Table 1.

Platelet-leukocyte aggregates changes in various clinical condition.

Clinical Condition	No. of Patients	Control Group	Outcome	Reference
Clinical Studies
CVD	yes, N = 345	no, N = 64	CVD cohort, including patients with CAD, PAD, carotid artery stenosis and abdominal aortic aneurysm, had significantly higher PLA-M compared with healthy controls (both groups received ASA). The association was true for each CVD separately, but after multivariant analysis it remained significant only for PAD. Patients with critical limb ischemia had significantly higher PLA-M than other PAD patients.	Allen et al., 2019 [19]
peripheral artery disease	critical limb ischemia, N = 20	intermittent claudication, N = 45 healthy, N = 20	PLA correlated with the disease severity. Critical limb ischemia was associated with significantly higher PLA-M with intermediate or non-classical monocytes, total PLA-M and PLA-N.	Dopheide et al., 2016 [20]
atrial fibrillation	with left atrium thrombus, N = 27	without left atrium thrombus, N = 80	Among patients assessed with transoesophageal echocardiography before electric cardioversion/ablation PLA-M over 170 cells/μl independently predicted left atrium thrombus with sensitivity of 93%.	Pfluecke et al., 2016 [21]
venous thromboembolism	VTE, N = 13	no VTE, N = 19	Patients who suffered VTE 30 days following orthopaedic surgery had significantly elevated PLA-M 24 h after the procedure compared with the group without this complication.	Shin et al., 2016 [22]
abdominal aortic aneurysm	AAA, N = 41	no AAA, N = 38	Although significantly more activated and newly released neutrophils circulated in blood of AAA group, it was not true for PLA-N or PLA-M.	Zagrapan et al., 2019 [23]
smoking	yes, N = 10	no, N = 10	PLA-M, PLA-N and PLA-Ly were significantly higher in smokers.	Badrnya et al., 2014 [41]
smoking	yes, N = 20	no, N = 20	Smokers had significantly higher PLA-M and PLA-N than controls. Three-weeks-long smoking cessation led to significantly reduced PLA-M formation.	Lupia et al., 2010 [42]
hypertension	yes, N = 22	no, N = 22	Hypertensive subjects had significantly higher PLA at baseline compared with controls. This effect persistent during 2 months’ observation despite the treatment with doxazosin and a trend towards pressure normalization.	Labios et al., 2006 [43]
depression	yes, N = 102	no, N = 44	Depressed patients had significantly higher PLA, PLA-M, PLA-G, than controls. This state persistent after 6 months of therapy despite concurrent improvement in platelet reactivity.	Morel-Kopp et al., 2009 [44]
exposure to biomass smoke	yes, N = 165	no, N = 155	PLA-M and platelet-polymorphonuclear aggregates were elevated in women cooking with biomass.	Ray et al., 2006 [45]
metabolic syndrome	yes, N = 18	no, N = 21	Metabolic syndrome was associated with elevated PLA-LyT and PLA-E.	Marques et al., 2019 [25]
type 2 diabetes mellitus	yes, N = 14	no, N = 14	Elevated PLA-M; its level correlated positively with plasma glucose and TG.	Patko et al., 2012 [26]
type 1 and type 2 diabetes mellitus	yes, N = 65	no, N = 25	Diabetic patients exhibited higher platelet–polymorphonuclear aggregates and PLA-M regardless of the disease type. Concentrations of aggregates were higher in diabetics with vascular lesions.	Elalamy et al., 2008 [27]
type 1 diabetes mellitus	yes, N = 35	no, N = 20	The patients had significantly higher PLA-M. Its level correlated with HbA1c, total cholesterol, LDL and TG.	Zahran et al., 2018 [72]
type 1 diabetes mellitus	T1D with microangiopathy, N = 20 T1D without microangiopathy, N = 19	healthy, N = 27	In T1D with microangiopathy baseline PLA-M and PLA-N were slightly higher that in controls. Total baseline PLA was comparable between all groups. TXA2 analogue-induced PLA formation was significantly greater in T1D subjects, whereas thrombin-induced PLA formation was significantly higher only in T1D with microangiopathy compared with controls.	Hu et al., 2004 [73]
type 1 diabetes mellitus, exercise	T1D, N = 16	healthy, N = 16	At rest T1D group showed higher PLA-G and PLA-Ly (spontaneous and TRAP-induced). Exercise significantly increased PLA-G, PLA-M, PLA-Ly in both groups; the effect of exercise was comparable between T1D and controls.	Hilberg et al., 2004 [74]
hyperlipidemia	yes, N = 8	no, N = 8	Hyperlipidaemic subjects had higher baseline PLA-M and PLA-N. Their PLA-M correlated positively with total cholesterol, LDL, and serum fibrinogen, and negatively with HDL.	Sener et al., 2005 [28]
primary hypercholesterolemia	yes, N = 22	no, N = 21	PLA-M, PLA-N and PLA-Ly were increased in PH compared with controls, which resulted in greater adhesiveness to arterial walls.	Collado et al., 2018 [76]
ACS	rosuvastatin, N = 21	placebo, N = 23	Administration of a high dose of rosuvastatin within 8 h of symptoms onset led to significantly lower PLA in rosuvastatin group compared with placebo at 8 and 24h after treatment.	Sexton et al., 2015 [77]
stable CAD with T2D/impaired glucose tolerance	simvastatin 80 mg, N = 16 ezetimibe 10 mg and simvastatin 10 mg, N = 16	without lipid-lowering treatment, N = 32	Patients received their assigned medication once daily for six weeks-no significant effect on baseline PLA was found.	Malmstrom et al., 2009 [78]
gallic acid	with gallic acid, N = 5	without gallic acid, N = 5	In vitro gallic acid significantly and dose-dependently decreased stimulation-induced (ADP, TXA2 analogue) PLA-M and PLA-G formation in blood from healthy subjects.	Chang et al., 2012 [79]
flavonolignans	with silychristin or silybin or silydianin, each N = 12	no flavonolignan, N = 12	In whole blood from healthy volunteers silychristin and silybin (but not silydianin) significantly and dose-dependently reduced PLA formation induced with IL-1β.	Bijak et al., 2017 [80]
anthocyanins	with 1 of 10 tested compounds, each N = 7	no anthocyanins added, N = 7	During in vitro ADP stimulation 2 of tested compounds (cyanidin-3-arabinoside, cyanidin-3-galactoside) significantly reduced PLA-N, ferulic acid reduced PLA-M and 4-hydroxybenzaldehyde reduced both.	Krga et al., 2018 [81]
cardiac surgery	supplementation, N = 7	no supplementation, N = 7	Preoperative supplementation of omega-3 fatty acids for 5 days resulted in significantly lower PLA on the day of operation and first post-operative day.	Iwase et al., 2014 [82]
exercise	after exercise, N = 20	before exercise, N = 20	PLA-G, PLA-M and PLA-Ly were significantly elevated after both moderate and strenuous exertion, although higher intensity caused significantly greater elevation of PLA-G and PLA-Ly.	Hilberg et al., 2008 [83]
exercise	after exercise, N = 21	before exercise, N = 21	PLA were significantly elevated after exertion; the effect was not abolished by enoxaparin nor argatroban administration. However, enoxaparin and argatroban did reduce PLA formation on thrombin or ADP stimulation post-exercise.	Li et al., 2007 [84]
exercise	after exercise, N = 14	before exercise, N = 14	PLA were significantly elevated after exertion, but percentage of PLA in regard to leukocyte count was similar. Increased PLA formation in response to exertion was not abolished by pretreatment with clopidogrel.	Perneby et al., 2004 [85]
exercise	MIH after exercise, N = 10 SIH after exercise, N = 10	MIH before exercise, N = 10 SIH before exercise, N = 10	After strenuous exercise PLA-E formation was significantly increased in response to LPS, shear stress and fMLP. Eight-weeks-long intermittent hypoxia regimen abolished this effect in both MIH and SIH group.	Wang et al., 2007 [86]
angiography for CAD evaluation	ACS, N = 125	No ACS, N = 437	PLA-M were significantly higher in ACS patients than those with and without angiographically-proven CAD. There was no association between baseline nor TRAP-induced PLA-M and adverse clinical outcomes (as defined in the study) in 2 years of observation.	Gremmel et al., 2016 [88]
	adverse clinical outcomes, N = 117	no adverse clinical outcomes, N = 445
CAD	FFR(+), N = 75	FFR(-), N = 70	PLA-M did not significantly differ dependent on FFR status. Higher PLA-M associated with coronary vasomotor dysfunction independent of FFR status.	Di Serafino et al., 2014 [90]
	vasoconstrictors in endothelial function test, N = 10	vasodilators in endothelial function test, N = 20
CAD	diffuse CAD, N = 50	healthy, N = 50 focal CAD, N = 40	PLA-M with M2 was significantly higher in the diffuse CAD group than the others; PLA-M with M2 independently and inversely correlated with endothelium-dependent vasodilation in patients with the diffuse disease.	Brown et al., 2018 [91]
CABG	after CABG, N = 15	before CABG, N = 15	PLA were increased for at least 1 week after the surgery and returned to baseline after 3 months. Immediately after CABG ADP- and TRAP-induced PLA formation was reduced, but at 1 week it was markedly increased. The effects were more evident for PLA-N and PLA-M.	Li et al., 2003 [95]
CABG for stable angina pectoris	after CABG, N = 54	before CABG, N = 54	One month after CABG PLA-M, PLA-N and PLA-Ly were significantly increased (unstimulated, ADP, thrombin, thromboxane analogue). At 3 months a significant decrease was observed in PLA-M (unstimulated, ADP, thromboxane analogue) and PLA-N (ADP, thromboxane analogue), whereas there was an increase for PLA-Ly (ADP).	Ivert et al. 2019 [96]
cardiac surgery (valve replacement for rheumatic heart disease)	AKI, N = 15	no AKI, N = 229	PLA in AKI group were significantly higher at all points throughout the perioperative period. High PLA (>6.8%) preoperatively entailed 18 times greater risk of AKI.	Yang et al., 2021 [97]
cardiac surgery (valve replacement for rheumatic heart disease)	high PLA, N = 151	low PLA, N = 93	Patients with high PLA (>6.8%) preoperatively, experienced significantly more major adverse events and perioperative complications than low PLA (<6.8%) group.	Liu et al., 2019 [98]
CABG	short-term intensive training after CABG, N = 19	conventional training after CABG, N = 21 healthy, N = 15	Strenuous exercise increased PLA-M and PLA-M subset with M1 in conventional training group but not in short-term intensive training group nor healthy controls.	Huang et al., 2017 [101]
AMI	AMI, N = 61	non-AMI chest pain, N = 150	In patients with chest pain PLA was significantly higher in AMI group. PLA level was the highest within 4 h from the onset of symptoms.	Furman et al., 2001 [114]
AMI	AMI, N = 9	non-AMI chest pain, N = 84 healthy, N = 10	Among patients with chest pain PLA-M were significantly higher in AMI group compared with non-AMI and control subjects.	Michelson et al., 2001 [115]
ACS	ACS, N = 43	non-ACS chest pain, N = 31	Among patients with chest pain PLA-M did not differ significantly between ACS and non-ACS subjects.	Levinas et al., 2012 [116]
AMI	AMI, N = 9	non-AMI chest pain, N = 21	Among patients suspected of AMI, those with actual AMI had significantly higher PLA-M. 100% of sensitivity and specificity for AMI with a PLA-M cut-off at 31.6%.	Lippi et al., 2007 [118]
UA	UA, N = 95	stable CAD, N = 30	UA patients had significantly higher total PLA-M, PLA-M with M2 and M3. Higher total PLA-M and PLA-M with M2 independently associated with intermediate-to-high risk by GRACE score.	Zeng et al., 2014 [119]
recurrent ischemia after UA/NSTEMI	yes, N = 7	no, N = 21	Patients who developed recurrent ischemia exhibited significantly greater PLA-N formation after adding TRAP. For every 1% increase in PLA-N the risk of recurrent ischemia rose about 7%.	Faraday et al., 2004 [121]
AMI treated with PCI	AMI, N = 31	healthy, N = 28	On admission AMI patients had higher PLA-M (in all subsets) than healthy controls. After adjustments higher PLA-M with M2 was significantly and independently associated with complications within 7 days of hospitalization in AMI patients.	Loguinova et al., 2018 [34]
AMI treated with PCI	STEMI, N = 100	stable CAD, N = 60 healthy controls, N = 35	PLA-M containing either M1 or M2 were significantly higher in STEMI group than in stable CAD at baseline. Higher accumulation of M2 and PLA-M with M2 on the seven days following AMI corresponded with the 2-years risk of serious cardiovascular adverse events.	Zhou et al., 2016 [122]
AMI treated with PCI	STEMI and successful reperfusion N = 35 STEMI and MVO, N = 13	stable CAD, N = 20	Baseline PLA-M were significantly higher in STEMI patients compared with stable CAD. MVO group had significantly higher PLA-M both on admission and 1-month post-treatment compared with good reperfusion group.	Aurigemma et al., 2014 [126]
AMI treated with PCI	poor perfusion, N = 28	good perfusion, N = 115	Patients with poor reperfusion after intervention had significantly higher PLA, PLA-M, PLA-N in aortic blood immediately after the procedure. PLA-N level showed independent correlation with sumSTR.	Huang et al., 2016 [127]
AMI treated with PCI	no-reflow, N = 19	successful reperfusion, N = 64	Patients with no-reflow after PCI had statistically greater preprocedural amount of PLA. High PLA appeared to be an independent risk factor for MVO.	Ren et al., 2016 [128]
ASA monotherapy	with, N = 6	without, N = 6	ASA did not affect spontaneous nor induced (with ADP, PAF) formation of PLA-N and PLA-M in vitro.	Zhao et al., 2001 [138]
ASA monotherapy	with, N = 15	without, N = 15	PLA amount did not differ before and after aspirin administration in healthy subjects.	Li et al., 2003 [140]
ASA monotherapy	with, N = 40	without, N = 40	Only PLA-M, but not PLA-G, amount differed significantly before and after ASA administration in healthy subjects. PLA formation in response to TRAP was not significantly affected.	Lukasik et al., 2011 [141]
clopidogrel treatment	Pro715-allele, N = 10 PSGL-1 B-allele, N = 5	without polymorphism, N = 10	PLA-M formation in response to ADP and TRAP was similarly reduced by clopidogrel both in controls and polymorphisms carriers.	Klinkhardt et al., 2005 [147]
clopidogrel for NSTEMI	NSTEMI on admission, N = 23	healthy, N = 20 NSTEMI group 24 h after clopidogrel, N = 23	NSTEMI patients had significantly higher PLA-M and PLA-N and enhanced PLA formation in response to ADP on admission. Clopidogrel counteracted both of these effects.	Xiao et al., 2004 [148]
coronary stenting	on clopidogrel after stenting, N = 51	before clopidogrel use and stenting, N = 51	Clopidogrel significantly reduced PLA formation in response to ADP, but not collagen. Nevertheless, PLA was significantly elevated for 30 days following the procedure.	Gurbel et al., 2002 [149]
P2Y12 and NHE-1 inhibitors treatment	with cariporide or AR-C 69331 or cariporide with AR-C 69331, each N = 8	no drug, N = 8	P2Y12 inhibitor AR-C69331 significantly limited PLA-M formation at normal and 7 pH, whereas NHE-1 inhibitor cariporide was only effective at 7 pH (conditions of NHE-1 activation). Combination of these agents had additive effect on PLA-M at 7 pH.	Klinkhardt et al., 2003 [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]
pneumonia	ticagrelor, N = 30	placebo, N = 30	PLA decreased significantly in patients on ticagrelor in 24-h, while it increased in placebo group after the same time. Ticagrelor group had better forced expiratory volume and needed less oxygen supplementation after the treatment.	Sexton et al., 2018 [158]
chronic antiplatelet treatment	clopidogrel group, N = 15 aspirin group, N = 15	healthy, N = 15	5 days after 7 days’ break in antiplatelet therapy and aortic surgery PLA were significantly elevated in the clopidogrel group, but not in the aspirin group.	Le Manach et al., 2014 [159]
ischemic stroke	clopidogrel with ASA, N = 284	ASA monotherapy, N = 286	Patients who experienced early neurological deterioration had higher baseline PLA-N, PLA-M, PLA-Ly. Significantly lower PLA levels at 7–10 days of treatment with clopidogrel + ASA, but only in a group with functionally deficient CYP2C19*2	Yi et al., 2018 [161]
stable CAD	ASA with clopidogrel, N = 16	ASA with placebo, N = 15	ASA with clopidogrel group showed lesser PLA and PLA-M formation in response to ADP and thrombin.	Perneby et al., 2007 [162]
UA treated with PCI and DAPT	clopidogrel, N = 23 prasugrel, N = 22	before clopidogrel, N = 23 before prasugrel, N = 22	After 3 months, prasugrel group showed 40% decrease in ADP-induced PLA formation. There was a moderate negative correlation between ADP-induced PLA formation and flow-mediated dilation (measurement of endothelial function).	Rudolph et al., 2017 [163]
acute ischemic stroke	ASA with clopidogrel, N = 284	ASA monotherapy, N = 286	Patients receiving DAPT had significantly lower PLA-N, PLA-M and PLA-Ly after 30 days of treatment. DAPT reduced recurrence of stroke more effectively than ASA monotherapy.	Yi et al., 2014 [164]
stable CAD	prasugrel, N = 55	clopidogrel, N = 55	PLA-M formation induced with ADP was significantly decreased in prasugrel group compared with clopidogrel. The effect persisted at least one month after starting the treatment.	Braun et al., 2008 [165]
glycoprotein IIb/IIIa inhibitor treatment	with SR121566 or c7E3, each N = 5	without SR121566 and c7E3, each N = 5	In vitro SR121566 and c7E3 (glycoprotein IIb/IIIa inhibitors) significantly reduced PAF-induced PLA-N formation but led to augmented PLA-N formation in response to ADP and TRAP.	Hu et al., 2003 [167]
epifibatide treatment	with epifibatide, N = 10	without epifibatide, N = 10	Epifibatide significantly augmented collagen-induced formation of PLA-M (stronger effect) and PLA-N in whole blood from healthy volunteers.	Scholz et al., 2002 [168]
hypothermic conditions	with epifibatide at 18 °C, N = 10	with epifibatide at 37 °C, N = 10	When epifibatide was added, after ADP stimulation PLA-G were significantly elevated in hypothermic conditions (18 °C) compared with normothermia (37 °C).	Straub et al., 2005 [169]
hypothermic conditions	with tirofiban or epifibatide or abciximab, each N = 4	no drug, N = 4	In normothermia (37 °C) each of three drugs partially reduced ADP-induced PLA-M and PLA-N formation compared with control. However, this effect disappeared in hypothermic conditions (32 °C).	Frelinger et al., 2003 [170]
AMI	with reteplase or reteplase with abciximab or tenecteplase with tirofiban, each N = 5	before drug, each N = 5	ADP-induced PLA formation was significantly reduced in patients treated with reteplase with abciximab as well as tenecteplase with tirofiban with the maximum effect at 2 h after administration.	Bertram et al., 2002 [172]
epoprostenol administration	epoprostenol, N = 5	PGE1 or ASA or cangrelor, each N = 4–6	Epoprostenol inhibited PLA-M and platelet-polymorphonuclear aggregates formation in blood from healthy subjects. In vitro concentration inhibiting 50% of the maximal response to stimulation (collagen with ADP) was smaller for epoprostenol (nanomolar range) than reference drugs in the study: PGE1, aspirin, cangrelor (micromolar range).	Tamburrelli et al., 2011 [174]
Preclinical studies
CHF and statin treatment	CHF, N = 22 rats CHF with rosuvastatin, N = 15 rats	healthy, N = 15 rats	Rat model of CHF was characterized by significantly increased PLA compared with controls. Amount of PLA in this animal was normalized on rosuvastatin treatment.	Schafer et al., 2005 [39]
alfrutamide, caffedymine	with alfrutamide or caffedymine, each N = 5	without alfrutamide and caffedymine, each N = 5	In vitro and in vivo both compounds significantly reduced unstimulated PLA formation in blood from mice.	Park et al., 2016 [40]
temporary ligation of the common carotid artery	ASA, N = 11 rivaroxaban, N = 12 ASA with rivaroxaban, N = 6	placebo, N = 11	Without stimulation ASA treated groups had significantly higher PLA compared with controls. No treatment reduced effect of 2-MeSADP stimulation on PLA, but rivaroxaban eliminated stimulating effect of PAR4 agonist.	Mastenbroek et al., 2020 [142]
ischemia-reperfusion injury in intestinal microcirculation	cilostazol, N = 14 mice ASA, N = 14 mice	placebo, N = 14 mice	Both cilostazol and ASA groups showed reduced PLA in intestinal circulation on microscopic assessment. However, only cilostazol group exhibited less endothelial damage.	Iba et al., 2006 [143]
clopidogrel treatment	mice treated with clopidogrel, N = 15–20	untreated mice, N = 15–20	Ability to form platelet–polymorphonuclear aggregates in response to ADP or thrombin was reduced in blood from treated mice. Platelets from untreated animals combined with polymorphonuclear cells from treated animals gave results like in untreated animals. Pretreating human blood with active metabolite of clopidogrel significantly reduced platelet–polymorphonuclear aggregates formation induced by ADP or PAR-1 agonist.	Evangelista et al., 2005 [146]
	human blood pretreated with clopidogrel 1, N = 3	human blood, N = 3
murine model of endotoxemic shock	prasugrel, N = 5	placebo, N = 5	In prasugrel group, platelet-polymorphonuclear aggregates formation was significantly reduced after stimulation with PAR-4 agonist peptide (only low concentrations) both after and without LPS injection.	Totani et al., 2012 [150]
murine model of endotoxemia	LPS with clopidogrel pretreatment, N = 10	placebo, N = 10 LPS, N = 10 clopidogrel, N = 10	LPS injection resulted in PLA-N elevation, but it was significantly lower in subjects pretreated with clopidogrel. Clopidogrel pretreatment group showed less histological change and less leukocyte and platelet infiltration in the lungs compared with LPS alone group.	Wang et al., 2019 [155]
murine model of hypertension and cardiac remodelling	with clopidogrel, N = 4	without clopidogrel, N = 4	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: 2-MeSADP—2-methylthioadenosine diphosphate; AAA—abdominal aortic aneurysm; ACS—acute coronary syndrome; ADP—adenosine diphosphate; AKI—acute kidney injury; AMI—acute myocardial infarction; ASA—acetylsalicylic acid; CABG—coronary artery bypass graft; CAD—coronary artery disease; CHF—congestive heart failure; CVD—cardiovascular diseases; DAPT—dual anti-platelet therapy; FFR—fractional flow reserve; FFR(+)—hemodynamically significant stenosis; FFR(-)—hemodynamically non-significant stenosis; fMLP—N-formyl-methionyl-leucyl-phenylalanine; GRACE—the Global Registry of Acute Coronary Events score; HbA1c—glycated haemoglobin; HDL—high-density lipoprotein; LDL—low-density lipoprotein; LPS—lipopolysaccharides; MIH—moderate induced hypoxia; MVO—coronary microvascular obstruction; M1—classical monocytes; M2—intermediate monocytes; M3—non-classical monocytes; NHE-1—sodium–hydrogen exchanger isoform 1; NSTEMI—non-ST elevation myocardial infarction; PAD—peripheral artery disease; PAF—platelet-activating factor; PAR-1,4—protease activated receptor 1,4; PCI—percutaneous coronary intervention; PLA—platelet-leukocyte aggregates; PLA-E—platelet-eosinophil aggregates; PLA-G—platelet-granulocyte aggregates; PLA-Ly—platelet-lymphocyte aggregates; PLA-LyT—platelet-lymphocyte T aggregates; PLA-M—platelet-monocyte aggregates; PLA-N—platelet-neutrophil aggregates; PGE1—prostaglandin E1; PH—primary hypercholesterolemia; SIH—severe induced hypoxia; STEMI—ST elevation myocardial infarction; sumSTR—sum-ST-segment resolution; T1D—type 1 diabetes mellitus; T2D—type 2 diabetes mellitus; TG—triglycerides; TLR-2,4—toll-like receptor 2,4; TRAP—thrombin receptor-activating peptide; TXA2—thromboxane A2; UA—unstable angina; VTE—venous thromboembolism. ¹ Active metabolite.