Kim Anh Nguyen

The Inflammatory Role of Platelets via Their TLRs and Siglec Receptors

Platelets are non-nucleated cells that play central roles in the processes of hemostasis, innate immunity, and inflammation; however, several reports show that these distinct functions are more closely linked than initially thought. Platelets express numerous receptors and contain hundreds of secretory products. These receptors and secretory products are instrumental to the platelet functional responses. The capacity of platelets to secrete copious amounts of cytokines, chemokines, and related molecules appears intimately related to the role of the platelet in inflammation. Platelets exhibit non-self-infectious danger detection molecules on their surfaces, including those belonging to the “toll-like receptor” family, as well as pathogen sensors of other natures (Ig- or complement receptors, etc.). These receptors permit platelets to both bind infectious agents and deliver differential signals leading to the secretion of cytokines/chemokines, under the control of specific intracellular regulatory pathways. In contrast, dysfunctional receptors or dysregulation of the intracellular pathway may increase the susceptibility to pathological inflammation. Physiological vs. pathological inflammation is tightly controlled by the sensors of danger expressed in resting, as well as in activated, platelets. These sensors, referred to as pathogen recognition receptors, primarily sense danger signals termed pathogen associated molecular patterns. As platelets are found in inflamed tissues and are involved in auto-immune disorders, it is possible that they can also be stimulated by internal pathogens. In such cases, platelets can also sense danger signals using damage associated molecular patterns (DAMPs). Some of the most significant DAMP family members are the alarmins, to which the Siglec family of molecules belongs. This review examines the role of platelets in anti-infection immunity via their TLRs and Siglec receptors.

Immune-reactive soluble OX40 ligand, soluble CD40 ligand, and interleukin-27 are simultaneously oversecreted in platelet components associated with acute transfusion reactions

Leukoreduction of labile blood components dramatically decreases the frequency of minor, intermediate, and severe adverse events (AEs), referred to as acute transfusion reactions (ATRs), especially after transfusion of platelet components (PCs). The pathophysiology of AEs may result from accumulation of soluble, secreted, platelet (PLT) factors with proinflammatory functions stored in PCs. Thus, several cosynergizing factors associated with PLT accumulation in PCs may contribute to clinically reported ATRs with inflammatory symptoms.

A computerized prediction model of hazardous inflammatory platelet transfusion outcomes.

Background Platelet component (PC) transfusion leads occasionally to inflammatory hazards. Certain BRMs that are secreted by the platelets themselves during storage may have some responsibility. Methodology/Principal Findings First, we identified non-stochastic arrangements of platelet-secreted BRMs in platelet components that led to acute transfusion reactions (ATRs). These data provide formal clinical evidence that platelets generate secretion profiles under both sterile activation and pathological conditions. We next aimed to predict the risk of hazardous outcomes by establishing statistical models based on the associations of BRMs within the incriminated platelet components and using decision trees. We investigated a large (n = 65) series of ATRs after platelet component transfusions reported through a very homogenous system at one university hospital. Herein, we used a combination of clinical observations, ex vivo and in vitro investigations, and mathematical modeling systems. We calculated the statistical association of a large variety (n = 17) of cytokines, chemokines, and physiologically likely factors with acute inflammatory potential in patients presenting with severe hazards. We then generated an accident prediction model that proved to be dependent on the level (amount) of a given cytokine-like platelet product within the indicated component, e.g., soluble CD40-ligand (>289.5 pg/109 platelets), or the presence of another secreted factor (IL-13, >0). We further modeled the risk of the patient presenting either a febrile non-hemolytic transfusion reaction or an atypical allergic transfusion reaction, depending on the amount of the chemokine MIP-1α (<20.4 or >20.4 pg/109 platelets, respectively). Conclusions/Significance This allows the modeling of a policy of risk prevention for severe inflammatory outcomes in PC transfusion.

Platelet components associated with adverse reactions: predictive value of mitochondrial DNA relative to biological response modifiers

Biological response modifiers (BRMs), secreted by platelets (PLTs) during storage, play a role in adverse events (AEs) associated with transfusion. Moreover, mitochondrial DNA (mtDNA) levels in PLT components (PCs) are associated with AEs. In this study we explore whether there is a correlation between pathogenic BRMs and mtDNA levels and whether these markers can be considered predictors of transfusion pathology.

Role of Siglec-7 in Apoptosis in Human Platelets

Background Platelets participate in tissue repair and innate immune responses. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are well-characterized I-type lectins, which control apoptosis. Methodology/Principal Findings We characterized the expression of Siglec-7 in human platelets isolated from healthy volunteers using flow cytometry and confocal microscopy. Siglec-7 is primarily expressed on α granular membranes and colocalized with CD62P. Siglec-7 expression was increased upon platelet activation and correlated closely with CD62P expression. Cross-linking Siglec-7 with its ligand, ganglioside, resulted in platelet apoptosis without any significant effects on activation, aggregation, cell morphology by electron microscopy analysis or secretion. We show that ganglioside triggered four key pathways leading to apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarization; (ii) elevated expression of pro-apoptotic Bax and Bak proteins with reduced expression of anti-apoptotic Bcl-2 protein; (iii) phosphatidylserine exposure and (iv), microparticle formation. Inhibition of NAPDH oxidase, PI3K, or PKC rescued platelets from apoptosis induced by Siglec-7 recruitment, suggesting that the platelet receptors P2Y1 and GPIIbIIIa are essential for ganglioside-induced platelet apoptosis. Conclusions/Significance The present work characterizes the role of Siglec-7 and platelet receptors in regulating apoptosis and death. Because some platelet pathology involves apoptosis (idiopathic thrombocytopenic purpura and possibly storage lesions), Siglec-7 might be a molecular target for therapeutic intervention/prevention.

Do manual and automated processes with distinct additive solutions affect whole blood-derived platelet components differently?

Dear Sir, Platelet component transfusions are still major contributors to adverse events, and avoiding storage lesions and the production of inflammatory products is of major importance during the production of these components. Ex vivo activation of platelets may lead to excess production of inflammatory factors that can cause acute transfusion reactions1. We compared the induction of inflammatory platelet storage lesion in platelet components produced by manual (m) and automated (a) procedures using the TACSI platform (Terumo France S.A., Guyancourt, France). Furthermore, for each procedure we compared platelet additive solutions (i.e., PASIII [Fenwal, La Châtre, France] versus PASIIIM [MacoPharma, Mouveaux, France]) with a mean range of 35% residual plasma. Differences in the composition of these platelet additive solutions have been described previously2. The comparison study involved five pooled whole blood buffy-coat-derived platelet components. The TACSI platform has proven suitable for clinical grade platelet component production with regards to primary haemostasis3 and platelet additive solutions have been developed to minimise the occurrence of transfusion-related acute lung injury (TRALI) and enable pathogen inactivation. However, little attention, if any, has been given to the impact of procedural changes on the pro-inflammatory lesions potentially inflicted by stored platelets. The results of the quality control for each product (mPC/PASIII, aPC/PASIII, mPC/PASIIIM, and aPC/PASIIIM) are shown in Table I. All products fell within the range acceptable for use. Some significant individual differences were found, but they were corrected by reporting the quantity of product per issued platelet component. According to past experience, we focused on two markers, the activation platelet surface markers CD62p and CD40L, along with their soluble counterparts, which were measured under each condition. For reference values we used thrombin-receptor activating peptide (TRAP), an analogue of thrombin (50 μg/mL; Saint Quentin-Fallavier, France). Measurements were made on the contents of the quality control sampling bag 24 h after whole blood collection; the products in this study were not destroyed, but issued to patients as authorised, because all four types of platelet component are licensed by the notifying and regulatory body (Affsaps). Platelet membrane activation was tested by flow cytometry using fluorescein isothiocyanate-conjugated anti-CD41 monoclonal antibody (BD Biosciences, Le Pont de Claix, France) for gating the whole platelet population and allophycocyanin-conjugated anti-CD62p and phycoerythrin-conjugated CD40L monoclonal antibodies (BD Biosciences) (FACSvantage SE flow-cytometer and CellQuestS-Pro software, BD Biosciences). Soluble proteins were measured in supernatant fractions using specific enzyme-linked immunosorbent assays. The monoclonal antibodies to CD62p and sCD40L were purchased from R&D Systems Europe Ltd, Lille, France and Bender MedSystems GmbH, Vienna, Austria, respectively. The reader was a Multiskan EX (Labsystem, Helsinki, Finland). Inter-experimental comparisons of data (10 platelet components in each arm) were performed using the Mann-Whitney U test. P-values <0.05 were considered statistically significant. Table I Comparison of platelets obtained from different processing systems on day 1. Results are expressed as mean±SD (n=10 in each arm). Next, we addressed the issue of whether all four types of platelet product were equivalent with regard to their propensity to be activated and secrete detectable inflammatory cytokines ex vivo. Slight differences in total CD40L and CD62p platelet surface expression were found between the products processed automatically or manually, irrespectively of the platelet additive solution (Table II). Secreted products were also significantly different between manually and automatically processed preparations. Platelets from all four types of preparations maintained the ability to express and secrete CD62p and CD40L following TRAP stimulation, demonstrating their viability and the absence of detrimental storage lesions (Table II). Significant differences were found but none was consistent with preferential conditions. Table II Mean fluorescence intensity (MFI) of CD40L/CD62p and sCD40L/sCD62p released from platelets with or without TRAP stimulation. None of the 40 platelet components considered led to any reported adverse transfusion reaction, and the individual levels of secreted sCD40L, a molecule that we found correlates with adverse transfusion reactions (R=0.986) (Nguyen et al., submitted), were far below the level necessary to mediate a bioactive effect on encountered cells4 or that has been associated with TRALI5. These findings validate the use of automated platelet component processing with commercially available platelet additive solutions, an advantage for homogenising the production of platelet components and increasing the production of pooled buffy coat-derived platelets compared to single donor aphaeresis platelet collection, which is currently considered advantageous for the donor and recipient.

Contribution of activated platelets to plasma IL-27 levels

Serum interleukin-27 (IL-27) protein concentration is predictive of bacterial infection in critically ill children. Here we show that upon activation platelets release functional IL-27 that is able to specifically induce B cell activation in vitro. These data highlight the inflammatory role of platelet-derived IL-27 and suggest that platelets could contribute to immune dysregulation in septic patients.

Specific activation, signalling and secretion profiles of human platelets following PAR-1 and PAR-4 stimulation

Abstract Blood platelets play a central haemostatic function; however, they also play a role in inflammation and are capable of secreting various cytokines, chemokines and related products. The purpose of this study was to identify subtle variations in platelet physiology using proteomics. We compared the levels of membrane proteins (n = 3), α and δ granule proteins (n = 18), and signalling proteins (n = 30) from unstimulated platelets with those of protease-activated receptor (PAR)-1- and PAR-4-stimulated platelets (n = 10). The vast majority of these proteins responded similarly to PAR-1 or PAR-4 engagement. However, differences were observed within membrane CD40L expressed, and α granule GRO-α and MDC secreted proteins.

Process automation in manufacturing of mesenchymal stromal cells.

T he remarkable progress over the past decade in mesenchymal stromal cell (MSC) translational and clinical research has vastly increased the understanding of the cell processing techniques necessary to deliver a safe cellular therapeutic product that is well characterized and potent. Marrow aspirates have proven to be the most widely utilized source of MSCs in current clinical trials. The cellular manufacturing unit operations that define marrow-derived MSC processing, whether autologous or allogeneic, typically includes separation of the mononuclear cell (MNC) fraction from the bulk marrow aspirate, ex vivo cell culture of the MSCs present in the MNC fraction, harvest of the MSCs from culture, washing and concentration of the harvested MSCs, and fill and finish of the washed and concentrated cells into the final drug product packaging, which may be destined for fresh infusion or cryopreservation. The majority of MSCs manufactured for current clinical trials is are obtained by manual methods. This typically includes density gradient separation of marrow aspirates via Ficoll density gradient separation and centrifugation to produce a MNC fraction, which is then placed into static 2D cell culture in plastic tissue culture flasks or cell factories. The MSCs are plastic adherent and proliferate in culture, while the other cellular constituents of the MNCs are nonadherent and will therefore not attach to the plastic cell culture substrate and subsequently may be removed from the culture vessel by a medium exchange, leaving the adherent MSCs to persist. Several passages of the MSCs from one flask or cell factory to another are required to achieve adequate cell yields for clinical doses. MSC doses are typically administered to patients in the 1 3 10 to 10 3 10 cells/kg range. Hanley and colleagues have reported that expansion of 200 3 10 MSCs from 25 mL of marrow aspirate in the good manufacturing practices facility at Baylor College of Medicine (Houston, TX) over a 30-day period requires four cell culture passages, the use of 340 T-175-cm tissue culture flasks, and 2040 pipettes, with 54,400 open events (manual manipulations) that must be performed in a Class 100 (ISO 5) biosafety cabinet. The washing and concentration of harvested MSCs are generally carried out by multiple centrifugation steps utilizing centrifuge tubes to remove the cell harvest medium, to exchange with wash fluid, and to resuspend the concentrated cell pellet up to the desired cellular concentration in either infusion solution or cryoprotectant. Finally, the resuspended MSCs at the desired final formulation concentration are filled into either cryovials or cryobags for controlled-rate freezing or else placed in other containment for fresh infusion, such as syringes, plastic or glass vials, or IV infusion bags. Due to the exceedingly labor-intensive nature of current MSC production, the risks of product contamination and product variability due to human error involved with open system manipulations, there is much interest in automating MSC manufacturing. Unfortunately, there are currently only a very limited number of automation devices commercially available that are specifically designed for MSC production. Many automated systems in use today for MSC production are either repurposed from technologies designed for established cellular therapies such as marrow transplantation or else from the biologics drug manufacturing industry. However, as MSCs as a therapeutic progress further from early-stage clinical trials to late-stage trials and ultimately commercialization, and as the need to both scale-up and scale-out manufacturing increases, it is anticipated that medical device, laboratory device, and bioprocessing equipment manufacturers will begin to design and commercialize automated systems expressly for MSC production. The majority of automated systems currently in use for MSC manufacturing utilize closed-system processing with single-use plastic disposables. This is to ensure the sterility of the cellular product as well as to potentially limit the need for processing within higher-grade good manufacturing practices cleanrooms, and the concomitant expense of operating and maintaining these facilities. In terms of the first common unit operation in MSC