Expression of platelet collagen receptor glycoprotein VI Clinical research

Clinical research
European Heart Journal (2006) 27, 2165–2169
doi:10.1093/eurheartj/ehl192
Coronary heart disease
Expression of platelet collagen receptor glycoprotein VI
is associated with acute coronary syndrome
Boris Bigalke1, Stephan Lindemann1*, Raila Ehlers1, Peter Seizer1, Karin Daub1, Harald Langer1,
Tanja Schonberger1, Elisabeth Kremmer2, Dorothea Siegel-Axel1, Andreas E. May1, and
Meinrad Gawaz1*
1
2
¨t Tu
¨bingen, Otfried-Mu
¨ller-Str. 10, D-72076 Tu
¨bingen, Germany; and
Medizinische Klinik III, Eberhard Karls-Universita
Institute for Molecular Immunology, GSF-National Research Center for Environment and Health, Munich, Germany
Received 4 March 2006; revised 23 June 2006; accepted 27 July 2006; online publish-ahead-of-print 21 August 2006
KEYWORDS
Platelets;
Glycoprotein VI;
Collagen;
Acute coronary syndrome
Introduction
Exposure of collagen to the bloodstream occurs at the site of
plaque rupture and initiates platelet activation and plateletmediated thrombus formation.1 Previously, several platelet collagen receptors have been identified, and it has become clear
by now that collagen binding to glycoprotein VI (GPVI) is the
initial trigger of platelet activation and aggregation at the
site of vascular injury where extracellular matrix components,
including collagen, are exposed.2–4 GPVI-mediated platelet
activation leads to firm platelet adhesion and secretion of procoagulative and proinflammatory compounds, which, in turn,
aggravate and consolidate thrombus formation.2 Further, collagen binding to platelets is followed by the expression of phosphatidylserine on the platelet membrane. Phosphatidylserines
provide prothrombinase activity that enhances plasmatic
thrombin formation.2 Moreover, the adhering platelets stay
alive for several hours or days at the site of vascular lesions
and initiate de novo protein synthesis and interleukin-1 beta
release through shedding of microvesicles,5–7 thereby inflammatory activity around a platelet thrombus.
Recently, GPVI has been described to be the major collagen receptor mediating platelet adhesion and aggregation
* Corresponding author. Tel: þ49 7071 29 83688; fax: þ49 7071 29 5749.
E-mail address: [email protected] or meinrad.
[email protected]
on human atherosclerotic plaque tissues.8,9 Inhibition of
GPVI results in the substantial attenuation of platelet
adhesion to atherosclerotic tissue.8 Although GPVI is constitutively surface-expressed, activation of platelets leads to
further release and enhanced plasma membrane expression
of GPVI.2,10 Preliminary data suggest that platelet collagen
receptor GPVI density may be related to myocardial infarction.11 Thus, an altered GPVI expression level on circulating
platelets may indicate a prothrombotic disease state such as
imminent myocardial infarction.
The present work evaluates the expression levels of platelet GPVI in a large cohort of patients with symptomatic coronary artery disease (CAD). The data imply that patients
with enhanced platelet GPVI are at increased risk for
acute coronary events. Determination of GPVI levels may
be an early marker of acute coronary syndrome (ACS)
before myocardial ischaemia is evident.
Methods
Study population and enrolment criteria
Our study population comprised 367 consecutive patients, in whom
CAD was suspected and thus received coronary angiography: 240
patients with stable angina pectoris (SAP) and 127 patients with
ACS/MI.
The exclusion criteria were age below 18 years, lack of informed
consent, and false positive elevation of creatine kinase (CK) and
& The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: [email protected]
Downloaded from by guest on October 21, 2014
Aims Platelet collagen receptor glycoprotein VI (GPVI) is critical for the formation of arterial
thrombosis. In this observational study, we examined the platelet surface expression of GPVI in
patients with symptomatic coronary artery disease (CAD).
Methods and results We evaluated a consecutive cohort of 367 patients with symptomatic CAD, who
underwent coronary angiography. The surface expression of platelet activation markers (GPVI, CD62P,
and CD42b) was determined by flow cytometry. Patients with acute coronary syndrome (ACS) showed
a significantly enhanced GPVI expression on admission when compared with patients with stable
angina pectoris (SAP) (ACS: 21.4 + 9.7; SAP: 18.6 + 7.1 mean fluorescence intensity + SD;
P ¼ 0.004). The expression of GPVI correlated with CD62P (r ¼ 0.702; P ¼ 0.001). Logistic regression
analysis demonstrated that on admission, elevated platelet GPVI expression was associated with ACS,
independent of markers of myocardial necrosis such as troponin and creatine kinase.
Conclusion Platelet GPVI surface expression is elevated in patients with ACS and is associated with
imminent acute coronary events. The determination of the platelet-specific thrombotic marker GPVI
may help to identify patients at risk before myocardial ischaemia is evident.
2166
CKMB. Twenty-six patients were excluded, among these, 19 refused
to give their consent and the other seven patients were excluded
because of exclusion criteria.
Definitions
SAP: stable angina pectoris, symptoms without progression and
stable intensity.
ACS: episodes of .20 min of angina pectoris with progress over
time or with recurrent episodes at rest or with minimal physical
strain within the last 24 h. The ECG shows at least one of the following criteria: new ST-depression of at least 0.05 mV, transient
(,20 min) ST-elevation of at least 0.1 mV, or negative T of at least
0.3 mV in two or more leads of standard electrocardiogram. CK
and troponin-I (Tn-I) levels may be increased, but can be normal
as well. If CK and Tn-I levels are increased, we also have a STEMI
or NSTEMI, depending on the ECG criteria (TNþACS, Figure 1B).
STEMI: episodes of .20 min of angina pectoris with the following
ECG and laboratory criteria: ST-segment elevation in two or more
leads with at least 0.1 mV or a two-fold elevation of CK when compared with the normal level and with a significant CKMB or an
increased Tn-I level.
NSTEMI: episodes of .20 min of angina pectoris with the following ECG and laboratory criteria: no ST-segment elevation in the
standard ECG. Two-fold elevation of CK when compared with the
normal level and with a significant CKMB or an increased Tn-I.
Controls
(22 men and eight women, age 31.8 + 8.3 years, no medication, and
except for four smokers, no cardiovascular risk factors). These controls did not receive coronary angiography.
We then examined a group of subjects in whom CAD was
excluded. These patients (nine men and six women, age 57.4 +
9.4 years) were angiographied because of chest pain but did not
have CAD. The patients had the following cardiovascular risk
profiles: hypertension (6x), diabetes (1x), family history for CAD
(1x), hypercholesterolaemia (1x), and smoking (1x). Medical
treatment includes b-blockers (5x), ASS (1x), statins (1x), and
ACE-inhibitors (1x).
Sample collection
Arterial blood was drawn from the sheath that was introduced into
the femoral artery at the beginning of cardiac catheterization.
Cardiac catheterization was started immediately after admission
to the hospital in patients with ACS. Patients with SAP were investigated on schedule in the morning. In the apparently healthy controls, the blood was obtained by venipuncture of the right
anticubital vein. The blood was filled into 5 mL citrate phosphate
dextrose adenine vials and analysed by flow cytometry.
FACS analysis
The surface expression of the platelet receptors GPVI, CD62P, and
GPIb was determined by two-colour whole blood flow cytometry,
as described previously.12,13 Each measurement was performed
in duplicate and mean fluorescence intensity (MFI) was used as
the index of receptor expression. Fluorescein (FITC)-conjugated
Figure 1 Platelet collagen receptor GPVI expression is elevated in patients with ACS. (A) Platelets of patients with SAP, ACS, and healthy control subjects (combined control level for GPVI; 15.8 + 4.3; n ¼ 45; dotted line) were stained for the collagen receptor (GPVI) and P-selectin (CD62P) using monoclonal antibodies
conjugated with either FITC or PE. Two-colour whole blood flow cytometry was performed according to the standardized protocol and the results were expressed
as MFI. (B) Surface expression of platelet GPVI and P-selectin (CD62P) in patients with Tn-I- positive ACS (TnþACS) and with myocardial infarction without/with
ST-segment elevation (NSTEMI/STEMI). P-value indicates statistical significance vs. SAP in (A). (C) Correlation of platelet GPVI and CD62P surface expression.
Downloaded from by guest on October 21, 2014
First, we wanted to check the platelet GPVI expression of apparently healthy controls. The personnel of our lab were investigated
B. Bigalke et al.
Platelet GPVI in ACS
anti-CD62P (clone CLB-Thromb/6) and phycoerythrin (PE)conjugated anti-CD42b (clone SZ2) monoclonal antibodies (mAb)
were purchased from Immunotec, Beckman Coulter, Inc., USA. The
anti-GPVI mAb 4C9 was generated and characterized, as described
previously.10,14
Statistics
Pairwise comparisons of ACS, Tnþ, NSTEMI, and STEMI with SAP were
evaluated with appropriate non-parametric tests (the Kruskall–
Wallis test). For corrections of multiple testing, a Bonferroni–
Holm correction was applied. A probability value of less than 0.05
was considered as statistically significant. Comparison between
GPVI and clinical parameters was made by the Spearman correlation. Adjustment by possible cofounders was performed by the
multifactorial analysis of covariance for the decadic logarithm of
GPVI. Association of the platelet GPVI surface expression was
found to be independent of medical treatment at the time of admission, classical cardiovascular risk factors, and laboratory markers.
The association of GPVI after adjustment for conventional laboratory markers was assessed by the logistic regression analysis. All
statistical analyses were performed using SPSS version 13.
Results
Table 1 Baseline patients’characteristics and medical treatment on admission
Characteristics
Total
(n ¼ 367)
SAP
(n ¼ 240)
ACS
(n ¼ 127)
Age, years (+SD)
Sex, no. (%)
Female
Male
67.1 + 10.1
66.9 + 9.4
67.5 + 11.3
96 (26.2)
271 (73.8)
59 (24.6)
181 (75.4)
37 (29.1)
90 (70.9)
31 (8.4)
260 (70.8)
17 (7.1)
176 (73.3)
14 (11)
84 (66.1)
201 (54.8)
134 (36.5)
88 (24.0)
151 (62.9)
88 (36.7)
59 (16.1)
50 (39.4)
46 (36.2)
29 (22.8)
116 (31.6)
75 (24.6)
41 (32.3)
CAD, no. (%)
One vessel
Two vessels
Three vessels
83 (22.6)
117 (31.9)
167 (45.5)
47 (19.6)
80 (33.3)
113 (47.1)
36 (28.4)
37 (29.1)
54 (42.5)
LVEF, no. (%)
Normal
Slightly reduced
Moderate
Low
201 (54.8)
92 (25.1)
47 (12.8)
27 (7.4)
135 (56.3)
61 (25.4)
27 (11.2)
17 (7.1)
66 (52)
31 (24.4)
20 (15.7)
10 (7.9)
113 (30.8)
174 (47.4)
30 (8.2)
52 (21.7)
129 (53.8)
28 (11.7)
61 (48)
45 (35.4)
2 (1.6)
216 (58.9)
204 (55.6)
216 (58.9)
75 (20.4)
21 (5.7)
165 (68.8)
159 (66.3)
162 (67.5)
63 (26.3)
15 (6.3)
51 (40.2)
45 (35.4)
54 (42.5)
12 (9.4)
6 (4.7)
Cardiovascular risk
factors, no. (%)
No risk factors
Arterial
hypertension
Hyperlipidaemia
Diabetes
Family history
of CAD
Smoking
Medication, no. (%)
No medication
ACE-inhibitors
Angiotensin
receptor blockers
Beta-blockers
Statins
Aspirin
Clopidogrel
Vitamin K
antagonist
Comparison of the decadic logarithm of GPVI between
ACS and SAP was adjusted by possible cofounders such
as medical treatment and cardiovascular risk factors
(Table 2). To test whether GPVI is associated with ACS independent of baseline conventional laboratory markers of ACS, we
performed a logistic regression analysis that included Tn-I,
CK, and C-reactive protein determined at the time of admission (Table 3). We found that the platelet surface expression
of GPVI was independently associated with ACS (Table 3).
Discussion
The present study is an observational study of 367 consecutive patients with SAP or ACS. We demonstrated that the
platelet GPVI expression levels are significantly elevated in
patients with ACS when compared with those with SAP.
The platelet GPVI surface expression determined on admission is associated with ACS, independent of markers of myocardial ischaemia (troponin and CK). Enhanced GPVI levels
on hospital admission are associated with the development
of ACS and myocardial infarction. The findings of the
present work imply that platelet GPVI may be an early
Downloaded from by guest on October 21, 2014
We consecutively investigated the platelet surface
expression of collagen receptor GPVI in a total of 367
patients with symptomatic CAD. In all patients, the diagnosis was verified and the severity of the disease was assessed
by coronary angiography. The demographic details are given
in Table 1.
The platelet surface expression of GPVI was significantly
enhanced in patients with ACS when compared with patients
with stable CAD (SAP) [ACS (MFI + SD): 21.4 + 9.7; SAP:
18.6 + 7.1; P ¼ 0.004] (Figure 1A). Patients with acute myocardial infarction (particularly STEMI) tended to reveal the
highest GPVI expression levels compared with patients
with minor myocardial ischaemia (troponin positive ACS),
although this did not reach statistical significance [Tn pos.
ACS (n ¼ 57): 21.6 + 9.2; NSTEMI (n ¼ 51): 20.7 + 8.2;
P ¼ 0.672; STEMI (n ¼ 19): 24 + 14.2; P ¼ 0.894] (Figure 1B).
Similarly, CD62P surface expression was significantly elevated
in patients with ACS compared with SAP [ACS (MFI + SD):
15.4 + 11.4; SAP: 13.5 + 8.3; P ¼ 0.036] (Figure 1A and B).
Platelet GPVI correlated with CD62P surface expression
levels (r ¼ 0.702; P ¼ 0.001) (Figure 1C). In order to obtain
the information about normal platelet GPVI expression in
apparently healthy people, we investigated our lab personnel
(n ¼ 30; MFI + SD: 15.6 + 4.7). These controls did not
receive coronary angiography. We then examined a group of
subjects in whom CAD was excluded. These patients were
angiographied because of chest pain but did not have CAD
(n ¼ 15; MFI + SD: 16.2 + 2.7). The mean MFI for GPVI
expression of both controls combined was 15.8 + 4.3
(n ¼ 45; Figure 1, dotted line).
Next, we asked whether platelet GPVI correlates with
markers of myocardial ischaemia. We found that GPVI levels
determined on admission did not correlate with the respective initial Tn-I or CK values (initial Tn: r ¼ 0.035, P ¼ 0.567;
initial CK: r ¼ 0.069, P ¼ 0.232) (Figure 2A). In contrast,
GPVI levels correlated with maximum Tn-I or CK values determined later on during hospital stay (maximum Tn: r ¼ 0.178,
P ¼ 0.031; maximum CK: r ¼ 0.181, P ¼ 0.047) (Figure 2A).
No correlation was found for GPVI and C-reactive protein
levels.
2167
2168
B. Bigalke et al.
Table 2
of GPVI
Multifactorial analysis of covariance for the logarithm
Category
Factor
P-value
Medication
ACE-inhibitors
Angiotensin
receptor blockers
Beta-blockers
Statins
Aspirin
Clopidogrel
Vitamin K antagonist
0.061
0.844
Cardiovascular
risk factors
Arterial hypertension
Hyperlipidaemia
Diabetes
History of CAD
Smoking
0.713
0.622
0.969
0.370
0.872
Conventional laboratory
markers
Initial troponin I (ng/mL)
Initial CK (U/L)
Initial C-reactive
protein (mg/dL)
0.110
0.484
0.216
Groups
ACS vs. SAP
0.024
0.546
0.912
0.456
0.121
0.586
biomarker for the identification of patients with ACS.
Because GPVI levels may be elevated when markers of myocardial necrosis are still within the normal range in patients
admitted to hospital with ACS, it is tempting to speculate
that a platelet-specific thrombotic marker may help to
define patients at risk before myocardial ischaemia is
evident.
Evaluation of patients who present at the hospital with a
complaint of chest pain or other signs or symptoms suggestive
of ACS is time-consuming and problematic. Recent investigations have indicated that increases in biomarkers upstream
from biomarkers of necrosis (cardiac troponins I and T), such
as markers of inflammation (e.g. C-reactive protein and
CD40L) or myocardial stretch (BNP), may provide an earlier
assessment of overall patient risk and help in identifying
patients with higher risk of an adverse event.
Platelet activation and platelet-mediated thrombus formation at the site of plaque rupture are the initial trigger
events in ACS.15 At the site of plaque rupture, subendothelial extracellular matrix proteins, namely collagen, are
exposed to the blood stream that are rapidly covered by a
layer of activated platelets.1 GPVI is the major collagen
receptor that mediates platelet adhesion and activation to
fibrillar collagen.2,14,16 GPVI expression is limited to the
megakaryocyte system and is, thus, platelet-specific.2
Limited data are available, to date, that document
changes in GPVI expression levels on circulating platelets
in humans. Previously, we found that GPVI is altered in
type 2 diabetes.10 Others found that GPVI levels are
enhanced in patients who suffered from myocardial infarction.11 Genetic elements seem to play an important role in
the development of coronary thrombosis and fatal myocardial infarction, as demonstrated by a study on GPVI
Downloaded from by guest on October 21, 2014
Figure 2 Correlation of platelet GPVI with markers of myocardial injury. (A) Correlation of platelet GPVI surface expression with initial Tn-I and initial CK at the
time of hospital admission. (B) Correlation of platelet GPVI surface expression on admission with maximum values of troponin and CK determined in the time
course after hospital admission.
Platelet GPVI in ACS
2169
Table 3 Association of GPVI after adjustment for conventional
laboratory markers
Parameters
GPVI (MFI)
Initial Tn-I (ng/mL)
Initial CK (U/L)
Initial C-reactive
protein (mg/dL)
P-value
0.013
0.167
0.025
0.882
OR
1.021
1.966
1.007
1.009
95% CI for OR
Lower
Upper
1.021
0.754
1.001
0.899
1.189
5.124
1.014
1.132
Acknowledgement
We thank Heike Runge, Iris Scha
¨fer, Felix Waibel, and Andreas
Scho
¨nfeld for expert technical assistance.
Conflict of interest: none declared.
References
1. Ruggeri ZM. Platelets in atherothrombosis. Nat Med 2002;8:1227–1234.
Downloaded from by guest on October 21, 2014
polymorphism.17 We did not investigate our cohort for GPVI
polymorphisms, but GPVI polymorphisms may significantly
contribute to the risk for a coronary event independently
of platelet GPVI expression. In the present study, we used
a specific anti-GPVI monoclonal antibody that was developed for flow cytometric analysis, as described recently.10
We found in a large consecutive cohort of patients with
symptomatic CAD that the platelet surface expression of
GPVI is enhanced in patients with ACS and myocardial infarction compared with those with stable CAD. Moreover, we
found that the GPVI expression levels determined on admission are associated with ACS, independent of myocardial
necrosis marker such as troponin or CK. Our data strengthen
the concept that platelet activation occurs in ACS and is
critical for disease progression. Further, we provide evidence that a platelet-specific activation marker that plays
a critical pathophysiological role in ACS (GPVI/collagen
interaction) may be a promising candidate for a biomarker
for risk stratification in patients with ACS. Determination
of elevated GPVI levels may indicate a prothrombotic state
in patients developing ACS and may serve as an early
biomarker of imminent myocardial ischaemia.
2. Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the
central receptor? Blood 2003;102:449–461.
3. Kahn ML. Platelet–collagen responses: molecular basis and therapeutic
promise. Semin Thromb Hemost 2004;30:419–425.
4. Moroi M, Jung SM. Platelet glycoprotein VI: its structure and function.
Thromb Res 2004;114:221–233.
5. Lindemann S, Tolley ND, Eyre JR, Kraiss LW, Mahoney TM, Weyrich AS.
Integrins regulate the intracellular distribution of eukaryotic initiation
factor 4E in platelets. A checkpoint for translational control. J Biol
Chem 2001;276:33947–33951.
6. Lindemann S, Tolley ND, Dixon DA, McIntyre TM, Prescott SM,
Zimmerman GA, Weyrich AS. Activated platelets mediate inflammatory
signaling by regulated interleukin 1beta synthesis. J Cell Biol 2001;
154:485–490.
7. Lindemann SW, Weyrich AS, Zimmerman GA. Signaling to translational
control pathways: diversity in gene regulation in inflammatory and
vascular cells. Trends Cardiovasc Med 2005;15:9–17.
8. Penz S, Reininger AJ, Brandl R, Goyal P, Rabie T, Bernlochner I, Rother E,
Goetz C, Engelmann B, Smethurst PA, Ouwehand WH, Farndale R,
Nieswandt B, Siess W. Human atheromatous plaques stimulate thrombus
formation by activating platelet glycoprotein VI. FASEB J 2005;19:
898–909.
9. Cosemans JM, Kuijpers MJ, Lecut C, Loubele ST, Heeneman S, JandrotPerrus M, Heemskerk JW. Contribution of platelet glycoprotein VI to
the thrombogenic effect of collagens in fibrous atherosclerotic lesions.
Atherosclerosis 2005;181:19–27.
10. Cabeza N, Li Z, Schulz C, Kremmer E, Massberg S, Bultmann A, Gawaz M.
Surface expression of collagen receptor Fc receptor-gamma/glycoprotein
VI is enhanced on platelets in type 2 diabetes and mediates release of
CD40 ligand and activation of endothelial cells. Diabetes 2004;53:
2117–2121.
11. Samaha FF, Hibbard C, Sacks J, Chen H, Varello MA, George T, Kahn ML.
Density of platelet collagen receptors glycoprotein VI and alpha2beta1
and prior myocardial infarction in human subjects, a pilot study. Med
Sci Monit 2005;11: CR224–CR229.
12. Gawaz M, Neumann FJ, Schomig A. Evaluation of platelet membrane glycoproteins in coronary artery disease: consequences for diagnosis and
therapy. Circulation 1999;99:E1–E11.
13. Fateh-Moghadam S, Bocksch W, Ruf A, Dickfeld T, Schartl M,
Pogatsa-Murray G, Hetzer R, Pleck E, Gawaz M. Changes in surface
expression of platelet membrane glycoproteins and progression of
heart transplant vasculopathy. Circulation 2000;102:890–897.
14. Massberg S, Konrad I, Bultmann A, Schulz C, Munch G, Peluso M, Lorenz M,
Schneider S, Bosta F, Muller I, Hu B, Langer H, Kremmer E, Rudelius M,
Heinzmann U, Ungerer M, Gawaz M. Soluble glycoprotein VI dimer inhibits
platelet adhesion and aggregation to the injured vessel wall in vivo.
FASEB J 2004;18:397–399.
15. Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis.
J Clin Invest 2005;115:3378–3384.
16. Massberg S, Gawaz M, Gruner S, Schulte V, Konrad I, Zohlnhofer D,
Heinzmann U, Nieswandt B. A crucial role of glycoprotein VI for platelet
recruitment to the injured arterial wall in vivo. J Exp Med 2003;197:
41–49.
17. Ollikainen E, Mikkelsson J, Perola M, Penttila A, Karhunen PJ. Platelet
membrane collagen receptor glycoprotein VI polymorphism is associated
with coronary thrombosis and fatal myocardial infarction in middle-age
men. Atherosclerosis 2004;176:95–99.