Vascular calcification is a marker of increased cardiovascular risk. Vitamin K–dependent matrix Gla protein (MGP) is important in inhibiting calcification. Because MGP activation is vitamin K dependent, we performed a cross-sectional study investigating the relationship between the use of vitamin K antagonists and extracoronary vascular calcification. From the Dutch thrombosis services we selected 19 patients younger than 55 years who had no other cardiovascular risk factors and who had used coumarins for more than 10 years, and compared these to 18 matched healthy controls. MGP was measured, and a plain x-ray of the thighs was taken to assess femoral arterial calcifications. The odds ratio for calcification in patients versus controls was 8.5 (95% confidence interval [CI] 2.01-35.95). Coumarin use and MGP were associated with calcification, even after adjusting for other risk factors. We conclude that long-term use of coumarins is associated with enhanced extracoronary vascular calcification, possibly through the inhibition of MGP carboxylation.

Vascular calcification is a marker of increased cardiovascular morbidity and mortality.1  Matrix Gla protein (MGP) is an important inhibitor of calcification.2-5  In animal studies, where carboxylation of MGP was blocked by vitamin K antagonists, excessive calcifications of the arteries were found.6  In humans, calcification of the coronary arteries (CaC) and heart valves is increased in patients on vitamin K antagonists, whereas intake of vitamin K is associated with less progression of CaC.7-10  Interestingly, the association between coumarin use and CaC is absent in an older population, and there are no reports on extracoronary arterial calcification in coumarin users.11  Therefore, we performed a cross-sectional study in middle-aged long-term coumarin users and a matched control group to test the hypothesis that chronic coumarin therapy is associated with femoral artery calcification as a proxy for coronary calcification along with decreased carboxylation of MGP.

We searched the database of the southern Dutch thrombosis services. For the present study, we selected patients younger than 55 years who had used coumarins for more than 10 years because of a cardiac valve operation or recurrent venous thrombosis, and who had no previous cardiovascular events. Spouses or close friends living in the same (social) environment were invited as control subjects. The study was approved by the Maastricht University Medical Center ethics committee, and all subjects gave informed consent in accordance with the Declaration of Helsinki.

Clinical assessments included data on smoking behavior, body mass index (kg/m2), and blood pressure (average of 3 office measurements [Accutor Plus; Datascope Corporation ]). Fasting glucose levels, lipid profile, calcium, phosphate, and creatinine were measured in serum with an automated analyzer (Beckmann Synchron CX 7-2). Endogenous creatinine clearance (ECC) was estimated using the Cockcroft and Gault formula.12  Desphospho-uncarboxylated MGP (dp-ucMGP) was measured in plasma using a sandwich enzyme-linked immunosorbent assay (ELISA; VitaK BV) as has been described previously.13  Femoral artery calcification was assessed by soft-tissue 50-kV x-ray of the left and right thigh (Siemens Aristos FX DR-Radiology system) in supine position and slight endorotation of the foot. The images were digitally processed to enhance soft-tissue structures (Diamond View; Siemens) and evaluated by an independent radiologist (T.L.) unaware of the clinical data. A subject was scored positive for calcification when calcium deposits were visible along one or both femoral artery regions.

Normally distributed variables are presented as means with standard deviation; otherwise, they are presented as medians with minimum and maximum values. Differences between groups were assessed using the Student t test or the Mann-Whitney U test for continuous variables and the χ2 test for ordinal and dichotomous variables. Calcification was correlated with age, sex, smoking, coumarin use, body mass index (BMI), systolic and diastolic blood pressure, fasting glucose, lipid profile, serum creatinine, ECC, calcium, phosphate, calcium-phosphate product, and dp-ucMGP using the Spearman test for nonnormally distributed variables, or the Pearson test for normally distributed data. Multiple logistic regression analyses were done with several models to evaluate the independent contribution of coumarins and dp-ucMGP to calcification. Coumarin use and dp-ucMGP were analyzed separately because of collinearity. The excess risk for calcification has been expressed as odds ratio. We used SPSS 16.0.1 (SPSS Inc) for statistical calculations; a P value less than .05 was considered statistically significant.

Of 21 identified patients, 2 refused to participate. A total of 18 control subjects volunteered. The characteristics of patients and controls are presented in Table 1. Median coumarin treatment duration was 13 years (range, 10-29 years). Target international normalized ratio (INR) in all patients (3 with aortic valve replacement) was 2.5 (range, 2.0-3.0) Univariate analysis showed a correlation between femoral artery calcification and coumarin use (r = .515, P < .001) and plasma dp-ucMGP levels (r = .585, P < .001). Coumarin use and plasma dp-ucMGP levels showed a strong correlation (r = .850, P < .001). Calcification was visible in 14 of 19 coumarin users compared with 4 of 18 controls (χ2 = 9.8, P = .002). The average dp-ucMGP level was 1439pM (± 481pM) versus 299pM (± 163pM) in coumarin users and controls, respectively (P < .001). The odds ratio for calcification in coumarin users was 8.49 (95% CI 2.01-35.95). In multiple regression analysis, coumarin use and dp-ucMGP levels were independently associated with the presence of calcification, and were also independent of known or accepted modifiers (Table 2).

Table 1

Characteristics of participants according to coumarin use

VariableCoumarin, N = 19No coumarin, N = 18P*
Sex, male/female 15/4 9/9 .065 
Age, y (range) 48 (33-56) 46 (36-53) .213 
(Ever) smoking, % 68 50 .254 
Body mass index, kg/m2 29 (± 5) 25 (± 4) .015 
Systolic blood pressure, mmHg 128 (± 13) 125 (± 19) .592 
Diastolic blood pressure, mmHg 81 (± 8) 80 (± 14) .676 
Glucose, mmol/L (range) 5.1 (4.3-9.0) 4.9 (4.2-6.2) .807 
Total cholesterol, mmol/L 5.9 (± 1.2) 5.5 (± 1.1) .303 
HDL cholesterol, mmol/L 1.18 (± 0.3) 1.40 (± 0.4) .058 
LDL cholesterol, mmol/L 4.1 (± 0.8) 3.7 (± 0.9) .173 
Triglycerides, mmol/l 1.57 (± 0.9) 0.98 (± 0.6) .023 
Creatinine, μmol/L 81 (± 11.5) 79 (± 16.4) .577 
Estimated creatinine clearance, mL/min 120 (± 27) 105 (± 24) .077 
Median calcium-phosphate product (range) 2.3 (1.5-4.4) 2.6 (2.3-3.4) .104 
VariableCoumarin, N = 19No coumarin, N = 18P*
Sex, male/female 15/4 9/9 .065 
Age, y (range) 48 (33-56) 46 (36-53) .213 
(Ever) smoking, % 68 50 .254 
Body mass index, kg/m2 29 (± 5) 25 (± 4) .015 
Systolic blood pressure, mmHg 128 (± 13) 125 (± 19) .592 
Diastolic blood pressure, mmHg 81 (± 8) 80 (± 14) .676 
Glucose, mmol/L (range) 5.1 (4.3-9.0) 4.9 (4.2-6.2) .807 
Total cholesterol, mmol/L 5.9 (± 1.2) 5.5 (± 1.1) .303 
HDL cholesterol, mmol/L 1.18 (± 0.3) 1.40 (± 0.4) .058 
LDL cholesterol, mmol/L 4.1 (± 0.8) 3.7 (± 0.9) .173 
Triglycerides, mmol/l 1.57 (± 0.9) 0.98 (± 0.6) .023 
Creatinine, μmol/L 81 (± 11.5) 79 (± 16.4) .577 
Estimated creatinine clearance, mL/min 120 (± 27) 105 (± 24) .077 
Median calcium-phosphate product (range) 2.3 (1.5-4.4) 2.6 (2.3-3.4) .104 

HDL indicates high-density lipoprotein; and LDL, low-density lipoprotein.

P values shown in bold indicate a significant difference.

*

The χ 2 test was used for dichotomous variables, the Student t test for normally distributed values, and the Mann-Whitney U test for not normally distributed variables.

Table 2

Regression analysis with femoral artery calcification as the dependent variable

ModelCoumarin use beta (95% CI)dp-ucMGP beta (95% CI)
0.515 (0.220-0.809) 0.661 (0.306-1.016) 
0.484 (0.186-0.782) 0.623 (0.261-0.985) 
0.443 (0.128-0.758) 0.584 (0.184-0.984) 
0.510 (0.166-0.854) 0.725 (0.284-1.165) 
0.509 (0.173-0.846) 0.680 (0.254-1.105) 
ModelCoumarin use beta (95% CI)dp-ucMGP beta (95% CI)
0.515 (0.220-0.809) 0.661 (0.306-1.016) 
0.484 (0.186-0.782) 0.623 (0.261-0.985) 
0.443 (0.128-0.758) 0.584 (0.184-0.984) 
0.510 (0.166-0.854) 0.725 (0.284-1.165) 
0.509 (0.173-0.846) 0.680 (0.254-1.105) 

Results are expressed as the beta for coumarin use and dp-ucMGP levels with their 95% confidence interval (CI). Model 1: crude; model 2: adjusted for age; model 3: adjusted for age and sex; model 4: adjusted for age, smoking, body mass index, and triglycerides; model 5: adjusted for age, fasting glucose, LDL cholesterol, estimated creatinine clearance, and calcium phosphate product.

This is the first study showing a relationship between coumarins and extracoronary calcification. Previous research already showed more calcification in animals with vitamin K deficiency due to a mutant vitamin K epoxide reductase subcomponent-1.14,15  In addition, coronary calcifications have been found in patients using vitamin K antagonists, but in contrast to our study, patients were relatively old, had other risk factors for atherosclerosis, or received coumarins for an atherosclerotic cardiovascular indication.7,16,17 

The dp-ucMGP assay described here specifically detects nonphosphorylated and noncarboxylated MGP, which has little or no affinity for calcium salts. It is thought that dp-ucMGP is easily set free in the circulation. Because ucMGP is only formed during vitamin K deficiency, dp-ucMGP serves as a biomarker for vascular vitamin K status. Our data demonstrate a strong association between circulating dp-ucMGP and coumarin use, which is consistent with increased synthesis of inactive ucMGP, and subsequently less inhibition of the calcification process. In response to progressive calcification, MGP production will be up-regulated, thus exhausting the local vitamin K stores even further, and explaining higher levels of plasma dp-ucMGP, in subjects using coumarins.18,19 

In this study, we only measured femoral vascular calcification. We cannot be sure that all vessels calcify in the same manner, although it has been shown that peripheral vascular calcification correlates with coronary calcification.20  Because all vascular calcifications are associated with increased cardiovascular risk, using coumarins may have an increased cardiovascular risk despite short-term benefits of decreased thrombosis tendency.1,21-23  This would indicate a hitherto unrecognized adverse side effect of long-term coumarin use in young subjects. However, prospective and long-term studies are necessary to clarify this issue further.

There are several limitations of this study. First, it is a cross-sectional analysis. A cause-and-effect relationship could therefore not be investigated. Second, the study result was obtained in a relatively small and selected population and should be reproduced in other cohorts. Third, plain X-ray is not the most sensitive technique to detect arterial calcification, but computer tomography would have involved more radiation and is more expensive. Last, there is a difference in sex distribution between the study groups. However, including sex as a confounder in multiple regression analysis did not significantly change the outcome, and there is no relationship between sex and MGP levels.16  The strength of our study was that we only studied patients without other cardiovascular disease, a relatively young population (so no age bias), and the demonstration that coumarin use affected calcification independently of other risk factors.

We conclude that long-term use of vitamin K antagonists is associated with femoral artery calcification, which is possibly enhanced through the inactivation of MGP.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Contribution: R.J.M.W.R., P.W.d.L., and A.A.K. designed the research and wrote the paper; R.J.M.W.R. and B.J.v.V. performed the actual study; L.J.S., K.H., and H.t.C. recruited patients and gave expert advice on interpreting MGP data and coumarin data; T.L. evaluated the radiologic data and contributed to the text about the radiologic aspects; and C.V. performed MGP testing and gave advice on interpreting the data.

Conflict-of-interest disclosure: C.V. is CEO of VitaK BV, The Netherlands. The remaining authors declare no competing financial interests.

Correspondence: Roger J. M. W. Rennenberg, Department of Internal Medicine, Maastricht University Medical Centre, P. Debyelaan 25, PO Box 5800, 6202 AZ Maastricht, The Netherlands; e-mail: rennen007@gmail.com.

1
Rennenberg
 
RJ
Kessels
 
AG
Schurgers
 
LJ
van Engelshoven
 
JM
de Leeuw
 
PW
Kroon
 
AA
Vascular calcifications as a marker of increased cardiovascular risk: a meta-analysis.
Vasc Health Risk Manag
2009
, vol. 
5
 
1
(pg. 
185
-
197
)
2
Dhore
 
CR
Cleutjens
 
JP
Lutgens
 
E
, et al. 
Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.
Arterioscler Thromb Vasc Biol
2001
, vol. 
21
 
12
(pg. 
1998
-
2003
)
3
Price
 
PA
Urist
 
MR
Otawara
 
Y
Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone.
Biochem Biophys Res Commun
1983
, vol. 
117
 
3
(pg. 
765
-
771
)
4
Zebboudj
 
AF
Imura
 
M
Bostrom
 
K
Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2.
J Biol Chem
2002
, vol. 
277
 
6
(pg. 
4388
-
4394
)
5
Zebboudj
 
AF
Shin
 
V
Bostrom
 
K
Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells.
J Cell Biochem
2003
, vol. 
90
 
4
(pg. 
756
-
765
)
6
Price
 
PA
Faus
 
SA
Williamson
 
MK
Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves.
Arterioscler Thromb Vasc Biol
1998
, vol. 
18
 
9
(pg. 
1400
-
1407
)
7
Schurgers
 
LJ
Aebert
 
H
Vermeer
 
C
Bultmann
 
B
Janzen
 
J
Oral anticoagulant treatment: friend or foe in cardiovascular disease?
Blood
2004
, vol. 
104
 
10
(pg. 
3231
-
3232
)
8
Koos
 
R
Mahnken
 
AH
Muhlenbruch
 
G
, et al. 
Relation of oral anticoagulation to cardiac valvular and coronary calcium assessed by multislice spiral computed tomography.
Am J Cardiol
2005
, vol. 
96
 
6
(pg. 
747
-
749
)
9
Lerner
 
RG
Aronow
 
WS
Sekhri
 
A
, et al. 
Warfarin use and the risk of valvular calcification.
J Thromb Haemost
2009
, vol. 
7
 
12
(pg. 
2023
-
2027
)
10
Shea
 
MK
O'Donnell
 
CJ
Hoffmann
 
U
, et al. 
Vitamin K supplementation and progression of coronary artery calcium in older men and women.
Am J Clin Nutr
2009
, vol. 
89
 
6
(pg. 
1799
-
1807
)
11
Villines
 
TC
O'Malley
 
PG
Feuerstein
 
IM
Thomas
 
S
Taylor
 
AJ
Does prolonged warfarin exposure potentiate coronary calcification in humans? Results of the Warfarin and Coronary Calcification Study.
Calcif Tissue Int
2009
, vol. 
85
 
6
(pg. 
494
-
500
)
12
Cockcroft
 
DW
Gault
 
MH
Prediction of creatinine clearance from serum creatinine.
Nephron
1976
, vol. 
16
 
1
(pg. 
31
-
41
)
13
Schurgers
 
LJ
Cranenburg
 
EC
Vermeer
 
C
Matrix Gla-protein: the calcification inhibitor in need of vitamin K.
Thromb Haemost
2008
, vol. 
100
 
4
(pg. 
593
-
603
)
14
Kohn
 
MH
Price
 
RE
Pelz
 
HJ
A cardiovascular phenotype in warfarin-resistant Vkorc1 mutant rats.
Artery Res
2008
, vol. 
2
 
4
(pg. 
138
-
147
)
15
Teichert
 
M
Visser
 
LE
van Schaik
 
RH
, et al. 
Vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphism and aortic calcification: the Rotterdam Study.
Arterioscler Thromb Vasc Biol
2008
, vol. 
28
 
4
(pg. 
771
-
776
)
16
Cranenburg
 
EC
Vermeer
 
C
Koos
 
R
, et al. 
The circulating inactive form of matrix Gla protein (ucMGP) as a biomarker for cardiovascular calcification.
J Vasc Res
2008
, vol. 
45
 
5
(pg. 
427
-
436
)
17
Koos
 
R
Krueger
 
T
Westenfeld
 
R
, et al. 
Relation of circulating Matrix Gla-Protein and anticoagulation status in patients with aortic valve calcification.
Thromb Haemost
2009
, vol. 
101
 
4
(pg. 
706
-
713
)
18
Shanahan
 
CM
Cary
 
NR
Metcalfe
 
JC
Weissberg
 
PL
High expression of genes for calcification-regulating proteins in human atherosclerotic plaques.
J Clin Invest
1994
, vol. 
93
 
6
(pg. 
2393
-
2402
)
19
Shanahan
 
CM
Cary
 
NR
Salisbury
 
JR
Proudfoot
 
D
Weissberg
 
PL
Edmonds
 
ME
Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification.
Circulation
1999
, vol. 
100
 
21
(pg. 
2168
-
2176
)
20
Reaven
 
PD
Sacks
 
J
Coronary artery and abdominal aortic calcification are associated with cardiovascular disease in type 2 diabetes.
Diabetologia
2005
, vol. 
48
 
2
(pg. 
379
-
385
)
21
Raggi
 
P
Shaw
 
LJ
Berman
 
DS
Callister
 
TQ
Prognostic value of coronary artery calcium screening in subjects with and without diabetes.
J Am Coll Cardiol
2004
, vol. 
43
 
9
(pg. 
1663
-
1669
)
22
Pidal
 
D
Sanchez Vidal
 
MT
Rodriguez
 
JC
, et al. 
Relationship between arterial vascular calcifications seen on screening mammograms and biochemical markers of endothelial injury.
Eur J Radiol
2009
, vol. 
69
 
1
(pg. 
87
-
92
)
23
Raggi
 
P
Callister
 
TQ
Cooil
 
B
, et al. 
Identification of patients at increased risk of first unheralded acute myocardial infarction by electron-beam computed tomography.
Circulation
2000
, vol. 
101
 
8
(pg. 
850
-
855
)
Sign in via your Institution