Chronic coumarin treatment is associated with increased extracoronary arterial calcification in humans

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).

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.

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.