It is well-established that under physiological conditions, platelets circulate in the bloodstream exhibiting a discoid shape in the resting state. Three types of secretory granules in platelets carry large amounts of active substances, including α-granules, dense granules, and lysosomes (42). Upon platelet activation, these granules undergo exocytosis and release contained substances, including platelet-specific derived mediators such as PF4, β-TG, soluble P-selectin, soluble CD40 ligand (sCD40L), and thrombospondin-1 (TSP-1). During this process, P-selectin translocates from α-granules to the platelet surface, and integrin αIIbβ3 (also known as glycoprotein IIbIIIa) undergoes conformational changes, obtaining a high affinity for its ligand (43–45). These findings suggest that platelet activation can be monitored by detecting platelet-specific derived mediators and platelet-specific surface markers by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively (43). The detailed information on platelet activation has been discussed in another review (43).

In 1981, a study reported that plasma PF4 was increased in patients with allergic asthma, providing the first evidence of the relationship between platelet activation and allergic asthma (13). There is ample evidence suggesting that platelet-specific products, including PF4, β-TG, and platelet microparticles, are elevated in allergic asthma subjects, confirming the presence of platelet activation in allergic asthma (14–20). Nowadays, a more detailed mechanism of platelet activation in allergic asthma has been documented, involving three different pathways for triggering platelet activation. 1) Inflammation induces platelet activation. Inflammatory reaction causes endothelial dysfunction, leading to increased production of thromboxane A₂ (TXA₂), von Willebrand factor (vWF), and thrombin, which are potent platelet agonists. Many inflammatory mediators such as proinflammatory cytokines or PAF also activate platelets (46–48). Additionally, CD40L-positive T cells in immune responses can activate platelets by interacting with CD40 on platelets (49, 50). 2) IgE generated during allergic reactions mediates platelet activation. Importantly, platelets express high- and low-affinity IgE receptors (FcϵRI and FcϵRII) (51–53). Moreover, current evidence suggests that in allergic asthmatic patients, the binding of IgE to platelets leads to their direct activation after contact with the corresponding allergens (50). 3) Different allergens may generate different mediators to activate platelets. In Candida albicans-induced airway infection, the fungi peptide toxin candidalysin was found to activate platelets by binding to GPIbα on platelets, resulting in the secretion of Wnt antagonist Dickkopf-1 (Dkk-1), which then drives allergic reactions (54).

Previous investigations on platelets have mainly focused on the biological processes in the vasculature. Accumulating evidence suggests that platelets migrate out of vessels and localize underneath the airways in allergic asthma subjects, enabling them to participate in tissue inflammation directly. Moreover, platelets form aggregates with leukocytes in the circulating blood of patients with allergic asthma, which contribute to the latter’s pulmonary recruitment (25). Surprisingly, further evidence showed that platelet migration precedes leukocyte trafficking into lung tissue and a high proportion of platelets are not adjacent to leukocytes, suggesting that platelets are not just accessory factors for leukocytes in tissue inflammation (22, 23).

The recruitment of platelets into the lung is largely dependent on the IgE receptor FcϵRI given that platelet penetration into the lungs and platelet chemotaxis toward ovalbumin (OVA) are severely impaired by FcϵRI deficiency (22). Moreover, platelets express chemokine receptors CCR3, CCR4, and CXCR4, which have been reported to control myeloid-cell migration during allergic inflammation. As a result, platelets undergo migration in response to the ligands of CCR3, CCR4, and CXCR4 (23). Paradoxically, in an allergic asthmatic mouse model, inhibition of CCR3 and CCR4 but not CXCR4 reduced platelet migration into the lungs. Besides, the expression of eotaxin, the chemokine ligand for CCR3, was increased in the airway smooth muscle of allergic asthmatic patients, which further promoted intrapulmonary recruitment of platelets. These findings suggest that the chemokine receptor CCR3 is essential in pulmonary platelet recruitment. However, it is noteworthy that CXCR4 inhibition significantly reduces the recruitment of leukocytes to the lungs, suggesting that the recruitment of platelet and leukocyte involves different pathways (23).

DCs have been documented to play a critical role in allergen sensitization (55). In this regard, DCs migrate to the secondary lymphatic organs after the uptake of inhaled allergens, presenting the processed antigens to naive CD4⁺ Th cells and promoting Th2 cell differentiation (56). A recent study documented that platelets and DCs were co-localized around the airway walls in allergen-sensitized mice, and transient platelet depletion could inhibit the pulmonary recruitment of DCs during the sensitization process (57). In addition, activated platelets can aggregate to thymic stromal lymphopoietin (TSLP)-stimulated DCs to induce the maturation of DCs and the production of CCL17 (a Th2 cell-attracting chemokine) (24). These studies demonstrated that interactions between platelets and DCs are crucial for allergen sensitization. Furthermore, Mac-1 (also known as integrin CD11b/CD18) on DCs and junctional adhesion molecules (JAM)-C on platelets, RANK on DCs and RANKL on platelets, as well as CD40 on DCs and CD40L on platelets, may contribute to these interactions (24, 58, 59) (Figure 2A). Interestingly, platelet-derived mediators such as ATP (35), histamine (60), and serotonin (61) have also been identified to promote the maturation and Th2 priming capacity of DCs.

Eosinophilic infiltration of the airway has been established as a hallmark of allergic asthma. Mounting evidence indicates that eosinophils migrate to the bronchial tissue during the late-phase asthmatic response (62, 63) and secrete large amounts of toxic proteins and proinflammatory mediators, leading to bronchoconstriction, airway hyperresponsiveness, and mucus overproduction (3). Importantly, eosinophil recruitment from the circulation requires their activation (64, 65), which has been reported to be positively associated with platelet activation in asthmatic airways (66, 67). Moreover, it has been shown that platelets can interact directly with eosinophils to mediate pulmonary eosinophil recruitment in a mouse model of OVA-induced asthma (25).

Mechanistic studies have demonstrated that platelet-eosinophil interactions involve platelet P-selectin and integrin αIIbβ3 ( Figure 2B ). P-selectin is an adhesion molecule, and its counter-receptor is P-selectin GP ligand-1 (PSGL-1) (68). It has been shown that platelets lacking P-selectin are unable to mediate eosinophil recruitment (25). Similarly, blockade by antibodies against P-selectin can reduce the binding and clustering abilities of eosinophils on the endothelium of allergic asthma patients (62). Further studies indicated that platelet P-selectin is required for the activation of eosinophil β1 integrin (69), which pairs with α4-integrin and then binds to the vascular cell adhesion molecule-1 (VCAM-1) of endothelial cells, mediating the rolling, adhesion, and migration of eosinophils on the endothelium (70). Current evidence suggests that integrin αIIbβ3 is a major membrane protein on platelets and is crucial for platelet aggregation (45, 71). Besides, a study documented that the inhibition of integrin αIIbβ3 by tirofiban could significantly reduce the platelet-eosinophil aggregates and pulmonary eosinophil infiltration in allergic airways and reduce airway hyperresponsiveness and inflammation (72). Mechanistic studies further indicated that Mac-1 on eosinophils could interact with integrin αIIbβ3 through a bridge molecule such as fibrinogen and sCD40L (73, 74).

It has been established that eosinophil extracellular traps (EETs), composed of DNA and major basic protein (MBP), are almost universally formed in the allergic asthmatic airways and associated with the severity of asthma (75). There is overwhelming evidence that EETs can increase proinflammatory cytokine production, airway epithelial permeability, goblet-cell hyperplasia, mucus production, and pulmonary inflammation during allergic asthma (75, 76). A negative correlation has also been found between the levels of EETs and pulmonary ventilatory function in asthmatic patients (76). Moreover, a mechanistic investigation revealed that EETs could activate pulmonary neuroendocrine cells (PNECs) and amplify allergic immune responses via neuropeptides and neurotransmitters (75). In recent years, platelet-eosinophil interactions have been shown to induce EETs formation to enhance thrombus stability in thrombosis (77). Accordingly, it is reasonable to speculate that these interactions may also be involved in the formation of EETs during allergic asthma.

Although allergic asthma is characterized by eosinophilic airway inflammation, neutrophils are also reportedly activated and critical in disease pathogenesis (78), especially in infection-related allergic asthma (79). Importantly, chemotaxis, phagocytic activity, and ROS production of neutrophils can be induced by allergen challenge in allergic asthma patients (80). Further evidence demonstrated that pulmonary recruitment of neutrophils is dependent on CXCR2 (the receptor for CXCL1, CXCL2, and CXCL5) during allergy challenges (78, 81). Importantly, blocking CXCR2 was found to inhibit lung recruitment of neutrophils and reduce allergic sensitization and airway inflammation (81). Beyond that, neutrophils have been shown to function as antigen-presenting cells (APCs) to activate T cells. Once the allergen is ingested, neutrophils present antigens to allergen-specific T cells to activate them and display a Th2-phenotype, producing a large amount of IL-4, IL-5, and IL-13 (82).

Interestingly, neutrophils recruited to the lungs function as APCs to present allergens to T cells and release neutrophil elastase (NE) and NETs to promote airway inflammation. As a major proteinase in primary granules in neutrophils (83), NE is released into the lungs after neutrophilic recruitment (84), inducing airway remodeling by upregulating the expression of chemokines CCL24 and IL-33 in epithelial cells (84), increasing mucus production and destroying ciliary motility (83). As expected, inhibition of NE could alleviate asthmatic airway inflammation (84). In addition, NETs have been documented in the airways of allergic asthma patients (85). Besides, a study found that exposure to a low dose of lipopolysaccharides (LPS) could upregulate CXCR4 expression on neutrophils recruited to the lungs, triggering the release of NETs by these neutrophils in mouse models of allergic asthma (79). It is widely believed that exacerbation of allergic asthma largely depends on the formation of NETs and NET-associated dsDNA. Intriguingly, NET-associated dsDNA contributes to pulmonary recruitment of monocyte-derived DCs (86), producing proinflammatory chemokines and presenting allergens to T cells (87). Moreover, injection of exogenous dsDNA has been reported to directly stimulate naive CD4⁺ Th cells to induce Th2 cell differentiation and activation and exhibit exacerbation features of type 2 inflammation and asthma pathology (86).

Platelets have been shown to interact with neutrophils and promote their activation and NETs formation in a wide range of inflammatory conditions (88). Despite the lack of relevant studies on platelet-neutrophil interactions in allergic asthma, we hypothesize that platelet-neutrophil interactions may exist and participate in the pathogenesis of allergic asthma given that platelet-leukocyte aggregates have been found in allergic asthmatic patients (89) and neutrophils are the most abundant leukocytes in the blood (78). The molecular mechanisms underlying platelet-neutrophil interactions have been discussed in detail in other reviews, involving several surface proteins on platelets and neutrophils, including P-selectin/PSGL-1, GPIbα/Mac-1, integrin αIIbβ3/Mac-1, intercellular adhesion molecule-2 (ICAM-2)/integrin αLβ2, etc (26, 88) ( Figure 2C ). In the context of allergic asthma, some platelet-derived mediators, such as ATP (35), serotonin (37), and PAF (39), can promote neutrophil activity. Substantial evidence suggests that ATP encourages neutrophil activation (90), pulmonary recruitment (91), and NETs formation (92). Besides, serotonin induces neutrophil degranulation (93) and recruitment to the inflammation sites (61), while PAF induces pulmonary recruitment of neutrophils in asthmatic patients (94) and plays a crucial role in neutrophil adhesion to platelets (95). Besides, platelets express pattern recognition receptors (PRRs), enabling them to recognize microbial structures during infection (96). The platelet toll-like receptor 4 (TLR4), belonging to the PRR family, is necessary for NETs formation for clearance of bacteria during sepsis (97). Accordingly, as a receptor for LPS (98), platelet TLR4 may also contribute to LPS-triggered NETs formation in infection-related allergic asthma.

Current evidence suggests that mediators secreted by platelets can regulate platelet function. As mentioned above, it has been established that three types of granules containing large amounts of biochemical molecules are present in platelets: α-granules, dense granules, and lysosomes. α-granules contain a wide variety of molecules, such as adhesive glycoproteins, coagulation factors, growth factors, cytokines, etc; dense granules contain several types of small molecules, including amines, cations, nucleotides, and polyphosphates; lysosomes contain various enzymes like many other cell types. Beyond that, platelets also synthesize multiple molecules upon activation, such as interleukin, PAF, and TXA₂ (27, 42). The secretion of platelet granules is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins and SNARE regulatory proteins (99). Moreover, Munc13 family proteins are well-recognized as essential SNARE regulatory proteins, and Munc13-4 is the only Munc13 family member expressed in platelets. Munc13-4 deficiency has been shown to inhibit dense granule secretion from platelets and severely impair α-granule and lysosomal secretion (100). Likewise, in an OVA-induced mouse allergic asthmatic model, the platelet-specific Munc13-4 knockout mice exhibited reduced airway hyperresponsiveness and eosinophilic inflammation. Taken together, these findings demonstrate that platelet granule secretion is vital for the progression of allergic asthma (101). Moreover, a large number of mediators involved in allergic asthma have been documented in platelet granules, such as ATP, histamine, serotonin, transforming growth factor β (TGFβ), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), etc (28–37). Several molecules, such as IL-33, PAF, and TXA₂, synthesized by platelets, have also been found to promote allergic reactions (27, 38–41) ( Figure 3 ).

As mentioned, Aspirin and Ozagrel inhibit the TXA₂ production (165). Aspirin is the most commonly used antiplatelet drug (166). It is well-established that the mechanism of aspirin involves acetylation of a serine residue on COX, leading to suppression of prostaglandins and TXA₂ synthesis (156). Importantly, low doses of aspirin (75 ~ 300 mg) can selectively inhibit platelet TXA₂ production and prevent cardiovascular disease (153). Existing evidence suggests that aspirin exerts a preventive effect on asthma: two large randomized, double-blind, placebo-controlled trials involving 22040 men and 37270 women revealed that aspirin could reduce the relative risk of newly diagnosed adult-onset asthma (22% lower in men and 10% lower in women) (157, 158). However, another study found that aspirin did not alleviate airway inflammation or pulmonary function in patients with mild-to-moderate persistent asthma (159). Ozagrel is widely used for asthma and stroke in Japan (167) and for stroke in China (165). On the contrary, it has not been approved by the Food and Drug Administration (FDA) or European Medicines Agency (EMA). It has been demonstrated that administration of Ozagrel can significantly decrease clinical symptom of asthma and reduce the dose of concomitant steroid therapy in Japanese clinical studies (160).

Nonetheless, it should be borne in mind that aspirin can induce a set of asthma-related symptoms in susceptible populations, referred to as aspirin-exacerbated respiratory disease or aspirin-induced asthma (168). Current evidence suggests that aspirin-induced asthma mainly results from dysregulation of arachidonic acid metabolism, leading to elevated levels of cysteinyl leukotrienes (CysLTs) and prostaglandin D₂ (PGD₂) and reduced levels of prostaglandin E₂ (PGE₂). It has been shown that elevated CysLTs and PGD₂ can trigger bronchoconstriction, vascular leakage, recruitment of inflammatory cells, and mucus overproduction. On the other hand, PGE₂ possesses anti-inflammatory properties and can induce bronchodilation (168). Surprisingly, aberrantly activated platelets have been documented in patients with aspirin-induced asthma rather than exhibiting an inhibition state as expected. Large numbers of platelet-adherent leukocytes have been reported in the peripheral blood and nasal polyp tissues of patients with aspirin-induced asthma. Indeed, low-dose aspirin (100 mg/d) does not reduce platelet-leukocyte aggregation. Moreover, adherent platelets contribute to the overproduction of CysLTs from leukocytes. Notwithstanding that it remains unclear whether platelets in patients with aspirin-induced asthma are intrinsic deficient, platelets have been demonstrated to act as effector cells in this disease (169). Therefore, aspirin does not inhibit platelet activity in this particular patient population; instead, it stimulates platelet activation and their interaction with leukocytes to promote airway inflammation.

Platelets express four types of purinergic receptors: P2Y1R, P2Y12R, P2X1R, and P2Y14R (170). As mentioned before, P2Y1R and P2Y12R represent ADP receptors, and P2X1R is an ATP receptor. P2Y14R is a receptor for UDP-glucose with no hemostatic function (171), while P2Y12R is a receptor for leukotriene E4 (LTE4), which is a powerful inducer of mucosal eosinophilia and airway hyperresponsiveness in asthma. It has been shown that platelet depletion could suppress the stimulatory effect of LTE4 on eosinophilia, goblet cell metaplasia, and IL-13 expression in OVA-induced allergic asthma mice (172). Furthermore, an association has been found between SNPs and haplotypes in P2Y12 and lung function in a large family-based asthma cohort; house dust mite exposure resulted in significant gene-by-environment effects (173). P2Y12R is well-recognized as the target of commonly used purinergic receptor antagonists, such as clopidogrel, prasugrel, and ticagrelor (156). The therapeutic effects of clopidogrel, prasugrel, and ticagrelor in allergic asthma have been extensively investigated over the years. Substantial evidence suggests that clopidogrel decreases airway hyperresponsiveness, eosinophilic infiltration, goblet cell hyperplasia, and type 2 cytokine production in an OVA-induced allergic asthmatic mouse model (161). Clopidogrel has also been found to decrease platelet-eosinophil aggregates (174). Likewise, a proof-of-concept, placebo-controlled, randomized, cross-over study (26 asthmatic patients) demonstrated that prasugrel treatment (10 mg/day for 15 days) could reduce airway hyperresponsiveness in asthma patients (162). However, no significant improvement in pulmonary function was observed with ticagrelor (a single 450 mg dose, 180 mg 12 hours later, twice daily for 2 days, and once on day 4) in mild asthmatic patients (n=11) (163). The different effects of clopidogrel, prasugrel, and ticagrelor on allergic asthma may be attributed to their different anti-P2Y12 mechanisms since clopidogrel and prasugrel are irreversible, competitive, thienopyridine P2Y12 receptor antagonists, and ticagrelor is a direct-acting, non-competitive, reversible, cyclopentyl-triazolopyrimidine P2Y12 receptor antagonist (156).

Interestingly, P2Y1R activation also participates in the pathogenesis of allergic asthma. Studies have shown that treatment with P2Y1R antagonists, MRS2179 and MRS2500, could decrease pulmonary leukocyte recruitment in OVA-induced allergic asthmatic mice. The formation of the platelet-leukocyte complex is also largely dependent on platelet P2Y1R. An in vitro mechanistic study showed that the RhoA signaling downstream of platelet P2Y1R is required for platelet-dependent leukocyte chemotaxis (164). In contrast with other findings in the literature (161, 174), Amison and colleagues found that P2Y12R antagonists yielded no effect on pulmonary leukocyte recruitment (164), which may be attributed to the heterogeneity in experimental methods.

Integrin αIIbβ3 is highly abundant on the platelet surface and plays key roles in platelet adhesion and aggregation, especially in the interaction between platelets, eosinophils (72), and neutrophils (26, 88). Current drugs targeting integrin αIIbβ3 include abciximab, eptifibatide, and tirofiban. However, these drugs have been associated with increased bleeding risk (156). Only tirofiban has been investigated in an OVA-induced allergic asthmatic mouse model to the best of our knowledge. Importantly, it has been found that tirofiban treatment could decrease airway hyperresponsiveness and cytokine production and attenuate platelet-eosinophil aggregates and subsequent eosinophilic infiltration (72).

The serine protease thrombin is well-recognized as one of the most potent platelet activators. Thrombin stimulates platelets by cleavage and activation of protease-activated receptors (PARs): PAR1 and PAR4. Moreover, vorapaxar is a common thrombin receptor antagonist, targeting PAR-1 (175). It has been suggested that thrombin generation is enhanced in blood serum and lungs of asthmatic patients (176, 177). Current reports suggest that thrombin promotes airway remodeling via PAR1 activation to induce TGFβ production in OVA-allergic rats (178). Moreover, in an OVA-induced allergic asthmatic mouse model, PAR1 deficiency decreased airway inflammation and Th2 cytokine levels, whereas PAR1 agonists could exacerbate findings. It should be borne in mind that thrombin plays a dual role in allergic asthma: low-dose thrombin yields an inhibitory effect, while high-dose thrombin stimulates airway inflammation. This phenomenon may be attributed to thrombin preferentially binding to thrombomodulin on endothelial and airway epithelial cells to limit inflammation and binding to PAR1 to promote inflammation until thrombomodulin are saturated with thrombin (177). However, the effect of vorapaxar in allergic asthma remains primarily understudied, warranting further investigation.

In sum, the application of multiple antiplatelet therapies in treating allergic asthma has achieved great success in either animal models or clinical trials, such as clopidogrel, prasugrel, and tirofiban, which provides new options for the treatment of allergic asthma. However, antiplatelet drugs have not been applied to treat allergic asthma, which may be attributed to two aspects. First of all, there is a lack of long-term and large-scale clinical trials to confirm the treatment effect of antiplatelet drugs in allergic asthma. Most relevant studies have been carried out in small-scale clinical studies or animal models. For instance, clinical studies of prasugrel or ticagrelor in the treatment of allergic asthma only included 26 or 11 patients, and they were conducted in a short time (162, 163). Indeed, the increased risk of bleeding associated with current antiplatelet drugs is a prominent side effect (156) and a limitation for long-term clinical trials. Accordingly, retrospective and prospective analyses evaluating the treatment effect of antiplatelet drugs in patients with asthma and thrombotic disorders are of significant importance in the future. Besides, platelet exhibit different phenotypes in prothrombotic and proinflammatory conditions (21, 170). During hemostasis/thrombosis, aggregation is the most important function of platelets (179). In contrast, the most important function of platelets is to interact with other leukocytes in allergic asthma (21). Currently available antiplatelet drugs are designed to inhibit platelet aggregation, which may not significantly influence the interactions between platelets and leukocytes. Developing new therapies to block these interactions will be a breakthrough in the treatment of allergic asthma. P-selectin is a great target because, as mentioned earlier, it plays a crucial role in mediating platelet-eosinophil/neutrophil interactions. Crizanlizumab, an anti-P-selectin monoclonal antibody, has been approved by FDA in the USA to prevent vascular occlusion crises (VOCs) in sickle cell disease patients who express high levels of P-selectin (180). We hypothesize that crizanlizumab has huge prospects for application in treating allergic asthma in the future. In parallel, we also expect other new therapies to target interactions between platelets and leukocytes.