Formation Of Heterotypic Aggregates Research Articles

The role of platelets and megakaryocytes in sepsis and ARDS.

Since the global COVID-19 pandemic, there has been a renewed focus on lung injury during infection. Systemic inflammatory responses such as acute respiratory distress syndrome (ARDS) and sepsis are a leading cause of morbidity and mortality for both adults and children. Improvements in clinical care have improved outcomes but mortality remains ∼40% and significant morbidity persists for those patients with severe disease. Mechanistic studies of the underlying biological processes remain essential to identifying therapeutic targets. Furthermore, methods for identifying the underlying drivers of organ failure are key to treating and preventing tissue injury. In this review, we discuss the contribution of megakaryocytes (MKs) and platelets to the pathogenesis of systemic inflammatory syndromes. We explore the role of MKs and the new identification of extramedullary MKs during sepsis. We describe the alterations in the platelet transcriptome during sepsis. Lastly, we explore platelet function as defined by aggregation, activation and the formation of heterotypic aggregates. Much more work is necessary to explore the contribution of platelets to these heterogenous syndromes, but the foundation of platelets as key contributors to inflammation has been laid.

Internalization of microparticles by platelets is partially mediated by toll-like receptor 4 and enhances platelet thrombogenicity

Background and aimsCirculating platelet microparticles (PMP) are the most abundant in bloodstream, are highly procoagulant and contribute to cross-talk with inflammatory cells. The aim of the present study was to investigate the interactions of PMP with platelets and explore the involvement of toll-like receptor 4 (TLR-4). MethodsPMP were separated by ultracentrifugation of expired platelet concentrates and added to: i) washed platelets, to confirm uptake, by flow cytometry and confocal and transmission electron microscopy, ii) platelet rich plasma (PRP), to assess changes in platelet function due to uptake by aggregometry in response to ADP; and iii) whole blood, to evaluate heterotypic aggregate (HA) formation by flow cytometry. Moreover, whole blood previously enriched with platelets with internalized PMP was used to explore modifications in thromboelastometry parameters (ROTEM). The inhibitory action of anti-TLR-4 was investigated. ResultsConfocal and ultrastructural microscopy studies revealed PMP internalization by platelets. Flow cytometry showed PMP-platelet association (p < 0.01 vs controls, at different PMP dilutions). PMP, at 1/20 dilution, increased HA (p < 0.05 vs controls), the percentage of maximal platelet aggregation to ADP (p < 0.05 vs controls), and accelerated clotting and clot formation times (p < 0.05 vs controls). Incubation of platelets with anti-TLR-4 prior to exposure to PMP reduced PMP-platelet association (p < 0.05 vs absence of the antibody), prevented HA formation, reduced maximal platelet aggregation and normalized ROTEM parameters. ConclusionsPlatelets exhibit internalization ability towards their own PMP, a process that potentiates their thrombogenicity and is partially mediated by the innate immunity receptor TLR-4.

Developmental endothelial locus-1 modulates platelet-monocyte interactions and instant blood-mediated inflammatory reaction in islet transplantation.

Platelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood-islet model and a syngeneic murine transplantation model. Indeed, Del-1 pre-treatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.

Lymphotoxin-β receptor signalling links steatohepatitis, hyper-lipidemia and atherosclerosis

Lymphotoxin-β receptor signalling links steatohepatitis, hyper-lipidemia and atherosclerosis

Equine platelets inhibit E. coli growth and can be activated by bacterial lipopolysaccharide and lipoteichoic acid although superoxide anion production does not occur and platelet activation is not associated with enhanced production by neutrophils

Activated platelets can contribute to host defense through release of products with bactericidal actions such as antimicrobial peptides and reactive oxygen species (ROS), as well as by forming heterotypic aggregates with neutrophils and enhancing their antimicrobial properties. Whilst release of vasoactive mediators from equine platelets in response to stimuli including bacterial lipopolysaccharide (LPS) has been documented, neither ROS production, nor the effects of activated platelets on equine neutrophil ROS production, have been reported. This study first sought evidence that activated equine platelets inhibit bacterial growth. Platelet superoxide production in response to stimuli including Escherichia coli-derived LPS and lipoteichoic acid (LTA) from Staphylococcus aureus was then determined. The ability of LPS and LTA to up-regulate platelet P-selectin expression and induce platelet–neutrophil aggregate formation was investigated and the effect of co-incubating activated platelets with neutrophils on superoxide production measured.Growth of E. coli was inhibited in a time-dependent manner, and to a similar extent, by addition of platelet rich plasma (PRP) or platelet poor plasma (PPP) obtained by centrifugation of PRP. Activation of platelets in PRP by addition of thrombin led to a significant increase in the inhibitory action between 0.5 and 2h.Although phorbol myristate acetate (PMA) caused superoxide production by equine platelets in a protein kinase C-dependent manner, thrombin, platelet activating factor (PAF), LPS, LTA and formyl-methionyl-leucyl phenylalanine (FMLP) were without effect. LPS and LTA did induce platelet activation, measured as an increase in P-selectin expression (% positive cells: 17±3 (un-stimulated); 63±6 (1μg/ml LPS); 64±6 (1μg/ml LTA); n=5) but not platelet superoxide production or heterotypic aggregate formation. Co-incubation of activated platelets with neutrophils did not increase neutrophil superoxide production.This study has demonstrated for the first time that when activated, equine platelets, like those of other species, are capable of releasing ROS that could assist in bacterial killing. However, the findings suggest that neither superoxide production by platelets nor enhancement of production by neutrophils is likely to play a significant role. Nevertheless, as has been reported in man, equine PPP and PRP did inhibit E. coli growth in vitro, and addition of thrombin significantly increased the inhibitory effect of PRP. This suggests that products released from activated platelets could contribute to antimicrobial activity in the horse. The factors in equine plasma and released by activated platelets that are responsible for inhibiting bacterial growth have yet to be determined.

Dangerous connections: neutrophils and the phagocytic clearance of activated platelets

Platelets and neutrophils co-localize at sites of vessel injury, hemorrhage and thrombosis. Moreover, circulating platelets and leukocytes interact productively, and the formation of heterotypic aggregates is a feature of acute coronary syndromes, systemic inflammatory, neoplastic and autoimmune diseases. We have summarized the evidence suggesting a homeostatic function of the interaction, culminating in the removal of activated platelets from the bloodstream. Anionic phospholipids, that is cell surface 'eat me' signals exposed both by activated platelets and dying cells, signals such as P-selectin (CD62P), specifically expressed by platelets, as well as of polarized clusters of neutrophils beta2 integrins play a role in the capture of platelets in vitro and in vivo. A bona-fide synapse assembles as a consequence of the interaction between P-selectin and its counter-receptor on neutrophils, with clustering of activated beta2 integrins into membrane microdomains and reorganization of cytoskeleton components that control cell motility and phagocytosis. Actual engulfment of the tethered platelet depends on the recognition of phosphatidylserine and/or of phosphatidylserine-associated molecules. This event may have a physiologic role in the regulation of the hemostatic potential of circulating blood; conversely, a failure may contribute to persistent vascular inflammation and thrombosis.

Neutrophil and platelet activation in equine recurrent airway obstruction is associated with increased neutrophil CD13 expression, but not platelet CD41/61 and CD62P or neutrophil–platelet aggregate formation

Recurrent airway obstruction (RAO) in mature horses is characterized by reversible airway obstruction and neutrophilic inflammation; there is also functional activation of circulating platelets and neutrophils. This study was undertaken to determine if changes in activation marker expression and heterotypic aggregate formation can be used as an indicator of this increased functional responsiveness. In vitro conditions for flow cytometric measurement of CD13, CD41/61 and CD62P expression on activated cells and heterotypic aggregate formation were established. Values were then compared before and after antigen challenge of RAO and healthy horses. Platelet adhesion to serum-coated plastic was measured as a functional marker of platelet activation. In vitro activation resulted in increased expression of neutrophil CD13 and platelet CD41/61 and CD62P. Activation of both cell types caused a significant increase in neutrophil–platelet aggregates. In horses with RAO, but not controls, there was a significant increase in the percentage of CD13 positive neutrophils at 10 h and 24 h and in the mean fluorescence intensity at 10 h. This was accompanied at 24 h by an increased mean platelet side scatter and thrombin-stimulated platelet adhesion. In conclusion, CD13 expression can be used as an indicator of equine neutrophil activation both in vitro and in vivo. Equine platelet activation in vitro can be detected by measuring CD41/61 or CD62P expression, and PAF-activated platelets and neutrophils form aggregates. However, despite evidence of circulating platelet activation, neither a change in expression of platelet activation markers, nor heterotypic aggregate aggregate formation could be detected.

Effect of adenosine A2 receptor stimulation on platelet activation–aggregation: Differences between canine and human models

IntroductionAdenosine A2 agonists improve arterial patency in experimental models of recurrent thrombosis, an effect purportedly triggered by stimulation of platelet A2 receptors and subsequent down-regulation of platelet function. However: (i) there is no direct evidence to substantiate this premise; and (ii) given the recognized differences among species in platelet signaling, it is possible that the mechanisms of A2 receptor stimulation may be model-dependent. Accordingly, we applied an integrated in vivo and in vitro approach, using both canine and human models, to test the hypothesis that the anti-thrombotic effects of A2 agonist treatment are due in part to inhibition of platelet activation. MethodsIn Protocol 1, recurrent coronary thrombosis was triggered in anesthetized dogs by application of a stenosis at a site of arterial injury. Coronary patency and flow cytometric indices of platelet activation (P-selectin expression; formation of heterotypic aggregates) were compared in dogs pre-treated with the A2 agonist CGS 21680 versus controls. In Protocols 2 and 3, blood samples were obtained from dogs and human volunteers. In vitro aggregation and platelet activation (assessed by impedance aggregometry and flow cytometry, respectively) were quantified in paired aliquots pre-incubated with CGS versus vehicle. ResultsIn the canine models, CGS improved in vivo coronary patency and attenuated in vitro aggregation but, contrary to our hypothesis, did not evoke a down-regulation in platelet activation. In contrast, in human blood samples, CGS attenuated both in vitro aggregation and flow cytometric markers of platelet activation–aggregation. ConclusionThe mechanisms contributing to the anti-thrombotic effect of A2 agonist treatment are species-dependent: adenosine A2 receptor stimulation inhibits platelet activation in human, but not canine, models.

Effects of etamsylate on equine platelets: In vitro and in vivo studies

The aim of this study was to investigate whether etamsylate produces equine platelet activation. In vitro and in vivo studies were designed in which seven and eight adult healthy horses were included, respectively. In the in vitro study, citrated blood was incubated with different concentrations of etamsylate, and P-selectin expression and annexin V binding were determined by flow cytometry. In the in vivo study, blood was collected before and 1 and 2 h after IV administration of etamsylate, and P-selectin expression was evaluated. In the in vitro study, a significant increase in P-selectin expression, leukocyte–platelet aggregate formation and annexin V binding were observed. In the in vivo study, a marked increase in P-selectin expression and heterotypic aggregate formation was seen in two and five horses, respectively, although no significant differences were detected when analyzing results from all the animals together. The results of the in vitro study indicate that etamsylate produces a pre-activation state in equine platelets, but this fact could be confirmed by the in vivo study.

The formation of platelet–leukocyte aggregates varies during the menstrual cycle

Platelet–leukocyte aggregates are considered to play a significant role in blood coagulation and inflammatory processes. We hypothesized that hormonal changes during the menstrual cycle affect the formation of heterotypic aggregates and therefore may constitute cycle-dependent variations of the susceptibility for thromboembolic events and inflammatory disease. We therefore measured platelet–leukocyte interaction by the determination of platelet–leukocyte aggregates (PLA), platelet P-Selectin expression, and platelet fibrinogen receptor activation by PAC-1 binding in 20 healthy women during their menstrual cycle by flow cytometry. The number of platelet–granulocyte aggregates (PGA) and platelet–monocyte aggregates (PMA) was higher at ovulation compared to any other time-point of the menstrual cycle (p = 0.005, p = 0.022, respectively). Likewise, P-Selectin expression peaked on day 14 (p = 0.040). The course of PLA formation during the menstrual cycle followed the course of estrogen levels, strongly suggesting direct effects of estrogen on platelet–leukocyte interaction. The susceptibility to form platelet–leukocyte aggregates that are inducible in vitro by a suboptimal concentration of thrombin receptor activating peptide-6 decreased slightly during the transition from day 1 to 14 (p = 0.040). These data indicate that platelet function varies during particular phases of the normal menstrual cycle.

Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule–1

The relative contributions of CD11a/CD18 and CD11b/CD18 to the dynamics and strength of neutrophil adhesion to intercellular adhesion molecule (ICAM)-1-transfected cells were examined over the time course of chemotactic stimulation. Suspensions of neutrophils and transfectants were sheared in a cone-plate viscometer, and formation of heterotypic aggregates was measured by 2-color flow cytometry. The 2-body collision theory was used to compute adhesion efficiency, defined as the proportion of collisions between neutrophils and target cells that resulted in capture. ICAM-1 surface density and shear rate both regulated adhesion efficiency. Target cells expressing approximately 1000 ICAM-1 sites/microm(2) (I(low)) were captured with an efficiency of 0.15 at 100 s(-1), which decreased to zero at 300 s(-1). At 8-fold higher ICAM-1 expression (I(high)) corresponding to levels measured on interleukin-1-stimulated endothelium, efficiency was 0.3 at 100 s(-1) and remained above background to 900 s(-1). Shear alone was sufficient for CD11a/CD18-mediated adhesion to ICAM-1, and stimulation with formyl-methionyl-leucyl-phenylalanine boosted capture efficiency through CD11a/CD18 by 4-fold. In comparison, CD11b/CD18 supported one third of this efficiency, but was necessary for aggregate stability over several minutes of shear and at shear stresses exceeding 5 dyne/cm(2). Hydrodynamics influenced capture efficiency predominantly through the collisional contact duration, predicted to be approximately 9 milliseconds for successful capture of I(low) and 4 milliseconds for I(high). The implication is that an increase in ICAM-1 from resting levels to those on inflamed endothelium effectively increases the permissible shear in which capture through beta(2)-integrins may occur. Neutrophil adhesion to ICAM-1 appears to be a cooperative and sequential process of CD11a-dependent capture followed by CD11b-mediated stabilization.

Platelet surface p-selectin, platelet-granulocyte heterotypic aggregates, and plasma-soluble p-selectin during plateletpheresis.

Plateletpheresis components have been shown to contain p-selectin-positive platelets after collection and storage. P-selectin mediates binding of activated platelets to granulocytes and monocytes. This study was undertaken to assess platelet activation, granulocyte activation, platelet-granulocyte heterotypic aggregate formation, and the plasma-soluble p-selectin level during plateletpheresis performed on a particular instrument (MCS+, Haemonetics). Flow cytometry was used to assay platelet surface p-selectin, granulocyte iC3b receptor, and platelet-granulocyte aggregates in the platelet component, residual blood in the disposable polycarbonate bowl of the MCS+, and in the donor blood with and without the addition of in vitro agonists before, during, and after plateletpheresis. The plasma-soluble p-selectin levels in the platelet component, disposable bowl, and donor venous blood were measured by an enzyme-linked immunosorbent assay. Levels of p-selectin-positive platelets, activated granulocytes, and platelet-granulocyte aggregates were greater in the disposable bowl than in the preapheresis donor blood. Levels of p-selectin-positive platelets, activated granulocytes, and platelet-granulocyte aggregates in the postapheresis donor blood were similar to those in the preapheresis donor blood. The platelet components contained no activated granulocytes or detectable platelet-granulocyte heterotypic aggregates, and only about 10-percent activated platelets. The plasma-soluble p-selectin level in the platelet component was significantly greater than that in the preapheresis donor blood, the residual blood in the disposable bowl, or the postapheresis donor blood. Measurements of platelet surface p-selectin, platelet-granulocyte heterotypic aggregates, and plasma-soluble p-selectin can be used to detect platelet activation during plateletpheresis.

Induction and Enhancement of FcεRI-Dependent Mast Cell Degranulation Following Coculture with Activated T Cells: Dependency on ICAM-1- and Leukocyte Function-Associated Antigen (LFA)-1-Mediated Heterotypic Aggregation

AbstractActivated mast cells are known to reside in close apposition to T cells in various inflammatory processes. In this regard, we have reported that activated mast cells form heterotypic aggregates with activated lymphocytes. To determine whether this interaction would result in mast cell degranulation, we examined the effect of EL-4, 2B4, or freshly isolated T cells, activated by PMA or immobilized anti-CD3 mAb, on histamine release from murine bone marrow-derived cultured mast cells (BMCMC). Coculturing BMCMC with activated but not with resting T cells resulted in significant histamine release. Also, FcεRI cross-linking-induced degranulation was augmented when BMCMC were cocultured with activated T cells. Supernatants of activated T cells failed to exert the stimulatory effect. Separation of the two cell populations with a porous membrane prevented degranulation, indicating that BMCMC activation was adhesion dependent. Indeed, the kinetics of histamine release paralleled the kinetics of the formation of heterotypic aggregates, which peaked after 12 h of coculture. Introduction of anti-LFA-1 and anti-intercellular adhesion molecule-1 mAb inhibited the adhesion-induced mast cell degranulation. These data suggest a heretofore unrecognized mast cell activation pathway induced by LFA-1/intercellular adhesion molecule-1-mediated heterotypic aggregation with activated T cells.

Activated T lymphocytes induce degranulation and cytokine production by human mast cells following cell-to-cell contact

Activated mast cells reside in close apposition to T cells in some inflammatory processes. In this study, we analyzed whether this close physical proximity affects human mast cell degranulation and cytokine release. Thus HMC-1 human mast cells or primary bone marrow-derived human mast cells were cocultured with activated and with resting T cells. Mast cells cocultured with activated T cells released histamine and beta-hexosaminidase and produced tumor necrosis factor alpha (TNF-alpha), an effect that peaked at 20 h. Kinetics of histamine release paralleled the formation of heterotypic aggregates. Separation of the two cell populations with a porous membrane prevented mediator release and TNF-alpha production. Addition of the PI3-kinase inhibitor, wortmannin, inhibited the heterotypic adhesion-associated degranulation but not TNF-alpha production. These data thus indicate a novel pathway through which human mast cells are activated to both release granule-associated mediators and to produce cytokines in association with heterotypic adhesion to activated human T cells.

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The agglutinabilities of the murine leukemia cell lines L1210, P388, and C1498 were determined in the presence of concanavalin A (Con A) by a quantitative cytoagglutination assay. The propensity of these cells to form heterotypic aggregates with normal syngeneic spleen cells, those obtained from mice carrying the respective leukemias in ascitic form, and syngeneic lung cells also were determined. Con A caused agglutination of all five types of leukemia cells and the resulting agglutination patterns had certain characteristics. Threshold concentrations of Con A, below which no significant cytoagglutination occurred, were very low. A steady increase in zeta, a previously defined index of agglutination that simultaneously takes into account the number of free cells and the number and size of the aggregates, was observed with increasing concentrations of Con A until a plateau was reached at 25-50 microgram/ml and this extended over a wide range of lectin concentrations (50-1,000 micrograms/ml). Self-aggregation of leukemia cells was not observed, and their propensity to form heterotypic aggregates with syngeneic lung cells was negligible. However, all leukemia cell lines formed measurable aggregates with spleen cells from both normal and leukemia-bearing mice; these aggregates usually reached a maximum plateau between 30 and 35 minutes of incubation and remained constant thereafter. Aggregation of leukemia cells with spleen cells from leukemic mice always was greater than that with spleen cells from normal mice. Con A agglutinability of leukemia cells was correlated with their propensity to form heterotypic aggregates, which suggests that Con A agglutinability of leukemia cells was correlated with their propensity to form heterotypic aggregates, which suggests that Con A receptor carbohydrate moieties may be involved in the intercellular adhesion leading to heterotypic aggregate formation.