Elevated Levels of Serum Fibrin and Fibrinogen Degradation Products Are Independent Predictors of Larger Coronary Plaques and Greater Plaque Necrotic Core

Abstract

Background: Co-existence of vulnerable plaque and pro-thrombotic state may provoke acute coronary events. It was hypothesized that elevated serum levels of fibrin and fibrinogen degradation products (FDP) are associated with larger total plaque and necrotic core (NC) areas.

Methods and Results: Seventy-five patients presenting with stable anginal symptoms (69%) or stabilized acute coronary syndrome (ACS; 31%), and found to have non-obstructive coronary artery disease (CAD) with a fractional flow reserve >0.8, were studied. Invasive virtual histology intravascular ultrasound (VH-IVUS) was performed in 68 LAD arteries, 6 circumflex arteries, and 1 right coronary artery. Serum FDP levels were measured using ELISA technique. Plaque volumetrics and composition were assessed in each VH-IVUS frame and averaged. The median age of patients was 56 (47–63) years; 52% were men and 23% had diabetes. The average length of coronary artery studied was 62 mm. After adjustment for systemic risk factors, medications, CRP levels and ACS, male gender (P<0.001) and serum FDP levels (P=0.02) were independent predictors of a larger NC area. Older age (P<0.001), male gender (P<0.0001) and increased serum FDP level (P=0.03) were associated with a larger plaque area.

Conclusions: In patients with CAD, a higher serum level of FDP is independently associated with larger plaques and greater plaque NC. (Circ J 2016; 80: 931–937)

Cardiovascular disease remains the leading cause of mortality in the USA and globally. Acute coronary syndromes (ACS) occur as a result of vulnerable atherosclerotic plaque rupture that triggers a cascade of reactions leading to platelet aggregation, thrombus formation and activation of the coagulation pathway at the site of coronary occlusion.¹ Early recognition of these plaques may improve outcomes. Biomarkers reflecting various pathways of atherogenesis, including inflammation, cell stress and coagulation, have been shown to add significant risk discrimination for incident cardiovascular events.^2,3 Only plasma glutathione, however, has been associated with intravascular imaging characteristics of “rupture-prone” plaques.⁴ Fibrin and fibrinogen degradation products (FDP) are byproducts of thrombin breakdown, and their increased circulating levels may reflect subclinical vascular thrombosis.⁵ Serum FDP levels are associated with coronary artery disease (CAD) incidence and adverse outcomes.^2,6–8

Intravascular ultrasound (IVUS) allows direct, real-time visualization of the coronary lumen and has well-recognized clinical applications in defining the distribution and morphology of coronary plaques. Virtual histology-IVUS (VH-IVUS), with its ability to differentiate plaque tissue types based on radiofrequency signals, has become a useful technique for precise characterization of plaque composition and assessment of coronary plaque vulnerability.⁹ We hypothesized that elevated FDP levels will be associated with VH-IVUS features of plaque vulnerability.

Methods

Patient Population

Patients who underwent a clinically indicated coronary angiography for anginal symptoms at our institution and enrolled in the Emory Cardiovascular Biobank Registry were screened for inclusion in this study. We included patients with atherosclerotic lesions significant enough to warrant further hemodynamic evaluation with fractional flow reserve (FFR) and interrogation with VH-IVUS. We also included patients who presented with non-ST elevation myocardial infarction and unstable angina who were observed for >48 h on optimal medical therapy without any further adverse events or hemodynamic instability prior to cardiac catheterization. We excluded patients presenting with ST-elevation myocardial infarction, cardiogenic shock or hemodynamic instability; patients with a history of severe valvular heart disease or coronary artery bypass grafting; or patients found to have obstructive CAD in any vessel requiring percutaneous coronary intervention (FFR <0.80). All study patients provided informed written consent prior to enrolment, and the study was approved by the Institutional Review Board of Emory University.

Invasive Physiologic and Imaging Protocol

All patients underwent angiography in a biplane cardiac catheterization system, Doppler blood flow and pressure measurements (ComboWire^® XT Guide Wire; Volcano Corporation, Rancho Cordova, CA, USA) and VH-IVUS imaging (Eagle Eye^® Platinum RX Digital IVUS Catheter; Volcano Corporation) evaluation. Per protocol, only one epicardial artery per patient was interrogated, which was determined at the discretion of the operator. Maximal hyperemia was induced by IV adenosine infusion at a rate of 140 mcg·kg^–1·min^–1 to calculate FFR as a ratio of distal to proximal (aortic) pressure, coronary flow reserve (CFR) as a ratio of hyperemic to basal average peak blood flow velocity, and hyperemic microvascular resistance as a ratio of distal pressure to average peak velocity during hyperemia. Microvascular dysfunction was defined as CFR <2.0.

Virtual histology-IVUS was performed by positioning the IVUS catheter in the mid to distal study vessel. Automated motorized pullback at a rate of 0.5 mm/s (R-100 Imaging Catheter Pullback Device; Volcano Corporation) was performed after administration of systemic heparin and 200 µg of intracoronary nitroglycerine, and IVUS images were continuously acquired up to the guide catheter in the aorta to sample approximately 60 mm of the proximal vessel.

Measurement of Serum FDP

Arterial blood samples were collected in BD Vacutainer^® Serum tubes (Becton Dickinson and Company; Franklin Lakes, NJ, USA) from fasting patients (at least 8 h). Whole blood was allowed to clot at room temperature for 30–60 min post-collection, followed by tube centrifugation at 3,000 RPM for 10 min at 4℃. Serum samples were labeled and stored at –80℃ until the time of FDP immunoassay analyses.

In contrast to the commonly used D-dimer assay, the FDP immunoassay (GenWay Biotech Inc, San Diego, CA, USA) detects the full complement of FDP, including fibrin mono- and oligomers, as well as fragments -X, -Y and -E generated from both fibrin and fibrinogen cleavage reactions. This FDP ELISA assay is a standard sandwich assay, which utilizes removable strips in a 96-well microtiter plate format. The wells are coated with affinity purified rabbit anti-FDP polyclonal antibodies. FDP in diluted sera (1:200) is captured from the sera by the antibodies immobilized onto the well of the microtiter plate. After a wash step, anti-human fibrinogen antibodies conjugated to horseradish peroxidase are added to the wells. If the FDP antigen is present, then the anti-human fibrinogen peroxidase complex will bind to FDP to form an immunological sandwich with the immobilized antibodies. After a second wash step, the enzyme substrate, 3,3’,5,5’-tetramethylbenzidine, is added to the well. The reaction is stopped with 0.1 N HCl and placed in a microplate reader at a 450 nm wavelength, where the color intensity is proportional to the amount of FDP in the serum. The amount is quantified by interpolation from a standard curve using the calibrators provided with the kit.

IVUS Analysis and Definitions

Analysis of the VH-IVUS cross-sections was conducted at the Imaging and Biomechanical Core Lab of Emory University and Georgia Institute of Technology. Intraobserver analysis was performed by an experienced analyst using random samples of 10 patients (n=886 frames) at least 2 weeks apart, demonstrating good reproducibility for plaque area (concordance correlation coefficient, 0.968; 95% confidence interval [CI], 0.965–0.971) and necrotic core (NC) area (concordance correlation coefficient, 0.978; 95% CI, 0.976–0.980). Measurements of the external elastic membrane (EEM), lumen cross-sectional area, and plaque area (plaque and media: EEM-lumen) were performed for every recorded VH-IVUS frame (0.5 mm thickness). Plaque burden was calculated as plaque area divided by EEM area×100%,¹⁰ and plaque composition was assessed as the absolute area of VH-IVUS parameters (fibro-fatty [FF] tissue, fibrous [FI] tissue, NC, and dense calcium [DC]) in each IVUS frame.^10–13 Segmental plaque cross-sectional area, plaque burden, and FF, FI, DC and NC areas measured in each patient were averaged over the length of the studied vessel. Features suggestive of plaque vulnerability were defined as larger plaques and greater plaque NC, and FF components.^14–17

Statistical Analysis

Continuous variables not normally distributed were reported as median and inter-quartile range (IQR) and compared using the Mann-Whitney U-test. As FDP levels and VH-IVUS parameters (except plaque burden) were highly right skewed, logarithmic transformation (using base 2 for FDP levels for the ease of interpretation and natural log for other variables) was applied to reduce heteroscedasticity and to achieve normality. Multivariate linear regression analysis was performed to examine the association between FDP levels and average NC area, as well as plaque area. Traditional cardiovascular risk factors, as well as medications affecting the parameter estimate of FDP by >10%, were included in the multivariate model. Regression diagnostics were performed to evaluate model fit and potential influential observations. Covariates in the multivariable regression model included age (centered at 55 years), gender (male), race (Caucasian vs. African American), smoking (current smoker or not), ACS, nitrate use, statin use, C-reactive protein (CRP) level, and history of hypertension, diabetes and dyslipidemia. Other medications (aspirin, clopidogrel, β-blockers, calcium channel blockers, ACE inhibitors) were considered but not included in the final model based on the above criteria. A 2-sided P-value of <0.05 was considered statistically significant. Statistical analyses were performed by using SPSS 20 (SPSS Inc, Chicago, IL, USA) and SAS 9.3 (SAS Institute Inc, Cary, NC, USA).

Results

The demographic and clinical characteristics of the 75 patients are presented in Table 1. The median age was 56 (47–63) years; 52% were men and 23% had diabetes. The majority of patients (69%) underwent cardiac catheterization for stable angina pectoris, while 31% presented with ACS. Among the 75 studied vessels, 68 were left anterior descending arteries, 6 were left circumflex arteries, and one was a right coronary artery. The average IVUS vessel segment studied was 62 mm. The median FDP level was 0.50 (0.32–0.76) mcg/ml, and the CRP level was 2.14 (0.90–4.57) mg/L. Vessel-level physiology, gray-scale IVUS and VH-IVUS findings are presented in Table 2.

Table 1. Demographic and Clinical Characteristics of the Study Population

	(Total number=75 patients)
Age (years)	56 (47–63)
Men	39 (52)
Caucasian race	47 (63)
African-American race	23 (31)
Body mass index	30 (26–36)
Cardiovascular risk factors
Hypertension	54 (72)
Systolic blood pressure (mmHg)	132 (121–145)
Diastolic blood pressure (mmHg)	74 (69–82)
Current smoking	15 (20)
Dyslipidemia	58 (77)
Diabetes mellitus	17 (23)
Obesity	34 (45)
Family history of CAD	33 (44)
Previous myocardial infarction	9 (12)
Lipid profile
Total cholesterol (mg/dl)	164 (133–190)
Low-density lipoprotein (mg/dl)	99 (70–120)
High-density lipoprotein (mg/dl)	42 (35–50)
Triglycerides (mg/dl)	97 (67–138)
Indication for cardiac catheterization
Stable angina	52 (69)
Acute coronary syndromes	23 (31)
Medications
Aspirin	50 (67)
Clopidogrel	15 (20)
Statin	39 (52)
β-blocker	32 (43)
ACE-inhibitor or ARB	33 (44)
Calcium channel blocker	21 (28)
Nitrates	18 (24)
Creatinine (mg/dl)	1.00 (0.80–1.10)
FDP (mcg/ml)	0.50 (0.32–0.76)
CRP (mg/L)	2.14 (0.90–4.57)

Data are presented as number (%) or median (interquartile range). ACE, angiotensin-converting enzyme; ARB, angiotensin-receptor blocker; CAD, coronary artery disease; CRP, C-reactive protein; FDP, fibrin and fibrinogen degradation products; body mass index, the weight in kilograms divided by the square of the height in meters.

Table 2. Vessel-Level Physiology, Gray-Scale IVUS and Virtual Histology-IVUS Findings of the Study Population

Parameter	(Total number=75 patients)
Fractional flow reserve	0.93 (0.86–0.98)
Coronary flow reserve	2.10 (1.75–2.65)
Hyperemic myocardial resistance index	1.74 (1.37–2.20)
External elastic membrane (mm2)	15.10 (12.63–17.87)
Lumen area (mm2)	9.18 (7.64–11.27)
Plaque area (mm2)	5.76 (3.93–7.68)
Plaque burden (%)	37.5 (28.2–49.0)
NC area (mm2)	0.46 (0.18–0.89)
DC area (mm2)	0.18 (0.06–0.45)
Fibrous area (mm2)	1.71 (0.78–2.65)
Fibro-fatty area (mm2)	0.21 (0.09–0.45)

Data are presented as median (interquartile range). DC, dense calcium; IVUS, intravascular ultrasound; NC, necrotic core.

The relationships between traditional cardiovascular risk factors and serum FDP levels are shown in Table 3. Median FDP levels were significantly higher in patients presenting with ACS as compared to those with stable angina pectoris (P=0.047). In contrast, FDP levels were not significantly different between males and females, patients of different races, patients who did or did not take aspirin, clopidogrel, statin, nitrate, β-blocker, calcium channel blocker or ACE inhibitor, or those with known systemic cardiovascular risk factors including hypertension, cigarette smoking, dyslipidemia, diabetes and obesity.

Table 3. Dichotomous Relationship Between FDP Level and Patient Characteristics

	FDP (mcg/ml)	P-value
Gender
Male	0.44 (0.28–0.67)
Female	0.51 (0.36–0.81)	0.261
Race
Caucasian	0.44 (0.30–0.65)
African American	0.62 (0.37–0.95)
Other	0.51 (0.27–0.51)	0.104
Hypertension
Yes	0.45 (0.29–0.76)
No	0.51 (0.39–0.77)	0.267
Current smoker
Yes	0.56 (0.33–0.82)
No	0.48 (0.32–0.75)	0.619
Dyslipidemia
Yes	0.50 (0.32–0.78)
No	0.50 (0.35–0.64)	0.889
Diabetes
Yes	0.46 (0.34–0.84)
No	0.50 (0.31–0.65)	0.512
Obesity
Yes	0.49 (0.29–0.78)
No	0.50 (0.34–0.73)	0.666
High CRP level (>3.0 mg/L)
Yes	0.56 (0.33–0.82)
No	0.48 (0.32–0.70)	0.602
Acute coronary syndrome
Yes	0.61 (0.42–0.76)
No	0.43 (0.29–0.75)	0.047
Coronary microvascular dysfunction
Yes	0.56 (0.36–0.87)
No	0.44 (0.29–0.64)	0.181

Data are presented as median (interquartile range). Abbreviations as in Table 1.

Gray-scale IVUS and VH-IVUS data categorized by FDP tertiles are represented in Table 4. After adjusting for age, race, gender, ACS, cigarette smoking, hypertension, diabetes mellitus, dyslipidemia, nitrate use, statin use, CRP and serum FDP level, male gender (P<0.001) and increased serum FDP level (P=0.02) were significantly associated with a larger NC area (Table 5). A doubling in serum FDP level was associated with a 6.5% (95% CI=[1.4%, 11.9%]) increase in average NC area. Similarly, Table 6 represents the multivariate regression analysis results for predictors of average plaque area. Older age (P<0.001), male gender (P<0.0001), and increased serum FDP level (P=0.03) were significantly associated with a larger plaque area. Specifically, doubling in serum FDP level was associated with an 8.9% (95% CI=[1.3%, 17.1%]) increase in average plaque area.

Table 4. Gray-Scale IVUS and Virtual Histology-IVUS Data Categorized by FDP Level Tertiles

	Tertile 1	Tertile 2	Tertile 3	P value
Plaque area (mm2)	5.44 (3.61–7.37)	5.17 (3.93–8.47)	6.70 (4.22–7.23)	0.601
Plaque burden (%)	35.96 (26.80–49.48)	37.07 (26.86–48.17)	40.85 (29.88–48.40)	0.598
NC area (mm2)	0.37 (0.15–0.75)	0.42 (0.18–0.94)	0.59 (0.30–1.10)	0.371
DC area (mm2)	0.17 (0.04–0.40)	0.18 (0.05–0.52)	0.2 (0.08–0.54)	0.770
FI area (mm2)	1.70 (0.69–2.82)	1.42 (0.58–2.76)	1.76 (0.93–2.14)	0.992
FF area (mm2)	0.19 (0.09–0.51)	0.21 (0.08–0.46)	0.26 (0.14–0.42)	0.764
NC %	11.35 (4.29–17.29)	12.52 (6.98–17.15)	14.62 (8.78–20.37)	0.354
DC %	4.80 (1.53–9.94)	5.21 (2.47–10.20)	5.09 (2.80–11.21)	0.779
FI %	58.31 (35.38–65.10)	43.31 (34.25–61.62)	49.49 (43.27–56.81)	0.553
FF %	6.57 (3.76–9.79)	5.43 (3.59–7.14)	7.44 (4.94–9.92)	0.267

Data are presented as median (interquartile range). FI, fibrous tissue; FF, fibro-fatty tissue. Other abbreviations as in Tables 1,2.

Table 5. Multivariate Linear Regression Model for Predictors of Plaque NC Area

Variable	Parameter estimate	Standard error	P value
Age*	0.006	0.004	0.099
Gender (Male)	−0.266	0.076	<0.001
Race (African American vs. Caucasian)	0.014	0.082	0.865
Current smoker	0.052	0.106	0.622
Hypertension	0.150	0.086	0.085
Diabetes	0.014	0.084	0.872
Dyslipidemia	0.099	0.099	0.323
Acute coronary syndrome	0.103	0.077	0.184
Nitrate use	0.098	0.089	0.275
CRP	0.011	0.008	0.158
Statin use	−0.087	0.077	0.264
Log2 FDP level	0.063	0.025	0.015

*Age was centered at 55 years. Abbreviations as in Tables 1,2.

Table 6. Multivariate Linear Regression Model for Predictors of Plaque Area

Variable	Parameter estimate	Standard error	P value
Age*	0.018	0.005	<0.001
Gender (Male)	−0.600	0.112	<0.0001
Race (African American vs. Caucasian)	0.115	0.122	0.349
Current smoker	0.148	0.156	0.347
Hypertension	0.214	0.127	0.097
Diabetes	0.058	0.125	0.642
Dyslipidemia	0.022	0.146	0.881
Acute coronary syndrome	0.086	0.113	0.453
Nitrate use	0.194	0.131	0.145
CRP	0.012	0.011	0.308
Statin use	−0.109	0.115	0.344
Log2 FDP level	0.085	0.037	0.027

*Age was centered at 55 years. Abbreviations as in Tables 1,2.

Discussion

This is, to the best of our knowledge, the first study demonstrating that elevated FDP levels are independently associated with larger plaques and greater plaque NC and FF components (Figure); these are all features suggestive of more advanced atherosclerosis and increased plaque vulnerability.^11,15,16,18 While a larger plaque NC area has previously been associated with diabetes, hypertension, history of myocardial infarction, low high-density lipoprotein cholesterol levels,¹⁹ and microvascular dysfunction,²⁰ this study demonstrates that the FDP level is also an independent predictor of larger lipid-rich plaques. This link between serum FDP and IVUS-defined vulnerable plaques lends credence to the suggestion that FDP can be a valid biomarker for predicting patients with non-obstructive CAD to be at a higher risk of developing ACS.

Figure.

Intravascular ultrasound examples of plaques from 2 patients with high and low serum FDP Levels. (A) Grayscale and VH-IVUS images of large lipid-rich plaque from a patient with a high serum FDP level: PA=9.53 mm², PB=59.5%, NC=2.66 mm², DC=1.58 mm², FF=0.40 mm², FI=1.88 mm², FDP level=10 mcg/ml. (B) Grayscale and VH-IVUS images of a smaller fibrous-rich plaque from a patient with a low serum FDP level: PA=3.79 mm², PB=29.6%, NC=0.02 mm², DC=0.00 mm², FF=0.21 mm², FI=1.09 mm², FDP level=0.25 mcg/ml. DC, dense calcium plaque area; FDP, fibrin and fibrinogen degradation products; FF, fibro-fatty plaque area; FI, fibrous plaque area; NC, necrotic core plaque area; PA, plaque area; PB, plaque burden; VH-IVUS, virtual histology intravascular ultrasound.

An elevated fibrinogen level was an independent predictor for the development of cardiovascular disease in the Framingham study.⁶ Similarly, an elevated D-dimer level in patients without known CAD was associated with risk of developing CAD events.^21,22 Furthermore, in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) study, high D-dimer levels were associated with an increased risk of cardiovascular events in diabetics with CAD.²³ Recently, we demonstrated that elevated levels of FDP were an independent predictor of all-cause death, myocardial infarction or revascularization in patients with suspected or known CAD.²

Interestingly, recent experimental studies demonstrated a mechanistic pro-inflammatory role for FDP at the molecular level. It has been suggested that fibrin and its degradation products play an important role in the inflammatory process, namely through stimulation of interleukin (IL)-1²⁴ and IL-8,²⁵ and expression of intracellular adhesion molecule-1 (ICAM-1).²⁶ Moreover, the specific pro-inflammatory role of fibrin E-fragments (part of the FDP extended complement spectrum measured for this study) has been recently described, whereby E1-fragments induce transmigration of inflammatory cells from the bloodstream to the subendothelial layer by forming a bridge between monocytes/neutrophils (binding to CD11c/CD18) and the endothelium (binding to VE-Cadherin).^27,28

Despite the association between FDP and IL-1, IL-8, ICAM-1, inflammatory cells, and endothelial cells, and the outcome studies describing the predictive value of serum FDP, no intravascular imaging data linking FDP level to coronary vessels with larger, more vulnerable plaques has been reported to date.

The prospective natural-history study of coronary atherosclerosis (PROSPECT) trial¹⁶ has shown a higher rate of major adverse cardiovascular events in patients with larger plaques or more vulnerable plaque phenotypes, namely thin-cap fibroatheroma (TCFA). Moreover, acute coronary events are thought to occur when a vulnerable plaque co-exists with circulating thrombosis-promoting factors.^1,15 An elevated FDP level may be an indication of subclinical plaque rupture and/or erosion, and thus an indicator of a pro-atherogenic coronary environment. Indeed, the results of this paper are in line with this interesting concept of vulnerable plaque. While the focus of the present investigation was the association of serum FDP with features of atherosclerosis in non-obstructive CAD, recent data suggests a higher relative frequency of TCFA in patients with more significant obstructive disease.²⁹ Whether the observed predictive value of serum FDP and plaque vulnerability is retained or becomes even stronger in patients with obstructive disease is not known and warrants further investigation.

Taken together, our finding that larger plaques with greater NC are associated with an elevated serum FDP level in the context of existing experimental data, demonstrating a pro-inflammatory role of FDP and the known association between a pro-thrombotic milieu and ACS, suggests that FDP may have a role in plaque development and transformation into a vulnerable phenotype.

Study Limitations

Several limitations require consideration when interpreting the results of the present study. First, this is a cross-sectional study at a single center with no outcome data, and therefore the investigation is limited to associations and cannot determine causality. Second, plaque composition data are derived from VH-IVUS and not true histology, which is the gold standard but not readily obtainable in clinical studies. While VH-IVUS has well-documented limitations,³⁰ its accuracy has been evaluated in both ex vivo human coronary histology studies^9,12 and in vivo directional coronary atherectomy specimens,¹³ with predictive accuracies of 87% to 97%, and it has been associated with clinical outcomes in the PROSPECT trial.¹⁶ Third, per protocol, only one vessel per patient was imaged and analyzed for plaque characteristics in this study. This limitation warrants further investigation into the association between FDP level and plaque vulnerability in the entire coronary system of each study subject. Finally, although vulnerable plaques have been defined as those with large plaque and NC areas in this study, it is worthwhile mentioning that our patient population had a low incidence of TCFA, which might limit the applicability of the study results to low- and intermediate-risk profile patients rather than the high-risk patient population.

Conclusions

Our study shows that higher serum levels of FDP is an independent predictor of larger plaques (P=0.03) and greater plaque NC (P=0.02), features suggestive of increased plaque vulnerability. The findings of this study provide mechanistic insight into our understanding of adverse outcomes associated with vulnerable plaques. Prospective trials are warranted to further explore the role of FDP in identifying patients with coronary atherosclerosis at risk of developing ACS.

Acknowledgments

The authors would like to thank Dr Sergey Sikora for his contribution to the Methods Section - Measurement of Serum Fibrin and Fibrinogen Degradation Products.

Funding Sources

Drs Hung and Eshtehardi are supported by the Ruth L. Kirschstein National Research Service Awards training grant (5T32HL007745), and receive research funding from Gilead Sciences (Foster City, CA, USA) and Medtronic, Inc (Minneapolis, MN, USA). Dr Mekonnen is supported by a supplement grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (P01HL101398). Dr Samady receives research funding from Volcano Corporation (San Diego, CA, USA) and St. Jude Medical (St. Paul, MN, USA). The other authors have no relationships with industries that are relevant to the content of this paper to disclose.

Disclosures

None declared.

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