Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca²⁺levels. Through this they act as Ca²⁺-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca²⁺-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.

Inflammation is the body’s way of defending itself against noxious stimuli and pathogens. Under normal circumstances, the body is able to eliminate the insult and subsequently promote the resolution of inflammation and the repair of damaged tissues. The concept of homeostasis is one that not only requires a fine balance between both pro-inflammatory mediators and pro-resolving/anti-inflammatory mediators, but also that this balance occurs in a time and space-specific manner. This review examines annexin A1, an anti-inflammatory protein that, when used as an exogenous therapeutic, has been shown to be very effective in limiting inflammation in a diverse range of experimental models, including myocardial ischemia/reperfusion injury, arthritis, stroke, multiple sclerosis, and sepsis. Notably, this glucocorticoid-inducible protein, along with another anti-inflammatory mediator, lipoxin A₄, is starting to help explain and shape our understanding of the resolution phase of inflammation. In so doing, these molecules are carving the way for innovative drug discovery, based on the stimulation of endogenous pro-resolving pathways.

Recent investigations conducted with human neutrophils have indicated an involvement for the receptor for formylated peptides, termed FPR, and its analog FPRL1 (or ALXR because it is the receptor for the endogenous ligand lipoxin A₄) in the in vitro inhibitory actions of the glucocorticoid-regulated protein annexin 1 and its peptidomimetics. To translate these findings in in vivo settings, we have used an ischemia/reperfusion (I/R) procedure to promote leukocyte-endothelium interactions in the mouse mesenteric microcirculation. In naive mice, the annexin 1 mimetic peptide Ac2-26 (20 to 100 μg administered intravenously prior to reperfusion) abolished I/R-induced cell adhesion and emigration, but not cell rolling. In FPR-deficient mice, peptide Ac2-26 retained significant inhibitory actions (about 50% of the effects in naive mice), and these were blocked by an FPR antagonist, termed butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe, or Boc2. In vitro, neutrophils taken from these animals could be activated at high concentrations of formyl-Met-Leu-Phe (30 μM; fMLP), and this effect was blocked by cell incubation with peptide Ac2-26 (66 μM) or Boc2 (100 μM). FPR-deficient neutrophils expressed ALXR mRNA and protein. Both ALXR agonists, lipoxin A₄ and peptide Ac2-26, provoked detachment of adherent leukocytes in naive as well as in FPR-deficient mice, whereas the CXC chemokine KC or fMLP were inactive. The present findings demonstrate that endogenous regulatory autocoids such as lipoxin A₄ and annexin 1–derived peptides function to disengage adherent cells during cell-cell interactions.

Background: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. Methods: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1 ^−/− ) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye–induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. Results: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1 ^−/− mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B ₂ and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease α _IIb β ₃ activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5–13 mice/group or 7–10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (α _IIb β ₃ ) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.

After injury or infection, neutrophils rapidly migrate from the circulation into tissues by means of an orderly progression of adhesion receptor engagements. Neutrophils have been previously considered to use selectins exclusively to roll on vessels before an adhesion step mediated by the β2 integrins, lymphocyte function–associated antigen (LFA)-1, and Mac-1. Here we use LFA-1−/− mice, function blocking monoclonal antibodies, and intravital microscopy to investigate the roles of LFA-1, Mac-1, and α4 integrins in neutrophil recruitment in vivo. For the first time, we show that LFA-1 makes a contribution to neutrophil rolling by stabilizing the transient attachment or tethering phase of rolling. In contrast, Mac-1 does not appear to be important for either rolling or firm adhesion, but instead contributes to emigration from the vessel. Blocking Mac-1 in the presence of LFA-1 significantly reduces emigration, suggesting cooperation between these two integrins. Low levels of α4β1 integrin can be detected on neutrophils from LFA-1+/+ and −/− mice. These cells make use of α4β1 during the rolling phase, particularly in the absence of LFA-1. Thus LFA-1 and α4β1, together with the selectins, are involved in the rolling phase of neutrophil recruitment, and, in turn, affect the later stages of the transmigration event.