Platelet–Leucocyte Aggregates as Novel Biomarkers in Cardiovascular Diseases

Simple Summary Cardiovascular diseases are the most common cause of death worldwide. Hence, novel biomarkers are urgently needed to improve diagnosis and treatment. Platelet–leucocyte aggregates are conglomerates of platelets and leucocytes and are widely investigated as biomarkers in cardiovascular diseases. Platelet–leucocytes aggregates are present in health, but increase in patients with cardiovascular risk factors and acute or stable coronary syndromes, making them a potential diagnostic marker. Moreover, platelet–leucocyte aggregates predict outcomes after surgery or percutaneous treatment and could be used to monitor antiplatelet therapy. Emerging data about the participation of platelet–leucocyte aggregates in cardiovascular diseases pathogenesis make them an attractive target for novel therapies. Furthermore, simple detection with conventional flow cytometry provides accurate and reproducible results, although requires specific sample handling. The main task for the future is to determine the standardized protocol to measure blood concentrations of platelet–leucocyte aggregates and subsequently establish their normal range in health and disease. Abstract Platelet–leucocyte aggregates (PLA) are a formation of leucocytes and platelets bound by specific receptors. They arise in the condition of sheer stress, thrombosis, immune reaction, vessel injury, and the activation of leukocytes or platelets. PLA participate in cardiovascular diseases (CVD). Increased levels of PLA were revealed in acute and chronic coronary syndromes, carotid stenosis cardiovascular risk factors. Due to accessible, available, replicable, quick, and low-cost quantifying using flow cytometry, PLA constitute an ideal biomarker for clinical practice. PLA are promising in early diagnosing and estimating prognosis in patients with acute or chronic coronary syndromes treated by percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). PLA were also a reliable marker of platelet activity for monitoring antiplatelet therapy. PLA consist also targets potential therapies in CVD. All of the above potential clinical applications require further studies to validate methods of assay and proof clinical benefits.


Introduction
Cardiovascular diseases (CVD) are the leading cause of death worldwide, responsible for over 17 million deaths in 2019 [1], which is the approximate number of inhabitants of the Netherlands [2]. Despite the recent development in the fields of prevention and treatment, CVD mortality increased globally from 27.9% in 2000 to 32.2% in 2019 [1]. Hence, novel biomarkers are urgently needed to predict CVD, improve early diagnostics, and monitor therapy effectiveness.
PLA have also been widely investigated in CVD. Levels of PLA, including monocytes or neutrophils, were higher in symptomatic coronary artery disease (CAD) than in healthy people [13]. PLA containing monocytes were increased in acute coronary syndromes (ACS) versus healthy subjects [14]. Similarly, levels of platelet-monocyte aggregates were higher in patients with non-ST elevation (NSTE-ACS), unstable angina (UA), acute myocardial infarction (AMI) or chronic coronary syndromes (CCS) compared with a group with noncardiac chest pain [15,16]. Moreover, increased percentage of PLA-containing monocytes was associated with elevated risk for future cardiovascular events in population with NSTE-ACS or in dialyzed patients [15,17]. Increased levels of PLA were also observed in patients with carotid stenosis [18], peripheral artery disease (in which it was significantly higher in critical limb ischemia) [19,20], and a high risk of thromboembolic events [21,22]. Data regarding PLA in patients with abdominal aortic aneurysm is mixed [19,23].
There are variable methods for the assessment of PLA. Conventional flow cytometry (CFC) is the best approach in clinical conditions because of its wide availability and its fast and accurate measurement. The main disadvantages of the cytometry-based methods are ex vivo observation, lack of evaluation of PLA placement in tissue, and no direct tracing of intracellular interactions. On the other hand, imaging flow cytometry (IFC) or intravital microscopy avoid the limitations of CFC, but these techniques are more complex than CFC [24].
Here, we present a comprehensive analysis of PLA as novel biomarkers in CVD by discussing the characteristics of platelet-leucocyte aggregates, and their clinical applications, including the impact of cardiovascular risk factors and CVD on PLA, the predictive value of PLA in patients after percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), and changes in PLA concentration during antiplatelet therapy. Finally, we discuss the methods of PLA measurement, including flow cytometry and microscopy. The mean size of PLA is about 9 micrometres (µm) [36]. Following platelet activation, large conjugates were observed, consisting of many platelets in the central part and leucocytes, especially granulocytes and monocytes on their perimeters [4,37]. PLA could occur as free complexes or bound to vessels walls [38] and were detected in animals [39,40] as well as humans [41][42][43][44][45]. They were revealed in health [37,38] and many pathologies, including CVD [13][14][15][16][17][18].
In healthy populations, platelets form aggregates mainly with monocytes (4.1-7.2%), followed by neutrophils (3.7-5.7%) and lymphocytes (2.4-5.2%) [4,35,46], but the referential ranges are not known because of a lack of a validated protocol for investigating the concentrations of PLA. PLA were detected in the paediatric population [47] as well as in adults [48]. In patients with chronic coronary syndromes (CCS), there were no changes in PLA level dependent on age [48]. On the other hand, in the population with CVD, women revealed a higher concentration of aggregates [48,49], but this difference was not observed in healthy people [49,50]. In healthy women, PLA levels changed collaterally with estrogenic profile during the menstrual cycle. The highest percentage of PLA-M and PLA-G were at ovulation (14th day) in comparison to the 1st, 7th, and 21st days. Interestingly, the ability to create PLA by stimulation with thrombin receptor agonist peptide (TRAP) was decreased on the 14th day of the cycle, but there was no significant difference on the 7th and 21st days [51].
Activation of platelets or leucocytes led to PLA formation [50]. Biochemic substances associated with thrombosis, such as adenosine diphosphate (ADP), collagen, In patients with CVD, the percentage of PLA-M, PLA-G, and PLA-Ly were increased compared with the group without CVD. In the population with CVD, PLA-M correlated with the number of atherosclerotic plaques and the mean intima-media thickness of the carotid artery. After 2 to 3 years of observation, people with CVD had decreased the percentage of PLA-M compared with their initial status, but still, PLA-M levels were higher than in a population without CVD at baseline. The percentage of PLA-M were similar in patients with CVD after 10 years of follow-up and without CVD at baseline [70].

Risk Factors of Cardiovascular Diseases
In a prospective cohort study including 155,722 participants from 21 countries followed-up for 9.5 years, 12 modifiable risk factors responsible for CVD development and mortality were shown: smoking, overusing alcohol, unhealthy diet, increased non -highdensity lipoproteins (non-HDL), an increased waist-hip ratio (WHR), too infrequent In patients with CVD, the percentage of PLA-M, PLA-G, and PLA-Ly were increased compared with the group without CVD. In the population with CVD, PLA-M correlated with the number of atherosclerotic plaques and the mean intima-media thickness of the carotid artery. After 2 to 3 years of observation, people with CVD had decreased the percentage of PLA-M compared with their initial status, but still, PLA-M levels were higher than in a population without CVD at baseline. The percentage of PLA-M were similar in patients with CVD after 10 years of follow-up and without CVD at baseline [70].

Risk Factors of Cardiovascular Diseases
In a prospective cohort study including 155,722 participants from 21 countries followedup for 9.5 years, 12 modifiable risk factors responsible for CVD development and mortality were shown: smoking, overusing alcohol, unhealthy diet, increased non -high-density lipoproteins (non-HDL), an increased waist-hip ratio (WHR), too infrequent physical activity, diabetes, hypertension, symptomatic depression, air pollution, decreased grip force, and lower education level [71].
In some of the above states, for example, in smokers [41,42], hypertension [43], symptoms of depression [44], or long-time exposure to biomass smoke, the concentrations of PLA were increased compared with the population without these states [45].
PLA-LyT, PLA-LyTh, PLA-LyTc, and PLA-E were increased in metabolic syndrome compared with no metabolic syndrome. In addition, PLA-LyTh positively correlated with plasma glucose concentration [25]. In patients with type 2 diabetes, PLA-M [26,27] and platelet-polymorphonuclear aggregates [27] were increased compared with healthy people and also correlated with glucose and triglycerides levels in plasma [26]. Similarly, in a population with type 1 diabetes, the levels of PLA-M [27,72,73] and platelet-polymorphonuclear aggregates were higher compared with the lack of diabetes [27]. In diabetes 1, PLA-M correlated with concentrations of glycated haemoglobin (HbA1c), triglycerides, total cholesterol (TC), and low-density lipoprotein (LDL) [72]. Concurrently, PLA-Ly and PLA-G were increased in insulin-dependent diabetes compared with healthy populations [74]. Cardiovascular risk increases with increasing plasma glucose concentrations [75], thus PLA-LyTh, PLA-M and platelet-polymorphonuclear aggregates correlating with glucose level could be considered a biomarker for cardiovascular risk stratification in patients with diabetes or metabolic syndrome [25][26][27].
An increased concentration of PLA-M, PLA-N, and PLA-Ly was noticed in hypercholesterolemia compared with a lack of dyslipidaemia [28,76]. What is more, PLA-M positively correlated with TC and LDL independent of dyslipidaemia. PLA-M or PLA-N negatively correlated with high-density lipoprotein (HDL) in hyperlipidaemic populations or healthy people, respectively [28].
The effect of lipid-lowering treatment on PLA is disputable. It seems to be dependent on concomitant states, leading to a decrease of this biomarker compared with placebo in patients with acute coronary syndromes [77] and an animal model of congestive heart failure [39], but in populations with coronary artery disease and impaired glucose tolerance or type 2 diabetes no changes compared with lack of treatment were observed [78].
Interestingly, item diet influences PLA percentage. In in vitro models, gallic acids occurring in red wines, tea, plants [79], and flavonolignans from the plant Silybum marianum lowered PLA level compared with a lack of supplementation [80]. In similar conditions, anthocyanins presenting in berry fruits and their metabolites decreased PLA-M and PLA-N compared with the lack of anthocyanins [81]. In mice, alfrutamide and caffedymide from plants like cocoa, coffee tree or garlic, inhibited PLA formation compared with lack of compounds from plants [40]. In people, supplementation of ω-3 fatty acids decreased PLA concentration compared with a lack of supplementation [82].
On the other hand, exhaustive exercises were connected with higher PLA levels [74,[83][84][85][86]. Still, less intensified strain led to gentler PLA enlargement [83] and suggested that only light workouts could benefit health.
To our best knowledge, no trials assess the influence of enlarged alcohol intake, decreased strength, or low education level, but other risk factors have a special connection with PLA.

Platelet-Leucocyte Aggregates in Chronic Coronary Syndromes
It was reported that the concentrations of PLA or, more specifically, PLA-M were higher in patients with CAD than in healthy controls. Besides, platelets from CAD patients form PLA-M more intensely in reaction to small concentrations of ADP or TRAP [87]. Other studies corroborate these findings [30,88]. Whether levels of PLA correlate with the severity of CAD is poorly documented. Two studies found no association between PLA and functional relevance of coronary stenosis quantified with fractional flow reserve (FFR) or several diseased vessels [89,90]. Nevertheless, one had a small sample size [89], and both assessed only the overall number of PLA-M [89,90], whereas PLA-M are heterogeneous concerning the phenotypes of monocytes [74]. In one study, a higher count of M2 containing PLA-M indicated a diffuse form of CAD. PLA-M, including M2, also negatively correlated with impaired endothelium-dependent vasodilation in these patients (the effect was stable over a 12-month observation), pointing to a possible background or the result of more advanced lesions [91]. Another study also demonstrated a positive correlation between endothelial dysfunction in coronary vessels (manifesting as paradoxical vasoconstriction in response to increased coronary flow) and PLA-M concentration in the context of CAD [90]. These findings encourage research into the specific roles of PLA-M and their subtypes in atherogenesis and as a potential marker of the extent of stable CAD.
More severe CAD in patients undergoing revascularization and valve replacement was linked with worse outcomes [92,93]. In a recent study high preprocedural M2 and CD-11b were independently associated with 3-month mortality after transcatheter aortic valve implantation. CD11b correlated with PLA-M (PLA-M subsets were not assessed) [94]. The interplay between CAD, PLA and prognosis after cardiac procedures remains to be understood better.

Changes in Platelet-Leucocyte Aggregates after Coronary Artery Bypass Grafting
The use of cardiopulmonary bypass during CABG and the surgery itself results in activation of platelets and leukocytes as reflected by amounts of P-selectin-positive platelets, CD11b positive leukocytes, platelet aggregation, and PLA. Ensuing in-pump aggregation of platelets results in their depletion immediately after the procedure, but their level culminates again on day 7-PLA amount also peaks at this time [95], stressing the role of platelets in initiating interaction with leukocytes. A recent study demonstrated increased platelet reactivity to agonists (ADP, thrombin, thromboxane analogue) up to 3 months after elective CABG in patients with stable angina compared with their state before operation. All PLA subtypes were elevated at 1 month, whereas at 3 months, PLA-Ly were elevated, but PLA-M and PLA-N were decreased compared with the baseline statement [96]. The significance of these findings for short-and long-term prognosis needs to be elucidated by further research.
We found no studies investigating the relationship between PLA formation in the post-CABG period and the patient outcome. Nevertheless, preoperative PLA was predictive of acute kidney injury (AKI) and 3-year risk of major adverse events in a cohort of surgical valve replacement due to rheumatic heart disease. The authors estimated that the optimal PLA cut-off to identify at-risk populations was 6.8%-patients with higher values before the operation were nearly 18 times more likely to suffer from AKI [97,98]. Cardiac surgery-associated acute kidney injury (CSA-AKI) is a complication common also in CABG patients [99]. However, its association with baseline PLA has not been studied so far. The advantage of PLA in predicting CSA-AKI is assessing the risk even before the procedure. In contrast, other known markers, such as neutrophil gelatinase-associated lipocalin, cystatin C, kidney injury molecule 1, or IL-18 are dependent on already existing damage to the kidney [100]. Identifying vulnerable patients before the operation might allow designing suitable interventions to reduce the additional risk. A certain type of exercise regime reduced PLA-M in post-CABG patients [101]. It remains an interesting question as to whether similar actions before cardiac surgery would effectively decrease the risk of AKI.

Platelet-Leucocyte Aggregates in Acute Coronary Syndromes
There are several ways by which PLA might contribute to ACS pathophysiology. The greatest platelet aggregability and concentrations of various coagulation markers are connected with morning hours and the start of activity [102,103], when blood catecholamines level and sympathetic tone are also high [104,105]. This is thought to play a role in the increased rates of cardiovascular events in the morning [105]. Anti-adrenergic medications lower PLA-M and PLA-N formation both in circulation and in response to ADP [106]. It suggests that PLA generation might be enhanced in the morning due to adrenergic signalling. Through PLA-M platelets induce cytokines [107] and tissue factor (TF) production in monocytes [108,109], an important source of microvesicle-borne TF, which promotes fibrin accumulation at the site of thrombus formation [110]. Disrupted P-selectin-PSGL-1 signalling led to reduced fibrin deposition within thrombus [110] and impaired aggregation of whole blood [61]. On monocytes, PLA-M also increased Mac-1 [3], which is involved in thrombosis and elevated after AMI [111]. In a mice model of carotid artery thrombosis, the lack of Mac-1 significantly prolonged time to occlusion [112]. In one study administration of abciximab to AMI patients undergoing PCI reduced both PLA-M and monocyte Mac-1 expression 24 and 72 hours (h) after the event [3]. Another important mechanism is the ability of platelets in PLA-N to cause the release of NETs from neutrophils [64,66]. Recently, interactions between platelets and leukocytes were even proposed as a possible pharmacological target to prevent atherothrombosis [113].
Among patients presenting with chest pain admitted to the emergency department, those undergoing acute myocardial infarction had a significantly higher level of PLA-M than the remaining non-infarction group [114,115]. However, this effect was not reported uniformly [116]. The evidence suggests that aggregate formation happens in the early myocardial infarction phase, when platelets are activated by ruptured plaque [115]. PLA-M elevation within 4 h from the onset of symptoms was over 1.5 times greater than between 4 and 8 h [114]. Additionally, intermediate monocytes, which are also candidates for AMI biomarkers, were raised due to monocyte-platelet interactions [117]. This means that PLA could be an even earlier marker of AMI. One small study showed that elevated PLA-M had strong discriminative power for AMI as early as 2 h from the onset of suspicious symptoms [118].
Total PLA-M and intermediate monocytes and their complexes with platelets were significantly higher on admission in patients with UA than stable coronary disease controls. These parameters correlated with the Global Registry of Acute Coronary Events (GRACE) score (which classifies patients into low-and intermediate-to-high risk) [119,120].
Additionally, patients with UA and non-ST elevation myocardial infarction (NSTEMI) predisposed for the relapse of ischemia could be distinguished based on the intensity of PLA-N formation in response to stimulation with TRAP. However, baseline levels of PLA-N were comparable between the groups [121]. This example stresses that a momentary level of PLA and the capacity for their formation may be of diagnostic or prognostic value as it testifies to the strength of inflammatory and atherogenic response.

Changes in Platelet-Leucocyte Aggregates after Percutaneous Coronary Intervention
Both short-and more distant-term prognoses after ST-elevation myocardial infarction (STEMI) treatment with PCI were associated with the intermediate monocytes at admission. It was observed that the concentration of PLA-M involving intermediate monocytes was independently associated with in-hospital complications (including left ventricle aneurysm, massive coronary thrombosis, and acute heart failure) over 7 days post event [34]. Similarly, it was reported that both intermediate monocytes and corresponding PLA-M, measured on day 2 following AMI (and standard treatment), independently predicted the 2-year incidence of major adverse cardiovascular events defined as cardiovascular death, nonfatal ischemic stroke, recurrent myocardial infarction, repeat revascularization, or rehospitalization for heart failure [122].
Evidence accumulates that elevated PLA in patients with STEMI may predict unsuccessful reperfusion after PCI termed coronary microvascular obstruction (MVO) or no-reflow phenomenon. Depending on the assessment method it was reported to complicate 2.3-56.9% of such cases, substantially increasing both early and late mortality [123][124][125]. One study found that the MVO group (MVO defined here as thrombolysis in myocardial infarction (TIMI) flow less than two or TIMI equal to three and a myocardial blush grade less than three despite residual stenosis less than 20%) compared with the successful reperfusion group had elevated PLA-M and their platelets expressed more activation markers in response to ADP, revealing a greater capacity for aggregation. Of note, those differences persisted at 1-month follow-up, despite dual antiplatelet therapy (DAPT) [126]. The authors suggested that these differences might underlie unfavourable outcomes associated with MVO [126]. In another study PLA, PLA-M, and PLA-N, but not PLA-Ly, were significantly associated with poor coronary perfusion in patients with STEMI treated with PCI within 12 h since the onset of symptoms [127]. Others also reported similar findings [128]. Although consistent, all the above studies included relatively few subjects and relied mainly on the angiographic diagnosis of MVO; we found no studies exploring the relationship between PLA and MVO defined based on cardiac magnetic resonance imaging. Hence, the continued effort to clarify the importance of PLA for MVO and no-reflow phenomenon should be warranted.

Treatment with β-Blockers in Acute Myocardial Infarction
It was observed that β-blockers, used chronically before AMI or in an early stage of the event improve the prognosis after STEMI [129]. Due to inhibition of corresponding receptors on leukocytes (and probably also platelets), the use of β-blockers was associated with reduced PLA-N and PLA-M formation, less inflammation, less microvascular Biology 2022, 11, 224 9 of 30 obstruction, and accordingly smaller infarct size [106,130]. Metoprolol limited infarct size in METOCARD-CNIC trial (Effect of Metoprolol in Cardioprotection During an Acute Myocardial Infarction) but did not show significant improvements in a similar EARLY-BAMI trial (early-beta blocker administration before reperfusion primary PCI in patients with ST-elevation myocardial infarction). However, the studies differed substantially in time from diagnosis to administration, and the effect of β-blockers strongly correlated with timing, which might explain the lack of effect in the EARLY-BAMI study (median of 10 min in METOCARD-CNIC) [131]. These observations once again underlie the connection between the PLA levels and outcomes after acute coronary events.

Monitoring Antiplatelet Treatment
Antiplatelet therapy, consisting of acetylsalicylic acid (ASA) alone or with a P2Y12inhibitor, constitutes a cornerstone of CVD treatment [132][133][134][135]. GP IIb/IIIa-blocker administration is advantageous in AMI with complications such as thrombosis or noreflow [133,135]. In turn, prostacyclin analogues used in patients with pulmonary hypertension to dilate pulmonary vessels, also inhibit platelet activation [136]. In monitoring platelet function, P-selectin was considered the gold standard [137], but PLA turned out to be much more sensitive [115]. Hence, they seem to be promising candidates for a biomarker in antiplatelet treatment monitoring.

Acetylsalicylic Acid in Monotherapy
The influence of ASA on PLA is not well stated. In in vitro trial, ASA was added to blood samples from half of 12 healthy participants, and there was no effect of this medicament on PLA-M and PLA-N, either in resting and activated by ADP or PAF specimens compared with an absence of medication [138]. Similar results were observed during oral intake of ASA. In 12 healthy men treated with 75 milligrams (mg) ASA daily for 10 days, there was no inhibition of PLA-M and PLA-N formation after stimulation with ADP compared with state before medicine administration [139]. Likewise, ASA did not inhibit spontaneous and induced by ADP, thrombin or PAF PLA, PLA-M, PLA-N, and PLA-Ly formation in 15 healthy men treated during a week in dose 75 mg and 500 mg for next 7 days compared with set point [140]. On the other hand, PLA-M percentage decreased in the healthy population of 40 after 10 days of 150 mg ASA intake compared with state before treatment, which suggested the necessity of more extended therapy with higher dose and possibly higher sensitivity of PLA-M as a marker than other PLA subtypes, but further studies are required due to the small group of participants [141]. The results from research in pathologies are inhomogeneous. In mice with temporal carotid artery strangulation treated with ASA, the percentage of PLA were increased in resting samples, and there were no changes after activation compared with placebo. However, in the same conditions, PLA as platelet to leucocyte ratio was decreased both with and without stimulation [142]. In another ischemia model, with an after-reperfusion effect on intestinal microcirculation in mice, there were less PLA after ASA intake than placebo [143]. Contrarily, there was no effect of 150 mg ASA prescription for 10 days on PLA in 63 patients directly after stroke [141]. Others reported that in patients who suffered a stroke within the previous 6 months, taking 81-650 mg of ASA daily for at least 1 month preceding testing was associated with significantly lower PLA compared with those who failed to take ASA at that time [144]. Subsequent studies are necessary to define the impact of ASA on PLA in particular diseases.

P2Y12-Receptor Antagonists
Clopidogrel, prasugrel, ticagrelor, and cangrelor are enumerated as available in clinical practice as P2Y12 inhibitors [145]. In mice treated with clopidogrel, plateletpolymorphonuclear aggregates formation after stimulation with ADP or thrombin was lower than without this medicament. This effect was dependent on platelets. Mixing platelets from untreated mice with polymorphonuclears from mice treated with clopidogrel did not influence the creation of platelet-polymorphonuclear aggregates. Likewise, in whole blood drawn from healthy people and then incubated with active metabolite of clopidogrel, less platelet-polymorphonuclear aggregates were observed after stimulation with ADP or PAR agonist than without any medicaments. Clopidogrel also decreased the formation of reactive oxygen species by human polymorphonuclears incubated with platelets subjected to clopidogrel action [146]. Despite the relationship between clopidogrel and platelets, the mechanism of clopidogrel's influence on PLA is not dependent on P-selection. In the human population with a deficient variant of P-selectin, clopidogrel inhibited PLA-M formation like in a group with accurate P-selectin [147]. In a clinical condition of ACS, clopidogrel successfully restored PLA-M and PLA-N to concentration nearly equal to those in healthy people and arrested the creation of PLA completely during ADP stimulation and to 50% after adding TRAP [148]. The impact of clopidogrel on PLA was stable during 30 days of observation [149].
On the other hand, patients with CCS and myocardial ischemia defined as FFR ≤ 0.75 treated with clopidogrel had a lower percentage of PLA-N than the population without ischemia. A similar trend occurred for PLA-M. This observation authors determine as paradoxical and explained by higher clearance of PLA, sequestration of leucocytes to the endothelium and migration in the direction of deeper located tissue [89].
The active metabolite of prasugrel, administrated orally for mice, inhibited connections between platelets and polymorphonuclears after stimulation [150]. Following combining prasugrel with blood from healthy volunteers with ADP or TRAP, this P2Y12 inhibitor contained platelet-polymorphonuclear aggregates [150], PLA-M, and PLA-N [151].
Similarly, the active form of cangrelor added in vitro [151] prevents an increase of PLA-M and PLA-N after stimulation with ADP [139]. Two other P2Y12 inhibitors reported in the literature but not applied in clinal practice presented variable action in adequate condition. AR-C69331 behaved like activated cangrelor [151,152], but the second one, AR-C69931, had no impact on PLA-M and PLA-N [115].
The pleiotropic role of P2Y12 inhibitors was previously described primarily in the context of supplementary impact on the immune system [153,154]. PLA are discussed as a critical mediator of anti-inflammatory action in this group of medicaments. In a mice model of sepsis, a lack of P2Y12 receptors caused lower PLA levels than in a population expressing P2Y12 [12]. On the other hand, clopidogrel inhibited PLA formation in mice with endotoxemia [155] or induced hypertension [156]. Additionally, an active form of prasugrel protected against platelet-activator-induced platelet polymorphonuclear formation in mice with endotoxic shock [150]. In healthy people treated with ticagrelor, PLA-M production was lower after in vitro exposure to toll-like receptor 2 and 4 (TLR-2, TLR-4) agonists than in the placebo group [157]. Ticagrelor significantly decreased PLA in patients with pneumonia after 24 h of treatment compared with their state before this pharmaceutical administration. Additionally, a higher level of PLA at baseline was connected with a more expressed effect of medicine [158].

Acetylsalicylic Acid in Comparison with P2Y12-Receptor Antagonists
As cited above, it was proven that the P2Y12 antagonists decreased PLA level [139,146,148,[150][151][152], but there is no direct comparison with ASA. However, in patients who had taken ASA or clopidogrel with a break of treatment for operation, the effect of ASA was more stable than clopidogrel during the lack of antiplatelet therapy [159].

Dual Antiplatelet Therapy
DAPT consists of ASA, and a P2Y12 inhibitor represents a baseline of treatment after PCI because of its efficacy in preventing recurrent thrombotic events, including stent thrombosis [160]. In in vitro assessment, ASA with AR-C66931 as P2Y12 antagonist did not influence PLA-M and PLA-N formation [115]. Nevertheless, in patients with ACS after PCI, combining ASA with clopidogrel or cangrelor prevented PLA-M and PLA-N stirring, and this action peaked when combing all three medical preparations, but data about bleeding complications were not reported [139]. The superiority of combination ASA with clopidogrel over ASA was observed even in a population with lower responsiveness to clopidogrel. In patients with ischemic stroke and lessened function of CYP2C19*2 alleles (decreasing response to clopidogrel), ASA coupled with clopidogrel were more potent in lowering PLA than was ASA in monotherapy [161]. In comparison with monotherapy with ASA, DAPT turned out to be more successful in PLA inhibition in patients with CCS (ASA with clopidogrel) [162], unstable angina (ASA with prasugrel) [163], and ischemic stroke (ASA with clopidogrel) [164]. Prasugrel is taken into consideration as the best P2Y12 inhibitor for DAPT according to its impact on PLA. It was observed that, in patients with CCS, PLA was similar at baseline to ASA with clopidogrel or prasugrel. However, ASA with prasugrel was significantly more potent in reducing PLA generating while stimulated with agonists than ASA with clopidogrel, and this effect was maintained through 29 days of follow up [165].

GP IIb/IIIa Inhibitors
Abciximab, eptifibatide, and tirofiban are used in clinical practice as medicaments inhibiting GP IIb/IIIa [135,136]. The impact of GP IIb/IIIa inhibitors on PLA is discussed and depends on subtypes of PLA, medicament, type of platelets stimulation, and conditions like temperature [166][167][168][169][170][171]. Abciximab decreased PLA production after douching by blood from healthy donors in a porcine model of damaged vessels compared with placebo [166]. Similarly, it was observed that less PLA-N in the blood from healthy humans when combined with non-peptide GP IIb/IIIa antagonist-SR121566 after stimulation with PAF, but SR121566 also led to an increase of PLA-N after ADP or adding TRAP compared with the absence of medicaments [167]. Likewise, eptifibatide increased mainly PLA-M and PLA-N after the exposure of blood from healthy volunteers to collagen compared with lack of pharmaceuticals [168]. In hypothermia (18 • C), eptifibatide elevated PLA-G creation in blood from healthy people stimulated with ADP compared with normothermia (37 • C) [169,170]. On the other hand, PLA-N levels did not change in blood from healthy populations after adding tirofiban and ADP or TRAP [171]. Interestingly, there were no changes in PLA-G and PLA-M in blood from healthy donors mixed with tirofiban in hypothermia compared with normothermia [170]. Higher PLA levels after exposure to GP IIb/IIIa are explained by the blocking of platelet-to-platelet binding and, thereby, more opportunity for platelet-leucocyte interactions [167,168].
There was also research conducted with patients with AMI treated orally with a GP IIb/IIIa inhibitor, and the results were more consistent than in vitro trials [3,[166][167][168][169][170][171][172]. Decreased PLA level was observed during intake of abciximab combined with heparin and DAPT and abciximab or tirofiban (more pronounced effect) in conjunction with ASA, heparin, and thrombolysis compared with the absence of a GP IIb/IIIa inhibitor [3,172].

Prostacyclin Analogues
Prostacyclin analogues have evidenced impact as an inhibitor of platelet reactivity and thrombus formation in pulmonary hypertension [173]. Epoprostenol, as a medicament from this group of pharmaceuticals, inhibited PLA creation after adding whole blood from healthy people and stimulation with ADP compared with ASA or cangrelor or prostaglandin E1 [174].
Results of PLA investigation in various clinical states are summarized in Table 1.      [152] ticagrelor in healthy subjects ticagrelor, N = 7 placebo, N = 7 Ticagrelor was associated with significant reduction in PLA-M formation in response to LPS and Pam3CSK4, but not spontaneous. Type of produced cytokines did not depend on ticagrelor use but seemed to differ due to receptor (TLR-2 or TLR-4).
Tunjungputri et al. 2014 [157]     High pressure (due to infusion of angiotensin II or phenylephrine) was associated with high PLA formation. Clopidogrel treatment counteracted this effect leading to a significant PLA reduction. Jia et al., 2013 [156] Abbreviations

Conventional Flow Cytometry
CFC is a method of choice for PLA assessment [24] and very handy for clinical practice [36,37]. The main advantages of CFC are no requirement for very advanced machine [24], availability in laboratories [36], easy access and a tiny amount (~5 microlitres [µL]) of whole blood as research material [47], quick measurement, high sensitivity, precision of assay [24], one-step detection, and quantifying PLA with distinction of leucocyte subsets, as well as assessment of platelet or leucocyte activity [31]. On the other hand, a very detailed test involves exceptional precision in handling and avoiding artifacts resulting from platelets' activation post drawing [175]. Figure 3 shows the possible traps in detecting and quantifying PLA with CFC. There are many published protocols for measuring PLA with CFC [4,36,37,47], but there are no standardized roles as in other biomarkers, such as EVs [176]. There is also a lack of regular extents in health and pathologies. These facts prohibit the effective comparing of results from various trials and the wide application of PLA as a marker in clinical practice.
Whole blood was usually taken for PLA assessment with CFC [4,24,36,37,46,47,50,175,177,178]. To the best of our knowledge, there is a lack of research proving the impact of applying a tourniquet, using a needle with a low diameter, or not rejecting the first blood portion on PLA level. For comparison in EVs also dependent on platelet or leucocyte, stasis, a needle diameter below 21 gauge and including the first 2-3 millilitres (mL) of blood to samples are not recommended [176]. On this account, it seems to be justified to avoid these activities during blood drawing, according to some authors [36,37,46,50,175]. What is more, it was proved that clean antecubital venepuncture, rather than intravenous cannula, is the best way for samples to be sourced because it was shown not to cause an increase of PLA level, even during repeated blood drawing [175].
On the other hand, arterial blood sampling with a catheter and a prick is equally safe for PLA assessment [178]. Sodium citrate or EDTA decreased, but heparin increased PLA level. Hence, direct thrombin inhibitors are recommended as anticoagulants [175]. sets, as well as assessment of platelet or leucocyte activity [31]. On the other hand, a very detailed test involves exceptional precision in handling and avoiding artifacts resulting from platelets' activation post drawing [175]. Figure 3 shows the possible traps in detecting and quantifying PLA with CFC. There are many published protocols for measuring PLA with CFC [4,36,37,47], but there are no standardized roles as in other biomarkers, such as EVs [176]. There is also a lack of regular extents in health and pathologies. These facts prohibit the effective comparing of results from various trials and the wide application of PLA as a marker in clinical practice.
Whole blood was usually taken for PLA assessment with CFC [4,24,36,37,46,47,50,175,177,178]. To the best of our knowledge, there is a lack of research proving the impact of applying a tourniquet, using a needle with a low diameter, or not rejecting the first blood portion on PLA level. For comparison in EVs also dependent on platelet or leucocyte, stasis, a needle diameter below 21 gauge and including the first 2-3 millilitres (mL) of blood to samples are not recommended [176]. On this account, it seems to be justified to avoid these activities during blood drawing, according to some authors [36,37,46,50,175]. What is more, it was proved that clean antecubital venepuncture, rather than intravenous cannula, is the best way for samples to be sourced because it was shown not to cause an increase of PLA level, even during repeated blood drawing [175]. Some authors reported using centrifugation and washing of the biological material, but these processes lead to cell loss and additional PLA formation [50]. For that reason, they are not recommended. In avoiding this process, the first step, most often, is marking platelets and leucocytes [4,36,50,175,179]. However, some authors fixed samples before following procedures [37,50]; however, latency over 10 min in labelling decreased repeatability in the number of PLA [175]. This reaction was weaker when sodium citrate was used as an anticoagulant instead of a direct thrombin inhibitor [175]. Fluorescing substances were applied for designating monoclonal antibodies (MA) connected with PLA [36]. It is important to mark cells with MA that do not influence molecules participating in the adhesion of leucocytes to platelets, especially when stimulation of PLA formation is planned. On this account, MA against GP Ib/IX (CD42) had the edge over against GP IIb/IIIa (CD41/CD61) [50]. Nevertheless, platelets can lose GP Ib (CD42b) from their surface [46,50]. For that reason, it is recommended to use MA against GPIX (CD42a) for labelling these blood-morphotic elements [46]. Except using MA for red-tagging platelets, at least one other MA specific for all leucocytes (CD45) [4] or their particular subtype [50] is required. One-step marking is possible with more than 2 MAs. CFC enables, additionally, the distinction of leucocyte subsets: lymphocytes B (CD19), lymphocytes T (CD3) helper (CD4) and cytotoxic (CD8), NK-cells (CD56), granulocytes (CD15), monocytes (CD14), neutrophils (CD16), eosinophils (lack of CD16) or assessment of platelet (CD62P), and leucocyte (CD11b) activation [4,25,36,37,46,47,50,74].
Although some authors fixed samples before labelling [36,50,179], most researchers followed steps in inverse order [4,36,46,50,175,179]. In unfixed specimens, at room temperature, the PLA level started increasing after 0.5 to 1 h. It is possible to use formaldehyde or paraformaldehyde to fix samples, but these substances could cause artefactual PLA formation [4,50]. In addition, their effect on blood stability is uncertain because, after using them, PLA increase was observed after 20 minutes (min) to 72 h [47]. After fixing samples with FACS-Lyse, PLA were stable at 4 • C over 24 h, but the percentage of PLA was not assessed with an unfixed control [175]. It is possible to use CellFix [36,180], but an increase of aggregates, at both 4 and 25 • C during 6 h in patients with CVD and healthy people, was observed [180]. On the other hand, ThromboFix protected against PLA formation for a week but guarded in vivo-ensuing PLA only for 4 h [181]. Some scholars chose erythrocytes lysis [4,50,175], but outcomes were inconsistent [50,175].
Many authors exploited in vivo stimulation to explore the ability of PLA formation in samples enhanced by platelets, leucocytes, or the activation of both [4,36,37,46,47,50,179]; as stimulation, substances like ADP, TRAP, collagen, epinephrine, LPS, TNFα, thrombin, PAF, and fMLP were used [36,50]. This step is not necessary but could yield extra information. It is suggested to store samples at 22 • C and avoiding stirring [46].
After preparation, probes are analysed by CFC. The flux speed is recommended to be as slow as possible (about 10 µL/min) because, during faster flow, more PLA were shown to form [182]. To detect PLA with CFC, it is essential to find platelets tethered with leucocytes. For this goal, gating based on fluorescence was applied [4]. Additionally, to avoid artifacts consequent to detecting EVs exposing the same markers, gating was employed based on objects' sizes [36].
CFC permits quantifying PLA and its subsets [4], but it is not possible to count directly the number of platelets using them [24]. The amount of PLA can be shown as a percentage of leucocytes (or subsets thereof, respectively) conjugated with platelets [4,46,177] or a percentage of platelet aggregates containing leucocytes [179]. Some authors used mean fluorescence index (MFI) as a gauge of PLA level [177], but this is only a semi-quantitative method [24,182]. Other researchers extrapolate the number of platelets tethering to leucocytes based on fluorescing, but this could vary from the percentage of PLA [141].
Despite various advantages of CFC in PLA assessment, the main obstacle to overcome is the lack of a unified protocol for drawing, handling, processing samples, presenting the results thereof, and a range of norms. This state precludes comparison between outcomes from various laboratories and clinical applications of CFC.

Imaging Flow Cytometry
IFC combines the advantages of CFC and fluorescence microscopy. It is possible to differentiate the proximity of platelets and leucocytes from PLA more effectively than with CFC. What is more, IFC enables higher throughput without significant over-detection of PLA [182].

Microscopy
Light microscopy also could be used to assess PLA [4,37]. Lower levels of PLA were observed in a sample during microscopic observation than with CFC measuring of the same sample [4], but a proportion of PLA subtypes correlated between these two methods [37]. The reason for this difference in observed PLA concentrations could be the lack of having excluded EVs [4], the random closeness of platelets and leucocytes without tethering [182], or trogocytosis [4] during assessment with CFC. Light microscopy also facilitates the denotation of PLA placement in tissue, but the picture is static. Alternatively, intravital microscopy, including wide-field epifluorescence, multiphoton, spinning-disk, or scanning confocal microscopy, allows assessing the live dynamics of platelet-leucocyte interaction in animal models [24,183]. Despite this advantage, microscopy is more suitable for experimental research than for clinical application [24], because it is less effective and more time-consuming than CFC [182].

Summary and Future Possibilities
Historically, over 40 years of research of PLA has provided increasingly more information about them, but still, it is not known how exactly they form, nor their detailed function in the pathogenesis of atherosclerosis and, thus, in CVD. The main reason for this missing information is the lack of a uniform protocol for PLA assessment using flow cytometry as the most common tool in clinical trials. On this account, researchers apply a variety of methods, and their results, in some cases, significantly diverge from others. Despite this, the available data allow us to consider PLA as the most sensitive marker of platelet activation. This development opens a wide extent of purpose. PLA could help to diagnose CVD, predict complications after PCI or CABG, monitor the response to antiplatelet therapy, and predict the overall risk of future cardiovascular events. As a biomarker dependent on platelets and leucocytes, they could also help discover and assess the pleiotropic results of treatment. In addition, PLA have a chance to be the target of new medications. Further studies are required, first of all, to introduce a consistent protocol for PLA assessments and the reporting of results thereof. Validation and the determination of normal ranges are also essential.
Finally, as mentioned before, PLA are now being widely investigated as potential cardiovascular complication markers in COVID-19. It is probably the most up-to-date field of interest in PLA research. Although SARS-CoV-2 vaccines induce spike protein overexpression, vaccination with BNT162b2 does not alter platelet protein expression, and reactivity [184] but SARS-CoV-2 infection induces PLA formation rather than platelet-platelet aggregates [185], and PLA are accused of forming blood clots in severe COVID-19 [186]. Altogether, they create a new concept of PLA' pathomechanism in COVID-19 cardiovascular complications, which deserves separate review.