Elevated Leukocyte Count and Platelet-Derived Thrombogenicity Measured Using the Total Thrombus-Formation Analysis System in Patients with ST-Segment Elevation Myocardial Infarction

Abstract

Aims: High platelet-derived thrombogenicity during the acute phase of ST-segment elevation myocardial infarction (STEMI) is associated with poor outcomes; however, the associated factors remain unclear. This study aimed to examine whether acute inflammatory response after STEMI affects platelet-derived thrombogenicity.

Methods: This retrospective observational single-center study included 150 patients with STEMI who were assessed for platelet-derived thrombogenicity during the acute phase. Platelet-derived thrombogenicity was assessed using the area under the flow-pressure curve for platelet chip (PL-AUC), which was measured using the total thrombus-formation analysis system (T-TAS). The peak leukocyte count was evaluated as an acute inflammatory response after STEMI. The patients were divided into two groups: the highest quartile of the peak leukocyte count and the other three quartiles combined.

Results: Patients with a high peak leukocyte count (＞15,222/mm³; n=37) had a higher PL-AUC upon admission (420 [386–457] vs. 385 [292–428], p=0.0018), higher PL-AUC during primary percutaneous coronary intervention (PPCI) (155 [76–229] vs. 96 [29–170], p=0.0065), a higher peak creatine kinase level (4200±2486 vs. 2373±1997, p＜0.0001), and higher PL-AUC 2 weeks after STEMI (119 [61–197] vs. 88 [46–122], p=0.048) than those with a low peak leukocyte count (≤ 15,222/mm³; n=113). The peak leukocyte count after STEMI positively correlated with PL-AUC during primary PPCI (r=0.37, p＜0.0001). A multivariable regression analysis showed the peak leukocyte count to be an independent factor for PL-AUC during PPCI (β=0.26, p=0.0065).

Conclusions: An elevated leukocyte count is associated with high T-TAS-based platelet-derived thrombogenicity during the acute phase of STEMI.

Introduction

Effective antiplatelet therapy is essential in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI) to support reperfusion and optimize clinical outcomes¹⁾. Platelet reactivity and aggregation in the acute phase of STEMI are high despite dual antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitor²⁾. High platelet reactivity during the acute phase of STEMI is associated with myocardial damage and impaired reperfusion^{3, 4)}. Moreover, platelet-derived thrombogenicity during PPCI, which was measured using the total thrombus-formation analysis system (T-TAS), is associated with the enzymatic infarct size and the slow-flow/no-reflow phenomenon in patients with STEMI⁵⁾. T-TAS is a novel automated microchip flow chamber system that assesses the platelet function in bleeding and the thrombosis risk⁶⁾. Although T-TAS is expected to have clinical value, the factors associated with high platelet-derived thrombogenicity measured using T-TAS remain unclear. To individualize optimal antiplatelet therapy for each patient, it is important to clarify the factors associated with high platelet-derived thrombogenicity after STEMI.

The acute inflammatory response plays an essential role in the pathophysiology of STEMI. STEMI is accompanied by a transient elevation of leukocytes as an acute inflammatory response⁷⁾. The association between an elevated leukocyte count and mortality in patients with STEMI has been reported for more than half a century⁸⁾. Even in the PPCI era, an elevated leukocyte count is an important factor in myocardial damage, impaired reperfusion, and mortality in patients with STEMI^9-11). Acute inflammatory responses after STEMI are associated with increased cardiovascular risk; however, the mechanisms underlying this association remain unclear.

Numerous crosslinks exist between thrombogenicity and inflammation. Experimental studies have demonstrated increased interactions between platelets and leukocytes in the pathophysiology of acute myocardial infarction (MI)^{12, 13)}. Acute inflammatory responses may result in high platelet-derived thrombogenicity following STEMI. This study aimed to examine the association between platelet-derived thrombogenicity, measured using T-TAS, and elevated leukocyte counts during the acute phase of STEMI.

Methods

Patient Population

This retrospective, observational, single-center study included Japanese patients with STEMI who underwent PPCI with stent implantation within 12 hours of symptom onset. STEMI was defined according to the fourth universal definition of MI as the presence of chest discomfort or other ischemic symptoms with a new ST-segment elevation at the j-point of more than 1 mm in two contiguous leads other than V2-3, including a newly diagnosed bundle branch block¹⁴⁾. The exclusion criteria were cardiopulmonary arrest upon admission, major bleeding events within 7 days prior to enrollment, hematologic or malignant disease, renal dysfunction on hemodialysis (HD), the simultaneous occlusion of multiple coronary arteries, and the use of any antiplatelet or oral anticoagulant drugs within 7 days prior to admission. Hypertension was defined as a history of hypertension with a prior use of antihypertensive drugs. Dyslipidemia was defined as low-density lipoprotein cholesterol (LDL-C) ≥ 140 mg/dL, high-density lipoprotein cholesterol (HDL-C) ＜40 mg/dL, triglyceride (TG) ≥ 150 mg/dL, or a history of dyslipidemia. Diabetes mellitus (DM) was diagnosed based on the HbA1_C level ≥ 6.5%, the 2-h plasma glucose level after a 75-g oral glucose tolerance test ≥ 200 mg/dL, or a history of DM. The study protocol was approved by the Ethics Committee of Yokohama City University, and written informed consent was obtained from all patients. This study was conducted in accordance with the Declaration of Helsinki.

Percutaneous Coronary Intervention (PCI) and Antiplatelet Therapy

All patients received a 200-mg loading dose of aspirin and unfractionated heparin at the time of presentation. PPCI was performed after the administration of a Japanese standard loading dose of 20 mg prasugrel or 300 mg clopidogrel. The P2Y12 inhibitor was selected at the discretion of the attending cardiologist. Newer P2Y12 inhibitors such as ticagrelor and cangrelor were not used in this study. An additional dose of unfractionated heparin was administered to maintain an activated clotting time (ACT) of ≥ 250 s during PPCI. Argatroban and monteplase were also used as bailout therapies in patients with a high thrombus burden on coronary angiography, such as acute stent thrombosis. Glycoprotein IIb/IIIa inhibitors were not approved for use in Japan at the time of the present study.

Measurement of Platelet-Derived Thrombogenicity Using the T-TAS

Platelet-derived thrombogenicity during the acute phase of STEMI was assessed using T-TAS (Fujimori Kogyo Co., Japan) on admission (before the administration of antithrombotic agents), during PPCI (1 h after loading of P2Y12 inhibitors), and 2 weeks after STEMI. T-TAS is an automated microchip flow chamber system for performing a quantitative analysis of the thrombus formation process under blood flow conditions^{15, 16)}. The platelet chip (PL chip) contains 25 capillary channels (width, 40 µm; depth, 40 µm) coated with type I collagen specifically designed for performing a quantitative analysis of the platelet thrombus formation process, involving platelet adhesion and aggregation, granule secretion, and thrombus growth in the absence of coagulation and fibrinolysis systems. Blood samples were collected in hirudin-containing blood sampling tubes (MP0600 [Verum Diagnostica]; final hirudin concentration, 25 µg/mL) and applied to the analytical path of the PL chip under a constant flow. The platelet aggregates gradually increased in size and occluded the capillaries, resulting in an increase in the flow pressure. In this study, platelet-derived thrombogenicity was expressed as the area under the flow-pressure curve for the first 10 min for the PL chip tested at a flow rate of 18 µL/min (PL-AUC). A high PL-AUC indicates high platelet-derived thrombogenicity.

Leukocyte Count and Enzymatic Infarct Size

Blood samples were obtained on admission at 3-h intervals until the indication of peak cardiac biomarker levels, and then at least every 24 h for 5 days after PPCI. The leukocyte counts, fractions (neutrophils, monocytes, eosinophils, and lymphocytes), and high-sensitivity C-reactive protein (hs-CRP) levels were measured simultaneously. We divided the patients into two groups: the group with the highest quartile of peak leukocyte count and the group with the other three quartiles combined. Several studies have demonstrated that the highest quartile of the leukocyte count and its fractions indicate poor clinical outcomes after STEMI^{9, 17-19)}.

In the present study, the peak levels of creatine kinase (CK) and CK-myocardial band (CK-MB) were used to estimate the infarct size.

Statistical Analysis

Data are reported as frequencies and percentages for categorical variables and as the mean±standard deviation (SD) for continuous variables. Categorical comparisons were performed using the chi-square test or Fisher’s exact test, and continuous values were compared using Student’s t-test. The PL-AUC values were reported as medians (interquartile ranges) and compared using the Mann-Whitney U test. Correlations between continuous variables were evaluated using a linear regression analysis. A multiple linear regression analysis was performed to determine the factors associated with PL-AUC. Variables with a p value ≤ 0.10 on a univariable analysis were included in the multivariable analysis. Statistical significance was set at p＜0.05. The data were analyzed using JMP Pro15 software program (SAS Institute Inc., Cary, NC, USA).

Results

Among the 457 patients with STEMI who underwent PPCI between September 2014 and December 2019, 150 who underwent PL-AUC assessment during the acute phase were included in this analysis (Supplementary Fig.1). The acute inflammatory responses after STEMI are shown in Table 1. The mean leukocyte count on admission was 10,245±3117/mm³ and with a peak level of 129,578±3205/mm³. The time from admission to peak leukocyte count was 10.4±11.8 h. Among the leukocyte fractions, the neutrophil and monocyte counts increased from the value on admission, whereas the eosinophil and lymphocyte counts decreased (Table 1). The hs-CRP level also increased from that on admission. The time from admission to the peak monocyte count and hs-CRP level was longer than that for the other parameters.

Supplementary Fig.1. Patient flow diagram

CPA, cardiopulmonary arrest; PPCI, primary percutaneous coronary intervention; STEMI, ST-segment elevation myocardial infarction; T-TAS, total thrombus formation analysis.

Table 1.Acute inflammatory responses after STEMI

	On-admission level	Peak level	Bottom level	Time from admission to peak/bottom level (h)
Leukocyte (/mm3)	10,244±3,117	12,958±3,205	-	10.4±11.8
Neutrophil (/mm3)	6,811±3,302	10,607±2,997	-	8.7±10.1
Monocyte (/mm3)	595±245	1,059±345	-	36.8±18.7
Eosinophil (/mm3)	192±231	-	30±65	10.5±10.1
Lymphocyte (/mm3)	2,647±1,309	-	1,073±467	5.7±5.7
hs-CRP (mg/dL)	0.240±0.351	6.759±4.105	-	59.4±20.3

Data are shown as mean±standard deviation.

hs-CRP, high-sensitivity C-reactive protein; STEMI, ST-segment elevation myocardial infarction

The patients were divided into two groups: the group with the highest quartile of peak leukocyte count (＞15,222/mm³; n=37) and the group with the other three quartiles combined (≤ 15,222/mm³; n=113). The baseline characteristics of the patients with high and low peak leukocyte counts are shown in Table 2. The patients with a high peak leukocyte count were younger and had a greater body weight, higher hemoglobin and hematocrit levels, and worse lipid and diabetic status (lower HDL-C, higher TG, and higher HbA1c and blood glucose levels) than those with a low peak leukocyte count. Although the door-to-device time was shorter in patients with a high peak leukocyte count, no significant difference in the onset-to-device time was observed between the two groups. Approximately 80% of the patients received a loading dose of prasugrel.

Table 2.Baseline characteristics according to peak leukocyte count

	Patients with a high peak leukocyte count (＞15,222/mm3; n= 37)	Patients with a low peak leukocyte count (≤ 15,222/mm3; n= 113)	p value
Clinical data
Age (years)	58±12	66±12	0.0013
Male	32 (86)	87 (77)	0.25
Weight (kg)	73.1±12.6	67.0±14.7	0.0026
BMI (kg/m2)	25.9±3.3	24.7±4.0	0.11
Hypertension	22 (59)	64 (57)	0.76
Dyslipidemia	33 (89)	91 (81)	0.23
Diabetes mellitus	16 (43)	45 (40)	0.71
Renal insufficiency	12 (32)	41 (36)	0.67
Current smoking	22 (59)	55 (49)	0.25
Laboratory data on admission
Hemoglobin (g/dL)	15.6±1.4	14.4±1.9	0.0002
Hematocrit (%)	46.8±4.2	43.0±5.7	0.0003
Platelet (×104/μL)	23.2±4.4	23.1±6.4	0.90
LDL-C (mg/dL)	141±53	133±32	0.23
HDL-C (mg/dL)	43±10	47±11	0.029
TG (mg/dL)	251±184	164±123	0.0013
HbA1c (%)	6.6±1.6	6.2±0.9	0.041
BG (mg/dL)	189±67	163±44	0.0077
eGFR (mL/min/1.73 m2)	70±24	69±18	0.84
Treatment time
Onset-to-door time (min)	143±150	116±119	0.27
Door-to-device time (min)	53±16	61±20	0.033
Onset-to-device time (min)	196±157	177±126	0.46
Antithrombotic agent
Aspirin	37 (100)	113 (100)	-
P2Y12 inhibitors			0.53
Prasugrel	28 (76)	91 (81)
Clopidogrel	9 (24)	22 (19)
Unfractionated heparin (units/kg)	116±28	125±30	0.094
Argatroban and/or monteplase use	0 (0)	4 (4)	0.57

Data are shown as mean±SD or n (%).

Renal insufficiency was defined as eGFR ≤ 60 mL/min/1.73 m² upon admission.

BG, blood glucose; BMI, body mass index; eGFR, estimated glomerular filtration rate; HbA1c, hemoglobin A1c; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

Angiography, PCI findings, and the enzymatic infarct size according to the peak leukocyte count are shown in Table 3. The percentage of initial thrombolysis in myocardial infarction (TIMI) flow grade of 0 or 1 (86% vs. 65%, p=0.012) and the percentage of the final TIMI grade (76% vs. 89%, p=0.056) were lower in patients with a high peak leukocyte count than in those with a low peak leukocyte count. Furthermore, patients with a high peak leukocyte count had higher peak CK and CK-MB levels than those with a low peak leukocyte count (4,200±2486 vs 2,373±1997, p＜0.0001 and 401±266 vs. 235±205, p=0.0001, respectively).

Table 3.Angiography and PCI findings and enzymatic infarct size according to peak leukocyte count

	Patients with a high peak leukocyte count (＞15,222/mm3; n= 37)	Patients with a low peak leukocyte count (≤ 15,222/mm3; n= 113)	p value
Angiographic and PCI findings
Culprit lesion of LAD	22 (59)	55 (49)	0.25
Initial TIMI flow grade 0 or 1	32 (86)	73 (65)	0.012
DES implantation	37 (100)	107 (95)	0.34
Final TIMI flow grade 3	28 (76)	100 (89)	0.056
Enzymatic infarct size
Peak CK (IU/L)	4,200±2,486	2,373±1,997	＜0.0001
Peak CK-MB (IU/L)	401±266	235±205	0.0001

Data are shown as mean±standard deviation or n (%).

CK, creatine kinase; CK-MB, CK-myocardial band; DES, drug-eluting stent; LAD, left anterior descending artery; PCI, percutaneous coronary intervention; TIMI, thrombolysis in myocardial infarction.

Patients with a high peak leukocyte count had a higher on-admission PL-AUC (420 [386–457] vs 385 [292–428], p=0.0018), higher PL-AUC during PPCI (155 [76–229] vs 96 [29–170], p=0.0065) than those of patients with a low peak leukocyte count (Fig.1A and 1B). Moreover, the PL-AUC 2 weeks after STEMI was higher in patients with a high peak leukocyte count than in those with a low peak leukocyte count (119 [61–197] vs 88 [46–122], p=0.048) (Fig.1C).

Fig.1. Platelet-derived thrombogenicity in patients with high and low peak leukocyte counts

PPCI, primary percutaneous coronary intervention; PL-AUC, area under the flow-pressure curve for platelet chip; STEMI, ST-segment elevation myocardial infarction

Acute Inflammatory Responses and Platelet-Derived Thrombogenicity

The on-admission leukocyte count positively correlated with on-admission PL-AUC (r=0.25, p=0.0028), whereas the fraction count and hs-CRP level on admission did not (Supplementary Fig.2A). The peak leukocyte count also positively correlated with on-admission PL-AUC (r=0.30, p=0.0003) (Supplementary Fig.2B). Among the fractions and hs-CRP levels, only the peak neutrophil count positively correlated with on-admission PL-AUC (r=0.27, p=0.0018) (Supplementary Fig.2C).

Supplementary Fig.2. Correlations between the acute inflammatory responses and on-admission platelet-derived thrombogenicity

(A) Correlation between on-admission leukocyte count and on-admission PL-AUC. (B and C) Correlations between peak inflammatory responses and on-admission PL-AUC

PL-AUC, area under the flow-pressure curve for platelet chip

The on-admission leukocyte count positively correlated with the PL-AUC during PPCI (r=0.21, p=0.0083) (Fig.2A). Among the leukocyte fractions, the on-admission monocyte count positively correlated with PL-AUC during PPCI (r=0.21, p=0.022) (Fig.2B); however, no significant correlations were observed between any other on-admission fraction counts and PL-AUC during PPCI. The on-admission hs-CRP levels also did not correlate with the PL-AUC during PPCI.

Fig.2. Correlations between the acute inflammatory responses and platelet-derived thrombogenicity

(A and B) Correlations between the on-admission inflammatory responses and PL-AUC during PPCI. (C to F) Correlations between the peak inflammatory responses and PL-AUC during PPCI. (G) Correlation between the peak leukocyte count and PL-AUC 2 weeks after STEMI.

hs-CRP, high-sensitivity C-reactive protein; PPCI, primary percutaneous coronary intervention; PL-AUC, area under the flow pressure curve for platelet chip; STEMI, ST-segment elevation myocardial infarction.

The peak inflammatory responses had stronger correlation coefficient values with the PL-AUC levels during PPCI than the on-admission inflammatory responses. The peak leukocyte count correlated with PL-AUC during PPCI (r=0.37, p＜0.0001) (Fig.2C). The peak neutrophil and monocyte counts correlated with PL-AUC during PPCI (r=0.34, p＜0.0001 and r=0.22, p=0.0073, respectively) (Fig.2D and 2E). The peak hs-CRP levels also correlated with PL-AUC during PPCI (r=0.19, p=0.0022) (Fig.2F). Conversely, the bottom eosinophil and lymphocyte counts did not correlate with PL-AUC during PPCI.

The peak leukocyte count also correlated with PL-AUC 2 weeks after STEMI (r=0.21, p=0.020) (Fig.2G), whereas the peak fraction count did not. Additionally, the peak hs-CRP levels did not correlate with PL-AUC 2 weeks after STEMI.

The correlation between the peak inflammatory responses and PL-AUC during PPCI and the peak CK levels is shown in Fig.3. The peak leukocyte count correlated with the peak CK levels (r=0.36, p＜0.0001) (Fig.3A). The peak neutrophil and monocyte counts correlated with the peak CK levels (r=0.41, p＜0.0001 and r=0.28, p=0.0007, respectively) (Fig.3B and 3C). The peak hs-CRP levels correlated with the peak CK levels (r=0.21, p=0.0096) (Fig.3D). There was a significant correlation between the PL-AUC during PPCI and the peak CK levels (r=0.18, p=0.025) (Fig.3E).

Fig.3. Correlations between the peak inflammatory responses and platelet-derived thrombogenicity during PPCI with the peak CK level

(A to D) Correlations between the peak inflammatory responses and the peak CK level. (E) Correlation between PL-AUC during PPCI and the peak CK level.

CK, creatine kinase; hs-CRP, high-sensitivity C-reactive protein; PPCI, primary percutaneous coronary intervention; PL-AUC, area under the flow pressure curve for the platelet chip

Factors Associated with Platelet-Derived Thrombogenicity

According to a univariate analysis, there were several differences in the factors associated with on-admission PL-AUC and PL-AUC during PPCI and 2 weeks after STEMI, although the on-admission and peak leukocyte counts were common associated factors (Table 4). Age, weight, and the hemoglobin and hematocrit levels were significantly associated with on-admission PL-AUC and PL-AUC during PPCI. The platelet count was associated only with on-admission PL-AUC. The diabetic status and peak CK levels were only associated with PL-AUC during PPCI. Only male sex, except for inflammatory responses, was associated with PL-AUC 2 weeks after STEMI.

Table 4.Univariable regression analysis for determinants of PL-AUC on admission, during PPCI, and 2 weeks after STEMI

Variables	On-admission		During PPCI		2 w after STEMI
	r	p value	r	p value	r	p value
On-admission leukocyte count (/mm3)	0.25	0.0028	0.21	0.0083	0.21	0.024
On-admission neutrophil count (/mm3)	0.18	0.053	0.14	0.12	0.13	0.20
On-admission monocyte count (/mm3)	0.12	0.20	0.21	0.022	0.20	0.040
On-admission eosinophil count (/mm3)	0.042	0.65	0.086	0.34	0.054	0.59
On-admission lymphocyte count (/mm3)	0.15	0.11	0.17	0.066	0.23	0.022
On-admission hs-CRP level (mg/dL)	-0.06	0.49	0.072	0.38	0.13	0.16
Peak leukocyte count (/mm3)	0.30	0.0003	0.37	＜0.0001	0.21	0.020
Peak neutrophil count (/mm3)	0.27	0.0018	0.34	＜0.0001	0.16	0.091
Peak monocyte count (/mm3)	0.15	0.091	0.22	0.0073	0.098	0.30
Bottom eosinophil count (/mm3)	0.078	0.38	0.074	0.37	0.014	0.88
Bottom lymphocyte count (/mm3)	0.11	0.19	-0.14	0.088	0.093	0.32
Peak hs-CRP level (mg/dL)	0.068	0.43	0.19	0.022	0.18	0.057
Age (years)	-0.28	0.0011	-0.24	0.0025	-0.18	0.057
Male	0.015	0.87	0.068	0.41	0.21	0.022
Weight (kg)	0.18	0.039	0.18	0.024	0.072	0.44
Hypertension	-0.064	0.46	-0.001	0.99	-0.12	0.20
LDL-C (mg/dL)	0.14	0.11	0.049	0.55	0.044	0.63
HDL-C (mg/dL)	-0.14	0.11	0.0073	0.93	-0.11	0.22
TG (mg/dL)	0.14	0.10	-0.0079	0.92	-0.021	0.82
HbA1c (%)	0.033	0.70	0.22	0.0075	0.034	0.71
BG on admission (mg/dL)	0.065	0.46	0.20	0.012	0.083	0.37
eGFR (ml/min/1.73 m2)	0.015	0.87	-0.025	0.76	0.12	0.20
Hemoglobin (g/dL)	0.26	0.0025	0.20	0.012	0.13	0.16
Hematocrit (%)	0.25	0.0031	0.22	0.0066	0.14	0.13
Platelet (×104/μL)	0.22	0.011	0.13	0.13	0.0033	0.97
Current smoking	0.12	0.16	0.10	0.21	0.021	0.82
Onset to device time (min)	-0.071	0.41	-0.055	0.50	0.012	0.90
Use of prasugrel	-	-	0.044	0.59	-0.045	0.63
Argatroban and/or monteplase use	-	-	0.12	0.15	-0.020	0.83
Unfractionated heparin (units/kg)	-	-	-0.15	0.061	-	-
Culprit lesions of LAD	-0.12	0.15	-0.085	0.30	0.020	0.83
Initial TIMI flow 0 or 1	-0.14	0.11	-0.041	0.62	0.038	0.68
DES implantation	-	-	0.047	0.57	0.13	0.15
Peak CK (IU/L)	0.020	0.82	0.18	0.025	-0.017	0.86

BG, blood glucose; CK, creatine kinase; DES, drug-eluting stent; eGFR, estimated glomerular filtration rate; HbA1c, hemoglobin A1c; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; LAD, left anterior descending artery; LDL-C, low-density lipoprotein cholesterol; PL-AUC, area under the flow pressure curve for the platelet chip; PPCI, primary percutaneous coronary intervention; STEMI, ST-segment elevation myocardial infarction; TG, triglyceride; TIMI, thrombolysis in myocardial infarction.

Multivariate regression analyses showed the peak leukocyte count to be a significant and independent factor for on-admission PL-AUC (β=0.19, p=0.037) and PL-AUC during PPCI (β=0.26, p=0.0065), but it was not an independent factor for PL-AUC 2 weeks after STEMI (β=0.16, p=0.11) (Table 5). Among the leukocyte fractions, the peak neutrophil count was a significant and independent factor of PL-AUC during PPCI (β=0.24, p=0.011 [model 2]) (Supplementary Table 1). The peak monocyte count (β=0.12, p=0.15 [model 3]) and peak hs-CRP level (β=0.089, p=0.28 [model 4]) were not independent factors for PL-AUC during PPCI according to multivariate regression analyses.

Table 5.Multivariable regression analysis for determinants of PL-AUC

Variables	On-admission		During PPCI		2 w after STEMI
	β	p value	β	p value	β	p value
Peak leukocyte count (/mm3)	0.19	0.037	0.26	0.0065	0.16	0.11
Age (years)	-0.13	0.25	-0.13	0.19	-0.065	0.52
Male					0.16	0.11
Weight (kg)	-0.018	0.86	-0.046	0.70
HbA1c (%)	-0.054	0.52	0.15	0.057
Hematocrit (%)	0.13	0.20	-0.0034	0.97
Platelet (×104/μL)	0.16	0.069
Unfractionated heparin (units/kg)			-0.073	0.49
Peak CK (IU/L)			0.12	0.17

CK, creatine kinase; HbA1c, hemoglobin A1c; PL-AUC, area under the flow-pressure curve for the platelet chip; PPCI, primary percutaneous coronary intervention; STEMI, ST-segment elevation myocardial infarction

Supplementary Table 1.Other models of multivariable regression analysis for determinants of PL-AUC during PPCI

Variables	Model 2		Model 3		Model 4
	β	p value	β	p value	β	p value
Peak neutrophil count (/mm3)	0.24	0.011
Peak monocyte count (/mm3)			0.12	0.15
Peak hs-CRP level (mg/dL)					0.089	0.28
Age (years)	-0.14	0.17	-0.18	0.090	-0.20	0.054
Weight (kg)	-0.039	0.75	-0.055	0.66	-0.061	0.62
HbA1c (%)	0.13	0.11	0.15	0.074	0.16	0.048
Hematocrit (%)	-0.013	0.89	0.034	0.73	0.060	0.53
Unfractionated heparin (units/kg)	-0.069	0.52	-0.048	0.66	-0.059	0.59
Peak CK (IU/L)	0.13	0.14	0.19	0.024	0.19	0.021

CK, creatine kinase; HbA1c, hemoglobin A1c; hs-CRP, high-sensitivity C-reactive protein; PL-AUC, area under the flow-pressure curve for platelet chip; PPCI, primary percutaneous coronary intervention

Discussion

The principal findings of this study are as follows. 1) Patients with a high peak leukocyte count, characterized by a larger infarct size, had higher platelet-derived thrombogenicity in the acute phase of STEMI than those with a low peak leukocyte count. 2) The peak leukocyte count was associated with on-admission PL-AUC and PL-AUC during PPCI and 2 weeks after STEMI. 3) The peak leukocyte count was an independent factor for on-admission PL-AUC and PL-AUC during PPCI. 4) Among the fractions of leukocytes, the peak neutrophil count was an independent factor of PL-AUC during PPCI.

Platelet-Derived Thrombogenicity and an Elevated Leukocyte Count after STEMI

Inflammation plays an important role in the development and progression of acute coronary syndrome (ACS)¹¹⁾. In patients with STEMI, leukocytes infiltrate the infarcted myocardium in the first few hours after onset²⁰⁾, cause proteolytic and oxidative damage to endothelial cells, plug the microvasculature, and induce hypercoagulability²¹⁾. In the STEMI setting, a higher leukocyte count on admission has been reported to be associated with an increased thrombus burden²²⁾. An elevated leukocyte count after STEMI is associated with infarct size, no-reflow phenomenon, and mortality^{11, 17, 23)}; However, the mechanisms associated with these phenomena have not yet been fully clarified. Although the association between leukocytes and thrombogenicity can explain worse outcomes in patients with STEMI who have higher leukocyte counts, few studies have so far investigated platelet-derived thrombogenicity during the acute phase of STEMI. Leukocyte count has been reported to be associated with increased platelet reactivity in patients at risk for coronary artery disease²⁴⁾, but the study population included healthy individuals. To the best of our knowledge, this is the first study to investigate the time course of platelet-derived thrombogenicity during the acute phase of STEMI and its association with the leukocyte count. The present study revealed that patients with a high peak leukocyte count had a high platelet-derived thrombogenicity. Moreover, high platelet-derived thrombogenicity in patients with a high peak leukocyte count was observed not only on admission, but also during PPCI and 2 weeks after STEMI. It has been suggested that an elevated leukocyte count may play an important role in platelet-derived thrombogenicity during the acute phase of STEMI and it can affect platelet-derived thrombogenicity even after the administration of antithrombotic drugs and after the stabilization of the patient’s condition.

Platelet-Derived Thrombogenicity and Leukocyte Subtypes

Each leukocyte fraction is also an important risk factor for coronary artery diseases. The neutrophil, monocyte, and eosinophil counts are associated with an increased risk of coronary artery diseases, whereas lymphocyte count has an inverse association^25-27). After STEMI onset, the neutrophil and monocyte counts transiently increase, and lymphocytes transiently decrease²⁸⁾. In particular, neutrophils and monocytes play important roles in the pathophysiology of STEMI. Elevated neutrophil counts after STEMI have been associated with infarct size, reperfusion effectiveness, LVEF, and cardiac events^{9, 18)}. An elevated monocyte count is also associated with infarct size and reperfusion effectiveness¹⁸⁾, and it also plays an important role in the progression of coronary plaque in patients with STEMI²⁸⁾. Neutrophils are the first subsets of leukocytes to be found in the damaged myocardium and massively infiltrate the infarct area in the first few hours following the onset of ischemia^{19, 20)}. Neutrophils are removed from the myocardium after the phagocytosis of debris¹⁹⁾. Monocytes migrate from capillaries to the extravascular space and are transformed into macrophages¹⁹⁾. Following neutrophils, monocyte-derived macrophages infiltrate the infarct area to remove cardiac tissue debris and apoptotic neutrophils²⁹⁾. The present study revealed that the peak neutrophil count was an independent factor platelet-derived thrombogenicity during PPCI. On-admission and peak monocyte counts also correlated with platelet-derived thrombogenicity during PPCI. Among the different leukocyte fractions, neutrophils and monocytes may be largely responsible for platelet-derived thrombogenicity during the acute phase of STEMI. Platelet-neutrophil aggregation and platelet-monocyte aggregation, but not platelet-lymphocyte aggregation, have been reported to be involved in the mechanisms of poor reperfusion in patients with STEMI³⁰⁾.

The hs-CRP level is also an independent factor in the poor prognosis of patients with STEMI³¹⁾. High hs-CRP levels are associated with thrombotic events, such as stent thrombosis and left ventricular thrombus^{32, 33)}. Although the peak hs-CRP level was not an independent factor of PL-AUC during PPCI in this study, it is considered to be an important factor in the acute inflammatory responses after STEMI.

Platelet-Derived Thrombogenicity, an Elevated Leukocyte Count, and Infarct Size

We previously reported that the PL-AUC during PPCI was associated with the peak CK level and slow-flow/no-reflow phenomenon in patients with STEMI⁵⁾. Other authors have also demonstrated that patients with poor myocardial perfusion have higher levels of platelet-leukocyte aggregation in patients with STEMI³⁰⁾. Although it was difficult to establish cause-and-effect relationships between the three factors (platelet-derived thrombogenicity, elevated leukocyte count, and infarct size) using this study design, these factors might be closely related and may create a vicious cycle. In patients with STEMI who have high peak leukocyte counts, increased platelet-derived thrombogenicity and spasm³⁴⁾ may be induced at the level of the arteriolar vessels embolized by thrombus and plaque contents from the culprit lesions, thus resulting in a large infarct size. Moreover, a large infarct size may lead to a further increase in the inflammatory response, because more leukocytes infiltrate the infarcted myocardium. The above phenomenon might be one of the mechanisms underlying the association between an elevated leukocyte count and poor clinical outcomes, including a large infarct size in patients with STEMI. Considering this relationship, the intervention targets to reduce the infarct size may be platelet-derived thrombogenicity and acute inflammatory responses in patients with STEMI.

A more potent antithrombotic therapy guided by T-TAS may be effective in reducing the infarct size; however, this remains unclear. In contrast, bleeding should be considered when performing antithrombotic therapy in patients with STEMI. Recently, shortening the duration of DAPT has been considered as a strategy to reduce bleeding events^{35, 36)}. However, in the STOPDAPT-2 ACS trial, 1-month DAPT did not meet the criteria for non-inferiority compared with 12-month DAPT for the composite ischemic and bleeding endpoints among patients with ACS who underwent drug-eluting stent implantation³⁷⁾. A short DAPT duration in patients with ACS may increase the risk of ischemic events³⁸⁾. Our study demonstrated that patients with a high peak leukocyte count had high platelet-derived thrombogenicity not only on admission and during PPCI, but also 2 weeks after STEMI. Moreover, patients with a high peak leukocyte count were younger and had a greater body weight, higher hemoglobin levels, worse lipid and diabetic status, and larger infarct size than those with a low peak leukocyte count. This finding suggests that these patients may require more potent antiplatelet therapy during the acute phase of STEMI. At least thrombogenicity in patients with STEMI with a high peak leukocyte count and low risk of bleeding events, adequate antiplatelet therapy during the acute phase should be recommended to reduce platelet-derived thrombogenicity.

The inhibition of acute inflammatory responses after STEMI may also reduce the infarct size. Whether the inhibition of acute inflammatory responses reduces platelet-derived thrombogenicity during the acute phase of STEMI remains unknown. Various drugs have been considered for the anti-inflammatory treatment of myocardial infarction; however, this remains controversial. Inflammation after STEMI contributes to not only acute ischemic cardiac injury but also repair and remodeling²⁰⁾. Neutrophils play an important role in cardiac repair by regulating the reparative processes²⁰⁾. This may be one of the mechanisms by which corticosteroids during acute myocardial infarction can cause delayed healing, left ventricular aneurysm, and cardiac rupture^{39, 40)}. In contrast, canakinumab, a monoclonal antibody targeting interleukin-1β, and colchicine reportedly reduce the risk of cardiovascular events in patients with previous and recent myocardial infarction^{41, 42)}. Recently, the cardiac protective effects of sodium-glucose co-transporter (SGLT)-2 inhibitors have been of great interest. SGLT receptors play an important role in the inflammatory response⁴³⁾. A multicenter international observational registry showed that diabetic patients with acute myocardial infarction receiving SGLT-2 inhibitors exhibited a reduced inflammatory response, which was evaluated by the leukocyte count and the fractions, and a smaller infarct size compared to those receiving other antidiabetic drugs⁴⁴⁾. The EMMY trial also showed that early treatment with empagliflozin for acute myocardial infarction was associated with a reduction in the N-terminal pro-hormone of brain natriuretic peptide, and the improvement in echocardiographic functional and structural parameters⁴⁵⁾. Further studies are necessary to evaluate the effect of these drugs, especially SGLT-2 inhibitors, during the acute phase of STEMI.

Factors Associated with PL-AUC

Previous studies have shown several factors to be associated with platelet-derived thrombogenicity measured using T-TAS. Oda et al. demonstrated that a high platelet count is most significantly associated with high PL-AUC in healthy volunteers, while a high hematocrit level and high leukocyte count, especially the monocyte count, are also associated⁴⁶⁾. Additionally, cardiovascular risk factors, such as high body mass index, high blood pressure, smoking history, and no habitual exercise were associated with high PL-AUC in their study. Nakanishi et al. demonstrated HD-related low PL-AUC in patients undergoing elective PCI, although there was no significant difference in PL-AUC between non-HD patients with eGFR ≥ 60 and ＜60 ⁴⁷⁾.

In the present study, the factors clarified in previous studies were not necessarily associated with PL-AUC. In addition, according to a univariate analysis of the present study, there were several differences in factors associated with PL-AUC on admission, during PPCI, and two weeks after STEMI, except for inflammatory responses. For example, the platelet count was associated with only on-admission PL-AUC, while the hematocrit level was associated with on-admission PL-AUC and PL-AUC during PPCI, but not PL-AUC 2 weeks after STEMI. In contrast, the present study demonstrated that the diabetes status and enzymatic infarct size were associated with PL-AUC during PPCI. The major difference from previous studies of PL-AUC is that the patient population in the present study was STEMI. A patient’s condition during the acute phase of STEMI can change minute by minute. On-admission PL-AUC represents platelet-derived thrombogenicity caused by STEMI onset. In addition, the PL-AUC during PCI is affected by the use of various antithrombotic agents, PCI procedures, and myocardial necrosis. Two weeks after STEMI, the patient’s condition was stable, and the efficacy of oral anti-platelet drugs such as P2Y12 inhibitors was sufficient. The complex and changing pathophysiology during the acute phase of STEMI may cause several differences in the factors associated with PL-AUC in previous studies and in the present study.

T-TAS in the Setting of STEMI

Although many methods are available to evaluate the platelet function, the PL-AUC measured using T-TAS was used in the present study. In the setting of STEMI, several pharmacological drugs such as aspirin, P2Y12 inhibitors, and unfractionated heparin are used to inhibit the cascade of thrombus formation. The VerifyNow system (Accumetrics, San Diego, CA, USA), a user-friendly point-of-care platelet function test system, allows measurement of the antiplatelet effect of antiplatelet drugs using different cartridges specific for aspirin or P2Y12 ^{48, 49)}. ACT is a test used to measure high doses of unfractionated heparin. However, these tests have limited utility for evaluating total platelet-derived thrombogenicity^{5, 50)}. This is because they evaluate only a part of the thrombus formation cascade. In our previous study, high on-treatment platelet reactivity measured by the VerifyNow System was not associated with the peak CK level and slow-flow/no-reflow phenomenon in patients with STEMI⁵⁾. On the other hand, T-TAS is a tool for simultaneously monitoring the antithrombotic effects of several pharmacological drugs. T-TAS can allow for the evaluation of total platelet-derived thrombogenicity even during the acute phase of STEMI, a period characterized by a complex chain of events such as acute hemodynamic instability, inflammation, and sympathetic stimulation⁵⁾.

In contrast, using T-TAS, it is difficult to determine which drug is responsible for PL-AUC in patients treated with several drugs⁵⁰⁾. Therefore, the combined use of the T-TAS and VerifyNow system might be beneficial to determine antithrombotic therapy using several drugs as needed. An additional limitation of T-TAS is that the method of measurement is more complex and takes longer than that of VerifyNow, which produces results rapidly using a simple method. In the present study, many patients were excluded from the analysis because of the lack of T-TAS data (Supplementary Fig.1). As a result, is not easy to measure platelet-derived thrombogenicity using T-TAS, especially in emergency settings such as STEMI.

Limitations

The present study is associated with several limitations. First, the sample size was too small to evaluate clinical outcomes. Moreover, the small sample size may have resulted in the limited statistical power. Second, although infarct size was evaluated based on peak CK and CK-MB levels, cardiac magnetic resonance imaging was not performed. Third, acute inflammatory responses after STEMI, T-TAS-based platelet-derived thrombogenicity, and the enzymatic infarct size correlated with each other; however, the correlation coefficients were low. Fourth, data on platelet-leukocyte aggregation, as measured using flow cytometry, were not obtained.

Conclusion

The peak leukocyte count was associated with T-TAS-based platelet-derived thrombogenicity during the acute phase of STEMI (on admission, during PPCI, and 2 weeks after STEMI). Patients with a high peak leukocyte count had high platelet-derived thrombogenicity and a large infarct size. Platelet-derived thrombogenicity may be involved in the mechanisms underlying the association between the acute inflammatory responses after STEMI and a poor clinical outcome.

Acknowledgments

The authors express their gratitude to the physicians and paramedics who participated in this study, particularly Takako Matsushita and Yuko Oda.

Sources of Funding

No funding supported the present study.

Conflict of Interest

The authors declare no conflicts of interest.

References

• 1)  Franchi F, Rollini F, Angiolillo DJ: Antithrombotic therapy for patients with STEMI undergoing primary PCI. Nat Rev Cardiol, 2017; 14: 361-379
• 2)  Funck-Jensen KL, Dalsgaard J, Grove EL, Hvas AM, Kristensen SD: Increased platelet aggregation and turnover in the acute phase of ST-elevation myocardial infarction. Platelets, 2013; 24: 528-537
• 3)  Frossard M, Fuchs I, Leitner JM, Hsieh K, Vlcek M, Losert H, Domanovits H, Schreiber W, Laggner AN, Jilma B: Platelet function predicts myocardial damage in patients with acute myocardial infarction. Circulation, 2004; 110: 1392-1397
• 4)  Bourdrel T, Bind MA, Bejot Y, Morel O, Argacha JF: Cardiovascular effects of air pollution. Arch Cardiovasc Dis, 2017; 110: 634-642
• 5)  Kikuchi S, Tsukahara K, Ichikawa S, Abe T, Minamimoto Y, Kimura Y, Akiyama E, Nakayama N, Okada K, Matsuzawa Y, Konishi M, Maejima N, Iwahashi N, Hibi K, Kosuge M, Ebina T, Tamura K, Kimura K: Platelet-Derived Thrombogenicity Measured by Total Thrombus-Formation Analysis System in Patients With ST-Segment Elevation Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention. Circ J, 2020; 84: 975-984
• 6)  Sikora J, Karczmarska-Wodzka A, Bugieda J, Sobczak P: The Use of Total Thrombus Formation Analysis System as a Tool to Assess Platelet Function in Bleeding and Thrombosis Risk-A Systematic Review. Int J Mol Sci, 2021; 22
• 7)  Coller BS: Leukocytosis and ischemic vascular disease morbidity and mortality: is it time to intervene? Arterioscler Thromb Vasc Biol, 2005; 25: 658-670
• 8)  Cole DR, Singian EB, Katz LN: The long-term prognosis following myocardial infarction, and some factors which affect it. Circulation, 1954; 9: 321-334
• 9)  Chia S, Nagurney JT, Brown DF, Raffel OC, Bamberg F, Senatore F, Wackers FJ, Jang IK: Association of leukocyte and neutrophil counts with infarct size, left ventricular function and outcomes after percutaneous coronary intervention for ST-elevation myocardial infarction. Am J Cardiol, 2009; 103: 333-337
• 10)  Karahan Z, Ucaman B, Ulug AV, Aydinalp O, Ugurlu M, Cevik K, Kaya I, Ozturk O: Effect of Hematologic Parameters on Microvascular Reperfusion in Patients With ST-Segment Elevation Myocardial Infarction Treated With Primary Percutaneous Coronary Intervention. Angiology, 2016; 67: 151-156
• 11)  Palmerini T, Mehran R, Dangas G, Nikolsky E, Witzenbichler B, Guagliumi G, Dudek D, Genereux P, Caixeta A, Rabbani L, Weisz G, Parise H, Fahy M, Xu K, Brodie B, Lansky A, Stone GW: Impact of leukocyte count on mortality and bleeding in patients with myocardial infarction undergoing primary percutaneous coronary interventions: analysis from the Harmonizing Outcome with Revascularization and Stent in Acute Myocardial Infarction trial. Circulation, 2011; 123: 2829-2837, 2827 p following 2837
• 12)  Falk E, Nakano M, Bentzon JF, Finn AV, Virmani R: Update on acute coronary syndromes: the pathologists’ view. Eur Heart J, 2013; 34: 719-728
• 13)  Vilahur G, Juan-Babot O, Pena E, Onate B, Casani L, Badimon L: Molecular and cellular mechanisms involved in cardiac remodeling after acute myocardial infarction. J Mol Cell Cardiol, 2011; 50: 522-533
• 14)  Thygesen K, Alpert JS, Jaffe AS, Chaitman BR, Bax JJ, Morrow DA, White HD, Executive Group on behalf of the Joint European Society of Cardiology /American College of Cardiology /American Heart Association /World Heart Federation Task Force for the Universal Definition of Myocardial I: Fourth Universal Definition of Myocardial Infarction (2018). J Am Coll Cardiol, 2018; 72: 2231-2264
• 15)  Hosokawa K, Ohnishi T, Kondo T, Fukasawa M, Koide T, Maruyama I, Tanaka KA: A novel automated microchip flow-chamber system to quantitatively evaluate thrombus formation and antithrombotic agents under blood flow conditions. J Thromb Haemost, 2011; 9: 2029-2037
• 16)  Hosokawa K, Ohnishi T, Fukasawa M, Kondo T, Sameshima H, Koide T, Tanaka KA, Maruyama I: A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process. Microvasc Res, 2012; 83: 154-161
• 17)  Kojima S, Sakamoto T, Ishihara M, Kimura K, Miyazaki S, Tei C, Hiraoka H, Sonoda M, Tsuchihashi K, Yamagishi M, Inoue T, Asada Y, Ikeda Y, Shirai M, Ogawa H, Japanese Acute Coronary Syndrome Study i: The white blood cell count is an independent predictor of no-reflow and mortality following acute myocardial infarction in the coronary interventional era. Ann Med, 2004; 36: 153-160
• 18)  Mariani M, Fetiveau R, Rossetti E, Poli A, Poletti F, Vandoni P, D’Urbano M, Cafiero F, Mariani G, Klersy C, De Servi S: Significance of total and differential leucocyte count in patients with acute myocardial infarction treated with primary coronary angioplasty. Eur Heart J, 2006; 27: 2511-2515
• 19)  Shen XH, Chen Q, Shi Y, Li HW: Association of neutrophil/lymphocyte ratio with long-term mortality after ST elevation myocardial infarction treated with primary percutaneous coronary intervention. Chin Med J (Engl), 2010; 123: 3438-3443
• 20)  Puhl SL, Steffens S: Neutrophils in Post-myocardial Infarction Inflammation: Damage vs. Resolution? Front Cardiovasc Med, 2019; 6: 25
• 21)  Madjid M, Awan I, Willerson JT, Casscells SW: Leukocyte count and coronary heart disease: implications for risk assessment. J Am Coll Cardiol, 2004; 44: 1945-1956
• 22)  Barron HV, Cannon CP, Murphy SA, Braunwald E, Gibson CM: Association between white blood cell count, epicardial blood flow, myocardial perfusion, and clinical outcomes in the setting of acute myocardial infarction - A Thrombolysis in Myocardial Infarction 10 substudy. Circulation, 2000; 102: 2329-2334
• 23)  Ferrari JP, Lueneberg ME, da Silva RL, Fattah T, Gottschall CAM, Moreira DM: Correlation between leukocyte count and infarct size in ST segment elevation myocardial infarction. Arch Med Sci Atheroscler Dis, 2016; 1: e44-e48
• 24)  Faraday N, Yanek LR, Vaidya D, Kral B, Qayyum R, Herrera-Galeano JE, Moy TF, Becker DM, Becker LC: Leukocyte count is associated with increased platelet reactivity and diminished response to aspirin in healthy individuals with a family history of coronary artery disease. Thromb Res, 2009; 124: 311-317
• 25)  Horne BD, Anderson JL, John JM, Weaver A, Bair TL, Jensen KR, Renlund DG, Muhlestein JB, Intermountain Heart Collaborative Study G: Which white blood cell subtypes predict increased cardiovascular risk? J Am Coll Cardiol, 2005; 45: 1638-1643
• 26)  Kounis NG, Soufras GD, Tsigkas G, Hahalis G: White blood cell counts, leukocyte ratios, and eosinophils as inflammatory markers in patients with coronary artery disease. Clin Appl Thromb Hemost, 2015; 21: 139-143
• 27)  Yamamoto E, Sugiyama S, Hirata Y, Tokitsu T, Tabata N, Fujisue K, Sugamura K, Sakamoto K, Tsujita K, Matsumura T, Kaikita K, Hokimoto S: Prognostic significance of circulating leukocyte subtype counts in patients with coronary artery disease. Atherosclerosis, 2016; 255: 210-216
• 28)  Nozawa N, Hibi K, Endo M, Sugano T, Ebina T, Kosuge M, Tsukahara K, Okuda J, Umemura S, Kimura K: Association Between Circulating Monocytes and Coronary Plaque Progression in Patients With Acute Myocardial Infarction. Circulation Journal, 2010; 74: 1384-1391
• 29)  Prabhu SD, Frangogiannis NG: The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis. Circ Res, 2016; 119: 91-112
• 30)  Huang GY, Yang LJ, Wang XH, Wang YL, Xue YZ, Yang WB: Relationship between platelet-leukocyte aggregation and myocardial perfusion in patients with ST-segment elevation myocardial infarction after primary percutaneous coronary intervention. Heart Lung, 2016; 45: 429-433
• 31)  van Diepen S, Newby LK, Lopes RD, Stebbins A, Hasselblad V, James S, Roe MT, Ezekowitz JA, Moliterno DJ, Neumann FJ, Reist C, Mahaffey KW, Hochman JS, Hamm CW, Armstrong PW, Granger CB, Theroux P, Investigators AA: Prognostic relevance of baseline pro- and anti-inflammatory markers in STEMI: an APEX AMI substudy. Int J Cardiol, 2013; 168: 2127-2133
• 32)  Park DW, Yun SC, Lee JY, Kim WJ, Kang SJ, Lee SW, Kim YH, Lee CW, Kim JJ, Park SW, Park SJ: C-reactive protein and the risk of stent thrombosis and cardiovascular events after drug-eluting stent implantation. Circulation, 2009; 120: 1987-1995
• 33)  Anzai T, Yoshikawa T, Kaneko H, Maekawa Y, Iwanaga S, Asakura Y, Ogawa S: Association between serum C-reactive protein elevation and left ventricular thrombus formation after first anterior myocardial infarction. Chest, 2004; 125: 384-389
• 34)  Hung MJ, Cherng WJ, Hung MY, Kuo LT, Cheng CW, Wang CH, Yang NI, Liao JK: Increased leukocyte Rho-associated coiled-coil containing protein kinase activity predicts the presence and severity of coronary vasospastic angina. Atherosclerosis, 2012; 221: 521-526
• 35)  Park DY, Wang P, An S, Grimshaw AA, Frampton J, Ohman EM, Rao SV, Nanna MG: Shortening the duration of dual antiplatelet therapy after percutaneous coronary intervention for acute coronary syndrome: A systematic review and meta-analysis. Am Heart J, 2022; 251: 101-114
• 36)  Angiolillo DJ, Galli M, Collet JP, Kastrati A, O’Donoghue ML: Antiplatelet therapy after percutaneous coronary intervention. EuroIntervention, 2022; 17: e1371-e1396
• 37)  Watanabe H, Morimoto T, Natsuaki M, Yamamoto K, Obayashi Y, Ogita M, Suwa S, Isawa T, Domei T, Yamaji K, Tatsushima S, Watanabe H, Ohya M, Tokuyama H, Tada T, Sakamoto H, Mori H, Suzuki H, Nishikura T, Wakabayashi K, Hibi K, Abe M, Kawai K, Nakao K, Ando K, Tanabe K, Ikari Y, Morino Y, Kadota K, Furukawa Y, Nakagawa Y, Kimura T, Investigators S-A: Comparison of Clopidogrel Monotherapy After 1 to 2 Months of Dual Antiplatelet Therapy With 12 Months of Dual Antiplatelet Therapy in Patients With Acute Coronary Syndrome: The STOPDAPT-2 ACS Randomized Clinical Trial. JAMA Cardiol, 2022; 7: 407-417
• 38)  Hahn JY, Song YB, Oh JH, Cho DK, Lee JB, Doh JH, Kim SH, Jeong JO, Bae JH, Kim BO, Cho JH, Suh IW, Kim DI, Park HK, Park JS, Choi WG, Lee WS, Kim J, Choi KH, Park TK, Lee JM, Yang JH, Choi JH, Choi SH, Gwon HC, investigators S-D: 6-month versus 12-month or longer dual antiplatelet therapy after percutaneous coronary intervention in patients with acute coronary syndrome (SMART-DATE): a randomised, open-label, non-inferiority trial. Lancet, 2018; 391: 1274-1284
• 39)  Bulkley BH, Roberts WC: Steroid therapy during acute myocardial infarction. A cause of delayed healing and of ventricular aneurysm. Am J Med, 1974; 56: 244-250
• 40)  Silverman HS, Pfeifer MP: Relation between use of anti-inflammatory agents and left ventricular free wall rupture during acute myocardial infarction. Am J Cardiol, 1987; 59: 363-364
• 41)  Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, Fonseca F, Nicolau J, Koenig W, Anker SD, Kastelein JJP, Cornel JH, Pais P, Pella D, Genest J, Cifkova R, Lorenzatti A, Forster T, Kobalava Z, Vida-Simiti L, Flather M, Shimokawa H, Ogawa H, Dellborg M, Rossi PRF, Troquay RPT, Libby P, Glynn RJ, Group CT: Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease. N Engl J Med, 2017; 377: 1119-1131
• 42)  Tardif JC, Kouz S, Waters DD, Bertrand OF, Diaz R, Maggioni AP, Pinto FJ, Ibrahim R, Gamra H, Kiwan GS, Berry C, Lopez-Sendon J, Ostadal P, Koenig W, Angoulvant D, Gregoire JC, Lavoie MA, Dube MP, Rhainds D, Provencher M, Blondeau L, Orfanos A, L’Allier PL, Guertin MC, Roubille F: Efficacy and Safety of Low-Dose Colchicine after Myocardial Infarction. N Engl J Med, 2019
• 43)  García-Ropero Á, Santos-Gallego CG, Badimon JJ: The anti-inflammatory effects of SGLT inhibitors. Aging (Albany NY), 2019; 11: 5866-5867
• 44)  Paolisso P, Bergamaschi L, Santulli G, Gallinoro E, Cesaro A, Gragnano F, Sardu C, Mileva N, Foa A, Armillotta M, Sansonetti A, Amicone S, Impellizzeri A, Casella G, Mauro C, Vassilev D, Marfella R, Calabro P, Barbato E, Pizzi C: Infarct size, inflammatory burden, and admission hyperglycemia in diabetic patients with acute myocardial infarction treated with SGLT2-inhibitors: a multicenter international registry. Cardiovasc Diabetol, 2022; 21: 77
• 45)  von Lewinski D, Kolesnik E, Tripolt NJ, Pferschy PN, Benedikt M, Wallner M, Alber H, Berger R, Lichtenauer M, Saely CH, Moertl D, Auersperg P, Reiter C, Rieder T, Siller-Matula JM, Gager GM, Hasun M, Weidinger F, Pieber TR, Zechner PM, Herrmann M, Zirlik A, Holman RR, Oulhaj A, Sourij H: Empagliflozin in acute myocardial infarction: the EMMY trial. Eur Heart J, 2022; 43: 4421-4432
• 46)  Oda Y, Ito T, Yamada Y, Koga T, Nagasato T, Ohnishi-Wada T, Hosokawa K, Fukase H, Hashiguchi T, Maruyama I: Cardiovascular risk factors are associated with augmented thrombogenicity in healthy individuals: analysis using the Total Thrombus-formation Analysis System. Thromb J, 2021; 19: 88
• 47)  Nakanishi N, Kaikita K, Ishii M, Kuyama N, Tabata N, Ito M, Yamanaga K, Fujisue K, Hoshiyama T, Kanazawa H, Hanatani S, Sueta D, Takashio S, Arima Y, Araki S, Usuku H, Nakamura T, Suzuki S, Yamamoto E, Soejimaa H, Matsushita K, Tsujita K: Hemodialysis-related low thrombogenicity measured by total thrombus-formation analysis system in patients undergoing percutaneous coronary intervention. Thromb Res, 2021; 200: 141-148
• 48)  Nielsen HL, Kristensen SD, Thygesen SS, Mortensen J, Pedersen SB, Grove EL, Hvas AM: Aspirin response evaluated by the VerifyNow Aspirin System and light transmission aggregometry. Thromb Res, 2008; 123: 267-273
• 49)  Price MJ, Angiolillo DJ, Teirstein PS, Lillie E, Manoukian SV, Berger PB, Tanguay JF, Cannon CP, Topol EJ: Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: a time-dependent analysis of the Gauging Responsiveness with a VerifyNow P2Y12 assay: Impact on Thrombosis and Safety (GRAVITAS) trial. Circulation, 2011; 124: 1132-1137
• 50)  Kaikita K, Hosokawa K, Dahlen JR, Tsujita K: Total Thrombus-Formation Analysis System (T-TAS): Clinical Application of Quantitative Analysis of Thrombus Formation in Cardiovascular Disease. Thromb Haemost, 2019; 19: 1554-1562