Journal of Clinical Lipidology (2014) 8, 494–500
Relation of circulating PCSK9 concentration to fibrinogen in patients with stable coronary artery disease Yan Zhang, MD, Cheng-Gang Zhu, MD, Rui-Xia Xu, PhD, Sha Li, MD, Yuan-Lin Guo, MD, Jing Sun, MS, Jian-Jun Li, MD, PhD* Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China KEYWORDS: PCSK9; Fibrinogen; Coronary artery disease; C-reactive protein; Inflammation
BACKGROUND: Both proprotein convertase subtilisin/kexin type 9 (PCSK9) and fibrinogen have been established as novel markers for atherosclerotic diseases. However, no data are available regarding the relationship between circulating PCSK9 and fibrinogen concentration up to now. OBJECTIVE: To explore the potential link of the circulating PCSK9 concentration to fibrinogen in patients with stable coronary artery disease (CAD). METHODS: We studied 219 eligible consecutive patients with angiographically proven stable CAD. Baseline clinical and laboratory data were collected. Plasma PCSK9 concentration was measured by enzyme-linked immunosorbent assay. High-sensitivity C-reactive protein (hs-CRP), erythrocyte sedimentation rate, D-dimer, and albumin were also measured in all subjects as inflammatory markers. The relation of the circulating PCSK9 concentration to fibrinogen was evaluated. RESULTS: The data indicated that the patients with high PCSK9 concentration tended to have higher fibrinogen levels according to PCSK9 tertiles (P 5 .037). Spearman correlation analysis revealed a positive relation between plasma PCSK9 concentration and fibrinogen (r 5 0.211, P 5 .002). Additionally, the circulating PCSK9 concentration also correlated positively with total cholesterol, low-density lipoprotein cholesterol, and hs-CRP levels (r 5 0.333, P , .001; r 5 0.302, P , .001; r 5 0.153, P 5 .023, respectively). In the stepwise multivariate regression analysis, the association between PCSK9 and fibrinogen remained significant (b 5 0.168, P 5 .011) after adjustment for conventional cardiovascular risk factors including lipid profiles, hs-CRP, and D-dimer. CONCLUSION: The present study first demonstrated that the circulating PCSK9 concentration was positively associated with fibrinogen in patients with stable CAD, suggesting that the interactions of PCSK9 and fibrinogen in the status of atherosclerosis may need further investigation. Ó 2014 National Lipid Association. All rights reserved.
* Corresponding author. Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, BeiLiShi Road 167, Beijing 100037, China. E-mail address: [email protected] Submitted May 14, 2014. Accepted for publication July 2, 2014.
Coronary artery disease (CAD) is a high-cost disease and remains the leading cause of death worldwide. More recently, there is convincing evidence that chronic inflammation plays a pivotal role in the initiation, development, and evolution of atherosclerosis.1,2 Fibrinogen is the major coagulation protein in blood and up-regulated in inflammatory conditions. Numerous epidemiological studies have
1933-2874/$ - see front matter Ó 2014 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2014.07.001
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PCSK9 and fibrinogen in CAD
documented the independent association of elevated plasma fibrinogen levels with increased incidence of cardiovascular disease.3–5 Long-term and large-scale prospective studies revealed that, even in the population of initially healthy individuals, circulating fibrinogen levels were related to the subclinical cardiovascular disease6,7 and could predict the severity of atherosclerosis and the mortality of CAD8,9 The Fibrinogen Studies Collaboration investigators reported that a long-term increase of 1 g/L in plasma fibrinogen level was associated with an approximate doubling the risk of major cardiovascular disease outcomes.10 Recently, proprotein convertase subtilisin/kexin type 9 (PCSK9) has been confirmed as a major factor regulating sterol homeostasis by targeting low-density lipoprotein receptor (LDLR) for degradation and consequently increasing low-density lipoprotein cholesterol (LDL-C) levels.11 Because elevated LDL-C has long been established as a predominant risk factor of CAD, manipulating PCSK9 concentration has been considered as the new promising strategy for the treatment of CAD.12 Additionally, several studies have indicated that PCSK9 is also involved in a variety of physiological and pharmacological processes, which may be LDLR-independent.13,14 For example, PCSK9 levels could be up-regulated by systemic inflammation induced by lipopolysaccharide,15 whereas PCSK9 small interfering RNA suppressed the inflammatory cytokine expression.16 Moreover, we have previously observed that plasma PCSK9 concentration is independently associated with white blood cell count and its subsets in patients with stable CAD.17 Nevertheless, there are no data concerning the association between circulating PCSK9 concentration and fibrinogen until now. Therefore, the aim of the present study was to evaluate the association of PCSK9 concentration with fibrinogen in patients with stable CAD without previous lipid-lowering drug therapy.
Methods Study design and population The study protocol complied with the Declaration of Helsinki and was approved by the hospital ethics review board (Fu Wai Hospital & National Center for Cardiovascular Diseases, Beijing, China). Each participant provided written, informed consent before enrollment. In the present study, we enrolled 262 consecutive patients with angiographically proven stable CAD between October 2012 and November 2013 in our division. Of these patients, 43 were excluded because of no fibrinogen measurement available. Therefore, there were 219 eligible cases finally studied. Stable CAD was defined as typical angina-like chest pain brought on by exertion and relieved by rest or sublingual nitrates or both, a positive treadmill exercise test (.1 mm ST-segment depression), and stable obstructive lesion .50% in at least 1 of the 3 major
495 coronary arteries or major branches assessed by at least 2 independent senior interventional cardiologists who had no knowledge of the patients’ clinical characteristics and biochemical results. Inclusion criteria were as follows: (1) definite clinical and angiographic evidence leading to a diagnosis of stable CAD; (2) no treatment history of statins and/or other lipid-lowering drugs at least 3 months before entering the study; and (3) assessment of clinical history, anthropometric characteristics, and standard cardiovascular risk factors. Exclusion criteria were the existence of any infectious or systematic inflammatory disease, acute coronary syndrome (ACS), heart failure (left ventricular ejection fraction ,45%), significant hematologic disorders (white blood cell count #3.0 ! 109/L or $20 ! 109/L), thyroid dysfunction, severe liver and/or renal insufficiency, and malignant tumors. Hypertension was defined as repeated systolic and/or diastolic blood pressure $140 and/or $90 mmHg on 2 different occasions or if patients were currently taking antihypertensive drugs. Diabetes mellitus was diagnosed as fasting serum glucose levels $6.99 mmol/L in multiple determinations or patients were being treated with insulin or oral hypoglycemic agents. Hyperlipidemia was considered to be present in patients if they had fasting total cholesterol (TC) $200 mg/dL or triglyceride (TG) $150 mg/dL.
Blood sample measurements Fasting blood samples were obtained from each patient at admission. The plasma levels of fibrinogen were quantitatively measured by the method of Clauss18 and a Stago autoanalyzer with STA Fibrinogen kit (Diagnostic Stago, Taverny, France). Plasma PCSK9 concentrations were measured using a high-sensitivity, quantitative sandwich enzyme immunoassay (Quantikine ELISA, R&D Systems Europe Ltd) according to our previous studies.19 The lower limit of detection was 0.096 ng/mL. Albumin concentration and lipid profiles were determined by automatic biochemistry analyzer (Hitachi 7150, Tokyo, Japan). In detail, the LDL-C concentration was analyzed by selective solubilization method (low-density lipid cholesterol test kit, Kyowa Medex, Tokyo). High-density lipoprotein cholesterol (HDL-C) concentration was determined by a homogeneous method (Determiner L HDL, Kyowa Medex, Tokyo). TC, TG, apolipoprotein AI, apolipoprotein B, and lipoprotein (a) were measured with commercial kits. Plasma D-dimer level was measured by Stago evolution (France). The concentrations of high-sensitivity C-reactive protein (hs-CRP) were determined using immunoturbidimetry (Beckmann Assay 360, Bera, CA, USA). The Westergren method was used for the measurement of erythrocyte sedimentation rate (ESR).
Statistical analysis Quantitative variables were expressed as mean 6 standard deviation or median with interquartile range, and were
496 analyzed by Student t tests, one-way analysis of variance, Mann-Whitney U tests, or Kruskal-Wallis tests as appropriate. The qualitative variables were expressed as numbers and percentages and were analyzed by chi-squared statistic tests. Correlations between variables with PCSK9 concentration were examined by Spearman correlation analysis. Stepwise multivariate regression analysis was used to determine the independent factors of PCSK9 concentration and factors that were significant in the correlation analysis were included in this analysis. A P value less than 0.05 was considered statistically significant. Statistical studies were carried out with the SPSS program (version 19.0, SPSS, Chicago, IL).
Results Baseline characteristics The current study consisted of 219 eligible angiographically proven stable CAD patients (160 males, mean age 57.85 6 10.01 years). The plasma PCSK9 levels ranged from 121.53 to 477.70 ng/mL (median 5 222.20 ng/mL) and the mean fibrinogen level was 3.19 6 0.82 g/L. The baseline demographic, clinical characteristics, and laboratory findings of the enrolled subjects were summarized in Table 1. To evaluate the levels of baseline clinical characteristics and laboratory data according to PCSK9 tertiles, as shown in Table 2, we observed that the gender characteristics and plasma fibrinogen levels (Fig. 1) were significantly different among the 3 groups (P 5 .004, P 5 .037, respectively). However, we did not found significant difference with regard to the variables including hs-CRP, ESR, D-dimer, albumin, age, body mass index (BMI), and smoking status.
Correlations of plasma fibrinogen and clinical variables with PCSK9 concentration To evaluate the associations of fibrinogen and other clinical variables with plasma PCSK9 concentration, Spearman correlation evaluation was performed in the current study. The results indicated that the circulating PCSK9 levels were positively associated with fibrinogen (r 5 0.211, P 5 .002) in patients with stable CAD (Table 3 and Fig. 2). In addition, PCSK9 concentration was significantly associated with TC (r 5 0.333, P , .001), LDL-C (r 5 0.302, P , .001), apolipoprotein AI (r 5 0.215, P 5 .001), apolipoprotein B (r 5 0.319, P , .001), and hs-CRP levels (r 5 0.153, P 5 .023, Fig. 2) respectively. Nevertheless, we did not observe statistically significant relationship between PCSK9 concentration and age, BMI, systolic blood pressure, diastolic blood pressure, fasting blood glucose, hemoglobin A1C, ESR, D-dimer, or albumin levels in patients with stable CAD (Table 3).
Journal of Clinical Lipidology, Vol 8, No 5, October 2014 Table 1 Baseline demographic, clinical, and laboratory characteristics of the study population Variables Risk factors Age (year) Male, n (%) BMI (kg/m2) Current smoking, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Hyperlipidemia, n (%) Family history of CAD, n (%) Lipid parameters Triglycerides (mg/dL) TC (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Apolipoprotein AI (mg/dL) Apolipoprotein B (mg/dL) Lipoprotein (a) (mg/dL) Free fatty acid (mg/dL) Laboratory test Albumin (g/L) D-dimer (mg/mL) ESR (mm/h) hs-CRP (mg/L) Fibrinogen (g/L) PCSK9 (ng/mL)
CAD (n 5 219) 57.85 6 10.01 160 (73.1) 25.91 6 3.28 111 (50.7) 147 (67.1) 56 (25.6) 151 (68.9) 33 (13.1) 143.49 (104.52–203.72) 185.86 6 42.31 42.29 6 15.00 117.42 6 36.20 139.75 6 26.51 99.19 6 27.28 13.71 (6.37–27.80) 12.00 6 5.14 41.05 (39.18–44.10) 0.31 (0.23–0.42) 7.00 (3.00–14.00) 1.77 (0.90–3.28) 3.19 6 0.82 222.20 (183.16–271.55)
BMI, body mass index; CAD, coronary artery disease; ESR, erythrocyte sedimentation rate; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high sensitivity C-reactive protein; LDL-C, lowdensity lipoprotein cholesterol; PCSK9, proprotein convertase subtilisin/Kexin type 9; TC, total cholesterol. Data are expressed as n (%), median (IQR) or mean 6 standard deviation.
Stepwise multivariate regression analysis of the relationship between fibrinogen and PCSK9 concentration Because of the potential associations of the plasma PCSK9 concentration and fibrinogen with age, gender, BMI, smoking, hypertension, and diabetes mellitus, a stepwise multivariate linear regression analysis was performed to evaluate the association of PCSK9 concentration with fibrinogen after adjusting for these confounders (Table 4). Briefly, in multivariate regression analysis, the positive association of PCSK9 concentration with fibrinogen remained significant after adjusting for age, gender, BMI, current smoking, systolic blood pressure, fasting blood glucose, and family history of CAD (b 5 0.199, P 5 .003). When TG, HDL-C, and LDL-C are added to the adjustment, the correlation was still statistically significant (b 5 0.173, P 5 .008). Because there is evidence that plasma fibrinogen levels may reflect systemic inflammation and coagulation status, we further added hs-CRP and D-dimer to the adjustment, respectively. Interestingly, the
Zhang et al Table 2
PCSK9 and fibrinogen in CAD
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The levels of clinical and laboratory data according to tertiles of PCSK9 concentration
Age (year) Sex Male, n (%) Female, n (%) BMI (kg/m2) Current smoking Albumin (g/L) D-dimer (mg/mL) ESR (mm/h) hs-CRP (mg/L) Fibrinogen (g/L)
PCSK9 (ng/mL) (121.53–193.38, n 5 71)
PCSK9 (ng/mL) (193.39–251.42, n 5 75)
PCSK9 (ng/mL) (251.43–477.70, n 5 73)
58.73 6 10.15
56.15 6 10.52
58.74 6 9.21
57 (35.63) 14 (23.73) 25.59 6 3.23 41 (57.75) 40.60 (39.00–44.45) 0.31 (0.25–0.40) 7.00 (3.00–13.00) 1.46 (0.71–2.86) 3.01 6 0.60
60 (37.50) 15 (25.42) 26.61 6 3.43 39 (52.00) 41.00 (39.10–43.80) 0.29 (0.20–0.40) 7.00 (3.00–13.00) 2.13 (1.13–4.28) 3.21 6 0.71
43 (26.88) 30 (50.85) 25.48 6 3.07 31 (42.47) 41.60 (39.40–44.10) 0.31 (0.23–0.47) 8.00 (3.00–15.00) 2.02 (0.92–3.18) 3.36 6 1.04
P value .192 .004
.067 .179 .726 .508 .41 .092 .037
BMI, body mass index; ESR, erythrocyte sedimentation rate; hs-CRP, high-sensitivity C-reactive protein; PCSK9 5 proprotein convertase subtilisin/ kexin type 9. Bold P values indicate statistical significance. Data are expressed as median (interquartile range) or mean 6 standard deviation.
results indicated that there remained statistically positive association between PCSK9 concentration and fibrinogen (b 5 0.168, P 5 .011).
Discussion The present study evaluated the association of the circulating PCSK9 concentration with fibrinogen in patients with stable CAD without previous lipid-lowering drug therapy. To our knowledge, this is the first study demonstrating that the plasma PCSK9 concentration was independently and positively associated with fibrinogen levels in patients with stable CAD. This association was independent of vascular risk factors because it remained significant after adjusting for these confounders. Therefore, in addition to LDLR-dependent effects, the current study might provide novel information with regard to PCSK9 in the development of atherosclerosis.
Figure 1 The fibrinogen concentration according to PCSK9 tertiles in patients with stable CAD. *P , .05 vs tertile 1. CAD, coronary artery disease; PCSK9, proprotein convertase subtilisin/ Kexin type 9.
An increasing number of studies have provided the compelling evidence that multiple inflammatory biomarkers were related to the progression and mortality of CAD20,21 Fibrinogen, a glycoprotein dimer, is a wellknown key coagulation factor that participates in the process of hemostasis in 2 pathways: first, it is part of the final Table 3 The correlations of clinical and laboratory data with plasma PCSK9 concentration CAD (n 5 219) Variables
Correlation coefficient
P value
Age (year) SBP (mmHg) DBP (mmHg) BMI (kg/m2) Glucose (mmol/L) HbA1c (%) Triglycerides (mg/dL) TC (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Apolipoprotein AI (mg/dL) Apolipoprotein B (mg/dL) Lipoprotein (a) (mg/dL) Free fatty acid (mg/dL) Albumin (g/L) D-dimer (mg/mL) ESR (mm/h) hs-CRP (mg/L) Fibrinogen (g/L)
0.033 20.101 0.000 20.039 20.028 0.127 0.106 0.333 0.106 0.302 0.215 0.319 0.086 20.052 0.059 0.011 0.123 0.153 0.211
.631 .137 .998 .565 .679 .061 .116 ,.001 .116 ,.001 .001 ,.001 .203 .441 .382 .877 .070 .023 .002
BMI, body mass index; DBP, diastolic blood pressure; ESR, erythrocyte sedimentation rate; HbA1C, hemoglobin A1C; HDLC, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol. Bold P values indicate statistical significance. Spearman and Pearson correlations are shown.
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Figure 2 The correlations of fibrinogen (A) and hs-CRP (B) with PCSK9 concentration in patients with stable CAD. CAD, coronary artery disease; hs-CRP, high-sensitivity C-reactive protein; PCSK9, proprotein convertase subtilisin/Kexin type 9.
common pathway of the coagulation cascade. Second, it is bound to platelet GpⅡb/Ⅲa membrane receptors and forms a web that provides stability to the newly formed thrombus.22,23 Besides, fibrinogen has been confirmed as an acute-phase reactant exclusively synthesized by the liver. It has been demonstrated that the circulating fibrinogen was not only involved in acute phase of ACS, but also acted as an inflammatory protein and increased in chronic inflammatory states.24 Multiple studies proved that fibrinogen could accelerate the progress of atherosclerosis inducing plaque growing, stimulating the adhesion of platelets and white blood cells to the vessels wall, and subsequently led to the development of clinical CAD.25,26 Furthermore, several lines of evidence revealed the positive association of elevated plasma fibrinogen with occurrence, severity, and adverse events of CAD.8–10 Thus, fibrinogen is recognized as an established marker of cardiovascular disease outcomes, even though the causal relation and molecular mechanisms are awaiting to test. Previous studies implied that increased basal fibrinogen expression could enhance cholesterol synthesis in HepG2 cells27 and elevated plasma fibrinogen was associated with increased lipid peroxidation.28 In this cross-sectional study, the results indicated that the circulating PCSK9 concentration was positively associated with fibrinogen, and the former is a novel marker regulating cholesterol homeostasis. More importantly, this association remained significant after adjusting for a variety of potential confounders including lipid profiles, hs-CRP, and D-dimer. Therefore, we postulated that the dyslipidemia related to fibrinogen may be related to PCSK9 pathway and that the interaction of fibrinogen and PCSK9 may be one of the involving reasons in the development of atherosclerosis, which may be needed to be testified in the future. PCSK9, as a new target for atherosclerosis, has gained tremendous attention during the past decade. Approaches to inhibit PCSK9 have emerged as a promising new strategy for lowering LDL-C levels.29 Monoclonal antibodies against PCSK9 have been shown to reduce LDL-C substantially, with acceptable safety and tolerability in 4 randomized, placebo-controlled, phase Ⅱ clinical trials.30
Although much progress has been obtained, physiological and pharmacological factors that related to PCSK9 concentration have been largely unknown. Recently, several clinical studies revealed that plasma PCSK9 concentration was related to multiple physiological metabolic factors, such as age, gender, lipid profiles, and so forth.13 Consistent with previous studies, our data also indicated that there were significant associations of plasma PCSK9 concentration with TC and LDL-C. Additionally, gender disparity was obvious and females tended to have higher PCSK9 concentrations in our study. Interestingly, we also found positive associations of PCSK9 with inflammatory markers, such as fibrinogen and hs-CRP, in stable CAD status. Therefore, our data confirmed and furthered previous studies. Besides the definite association with LDL-C levels, recent studies uncovered a certain relationship between systemic inflammation15 and PCSK9 concentration. In addition, experimental studies indicated that PCSK9 might accelerate atherosclerosis by promoting inflammation,12,31 which may be through mechanisms independent of the LDLR.12 However, most of previous studies exploring the relationship between PCSK9 and inflammation using acute inflammation and animal models, and clinical data referring to PCSK9 and chronic inflammation were obscure. There were numerous studies demonstrated that the multiple inflammatory markers were up-regulated in chronic inflammatory status and could predict risk of CAD. According to the meta-analysis conducted by Lowe32 suggested the associations of various inflammatory markers with risk of CAD: fibrinogen (odds ratio [OR] 1.8; 95% confidence intervals [95%CI]: 1.6–2.0); hs-CRP (OR 1.49; 95%CI: 1.37– 1.62); ESR (OR 1.33; 95%CI: 1.22–1.44); D-dimer (OR 1.7; 95%CI: 1.3–2.2), and albumin (OR 1.5; 95%CI: 1.3– 1.7). In the present study, therefore, we additionally investigated the clinical readily measurable inflammatory markers, hs-CRP, ESR, D-dimer, and albumin, with PCSK9 concentration and found positive associations of PCSK9 with hs-CRP as well as fibrinogen. However, the elaborate mechanisms and causal relationship need further investigations.
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Table 4 The independent correlation between circulating fibrinogen and PCSK9 concentration in patients with stable CAD
4.
Fibrinogen Models
Other variables
b
P value
1 2 3 4
Covariates Model 1 1 TG 1 HDL-C 1 LDL-C Model 2 1 hs-CRP Model 2 1 D-dimer
0.199 0.173 0.168 0.168
.003 .008 .011 .011
BMI, body mass index; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein–cholesterol; SBP, systolic blood pressure; TC, total cholesterol. Bold P values indicate statistical significance. Multivariable regression stepwise models are shown. The dependent variable was PCSK9. Covariates including age, gender, BMI, current smoking, family history of CAD, SBP, and fasting blood glucose.
There were several limitations in our study. First, because the sample size is so small the findings could also be from chance. Moreover, this is an observational study and so there could be residual confounding. Finally, whether such association is present in other populations, such as those without known CAD or those with ACS, should be studied in the future.
5.
6.
7.
8.
9.
10.
11.
Conclusions In summary, PCSK9 concentration was independently and positively associated with fibrinogen in patients with stable CAD, suggesting that the interactions of PCSK9 and fibrinogen in the status of atherosclerosis may be needed further investigations.
12.
13.
14.
Acknowledgments This work was partially supported by the National Natural Scientific Foundation (81070171, 81241121), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20111106110013), the Capital Special Foundation of Clinical Application Research (Z121107001012015), the Capital Health Development Fund (2011400302), and the Beijing Natural Science Foundation (7131014) awarded to Dr. Jian-Jun Li. Conflicts of interest: The authors declare that they have no conflicts of interests.
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Journal of Clinical Lipidology, Vol 8, No 5, October 2014 27. Redman CM, Xia H. Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion. Ann N Y Acad Sci. 2001;936:480–495. 28. Rankinen T, Hietanen E, V€ais€anen S, Lehti€o M, Penttil€a I, Bouchard C, et al. Relationship between lipid peroxidation and plasma fibrinogen in middle-aged men. Thromb Res. 2000;99:453–459. 29. Stein EA, Swergold GD. Potential of proprotein convertase subtilisin/kexin type 9 based therapeutics. Curr Atheroscler Rep. 2013;15:310. 30. Lee P, Hegele RA. Current Phase II proprotein convertase subtilisin/kexin 9 inhibitor therapies for dyslipidemia. Expert Opin Investig Drugs. 2013;22:1411–1423. 31. Denis M, Marcinkiewicz J, Zaid A, Gauthier D, Poirier S, Lazure C, et al. Gene inactivation of proprotein convertase subtilisin/kexin type 9 reduces atherosclerosis in mice. Circulation. 2012;125:894–901. 32. Lowe GD. Circulating inflammatory markers and risks of cardiovascular and non-cardiovascular disease. J Thromb Haemost. 2005;3:1618–1627.
Relation of circulating PCSK9 concentration to fibrinogen in patients with stable coronary artery disease Yan Zhang, MD, Cheng-Gang Zhu, MD, Rui-Xia Xu, PhD, Sha Li, MD, Yuan-Lin Guo, MD, Jing Sun, MS, Jian-Jun Li, MD, PhD* Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China KEYWORDS: PCSK9; Fibrinogen; Coronary artery disease; C-reactive protein; Inflammation
BACKGROUND: Both proprotein convertase subtilisin/kexin type 9 (PCSK9) and fibrinogen have been established as novel markers for atherosclerotic diseases. However, no data are available regarding the relationship between circulating PCSK9 and fibrinogen concentration up to now. OBJECTIVE: To explore the potential link of the circulating PCSK9 concentration to fibrinogen in patients with stable coronary artery disease (CAD). METHODS: We studied 219 eligible consecutive patients with angiographically proven stable CAD. Baseline clinical and laboratory data were collected. Plasma PCSK9 concentration was measured by enzyme-linked immunosorbent assay. High-sensitivity C-reactive protein (hs-CRP), erythrocyte sedimentation rate, D-dimer, and albumin were also measured in all subjects as inflammatory markers. The relation of the circulating PCSK9 concentration to fibrinogen was evaluated. RESULTS: The data indicated that the patients with high PCSK9 concentration tended to have higher fibrinogen levels according to PCSK9 tertiles (P 5 .037). Spearman correlation analysis revealed a positive relation between plasma PCSK9 concentration and fibrinogen (r 5 0.211, P 5 .002). Additionally, the circulating PCSK9 concentration also correlated positively with total cholesterol, low-density lipoprotein cholesterol, and hs-CRP levels (r 5 0.333, P , .001; r 5 0.302, P , .001; r 5 0.153, P 5 .023, respectively). In the stepwise multivariate regression analysis, the association between PCSK9 and fibrinogen remained significant (b 5 0.168, P 5 .011) after adjustment for conventional cardiovascular risk factors including lipid profiles, hs-CRP, and D-dimer. CONCLUSION: The present study first demonstrated that the circulating PCSK9 concentration was positively associated with fibrinogen in patients with stable CAD, suggesting that the interactions of PCSK9 and fibrinogen in the status of atherosclerosis may need further investigation. Ó 2014 National Lipid Association. All rights reserved.
* Corresponding author. Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, BeiLiShi Road 167, Beijing 100037, China. E-mail address: [email protected] Submitted May 14, 2014. Accepted for publication July 2, 2014.
Coronary artery disease (CAD) is a high-cost disease and remains the leading cause of death worldwide. More recently, there is convincing evidence that chronic inflammation plays a pivotal role in the initiation, development, and evolution of atherosclerosis.1,2 Fibrinogen is the major coagulation protein in blood and up-regulated in inflammatory conditions. Numerous epidemiological studies have
1933-2874/$ - see front matter Ó 2014 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2014.07.001
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PCSK9 and fibrinogen in CAD
documented the independent association of elevated plasma fibrinogen levels with increased incidence of cardiovascular disease.3–5 Long-term and large-scale prospective studies revealed that, even in the population of initially healthy individuals, circulating fibrinogen levels were related to the subclinical cardiovascular disease6,7 and could predict the severity of atherosclerosis and the mortality of CAD8,9 The Fibrinogen Studies Collaboration investigators reported that a long-term increase of 1 g/L in plasma fibrinogen level was associated with an approximate doubling the risk of major cardiovascular disease outcomes.10 Recently, proprotein convertase subtilisin/kexin type 9 (PCSK9) has been confirmed as a major factor regulating sterol homeostasis by targeting low-density lipoprotein receptor (LDLR) for degradation and consequently increasing low-density lipoprotein cholesterol (LDL-C) levels.11 Because elevated LDL-C has long been established as a predominant risk factor of CAD, manipulating PCSK9 concentration has been considered as the new promising strategy for the treatment of CAD.12 Additionally, several studies have indicated that PCSK9 is also involved in a variety of physiological and pharmacological processes, which may be LDLR-independent.13,14 For example, PCSK9 levels could be up-regulated by systemic inflammation induced by lipopolysaccharide,15 whereas PCSK9 small interfering RNA suppressed the inflammatory cytokine expression.16 Moreover, we have previously observed that plasma PCSK9 concentration is independently associated with white blood cell count and its subsets in patients with stable CAD.17 Nevertheless, there are no data concerning the association between circulating PCSK9 concentration and fibrinogen until now. Therefore, the aim of the present study was to evaluate the association of PCSK9 concentration with fibrinogen in patients with stable CAD without previous lipid-lowering drug therapy.
Methods Study design and population The study protocol complied with the Declaration of Helsinki and was approved by the hospital ethics review board (Fu Wai Hospital & National Center for Cardiovascular Diseases, Beijing, China). Each participant provided written, informed consent before enrollment. In the present study, we enrolled 262 consecutive patients with angiographically proven stable CAD between October 2012 and November 2013 in our division. Of these patients, 43 were excluded because of no fibrinogen measurement available. Therefore, there were 219 eligible cases finally studied. Stable CAD was defined as typical angina-like chest pain brought on by exertion and relieved by rest or sublingual nitrates or both, a positive treadmill exercise test (.1 mm ST-segment depression), and stable obstructive lesion .50% in at least 1 of the 3 major
495 coronary arteries or major branches assessed by at least 2 independent senior interventional cardiologists who had no knowledge of the patients’ clinical characteristics and biochemical results. Inclusion criteria were as follows: (1) definite clinical and angiographic evidence leading to a diagnosis of stable CAD; (2) no treatment history of statins and/or other lipid-lowering drugs at least 3 months before entering the study; and (3) assessment of clinical history, anthropometric characteristics, and standard cardiovascular risk factors. Exclusion criteria were the existence of any infectious or systematic inflammatory disease, acute coronary syndrome (ACS), heart failure (left ventricular ejection fraction ,45%), significant hematologic disorders (white blood cell count #3.0 ! 109/L or $20 ! 109/L), thyroid dysfunction, severe liver and/or renal insufficiency, and malignant tumors. Hypertension was defined as repeated systolic and/or diastolic blood pressure $140 and/or $90 mmHg on 2 different occasions or if patients were currently taking antihypertensive drugs. Diabetes mellitus was diagnosed as fasting serum glucose levels $6.99 mmol/L in multiple determinations or patients were being treated with insulin or oral hypoglycemic agents. Hyperlipidemia was considered to be present in patients if they had fasting total cholesterol (TC) $200 mg/dL or triglyceride (TG) $150 mg/dL.
Blood sample measurements Fasting blood samples were obtained from each patient at admission. The plasma levels of fibrinogen were quantitatively measured by the method of Clauss18 and a Stago autoanalyzer with STA Fibrinogen kit (Diagnostic Stago, Taverny, France). Plasma PCSK9 concentrations were measured using a high-sensitivity, quantitative sandwich enzyme immunoassay (Quantikine ELISA, R&D Systems Europe Ltd) according to our previous studies.19 The lower limit of detection was 0.096 ng/mL. Albumin concentration and lipid profiles were determined by automatic biochemistry analyzer (Hitachi 7150, Tokyo, Japan). In detail, the LDL-C concentration was analyzed by selective solubilization method (low-density lipid cholesterol test kit, Kyowa Medex, Tokyo). High-density lipoprotein cholesterol (HDL-C) concentration was determined by a homogeneous method (Determiner L HDL, Kyowa Medex, Tokyo). TC, TG, apolipoprotein AI, apolipoprotein B, and lipoprotein (a) were measured with commercial kits. Plasma D-dimer level was measured by Stago evolution (France). The concentrations of high-sensitivity C-reactive protein (hs-CRP) were determined using immunoturbidimetry (Beckmann Assay 360, Bera, CA, USA). The Westergren method was used for the measurement of erythrocyte sedimentation rate (ESR).
Statistical analysis Quantitative variables were expressed as mean 6 standard deviation or median with interquartile range, and were
496 analyzed by Student t tests, one-way analysis of variance, Mann-Whitney U tests, or Kruskal-Wallis tests as appropriate. The qualitative variables were expressed as numbers and percentages and were analyzed by chi-squared statistic tests. Correlations between variables with PCSK9 concentration were examined by Spearman correlation analysis. Stepwise multivariate regression analysis was used to determine the independent factors of PCSK9 concentration and factors that were significant in the correlation analysis were included in this analysis. A P value less than 0.05 was considered statistically significant. Statistical studies were carried out with the SPSS program (version 19.0, SPSS, Chicago, IL).
Results Baseline characteristics The current study consisted of 219 eligible angiographically proven stable CAD patients (160 males, mean age 57.85 6 10.01 years). The plasma PCSK9 levels ranged from 121.53 to 477.70 ng/mL (median 5 222.20 ng/mL) and the mean fibrinogen level was 3.19 6 0.82 g/L. The baseline demographic, clinical characteristics, and laboratory findings of the enrolled subjects were summarized in Table 1. To evaluate the levels of baseline clinical characteristics and laboratory data according to PCSK9 tertiles, as shown in Table 2, we observed that the gender characteristics and plasma fibrinogen levels (Fig. 1) were significantly different among the 3 groups (P 5 .004, P 5 .037, respectively). However, we did not found significant difference with regard to the variables including hs-CRP, ESR, D-dimer, albumin, age, body mass index (BMI), and smoking status.
Correlations of plasma fibrinogen and clinical variables with PCSK9 concentration To evaluate the associations of fibrinogen and other clinical variables with plasma PCSK9 concentration, Spearman correlation evaluation was performed in the current study. The results indicated that the circulating PCSK9 levels were positively associated with fibrinogen (r 5 0.211, P 5 .002) in patients with stable CAD (Table 3 and Fig. 2). In addition, PCSK9 concentration was significantly associated with TC (r 5 0.333, P , .001), LDL-C (r 5 0.302, P , .001), apolipoprotein AI (r 5 0.215, P 5 .001), apolipoprotein B (r 5 0.319, P , .001), and hs-CRP levels (r 5 0.153, P 5 .023, Fig. 2) respectively. Nevertheless, we did not observe statistically significant relationship between PCSK9 concentration and age, BMI, systolic blood pressure, diastolic blood pressure, fasting blood glucose, hemoglobin A1C, ESR, D-dimer, or albumin levels in patients with stable CAD (Table 3).
Journal of Clinical Lipidology, Vol 8, No 5, October 2014 Table 1 Baseline demographic, clinical, and laboratory characteristics of the study population Variables Risk factors Age (year) Male, n (%) BMI (kg/m2) Current smoking, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Hyperlipidemia, n (%) Family history of CAD, n (%) Lipid parameters Triglycerides (mg/dL) TC (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Apolipoprotein AI (mg/dL) Apolipoprotein B (mg/dL) Lipoprotein (a) (mg/dL) Free fatty acid (mg/dL) Laboratory test Albumin (g/L) D-dimer (mg/mL) ESR (mm/h) hs-CRP (mg/L) Fibrinogen (g/L) PCSK9 (ng/mL)
CAD (n 5 219) 57.85 6 10.01 160 (73.1) 25.91 6 3.28 111 (50.7) 147 (67.1) 56 (25.6) 151 (68.9) 33 (13.1) 143.49 (104.52–203.72) 185.86 6 42.31 42.29 6 15.00 117.42 6 36.20 139.75 6 26.51 99.19 6 27.28 13.71 (6.37–27.80) 12.00 6 5.14 41.05 (39.18–44.10) 0.31 (0.23–0.42) 7.00 (3.00–14.00) 1.77 (0.90–3.28) 3.19 6 0.82 222.20 (183.16–271.55)
BMI, body mass index; CAD, coronary artery disease; ESR, erythrocyte sedimentation rate; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high sensitivity C-reactive protein; LDL-C, lowdensity lipoprotein cholesterol; PCSK9, proprotein convertase subtilisin/Kexin type 9; TC, total cholesterol. Data are expressed as n (%), median (IQR) or mean 6 standard deviation.
Stepwise multivariate regression analysis of the relationship between fibrinogen and PCSK9 concentration Because of the potential associations of the plasma PCSK9 concentration and fibrinogen with age, gender, BMI, smoking, hypertension, and diabetes mellitus, a stepwise multivariate linear regression analysis was performed to evaluate the association of PCSK9 concentration with fibrinogen after adjusting for these confounders (Table 4). Briefly, in multivariate regression analysis, the positive association of PCSK9 concentration with fibrinogen remained significant after adjusting for age, gender, BMI, current smoking, systolic blood pressure, fasting blood glucose, and family history of CAD (b 5 0.199, P 5 .003). When TG, HDL-C, and LDL-C are added to the adjustment, the correlation was still statistically significant (b 5 0.173, P 5 .008). Because there is evidence that plasma fibrinogen levels may reflect systemic inflammation and coagulation status, we further added hs-CRP and D-dimer to the adjustment, respectively. Interestingly, the
Zhang et al Table 2
PCSK9 and fibrinogen in CAD
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The levels of clinical and laboratory data according to tertiles of PCSK9 concentration
Age (year) Sex Male, n (%) Female, n (%) BMI (kg/m2) Current smoking Albumin (g/L) D-dimer (mg/mL) ESR (mm/h) hs-CRP (mg/L) Fibrinogen (g/L)
PCSK9 (ng/mL) (121.53–193.38, n 5 71)
PCSK9 (ng/mL) (193.39–251.42, n 5 75)
PCSK9 (ng/mL) (251.43–477.70, n 5 73)
58.73 6 10.15
56.15 6 10.52
58.74 6 9.21
57 (35.63) 14 (23.73) 25.59 6 3.23 41 (57.75) 40.60 (39.00–44.45) 0.31 (0.25–0.40) 7.00 (3.00–13.00) 1.46 (0.71–2.86) 3.01 6 0.60
60 (37.50) 15 (25.42) 26.61 6 3.43 39 (52.00) 41.00 (39.10–43.80) 0.29 (0.20–0.40) 7.00 (3.00–13.00) 2.13 (1.13–4.28) 3.21 6 0.71
43 (26.88) 30 (50.85) 25.48 6 3.07 31 (42.47) 41.60 (39.40–44.10) 0.31 (0.23–0.47) 8.00 (3.00–15.00) 2.02 (0.92–3.18) 3.36 6 1.04
P value .192 .004
.067 .179 .726 .508 .41 .092 .037
BMI, body mass index; ESR, erythrocyte sedimentation rate; hs-CRP, high-sensitivity C-reactive protein; PCSK9 5 proprotein convertase subtilisin/ kexin type 9. Bold P values indicate statistical significance. Data are expressed as median (interquartile range) or mean 6 standard deviation.
results indicated that there remained statistically positive association between PCSK9 concentration and fibrinogen (b 5 0.168, P 5 .011).
Discussion The present study evaluated the association of the circulating PCSK9 concentration with fibrinogen in patients with stable CAD without previous lipid-lowering drug therapy. To our knowledge, this is the first study demonstrating that the plasma PCSK9 concentration was independently and positively associated with fibrinogen levels in patients with stable CAD. This association was independent of vascular risk factors because it remained significant after adjusting for these confounders. Therefore, in addition to LDLR-dependent effects, the current study might provide novel information with regard to PCSK9 in the development of atherosclerosis.
Figure 1 The fibrinogen concentration according to PCSK9 tertiles in patients with stable CAD. *P , .05 vs tertile 1. CAD, coronary artery disease; PCSK9, proprotein convertase subtilisin/ Kexin type 9.
An increasing number of studies have provided the compelling evidence that multiple inflammatory biomarkers were related to the progression and mortality of CAD20,21 Fibrinogen, a glycoprotein dimer, is a wellknown key coagulation factor that participates in the process of hemostasis in 2 pathways: first, it is part of the final Table 3 The correlations of clinical and laboratory data with plasma PCSK9 concentration CAD (n 5 219) Variables
Correlation coefficient
P value
Age (year) SBP (mmHg) DBP (mmHg) BMI (kg/m2) Glucose (mmol/L) HbA1c (%) Triglycerides (mg/dL) TC (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Apolipoprotein AI (mg/dL) Apolipoprotein B (mg/dL) Lipoprotein (a) (mg/dL) Free fatty acid (mg/dL) Albumin (g/L) D-dimer (mg/mL) ESR (mm/h) hs-CRP (mg/L) Fibrinogen (g/L)
0.033 20.101 0.000 20.039 20.028 0.127 0.106 0.333 0.106 0.302 0.215 0.319 0.086 20.052 0.059 0.011 0.123 0.153 0.211
.631 .137 .998 .565 .679 .061 .116 ,.001 .116 ,.001 .001 ,.001 .203 .441 .382 .877 .070 .023 .002
BMI, body mass index; DBP, diastolic blood pressure; ESR, erythrocyte sedimentation rate; HbA1C, hemoglobin A1C; HDLC, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol. Bold P values indicate statistical significance. Spearman and Pearson correlations are shown.
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Figure 2 The correlations of fibrinogen (A) and hs-CRP (B) with PCSK9 concentration in patients with stable CAD. CAD, coronary artery disease; hs-CRP, high-sensitivity C-reactive protein; PCSK9, proprotein convertase subtilisin/Kexin type 9.
common pathway of the coagulation cascade. Second, it is bound to platelet GpⅡb/Ⅲa membrane receptors and forms a web that provides stability to the newly formed thrombus.22,23 Besides, fibrinogen has been confirmed as an acute-phase reactant exclusively synthesized by the liver. It has been demonstrated that the circulating fibrinogen was not only involved in acute phase of ACS, but also acted as an inflammatory protein and increased in chronic inflammatory states.24 Multiple studies proved that fibrinogen could accelerate the progress of atherosclerosis inducing plaque growing, stimulating the adhesion of platelets and white blood cells to the vessels wall, and subsequently led to the development of clinical CAD.25,26 Furthermore, several lines of evidence revealed the positive association of elevated plasma fibrinogen with occurrence, severity, and adverse events of CAD.8–10 Thus, fibrinogen is recognized as an established marker of cardiovascular disease outcomes, even though the causal relation and molecular mechanisms are awaiting to test. Previous studies implied that increased basal fibrinogen expression could enhance cholesterol synthesis in HepG2 cells27 and elevated plasma fibrinogen was associated with increased lipid peroxidation.28 In this cross-sectional study, the results indicated that the circulating PCSK9 concentration was positively associated with fibrinogen, and the former is a novel marker regulating cholesterol homeostasis. More importantly, this association remained significant after adjusting for a variety of potential confounders including lipid profiles, hs-CRP, and D-dimer. Therefore, we postulated that the dyslipidemia related to fibrinogen may be related to PCSK9 pathway and that the interaction of fibrinogen and PCSK9 may be one of the involving reasons in the development of atherosclerosis, which may be needed to be testified in the future. PCSK9, as a new target for atherosclerosis, has gained tremendous attention during the past decade. Approaches to inhibit PCSK9 have emerged as a promising new strategy for lowering LDL-C levels.29 Monoclonal antibodies against PCSK9 have been shown to reduce LDL-C substantially, with acceptable safety and tolerability in 4 randomized, placebo-controlled, phase Ⅱ clinical trials.30
Although much progress has been obtained, physiological and pharmacological factors that related to PCSK9 concentration have been largely unknown. Recently, several clinical studies revealed that plasma PCSK9 concentration was related to multiple physiological metabolic factors, such as age, gender, lipid profiles, and so forth.13 Consistent with previous studies, our data also indicated that there were significant associations of plasma PCSK9 concentration with TC and LDL-C. Additionally, gender disparity was obvious and females tended to have higher PCSK9 concentrations in our study. Interestingly, we also found positive associations of PCSK9 with inflammatory markers, such as fibrinogen and hs-CRP, in stable CAD status. Therefore, our data confirmed and furthered previous studies. Besides the definite association with LDL-C levels, recent studies uncovered a certain relationship between systemic inflammation15 and PCSK9 concentration. In addition, experimental studies indicated that PCSK9 might accelerate atherosclerosis by promoting inflammation,12,31 which may be through mechanisms independent of the LDLR.12 However, most of previous studies exploring the relationship between PCSK9 and inflammation using acute inflammation and animal models, and clinical data referring to PCSK9 and chronic inflammation were obscure. There were numerous studies demonstrated that the multiple inflammatory markers were up-regulated in chronic inflammatory status and could predict risk of CAD. According to the meta-analysis conducted by Lowe32 suggested the associations of various inflammatory markers with risk of CAD: fibrinogen (odds ratio [OR] 1.8; 95% confidence intervals [95%CI]: 1.6–2.0); hs-CRP (OR 1.49; 95%CI: 1.37– 1.62); ESR (OR 1.33; 95%CI: 1.22–1.44); D-dimer (OR 1.7; 95%CI: 1.3–2.2), and albumin (OR 1.5; 95%CI: 1.3– 1.7). In the present study, therefore, we additionally investigated the clinical readily measurable inflammatory markers, hs-CRP, ESR, D-dimer, and albumin, with PCSK9 concentration and found positive associations of PCSK9 with hs-CRP as well as fibrinogen. However, the elaborate mechanisms and causal relationship need further investigations.
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Table 4 The independent correlation between circulating fibrinogen and PCSK9 concentration in patients with stable CAD
4.
Fibrinogen Models
Other variables
b
P value
1 2 3 4
Covariates Model 1 1 TG 1 HDL-C 1 LDL-C Model 2 1 hs-CRP Model 2 1 D-dimer
0.199 0.173 0.168 0.168
.003 .008 .011 .011
BMI, body mass index; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein–cholesterol; SBP, systolic blood pressure; TC, total cholesterol. Bold P values indicate statistical significance. Multivariable regression stepwise models are shown. The dependent variable was PCSK9. Covariates including age, gender, BMI, current smoking, family history of CAD, SBP, and fasting blood glucose.
There were several limitations in our study. First, because the sample size is so small the findings could also be from chance. Moreover, this is an observational study and so there could be residual confounding. Finally, whether such association is present in other populations, such as those without known CAD or those with ACS, should be studied in the future.
5.
6.
7.
8.
9.
10.
11.
Conclusions In summary, PCSK9 concentration was independently and positively associated with fibrinogen in patients with stable CAD, suggesting that the interactions of PCSK9 and fibrinogen in the status of atherosclerosis may be needed further investigations.
12.
13.
14.
Acknowledgments This work was partially supported by the National Natural Scientific Foundation (81070171, 81241121), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20111106110013), the Capital Special Foundation of Clinical Application Research (Z121107001012015), the Capital Health Development Fund (2011400302), and the Beijing Natural Science Foundation (7131014) awarded to Dr. Jian-Jun Li. Conflicts of interest: The authors declare that they have no conflicts of interests.
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