Measuring and interpreting platelet-leukocyte aggregates

Abstract

Platelets, besides their specialised role in haemostasis and atherothrombosis, actively modulate innate and adaptive immune responses with crucial roles in immune surveillance, inflammation and host defence during infection. An important prerequisite for platelet-mediated changes of immune functions involves direct engagement with different types of leukocytes. Indeed, increased platelet-leukocyte aggregates (PLAs) within the circulation and/or locally at the site of inflammation represent markers of many thrombo-inflammatory diseases, such as cardiovascular diseases, acute lung injury, renal and cerebral inflammation. Therefore, measurement of PLAs could provide an attractive and easily accessible prognostic and/or diagnostic tool for many diseases. To measure PLAs in different (patho-)physiological settings in human and animal models flow cytometric and microscopic approaches have been applied. These techniques represent complementary tools to study different aspects relating to the involvement of leukocyte subtypes and molecules, as well as location of PLAs within tissues, dynamics of their interactions and/or dynamic changes in leukocyte and platelet behaviour. This review summarises various approaches to measure and interpret PLAs and discusses potential experimental factors influencing platelet binding to leukocytes. Furthermore, we summarise insights gained from studies regarding the underlying mechanism of platelet-leukocyte interactions and discuss implications of these interactions in health and disease.

Keywords: Platelet-leukocyte aggregates; flow cytometry; inflammation; live cell imaging.

Figure 1.

Methods to investigate platelet-leukocyte aggregate (PLA) formation. (A) Flow cytometry represents a high-throughput approach to quantify circulating PLAs in human and animal blood and to investigate expression of involved molecules. Due to its fast protocol and independence of highly specific machines, standard flow cytometry is readily usable in clinical settings. Panels show gating strategy to determine platelet hetero-aggregates with distinct leukocyte subtypes in stimulated murine blood. CD45 (pan-leukocyte marker), Ly6G (neutrophil marker), CD11b (marker to exclude cells other than lymphocytes) and CD41 (pan-platelet marker). (B) Tissue sectioning adds information about location of PLAs within human or animal tissues in conjunction with quantitative and qualitative evaluation of PLAs. However, preparation and evaluation of samples is more time-consuming and does not allow for high-throughput analysis, making tissue sectioning less attractive for clinical laboratories. Images show PLAs in inflamed murine lung histology sections (neutrophils labelled with anti-CD45 and platelets with anti-CD41). C) Concomitant live analysis of multiple cell types in animals in vivo or of human or animal blood cells in microfluidic devices provides information about dynamic changes of platelet-leukocyte interactions and their effect on cellular effector functions within physiologic microenvironments or under semi-physiological conditions under flow. Live cell imaging to investigate PLAs requires state-of-the-art microscopy facilities and areas of interest that are accessible for microscopy, thus making it well suited for basic research but not for clinical applications. Panel shows multiphoton image sequences of a platelet (labelled with anti-CD49b) interacting with a GFP⁺ neutrophil within glomerular capillaries of a LysM-GFP mouse over time (endothelial cells labelled with anti-CD31). PLA: platelet-leukocyte aggregate, SSC-A: side scatter––area, DAPI: 4′,6-Diamidin-2-phenylindol.

Figure 2.

Overview of PLA formation and their effects in (thrombo-) inflammatory pathologies of selected organs. Physical interactions of platelets with neutrophils, monocytes/macrophages, tissue-resident leukocytes or lymphocytes have been reported in a number of (thrombo-)inflammatory diseases of the circulatory system, brain, liver, kidney, and lung. Effects of platelet-leukocyte binding on cellular and/or molecular responses are summarised for individual leukocyte subtypes. COPD: chronic obstructive pulmonary disease, I/R: ischemia/reperfusion, JAM-C: Junctional adhesion molecule-C, NET: neutrophil extracellular trap, PMA: platelet-monocyte aggregate, PNA: platelet-neutrophil aggregate, PSGL-1: P-selectin glycoprotein ligand 1, ROS: reactive oxygen species, vWF: von Willebrand factor.