Expression and Clinical Prognostic Value of Platelet NLRP3 in Acute Coronary Syndrome

Figures & data

Figure 1 Expression of platelet NLRP3 in health subjects and coronary heart disease patients. (A) The relative platelet NLRP3 mRNA level in healthy subjects (n=25), patients with SAP (n=23), and patients with ACS (n=72). (B) The relative platelet NLRP3 mRNA level in patients with UAP (n=23), NSTEMI (n=26), and STEMI (n=23). (C) Representative expression pattern of NLRP3 protein by Western blotting (Left panel) and relative quantification (Right panel) of NLRP3 protein in healthy subjects (n=25), patients with SAP (n=23), and patients with ACS (n=72). (D) Representative expression pattern of NLRP3 protein by Western blotting (Left panel) and relative quantification (Right panel) of NLRP3 protein in healthy subjects (n=25), and patients with UAP (n=23), NSTEMI (n=26), and STEMI (n=23). Data are presented as mean ± SD, P<0.05*, P<0.01**.

Abbreviations: ACS, acute coronary syndrome; SAP, stable angina pectoris; UAP, unstable angina pectoris; NSTEMI, non-ST-segment elevation myocardial infarction; STEMI: ST-segment elevation myocardial infarction.

Table 1 Baseline Characteristics and the Level of Platelet Parameters in the Study Population

Characteristics	Health N=25	SAP N=23	ACS			P1	P2
			UAP N=23	NSTEMI N=26	STEMI N=23
Age	65.1±4.3	67.0±4.9	66.0±2.1	66.6±4.0	68.4±4.3	0.123	0.071
Male	16(64.0%)	14(60.9%)	11(47.8%)	14(53.8%)	15(65.2%)	0.734	0.797
BMI	22.8±1.7	22.7±2.5	23.0±1.8	21.7±2.0	22.3±2.0	0.551	0.188
Hypertension	10(40.0%)	13(56.5%)	11(47.8%)	14(53.8%)	12(52.2%)	0.485	0.773
Diabetes mellitus	10(40.0%)	8(34.8%)	8(34.8%)	9(34.6%)	9(39.1%)	0.933	0.996
HC	9(36.0%)	8(34.8%)	9(39.1%)	9(34.6%)	8(34.8%)	0.970	0.961
Current smoking	8(32.0%)	9(39.1%)	10(43.5%)	7(26.9%)	9(39.1%)	0.849	0.973
Platelet parameter
MPV	8.4 (7.8–9.6)	22.7 (20.5–25.0)	21.4 (20.0–23.4)	22.6 (20.7–23.7)	23.2 (21.8–24.8)	0.523	0.044
PLT	247.5±47.6	255.6±59.1	274.3±48.4	247.1±56.5	269.0±64.6	0.485	0.307
PDW	21.4±1.7	21.6±1.5	20.7±0.9	21.14±1.50	20.5±1.7	0.052	0.091
PCT	0.192 (0.168–0.208)	0.183 (0.164–0.211)	0.194 (0.162–0.209)	0.190 (0.155–0.209)	0.182 (0.134–0.206)	0.772	0.665

Notes: Values are presented as mean ± SD, as median (interquartile range), or as n (%). A p-value smaller than 0.05, indicated by the bold-italic text. P1: the comparison was made between the healthy, SAP and ACS groups; P2: the comparison was made between the UAP, NSTEMI and STEMI groups.

Abbreviations: BMI, body mass index; SAP, stable angina pectoris; ACS, acute coronary syndrome; UAP, unstable angina pectoris; NSTEMI, non-ST-segment elevation myocardial infarction; STEMI, ST-segment elevation myocardial infarction; HC, hypercholesterolemia; PLT, platelet count; MPV, mean platelet volume; PDW, volume distribution width; PCT, the platelet crit.

Figure 2 Correlation between platelet NLRP3 expression and clinical parameters. (A) Correlation between platelet NLRP3 mRNA level and mean platelet volume (MPV, n=252). (B) Correlation between platelet NLRP3 mRNA level and platelet count (PLT, n=252). (C) Correlation between platelet NLRP3 expression and platelet volume distribution width (PDW, n=252). (D) Correlation between platelet NLRP3 expression and the plateletcrit (PCT, n=252). (E) Correlation between platelet NLRP3 mRNA level and left ventricular ejection fraction (LVEF, n=252). (F) Correlation between platelet NLRP3 mRNA level and high-density lipoprotein cholesterol (HDL-C, n=252). (G) Correlation between platelet NLRP3 expression and low-density liptein cholesterol (LDL-C, n=252). (H) Correlation between platelet NLRP3 expression and white blood cell count (WBC, n=252).

Figure 3 ROC curves of platelet NLRP3 that predicted adverse cardiovascular events in patients who underwent PCI. (A) ROC curves with an AUC of 0.757 (95% CI, 0.680–0.835; P<0.001) of platelet NLRP3 that predicted adverse cardiovascular events in patients who underwent PCI. (B) Division of relative expression of NLRP3 in patients with ACS. The boundary value for NLRP3 is indicated by the black straight line (3.885); Low (n=81): The relative expression of NLRP3 ≤3.885; High (n=71): The relative expression of NLRP3 >3.885.

Abbreviations: ROC, receiver operating characteristic; AUC, area under the ROC curve; CI, confidence interval.

Table 2 Baseline Demographic and Clinical Characteristics of Low and High NLRP3 Groups

Characteristics	Low NLRP3 (≤3.8850) N=81	High NLRP3 (>3.8850) N=71	P value
Age	66.0±4.6	66.7±4.7	0.307
Male	45 (55.6%)	42 (59.2%)	0.655
BMI	22.7 (20.8–24.0)	22.1 (20.8–23.7)	0.440
Hypertension	42 (51.9%)	35 (49.3%)	0.753
Diabetes mellitus	26 (32.1%)	21 (29.6%)	0.737
Smoking	28 (34.6%)	20 (28.2%)	0.397
Hypercholesterolemia	22 (27.2%)	21 (29.6%)	0.741
Stroke	4 (4.94%)	6 (8.45%)	0.383
The medications during the study, n (%)
Aspirin	78 (96.3%)	68 (95.8%)	0.545
P2Y12 Inhibitor
Clopidogrel	77 (95.1%)	62 (87.3%)	0.089
Ticagrelor	3 (3.70%)	4 (5.63%)	0.571
β receptor blocker	42 (51.9%)	36 (50.7%)	0.888
ACE1	40 (49.4%)	37 (52.1%)	0.737
ARB	14 (17.3%)	12 (16.9%)	0.950
PPIs	42 (51.9%)	35 (49.3%)	0.753
H2RA	22 (27.2%)	16 (22.5%)	0.511
Statins	78 (96.3%)	69 (97.2%)	0.760
Nitrates	48 (59.3%)	43 (60.6%)	0.870
ACS
UAP	61 (75.3%)	46 (64.8%)	0.243
NSTEMI	10 (12.3%)	6 (8.45%)	0.435
STEMI	10 (12.3%)	19 (26.8%)	0.024
Platelet parameters
PLT (×10^9/L)	319.8±60.5	321.8±73.7	0.854
MPV (fL)	9.2±1.3	10.7±0.9	<0.001
PDW (fL)	21.1±1.8	21.4±1.8	0.252
PCT (%)	0.18±0.03	0.18±0.03	0.751
Target lesion
2-vessel disease	6 (7.4%)	5 (7.0%)	0.950
3-vessel disease	3 (3.7%)	3 (4.2%)	0.588
Multi-vessel disease	10 (12.3%)	9 (12.7%)	0.558
Stent length
Average number of stents per lesion	1.3±0.3	1.4±0.3	0.230
Average stent length per lesion (mm)	24.9±4.3	25.3±6.0	0.562
Cr (mg/dL)	2.1±2.6	2.2±2.1	0.163
hs-CRP (mg/L)	160.4±6.8	165.4±5.2	0.026
Total cholesterol (mg/dL)	4.7 (4.4–5.3)	4.5 (4.2–5.0)	0.088
IL-1β (pg/mL)	7.3±0.6	7.5±0.6	0.023
LDL-C (mg/dL)	54.5±12.8	58.1±10.4	0.066
Blood glucose (mg/dL)	173.8±82.5	165.7±82.9	0.511
LVEF (%)	51.1 (42.8–60.1)	56.0 (45.9–63.1)	0.046

Note: Date are presented as mean ± SD or N (%). A p-value smaller than 0.05, indicated by the bold-italic text.

Abbreviations: ACS, acute coronary syndrome; BMI: body mass index; Cr, creatinine; hs-CRP, high-sensitivity C-reactive protein; LVEF, left ventricular ejection fraction; LDL-C, low density lipoprotein cholesterol; ACE1: angiotensin converting enzyme 1; ARB: angiotensin receptor blocker; PPIs: proton pump inhibitors; H2RA: H2 receptor antagonist; UAP: unstable angina pectoris; NSTEMI: non-ST-segment elevation myocardial infarction; STEMI: ST-segment elevation myocardial infarction.

Table 3 Univariate and Multivariate Cox Regression Analysis in ACS Patient with PCI

Variable	Single Factor Cox Regression Analysis			Multivariate Cox Regression Analysis
	OR	95% CI	P value	OR	95% CI	P value
Age	1.056	1.003–1.113	0.039	1.039	0.984–1.098	0.169
Male	1.281	0.769–2.135	0.342
Hypertension	1.737	1.046–2.883	0.033	1.645	0.988–2.740	0.056
Diabetes	1.257	0.754–2.096	0.380
Smoke	0.954	0.562–1.619	0.862
BMI	0.958	0.848–1.082	0.489
TG	0.929	0.651–1.326	0.685
TC	0.964	0.173–5.356	0.966
HDL-C	0.348	0.067–1.822	0.212
LDL-C	0.993	0.685–1.439	0.971
Platelet NLRP3	1.084	1.035–1.136	0.001	1.071	1.022–1.123	0.004

Note: A p-value smaller than 0.05, indicated by the bold-italic text.

Abbreviations: OR, odds ratio; CI, confidence interval; BMI, body mass index; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

Figure 4 Kaplan-Meier curve analysis for estimated proportion of patients remaining free of adverse cardiovascular events. (A) The proportion of patients that underwent PCI remaining free of events of in the 3-year follow up was significantly different between the NLRP3-low group (n=81) and NLRP3-high group (n=71) (P=0.024). (B) The percentage of IL-1β–expressing platelets was assessed in platelets that were isolated from NLRP3-low group (n=81) and NLRP3-high group (n=71). (C) The expression of PAC-1 in the platelet NLRP3-low group (n=81) and NLRP3-high group (n=71). (D) The expression of CD62p in the platelet NLRP3-low group (n=81) and NLRP3-high group (n=71). Red line, high NLRP3 high expression; blue line, low NLRP3 expression. P<0.01**.