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Key Event Title
Platelet-neutrophil interactions, Increased
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|TLR9 activation leading to ARDS and MOD||KeyEvent||Gillina Bezemer (send email)||Under development: Not open for comment. Do not cite|
|SARS-CoV-2 infection leading to thromboinflammation||KeyEvent||Luigi Margiotta-Casaluci (send email)||Under development: Not open for comment. Do not cite|
Key Event Description
Neutrophils represent 50–70% of all circulating leukocytes in humans and are the foot soldiers of the immune response, being the first immune cell on the scene of an infection or inflammation. They exert their functional roles via a combination of processes, including the generation of reactive oxygen species (ROS), degranulation, phagocytosis, and release of neutrophil extracellular traps (NETs). On the other hand, approximately 750 billion platelets circulate in humans and these cells are traditionally known for their role in haemostasis and thrombosis and are first responders at sites of vascular damage. More recently investigations have shown that platelets play important roles in inflammatory and immune processes (Gu et al., 2019), including resolution (Senchenkova et al., 2019). Platelets have a range of different receptors including Toll-like receptors (TLRs), C-type lectin receptors, and nucleotide-binding and oligomerization domain–like receptors, which are known to recognise infection (Semple et al., 2011) and are effectors of injury in a variety of pulmonary disorders and syndromes, and facilitate tissue repair (Xu et al., 2016).
These two cell types modulate each other’s functions, and neutrophil-platelet interaction and platelet-platelet interactions are increased during inflammation (Lisman, 2018). Circulating platelet–neutrophil complexes have been observed in many different inflammatory conditions including bacterial infections (Gawaz et al. 1995) and pulmonary syndromes involving inflammation (Caudrillier et al. 2012). The physical interaction between neutrophils and platelets is mediated by multiple molecular mechanisms including platelet P-selectin binding to neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) (Moore et al. 1995) and platelet glycoprotein Ibα binding to neutrophil MAC-1 (Simon et al. 2000).
A first important example of functional interaction between the two cell types is the ability of platelets to facilitate neutrophil adhesion to activated endothelial cells (i.e. at the site of inflammation or injury) and to promote their transmigration across the endothelium. Many studies have demonstrated that neutrophils patrol the vasculature for activated platelets to initiate inflammatory responses (Sreeramkumar et al. 2014), hence activated platelets are crucial in neutrophil-mediated inflammatory responses (Senchenkova et al., 2019). The depletion of platelets decreases neutrophil recruitment to the site of inflammation, and the depletion of neutrophils decreases platelet recruitment (Sreeramkumar et al. 2014). During activation, both neutrophils and platelets are capable of releasing microparticles, (MPs, also termed ectosomes) which carry markers of the parent cells outer membrane e.g. neutrophil MPs carry different cell adhesion molecules and proteases such as proteinase 3 or elastase (Ramirez et al., 2019).
Beyond recruitment and localization processes, activated platelets are able to modulate (i.e. both enhancing and inhibiting) neutrophil responses, including phagocytosis, production of ROS, and production of NETs. These effects can be mediated by direct cell-cell contact or by the release of soluble mediators such as CCL5 and platelet factor 4 (von Hundelshausen et al. 2005). On the other hand, neutrophils can also release soluble mediators (e.g. cathepsin G, elastase) that enhance or inhibit platelet responses (Mihara et al. 2013; Bonnefoy and Legrand 2000).
Platelets can stimulate the synthesis of various leukotrienes by neutrophils via transcellular transfer of the arachidonic acid metabolite 12-HETE, which is further processed by neutrophils into bioactive leukotrienes (Rossaint et al., 2018). Platelets also store preformed and immunomodulatory molecules that can affect neutrophils (e.g. affecting chemotaxis) and the immune response, including interleukin-1 and platelet-derived growth factor (Semple et al., 2011).
Over the past several years, the interactions between immune cells such as neutrophils and platelets have been given different names, including: thromboinflammatoin, immunothrombosis and immunohemostasis (Guo et al., 2019). These names have been introduced to try to capture the responses and mechanisms of both cell types (independently and collectively) in thrombosis and inflammation.
How It Is Measured or Detected
Neutrophil-platelet complexes (sometimes called platelet-leukocyte aggregation, PLA) can be measured using the following techniques (see Finsterbusch et al. (2018) for a comprehensive review):
- Microfluidic assays can allow to visualize neutrophil-platelet interactions in real time in vitro. This approach involves the use of isolated cells or whole blood that are perfused through a chamber coated with immobilized proteins or cells. Specific cells are visualized thanks to the use of specific antibodies. This technique allows a fine tuning of experimental conditions and the use of human cells.
- Flow cytometry is the method of choice to measure circulating neutrophil-platelet complexes in both humans and animals. This technique is based on fluorescence-labelling of platelets and leukocytes in whole blood. Characteristic side scatter properties of leukocyte subtypes already permit basic discrimination of platelets binding to neutrophils, monocytes and lymphocytes. However, leukocyte subtype-specific antibodies generate more accurate data. The endpoint is quantified as the percentage of total leukocytes (i.e. neutrophils) that also stain for a platelet-specific marker. A limitation of standard flow cytometry is that potential platelet-derived macrovesicles fused to leukocytes could influence results.
- Flow cytometry coupled with fluorescence microscopy (imaging flow cytometry). This approach enables direct assessment of both number of bound platelets as well as involved interaction molecules and coincidental events.
- Histochemical and immunofluorescent imaging of frozen or paraffin-embedded tissue sections. This approach can provide valuable information about the location of platelet recruitment and neutrophil-platelet complexes within tissue microenvironments of most organs. These techniques can be coupled with confocal or electron microscopy to provide higher resolution visualization of platelet-neutrophil interactions. A major limitation of this approach is that it only provides a static assessment of the endpoint.
- Live cell imaging coupled with intravital microscopy (IVM) allows to track labeled cells in live animals over longer time periods in organs including liver, lung, and brain. This approach can be used to analyze the dynamic interactions between platelets and neutrophils (e.g., duration of interactions and behavioural changes in neutrophils and/or platelets upon contact).
- Intravenous administration of fluorochrome-conjugated monoclonal antibodies targeting specific protein markers on platelets.
Domain of Applicability
Bonnefoy, A., Legrand, C., 2000. Proteolysis of Subendothelial Adhesive Glycoproteins (Fibronectin, Thrombospondin, and von Willebrand Factor) by Plasmin, Leukocyte Cathepsin G, and Elastase. Thrombosis Research 98, 323-332.
Caudrillier, A., Kessenbrock, K., Gilliss, B.M., Nguyen, J.X., Marques, M.B., Monestier, M., Toy, P., Werb, Z., Looney, M.R., 2012. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest 122, 2661-2671.
Finsterbusch, M., Schrottmaier, W.C., Kral-Pointner, J.B., Salzmann, M., Assinger, A., 2018. Measuring and interpreting platelet-leukocyte aggregates. Platelets 29, 677-685.
Gawaz, M., Fateh‐Moghadam, S., Pilz, G., Gurland, H.-., Werdan, K., 1995. Platelet activation and interaction with leucocytes in patients with sepsis or multiple organ failure. European Journal of Clinical Investigation 25, 843-851.
Guo, L., Rondina, M.T., 2019. The Era of Thromboinflammation: Platelets Are Dynamic Sensors and Effector Cells During Infectious Diseases. Front Immunol 10.
Lisman, T., 2018. Platelet–neutrophil interactions as drivers of inflammatory and thrombotic disease. Cell Tissue Res 371, 567-576.
Mihara, K., Ramachandran, R., Renaux, B., Saifeddine, M., Hollenberg, M.D., 2013. Neutrophil Elastase and Proteinase-3 Trigger G Protein-biased Signaling through Proteinase-activated Receptor-1 (PAR1). J Biol Chem 288, 32979-32990.
Moore, K.L., Patel, K.D., Bruehl, R.E., Li, F., Johnson, D.A., Lichenstein, H.S., Cummings, R.D., Bainton, D.F., McEver, R.P., 1995. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. Journal of Cell Biology 128, 661-671.
Ramirez, G.A., Manfredi, A.A., Maugeri, N., 2019. Misunderstandings Between Platelets and Neutrophils Build in Chronic Inflammation. Front Immunol 10.
Rossaint, J., Margraf, A., Zarbock, A., 2018. Role of Platelets in Leukocyte Recruitment and Resolution of Inflammation. Front Immunol 9.
Semple, J.W., Italiano, J.E., Freedman, J., 2011. Platelets and the immune continuum. Nature Reviews Immunology 11, 264-274.
Senchenkova, E.Y., Ansari, J., Becker, F., Vital, S.A., Al-Yafeai, Z., Sparkenbaugh, E.M., Pawlinski, R., Stokes, K.Y., Carroll, J.L., Dragoi, A., Qin, C.X., Ritchie, R.H., Sun, H., Cuellar-Saenz, H.H., Rubinstein, M.R., Han, Y.W., Orr, A.W., Perretti, M., Granger, D.N., Gavins, F.N.E., 2019. Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation. Circulation 140, 319-335.
Simon, D.I., Chen, Z., Xu, H., Li, C.Q., Dong, J., McIntire, L.V., Ballantyne, C.M., Zhang, L., Furman, M.I., Berndt, M.C., López, J.A., 2000. Platelet Glycoprotein Ibα Is a Counterreceptor for the Leukocyte Integrin Mac-1 (Cd11b/Cd18). J Exp Med 192, 193-204.
Sreeramkumar, V., Adrover, J.M., Ballesteros, I., Cuartero, M.I., Rossaint, J., Bilbao, I., Nácher, M., Pitaval, C., Radovanovic, I., Fukui, Y., McEver, R.P., Filippi, M., Lizasoain, I., Ruiz-Cabello, J., Zarbock, A., Moro, M.A., Hidalgo, A., 2014. Neutrophils scan for activated platelets to initiate inflammation. Science 346, 1234-1238.
von Hundelshausen, P., Koenen, R.R., Sack, M., Mause, S.F., Adriaens, W., Proudfoot, A.E.I., Hackeng, T.M., Weber, C., 2005. Heterophilic interactions of platelet factor 4 and RANTES promote monocyte arrest on endothelium. Blood 105, 924-930.
Xu, X.R., Zhang, D., Oswald, B.E., Carrim, N., Wang, X., Hou, Y., Zhang, Q., Lavalle, C., McKeown, T., Marshall, A.H., Ni, H., 2016. Platelets are versatile cells: New discoveries in hemostasis, thrombosis, immune responses, tumor metastasis and beyond. Critical Reviews in Clinical Laboratory Sciences 53, 409-430.