Hirudin-anticoagulated human blood was stained with 3,3′-dihexyloxacarbocyanine iodide and circulated through a collagen-coated microchannel with and without PDMPs at defined shear stress magnitudes (13.5, 49.5, or 72 dyne/cm2) for 4 min. Platelet adhesion was visualized by fluorescence microscopy; images were analyzed using MATLAB software. Platelet adhesion and thrombogenicity was assessed via four parameters: 1. surface coverage (SC) – indicative of overall surface occupied by platelet thrombi; 2. number of thrombi (NT) – indicative of the number of sites of platelet adhesion on a collagen-covered surface; 3. mean thrombus area (MTA) and 4. mean fluorescence intensity (MFI) – both characterizing thrombus size. Results: Whole blood platelet exposure to increasing shear stress facilitated platelet adhesion to collagen, with increased SC, MTA and MFI (Fig. 1, WB group). Interestingly, the number of thrombi decreased (846±148 vs. 256±81 at 13.5 and 72 dyne/cm2, respectively) with increasing shear suggesting that increased platelet adhesion was due to increased thrombus size, rather than the number of sites of platelet adhesion. Circulating PDMPs significantly inhibited platelet adhesion to collagen, with a decrease of SC, MTI and MFI (but not NT) at all shear stress magnitudes (Fig. 1, WB+PDMPs vs. WB group, ANOVA: p<0.05). At high shear, PDMP-mediated inhibition of platelet adhesion was pronounced with a more than a 2-fold decrease of thrombus size, as indicated by MTA and MFI. Notably, adding PDMPs to recirculated whole blood increased NT adhered to collagen only at lower shear magnitudes. Conclusion: Shear stress of increasing magnitude promotes increased platelet adhesion to collagen, platelet thrombus size and surface coverage; while the number of sites of platelet adhesion decreases. Circulating PDMPs inhibit platelet adhesion under flow, resulting in a significant decrease in platelet thrombi size. Adding PDMPs to circulating blood increases the number of thrombi formed on collagen, but only at lower shear magnitudes. We speculate that increased levels of circulating PDMPs facilitate platelet adhesion and primary thrombus formation, contributing to an overall prothrombotic phenotype, yet with associated microthrombosis with embolic potential, imposing increased risk of distal ischemic and occlusive events.Figure 1. Circulating platelet-derived microparticles (PDMPs) inhibit platelet adhesion to collagen under shear stress: WB – whole blood, circulated without PDMPs; WB+PDMPs - whole blood, circulated without PDMPs (9:1). Mean ± SD, n = 7, ANOVA: * - p < 0.05, ** - p < 0.01.

Exploitation of platelets for antitumor drug delivery and modulation of the tumor immune microenvironment

Platelets are blood components traditionally believed to have fundamental roles in vascular hemostasis and thrombosis. In recent years, platelets have received new attention for their roles in tumorigenesis and progression. On the one hand, platelets are actively recruited by various tumors and comprise a crucial part of the tumor microenvironment (TME), thus inspiring the use of platelets for tumor-targeted drug delivery. To this end, various platelet-based devices have been proposed, such as natural platelets, engineered platelets, platelet membranes, and platelet-derived microparticles. On the other hand, platelets are involved in tumor immunosuppression mechanisms, by directing and/or assisting various tumor-associated immune cells. However, in the context of inflammation and autoimmune diseases, platelets can amplify immune responses by promoting immune cell mobilization and activation, thereby exacerbating tissue damage. Thus, interest is growing in the use of tumor-associated platelets as targets for therapeutic modulation of the TME and augmenting anti-tumor immune responses. In this review, we summarize current advances in exploiting platelets for both antitumor drug delivery and immune modulation of the TME.

Novel Knowledge about Molecular Mechanisms of Heparin-Induced Thrombocytopenia Type II and Treatment Targets.

Heparin-induced thrombocytopenia type II (HIT II), as stated in the literature, occurs in about 3% of all patients and in 0.1-5% of surgical patients. Thrombosis develops in 20-64% of patients with HIT. The mortality rate in HIT II has not decreased using non-heparin treatment with anticoagulants such as argatroban and lepirudin. An improved understanding of the pathophysiology of HIT may help identify targeted therapies to prevent thrombosis without subjecting patients to the risk of intense anticoagulation. The review will summarize the current knowledge about the pathogenesis of HIT II, potential new therapeutic targets related to it, and new treatments being developed. HIT II pathogenesis involves multi-step immune-mediated pathways dependent on the ratio of PF4/heparin and platelet, monocyte, neutrophil, and endothelium activation. For years, only platelets were known to take part in HIT II development. A few years ago, specific receptors and signal-induced pathways in monocytes, neutrophils and endothelium were revealed. It had been shown that the cells that had become active realised different newly formed compounds (platelet-released TF, TNFα, NAP2, CXCL-7, ENA-78, platelet-derived microparticles; monocytes-TF-MPs; neutrophils-NETs), leading to additional cell activation and consequently thrombin generation, resulting in thrombosis. Knowledge about FcγIIa receptors on platelets, monocytes, neutrophils and FcγIIIa on endothelium, chemokine (CXCR-2), and PSGL-1 receptors on neutrophils could allow for the development of a new non-anticoagulant treatment for HIT II. IgG degradation, Syk kinase and NETosis inhibition are in the field of developing new treatment possibilities too. Accordingly, IdeS and DNases-related pathways should be investigated for better understanding of HIT pathogenesis and the possibilities of being the HIT II treatment targets.

Microparticles: potential new contributors to the pathogenesis of systemic sclerosis?

BackgroundMicroparticles (MPs) are membrane-derived vesicles released from cells undergoing activation or apoptosis with diverse proinflammatory and prothrombotic activities, that have been implicated in the pathogenesis of systemic sclerosis (SSc). We aimed to evaluate the plasma levels of platelet-derived microparticles (PMPs), endothelial cell-derived microparticles (EMPs), and monocyte-derived microparticles (MMPs) in SSc patients, and the association between MPs and the clinical features of SSc.MethodsIn this cross-sectional study, 70 patients with SSc and 35 age- and sex-matched healthy controls were evaluated. Clinical and nailfold capillaroscopy (NFC) data were obtained from all patients. Plasma levels of PMPs (CD42+/31+), EMPs (CD105+), and MMPs (CD14+) were quantified by flow cytometry.ResultsPatients were mainly females (90%), with a mean age of 48.9 years old. PMP, EMP, and MMP levels were significantly increased in SSc patients compared to controls (79.2% ± 17.3% vs. 71.0% ± 19.8%, p = 0.033; 43.5% ± 8.7% vs. 37.8% ± 10.4%, p = 0.004; and 3.5% ± 1.3% vs. 1.1% ± 0.5%, p < 0.0001, respectively). PMP levels were significantly higher in patients with positive anti-topoisomerase-I antibodies (p = 0.030) and in patients with a disease duration > 3 years (p = 0.038). EMP levels were lower in patients with a higher modified Rodnan skin score (p = 0.015), and in those with an avascular score > 1.5 in NFC (p = 0.042).ConclusionThe increased levels of PMPs, EMPs and MMPs in scleroderma patients might indicate a possible role for these agents in the pathogenesis of this challenging disease.

Shear-Mediated Platelet Microparticles Demonstrate Phenotypic Heterogeneity as to Morphology, Receptor Distribution, and Hemostatic Function.

Implantable Cardiovascular Therapeutic Devices (CTD), while lifesaving, impart supraphysiologic shear stress to platelets, resulting in thrombotic and bleeding coagulopathy. We previously demonstrated that shear-mediated platelet dysfunction is associated with downregulation of platelet GPIb-IX-V and αIIbβ3 receptors via generation of Platelet-Derived MicroParticles (PDMPs). Here, we test the hypothesis that sheared PDMPs manifest phenotypical heterogeneity of morphology and receptor surface expression and modulate platelet hemostatic function. Human gel-filtered platelets were exposed to continuous shear stress. Alterations of platelet morphology were visualized using transmission electron microscopy. Surface expression of platelet receptors and PDMP generation were quantified by flow cytometry. Thrombin generation was quantified spectrophotometrically, and platelet aggregation was measured by optical aggregometry. Shear stress promotes notable alterations in platelet morphology and ejection of distinctive types of PDMPs. Shear-mediated microvesiculation is associated with the remodeling of platelet receptors, with PDMPs expressing significantly higher levels of adhesion receptors (αIIbβ3, GPIX, PECAM-1, P-selectin, and PSGL-1) and agonist receptors (P2Y12 and PAR1). Sheared PDMPs promote thrombin generation and inhibit platelet aggregation induced by collagen and ADP. Sheared PDMPs demonstrate phenotypic heterogeneity as to morphology and defined patterns of surface receptors and impose a bidirectional effect on platelet hemostatic function. PDMP heterogeneity suggests that a range of mechanisms are operative in the microvesiculation process, contributing to CTD coagulopathy and posing opportunities for therapeutic manipulation.

Abstract 3708: Platelet-derived microparticles modulate breast cancer malignant processes

Abstract Breast cancer is one of the leading causes of morbidity and mortality among women, where metastasis accounts for the majority of deaths associated with this disease. Thus, the potential to effectively target tumor malignancy offers hope to mitigate disease progression and improve patient outcomes. It is well established that platelets promote multiple processes of metastasis cascade. Recently, platelets have received new attention for their impact in cancer through the production of platelet-derived microparticles (PMPs). Interestingly, PMPs allow intercellular exchange and trafficking of bioactive material through the internalization of these vesicles into recipient cells. As a result, the delivery of the intravesicular cargo can modulate signaling and activation processes of recipient cells. We recently identified a new subpopulation of these vesicles (termed mitoMPs) containing functional mitochondria. Given the predominant role of mitochondria in cancer malignancy, we believe that mitoMPs provide an important source of foreign mitochondria to support recipient breast cancer cells in malignancy and disease progression. We therefore set out to study the impact of mitoMPs on breast cancer metabolic and phenotypic processes involved in metastasis. Technically, PMPs were generated and purified from human blood platelets and co-incubated with various breast cell models (MB231, MCF7 and MCF10A). The physiological significance of mitoMPs in breast cancer disease was then assessed using various cellular and molecular assays. We demonstrate that the level of PMP internalization is highly dependent upon the type of breast cancer recipient cells. Furthermore, we show that the cargo of mitoMPs (notably mitochondria) is biologically active where recipient breast cancer cells acquired mitochondria-dependent functions, such as increased oxygen consumption rates and intracellular ATP production. Finally, we observe that mitoMPs promote malignant features such as cancer cell migration and invasion. Overall, we demonstrate that PMPs can modulate cancer cell activation and behaviour. These findings provide a better understanding of the extracellular tumor environment and the contribution of mitoMPs in supporting breast cancer cells through the metastatic landscape. The knowledge gained will further provide new avenues for therapeutic strategies in breast cancer patients. Citation Format: Vanessa Veilleux, Nicolas Pichaud, Luc H. Boudreau, Gilles A. Robichaud. Platelet-derived microparticles modulate breast cancer malignant processes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3708.

Abstract 4846: Mitochondria packaged from platelet-derived microvesicles modulate the metabolism and mitochondrial dynamics of breast cancer cells

Abstract The incidence of breast cancer among women far exceeds those of other cancers where metastasis accounts for the majority of deaths associated with the disease. It is well established that platelets play a prominent role in breast cancer malignancy and disease progression. Recently, activated platelets have been characterized to release platelet-derived microparticles (PMPs), which can also package functional mitochondria of platelet origin (termed mitoMPs). We recently demonstrated that mitoMPs can interact with breast cancer cells where they transfer their cargo (i.e., mitochondria) to recipient cancer cells upon their internalization. Given that metabolic reprogramming through mitochondrial dynamics represents a hallmark trait of cancer progression, the intercellular trafficking of foreign mitochondria provided by mitoMPs represent a new mechanistic avenue for cancer cell metabolic plasticity and disease progression.Therefore, the aim of this study was to evaluate the impact of mitoMP-packaged mitochondria on recipient breast cancer cell mitochondrial dynamics upon internalization. Technically, a series of breast cancer cell models (MB231, MCF7 and MCF10A) were co-cultured with mitoMPs and profiled for mitochondrial dynamics and function using complementing experimental approaches, such as flow cytometry, confocal microscopy, and RT-qPCR. We show that mitoMPs and their mitochondria cargo internalize primarily into the malignant MB231 cell model. To investigate the impact of mitoMP uptake by cancer cells on the mitochondrial dynamics, RT-qPCR and Western blot analyses were performed to profile the expression levels of several fusion and fission proteins, including OPA1, MFF, DRP1, MitoFusin 1 and MitoFusin 2. Finally, using fluorescent-based assays, we confirmed that mitoMPs modulated ATP production in MB231 breast cancer cells through upregulation of the OXPHOS pathway. Our research provides a new mechanism by which extracellular vesicles (mitoMPs) play an important role in cancer cell mitochondrial dynamics and function to support metabolic plasticity during the metastatic journey of cancer cells. The knowledge gained from these studies will further provide new avenues for strategic intervention to mitigate the morbidity and mortality associated with breast cancer disease. Citation Format: Vanessa L. Gauvin, Vanessa Veilleux, Nicolas Pichaud, Gilles A. Robichaud, Luc H. Boudreau. Mitochondria packaged from platelet-derived microvesicles modulate the metabolism and mitochondrial dynamics of breast cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4846.

Platelets at the Vessel Wall in Non-Thrombotic Disease.

Platelets are small, anucleate entities that bud from megakaryocytes in the bone marrow. Among circulating cells, platelets are the most abundant cell, traditionally involved in regulating the balance between thrombosis (the terminal event of platelet activation) and hemostasis (a protective response to tissue injury). Although platelets lack the precise cellular control offered by nucleate cells, they are in fact very dynamic cells, enriched in preformed RNA that allows them the capability of de novo protein synthesis which alters the platelet phenotype and responses in physiological and pathological events. Antiplatelet medications have significantly reduced the morbidity and mortality for patients afflicted with thrombotic diseases, including stroke and myocardial infarction. However, it has become apparent in the last few years that platelets play a critical role beyond thrombosis and hemostasis. For example, platelet-derived proteins by constitutive and regulated exocytosis can be found in the plasma and may educate distant tissue including blood vessels. First, platelets are enriched in inflammatory and anti-inflammatory molecules that may regulate vascular remodeling. Second, platelet-derived microparticles released into the circulation can be acquired by vascular endothelial cells through the process of endocytosis. Third, platelets are highly enriched in mitochondria that may contribute to the local reactive oxygen species pool and remodel phospholipids in the plasma membrane of blood vessels. Lastly, platelets are enriched in proteins and phosphoproteins which can be secreted independent of stimulation by surface receptor agonists in conditions of disturbed blood flow. This so-called biomechanical platelet activation occurs in regions of pathologically narrowed (atherosclerotic) or dilated (aneurysmal) vessels. Emerging evidence suggests platelets may regulate the process of angiogenesis and blood flow to tumors as well as education of distant organs for the purposes of allograft health following transplantation. This review will illustrate the potential of platelets to remodel blood vessels in various diseases with a focus on the aforementioned mechanisms.

Platelet-derived microparticles stimulate the invasiveness of colorectal cancer cells via the p38MAPK-MMP-2/MMP-9 axis

BackgroundMetastasis is the main cause of death in patients with colorectal cancer (CRC). Apart from platelets, platelet-derived microparticles (PMPs) are also considered important factors that can modify the activity of cancer cells. PMPs are incorporated by cancer cells and can also serve as intracellular signalling vesicles. PMPs are believed to affect cancer cells by upregulating their invasiveness. To date, there is no evidence that such a mechanism occurs in colorectal cancer. It has been shown that platelets can stimulate metalloproteases (MMPs) expression and activity via the p38MAPK pathway in CRC cells, leading to their elevated migratory potential. This study aimed to investigate the impact of PMPs on the invasive potential of CRC cells of various phenotypes via the MMP-2, MMP-9 and p38MAPK axis.MethodsWe used various CRC cell lines, including the epithelial-like HT29 and the mesenchymal-like SW480 and SW620. Confocal imaging was applied to study PMP incorporation into CRC cells. The presence of surface receptors on CRC cells after PMP uptake was evaluated by flow cytometry. Transwell and scratch wound-healing assays were used to evaluate cell migration. The level of C-X-C chemokine receptor type 4 (CXCR4), MMP-2, and MMP-9 and the phosphorylation of ERK1/2 and p38MAPK were measured by western blot. MMP activity was determined using gelatine-degradation assays, while MMP release was evaluated by ELISA.ResultsWe found that CRC cells could incorporate PMPs in a time-dependent manner. Moreover, PMPs could transfer platelet-specific integrins and stimulate the expression of integrins already present on tested cell lines. While mesenchymal-like cells expressed less CXCR4 than epithelial-like CRC cells, PMP uptake did not increase its intensity. No significant changes in CXCR4 level either on the surface or inside CRC cells were noticed. Levels of cellular and released MMP-2 and MMP-9 were elevated in all tested CRC cell lines after PMP uptake. PMPs increased the phosphorylation of p38MAPK but not that of ERK1/2. Inhibition of p38MAPK phosphorylation reduced the PMP-induced elevated level and release of MMP-2 and MMP-9 as well as MMP-dependent cell migration in all cell lines.ConclusionsWe conclude that PMPs can fuse into both epithelial-like and mesenchymal-like CRC cells and increase their invasive potential by inducing the expression and release of MMP-2 and MMP-9 via the p38MAPK pathway, whereas CXCR4-related cell motility or the ERK1/2 pathway appears to not be affected by PMPs.A1m_Mu21TUHDLKeRdJvkXhVideo

Coagulopathy is Initiated with Endothelial Dysfunction and Disrupted Fibrinolysis in Patients with COVID-19 Disease.

The online version contains supplementary material available at 10.1007/s12291-023-01118-3.

Platelets derived citrullinated proteins and microparticles are potential autoantibodies ACPA targets in RA patients.

Citrullinated neoepitopes have emerged as key triggers of autoantibodies anti-citrullinated protein antibodies (ACPA) synthesis in rheumatoid arthritis (RA) patients. Apart from their critical role in homeostasis and thrombosis, platelets have a significant contribution to inflammation as well. Although anuclear in nature, platelets have an intricate post-translational modification machinery. Till now, citrullination in platelets and its contribution to trigger autoantibodies ACPA production in RA is an unexplored research direction. Herein, we investigated the expression of peptidylarginine deiminase (PAD) enzymes and citrullinated proteins/peptides in the human platelets and platelet derived microparticles (PDP). Both PAD4 mRNA and protein, but not the other PAD isoforms, are detectable in the human platelets. With a strict filtering criterion,108 citrullination sites present on 76 proteins were identified in the human platelets, and 55 citrullinated modifications present on 37 different proteins were detected in the PDPs. Among them, some are well-known citrullinated autoantigens associated with RA. Citrullinated forms of thrombospondin-1, β-actin, and platelet factor-4 (also known as CXCL4) are highly immunogenic and bound by autoantibodies ACPA. Furthermore, ACPA from RA sera and synovial fluids recognized citrullinated proteins from platelets and significantly activated them as evidenced by P-selectin upregulation and sCD40 L secretion. These results clearly demonstrate the presence of citrullinated autoantigens in platelets and PDPs, thus could serve as potential targets of ACPA in RA.

Association of High Mobility Group Box-Protein 1 and Platelet Microparticles in Patients After Hematopoietic Stem Cell Transplantation.

Thrombotic complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly impact transplant outcomes. We focused on high mobility group box-protein (HMGB)1, one causative agent of thrombotic lesions in allo-HSCT, and investigated its association with platelets. We statistically analyzed available data from 172 patients with hematopoietic malignancies receiving allo-HSCT. A significant enhancement of monocyte-chemotactant protein-1, HMGB1, and platelet-derived microparticle (PDMP) levels was observed at day 0 after transplantation as compared to pre-transplantation. Multivariate analysis of the association among HMGB1 and 16 factors on day 0 revealed a significant correlation of HMGB1 levels with thrombin-antithrombin complex, interleukin-6, and PDMPs. High mobility group box-protein 1-induced procoagulant platelet induction and PDMP generation were performed in vitro using healthy platelets. High mobility group box-protein 1-induced PDMP generation was suppressed by toll-like receptor inhibitors and recombinant thrombomodulin. These results suggest that HMGB1 contributes to platelet activation in patients after allo-HSCT and is associated with PDMP-related thrombotic complications.

Hepatocyte-derived Microparticles as Novel Biomarkers for the Diagnosis of Deep Venous Thrombosis in Trauma Patients.

Venous thromboembolism is a common complication following trauma. We investigated the dynamics of plasma microparticles (MPs) levels and explored their potential as biomarkers of deep vein thromboembolism (DVT) after trauma. A total of 775 patients with traumatic fractures were recruited in this nested study. About 106 trauma patients (53 DVT subjects and 53 age-, sex-, and fracture site-matched non-DVT subjects) and 53 healthy volunteers met the enrollment criteria. MPs were characterized by transmission electron microscope, nanoparticle tracking analysis, and western blotting. Circulating levels of MPs were measured using a flow cytometer. Meanwhile, routine laboratory parameters were examined in all patients. Compared to non-DVT patients, DVT patients had higher circulating phosphatidylserine (PS) + MPs, hepatocyte-derived MPs (HMPs), PS + HMPs, and platelet-derived MPs (PMPs). Notably, PS + HMPs had the best predictive value for DVT diagnosis in trauma patients (area under the curve [AUC] 0.8939, 95% CI 0.8326 to 0.9552), which was superior to d-dimer (AUC 0.5881). The Hepatic Procoagulant Index combined plasma levels of PS + HMPs and albumin, increasing the AUC to 0.8978 (95% CI 0.8396 to 0.9561). This is the first study that addressed circulating PS + HMPs are promising biomarkers with high performance in diagnosing DVT. The Hepatic Procoagulant Index is a potential predictor of DVT in trauma patients.

Platelet-derived microparticles stimulated by anti-β2GPI/β2GPI complexes induce pyroptosis of endothelial cells in antiphospholipid syndrome

Platelet microparticles (PMPs) are vesicles that are released by platelets into the extracellular space and play a role in antiphospholipid antibody syndromes. PMPs have recently been recognized as a new and viable cell. There is growing evidence that the anti-β2 glycoprotein (GPI)/β2GPI complex is associated with aberrant activation of PMPs. Although studies suggest that aberrant activation of PMPs may lead to inflammatory necrosis of endothelial cells, the underlying mechanisms remain unclear. We found that although the difference in the number of PMPs was not statistically significant, NLR family pyrin domain containing 3 (NLRP3) within PMPs was increased during stimulation of anti-β2GPI/β2GPI complexes. Furthermore, we demonstrated that anti-β2GPI/β2GPI complex-induced PMPs effectively stimulated endothelial cell pyroptosis via the NLRP3/nuclear factor (NF)-κB/gasdermin D (GSDMD) signaling pathway as well as the NLRP3/Caspase-1 signaling pathway. Additionally, inhibition of NLRP3 expression in PMPs effectively reduced the inflammatory response and pyroptosis in endothelial cells. Our data suggest that PMPs aberrantly activated by anti-β2GPI/β2GPI complexes play a vital role in endothelial cell pyroptosis, and these studies provide major insights into the mechanisms of thrombosis during the treatment of antiphospholipid antibody syndrome.