Circulating uncarboxylated matrix Gla protein, a marker of vitamin K status, as a risk factor of cardiovascular disease.

OBJECTIVES: Vitamin K plays a pivotal role in the synthesis of Matrix Gla protein (MGP), a calcification inhibitor in vascular tissue. Vascular calcification has become an important predictor of cardiovascular disease. The aim of the current study was to examine the potential association of circulating desphospho-carboxylated and -uncarboxylated MGP (dp-cMGP and dp-ucMGP), reflecting vitamin K status, with the incidence of cardiovascular events and disease (CVD) in older individuals. STUDY DESIGN: The study was conducted in 577 community-dwelling older men and women of the Longitudinal Aging Study Amsterdam (LASA), aged >55 year, who were free of cardiovascular disease at baseline. Multivariate Cox proportional hazards models were used to analyze the data. MAIN OUTCOME MEASURES: Incidence of CVD. RESULTS: After a mean follow-up of 5.6±1.2 year, we identified 40 incident cases of CVD. After adjustment for classical confounders and vitamin D status, we observed a more than 2-fold significantly higher risk of CVD for the highest tertile of dp-ucMGP with a HR of 2.69 (95% CI, 1.09-6.62) as compared with the lowest tertile. Plasma dp-cMGP was not associated with the risk of CVD. CONCLUSIONS: Vitamin K insufficiency, as assessed by high plasma dp-ucMGP concentrations is associated with an increased risk for cardiovascular disease independent of classical risk factors and vitamin D status. Larger epidemiological studies on dp-ucMGP and CVD incidence are needed followed by clinical trials to test whether vitamin K-rich diets will lead to a decreased risk for cardiovascular events.

Matrix Gla protein species and risk of cardiovascular events in type 2 diabetic patients.

OBJECTIVE: To investigate the relationship of circulating matrix Gla protein (MGP) species with incident cardiovascular disease (CVD) or coronary heart disease (CHD) in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: EPIC-NL is a prospective cohort study among 40,011 Dutch men and women. At baseline (1993-1997), 518 participants were known to have type 2 diabetes. MGP levels were measured by ELISA techniques in baseline plasma samples. The incidence of fatal and nonfatal CVD and CVD subtypes-CHD, peripheral arterial disease (PAD), heart failure, and stroke-were obtained by linkage to national registers. Cox proportional hazard models were used to calculate hazard ratios (HRs), adjusted for sex, waist-to-hip ratio, physical activity, and history of CVD. RESULTS: During a median 11.2 years of follow-up, 160 cases of CVD were documented. Higher circulating desphospho-uncarboxylated MGP (dp-ucMGP) levels were significantly associated with higher risk of CVD, with an HR per SD (HRSD) of 1.21 (95% CI 1.06-1.38), PAD (HRSD 1.32 [95% CI 1.07-1.65]), and heart failure (HRSD 1.75 [95% CI 1.42-2.17]) after adjustment. Higher circulating dp-ucMGP levels were not related to risk of CHD (HRSD 1.12 [95% CI 0.94-1.34]) or stroke (HRSD 1.05 [95% CI 0.73-1.49]). Circulating desphospho-carboxylated MGP and circulating total-uncarboxylated MGP levels were not associated with CVD or CVD subtypes. CONCLUSIONS: High dp-ucMGP levels were associated with increased CVD risk among type 2 diabetic patients, especially with the subtypes PAD and heart failure, while other MGP species were not related to CVD risk. These results suggest that a poor vitamin K status is associated with increased CVD risk.

Circulating matrix Gla protein is associated with coronary artery calcification and vitamin K status in healthy women.

Matrix Gla protein (MGP) is a vitamin K-dependent protein and an inhibitor of vascular calcification. Vitamin K is required for the carboxylation of MGP and can thereby reduce calcification. Circulating MGP species with different conformations have been investigated as markers for coronary artery calcification (CAC). In high-risk populations, high total uncarboxylated MGP (t-ucMGP) was associated with decreased CAC, while high non-phosphorylated uncarboxylated MGP (dp-ucMGP) was associated with a poor vitamin K status. This cross-sectional study investigated the association of MGP species with CAC, vitamin K status among 200 healthy women. Circulating dp-ucMGP, t-ucMGP and, non-phosphorylated carboxylated MGP (dp-cMGP) levels were measured by ELISA techniques and Agatston score by multi-detector computed tomography. The ratio of uncarboxylated to carboxylated osteocalcin was used as proxy of vitamin K status. A borderline significant (P=.06) association between higher circulating dp-ucMGP levels and high CAC was observed (ß=0.091, 95% CI-0.01; 0.19). In the entire study population, high t-ucMGP levels tended to be associated (P=.09) with lower CAC (ß=-0.36, 95% CI:-0.78; 0.06). This association strengthened amongst women with CAC to a significant relation between high t-ucMGP levels and lower CAC (ß=-0.55, 95% CI-1.01;-0.10). Dp-cMGP was not associated with CAC. Low vitamin K-status was associated with high dp-ucMGP concentrations (ß=0.138, 95% CI 0.09; 0.19) but not with other MGP species. These results show that dp-ucMGP may serve as a biomarker of vitamin K status. Circulating dp-ucMGP and t-ucMGP may serve as markers for the extent of CAC, but these findings need to be confirmed.

Vitamin K intake and status are low in hemodialysis patients.

Vitamin K is essential for the activity of γ-carboxyglutamate (Gla)-proteins including matrix Gla28 protein and osteocalcin; an inhibitor of vascular calcification and a bone matrix protein, respectively. Insufficient vitamin K intake leads to the production of non-carboxylated, inactive proteins and this could contribute to the high risk of vascular calcification in hemodialysis patients. To help resolve this, we measured vitamin K(1) and K(2) intake (4-day food record), and the vitamin K status in 40 hemodialysis patients. The intake was low in these patients (median 140 µg/day), especially on days of dialysis and the weekend as compared to intakes reported in a reference population of healthy adults (mean K(1) and K(2) intake 200 µg/day and 31 µg/day, respectively). Non-carboxylated bone and coagulation proteins were found to be elevated in 33 hemodialysis patients, indicating subclinical hepatic vitamin K deficiency. Additionally, very high non-carboxylated matrix Gla28 protein levels, endemic to all patients, suggest vascular vitamin K deficiency. Thus, compared to healthy individuals, hemodialysis patients have a poor overall vitamin K status due to low intake. A randomized controlled trial is needed to test whether vitamin K supplementation reduces the risk of arterial calcification and mortality in hemodialysis patients.

Low-dose menaquinone-7 supplementation improved extra-hepatic vitamin K status, but had no effect on thrombin generation in healthy subjects.

Vitamin K is required for the carboxylation of Gla-proteins in the liver (coagulation factors) and extra-hepatic tissues, such as bone (osteocalcin, OC), and arterial wall (matrix Gla-protein, MGP). Although the coagulation factors are essentially fully carboxylated under normal conditions, 10-40 % of OC and MGP remains undercarboxylated. We were therefore interested to study the dose-response effects of extra intake of menaquinones on the carboxylation of the extra-hepatic Gla-proteins. A total of forty-two healthy Dutch men and women aged between 18 and 45 years were randomised into seven groups to receive: placebo capsules or menaquinone-7 (MK-7) capsules at a daily dose of 10, 20, 45, 90, 180 or 360 µg. Circulating uncarboxylated OC (ucOC), carboxylated OC (cOC) and desphospho-uncarboxylated MGP were measured by ELISA. The ucOC:cOC ratio was calculated from circulating ucOC and cOC values. Endogenous thrombin potential and peak height were determined by calibrated automated thrombography. To increase the statistical power, we collapsed the treatment groups into three dosage groups: placebo, low-dose supplementation (doses below RDA, Commission Directive 2008/100/EC), and high-dose supplementation (doses around RDA, Commission Directive 2008/100/EC). MK-7 supplementation at doses in the order of the RDA (Commission Directive 2008/100/EC) increased the carboxylation of circulating OC and MGP. No adverse effects on thrombin generation were observed. Extra MK-7 intake at nutritional doses around the RDA (Commission Directive 2008/100/EC) improved the carboxylation of the extra-hepatic vitamin K-dependent proteins. Whether this improvement contributes to public health, i.e. increasing the protection against age-related diseases needs further investigation in specifically designed intervention trials.

Effect of vitamin K2 supplementation on functional vitamin K deficiency in hemodialysis patients: a randomized trial.

BACKGROUND: Vascular calcification is a predictor of cardiovascular morbidity and mortality. Hemodialysis patients experience severe vascular calcifications. Matrix Gla protein (MGP) is a central calcification inhibitor of the arterial wall; its activity depends on vitamin K-dependent γ-glutamate carboxylation. Uncarboxylated MGP, formed as a result of vitamin K deficiency, is associated with cardiovascular disease. Recent studies suggest poor vitamin K status in hemodialysis patients. We therefore aimed to investigate whether daily vitamin K supplementation improves the bioactivity of vitamin K-dependent proteins in hemodialysis patients, assessed by circulating dephosphorylated-uncarboxylated MGP, uncarboxylated osteocalcin, and uncarboxylated prothrombin (PIVKA-II [protein induced by vitamin K absence II]). STUDY DESIGN: Interventional randomized non-placebo-controlled trial with 3 parallel groups. SETTING & PARTICIPANTS: 53 long-term hemodialysis patients in stable conditions, 18 years or older. 50 healthy age-matched individuals served as controls. INTERVENTIONS: Menaquinone-7 (vitamin K(2)) treatment at 45, 135, or 360 µg/d for 6 weeks. OUTCOMES: Plasma levels of dephosphorylated-uncarboxylated MGP, uncarboxylated osteocalcin, and PIVKA-II. MEASUREMENTS: Plasma levels were assessed using enzyme-linked immunosorbent assays. RESULTS: At baseline, hemodialysis patients had 4.5-fold higher dephosphorylated-uncarboxylated MGP and 8.4-fold higher uncarboxylated osteocalcin levels compared with controls. PIVKA-II levels were elevated in 49 hemodialysis patients. Vitamin K(2) supplementation induced a dose- and time-dependent decrease in circulating dephosphorylated-uncarboxylated MGP, uncarboxylated osteocalcin, and PIVKA-II levels. Response rates in the reduction in dephosphorylated-uncarboxylated MGP levels were 77% and 93% in the groups receiving 135 µg and 360 µg of menaquinone-7, respectively. LIMITATIONS: Small sample size. CONCLUSIONS: This study confirms that most hemodialysis patients have a functional vitamin K deficiency. More importantly, it is the first study showing that inactive MGP levels can be decreased markedly by daily vitamin K(2) supplementation. Our study provides the rationale for intervention trials aimed at decreasing vascular calcification in hemodialysis patients by vitamin K supplementation.

Circulating uncarboxylated matrix gla protein is associated with vitamin K nutritional status, but not coronary artery calcium, in older adults.

Matrix Gla protein (MGP) is a calcification inhibitor in vascular tissue that must be carboxylated by vitamin K to function. Evidence suggests circulating uncarboxylated MGP (ucMGP) is elevated in persons with disease characterized by vascular calcification. The primary purpose of this study was to determine cross-sectional and longitudinal associations between plasma ucMGP, vitamin K status, and coronary artery calcium (CAC) in older adults without coronary heart disease. Genetic determinants of ucMGP were also explored. Cross-sectional associations among baseline plasma ucMGP, vitamin K status biomarkers [plasma phylloquinone, uncarboxylated prothrombin (PIVKA-II), serum uncarboxylated osteocalcin (%ucOC)], CAC, and plausible genetic polymorphisms were examined in 438 community-dwelling adults (60-80 y, 59% women). The effect of phylloquinone supplementation (500 µg/d) for 3 y on plasma ucMGP was determined among 374 participants. At baseline, plasma phylloquinone was lower and %ucOC and PIVKA-II were greater across higher plasma ucMGP quartiles (all P < 0.001, age-adjusted). Major allele homozygotes for MGP rs1800801 and rs4236 had higher plasma ucMGP than heterozygotes or minor allele homozygotes. (P ≤ 0.004). The decrease in plasma ucMGP was greater in the 190 participants who received phylloquinone (mean ± SD) (-345 ± 251 pmol/L) than in the 184 who did not (-40 ± 196 pmol/L) (P < 0.0001). CAC did not differ according to ucMGP quartile (P = 0.35, age-adjusted). In the phylloquinone-supplemented group, the 3-y change in ucMGP was not associated with the 3-y change in CAC [unstandard ß (SE) = -0.02 (0.02); P = 0.44]. Plasma ucMGP was associated with vitamin K status biomarkers and was reduced following phylloquinone supplementation, suggesting it may be a useful marker of vitamin K status in vascular tissue. Plasma ucMGP did not reflect CAC in healthy older adults.

Circulating nonphosphorylated carboxylated matrix gla protein predicts survival in ESRD.

The mechanisms for vascular calcification and its associated cardiovascular mortality in patients with ESRD are not completely understood. Dialysis patients exhibit profound vitamin K deficiency, which may impair carboxylation of the calcification inhibitor matrix gla protein (MGP). Here, we tested whether distinct circulating inactive vitamin K-dependent proteins associate with all-cause or cardiovascular mortality. We observed higher levels of both desphospho-uncarboxylated MGP (dp-ucMGP) and desphospho-carboxylated MGP (dp-cMGP) among 188 hemodialysis patients compared with 98 age-matched subjects with normal renal function. Levels of dp-ucMGP correlated with those of protein induced by vitamin K absence II (PIVKA-II; r = 0.62, P < 0.0001). We found increased PIVKA-II levels in 121 (64%) dialysis patients, indicating pronounced vitamin K deficiency. Kaplan-Meier analysis showed that patients with low levels of dp-cMGP had an increased risk for all-cause and cardiovascular mortality. Multivariable Cox regression confirmed that low levels of dp-cMGP increase mortality risk (all-cause: HR, 2.2; 95% CI, 1.1 to 4.3; cardiovascular: HR, 2.7; 95% CI, 1.2 to 6.2). Furthermore, patients with higher vascular calcification scores showed lower levels of dp-cMGP. In 17 hemodialysis patients, daily supplementation with vitamin K2 for 6 weeks reduced dp-ucMGP levels by 27% (P = 0.003) but did not affect dp-cMGP levels. In conclusion, the majority of dialysis patients exhibit pronounced vitamin K deficiency. Lower levels of circulating dp-cMGP may serve as a predictor of mortality in dialysis patients. Whether vitamin K supplementation improves outcomes requires further study.

Characterisation and potential diagnostic value of circulating matrix Gla protein (MGP) species.

Matrix γ-carboxyglutamate (Gla) protein (MGP) is an important local inhibitor of vascular calcification, which can undergo two post-translational modifications: vitamin K-dependent γ-glutamate carboxylation and serine phosphorylation. While carboxylation is thought to have effects upon binding of calcium-ions, phosphorylation is supposed to affect the cellular release of MGP. Since both modifications can be exerted incompletely, various MGP species can be detected in the circulation. MGP levels were measured with two commercially available competitive and two novel sandwich assays in healthy controls, in patients with rheumatic disease, aortic valve disease, and end-stage renal disease, as well as in volunteers after vitamin K supplementation (VKS) and treatment with vitamin K antagonists (VKA). Major differences were found between the MGP assays, including significantly different behaviour with regard to vascular disease and the response to VKA and VKS. The dual-antibody assay measuring non-phosphorylated, non-carboxylated MGP (dp-ucMGP) was particularly sensitive for these changes and would be suited to assess the vascular vitamin K status. We conclude that the different assays for particular circulating MGP species allows the assessment of various aspects of the MGP system.

The circulating inactive form of matrix gla protein is a surrogate marker for vascular calcification in chronic kidney disease: a preliminary report.

BACKGROUND AND OBJECTIVES: Vitamin K-dependent matrix Gla protein (MGP) acts as a calcification inhibitor in vitro and in vivo. The present study was performed to (1) determine plasma levels of the inactive, dephosphorylated, uncarboxylated MGP (dp-ucMGP) in a cohort of patients at different stages of chronic kidney disease (CKD) and (2) evaluate the association between dp-ucMGP levels on one hand and aortic calcification and mortality on the other. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: 107 patients (67 +/- 13 years; 60% male; 32% at CKD stages 2 to 3, 31% at stages 4 to 5, 37% at stage 5D) were assayed for dp-ucMGP and underwent multislice spiral computed tomography scans to quantify aortic calcification at baseline. They were prospectively monitored for mortality. RESULTS: Plasma dp-ucMGP levels augmented progressively with CKD stage, with a significant difference from CKD stage 4. CKD stage, hemoglobin, age, and coumarin use were independently associated with plasma dp-ucMGP levels. Furthermore, plasma dp-ucMGP and age were positively and independently associated with the aortic calcification score. During follow-up (802 +/- 311 days), 34 patients died (20 from cardiovascular events). In a crude analysis, [plasma dp-ucMGP] > 921 pM was associated with overall mortality; this association was lost after adjusting for both age and the calculated propensity score. CONCLUSIONS: Plasma dp-ucMGP increased progressively in a CKD setting and was associated with the severity of aortic calcification. Plasma dp-ucMGP could thus be a surrogate marker for vascular calcification in CKD.

Factor Va, bound to microparticles released during platelet storage, is resistant to inactivation by activated protein C.

BACKGROUND: Microparticles (MPs) support coagulation and can be helpful in restoring the hemostatic system in thrombocytopenic patients. The anticoagulant properties of MPs shed during storage of platelets (PLTs) have not been studied yet. STUDY DESIGN AND METHODS: Storage-induced MPs were harvested from outdated PLT concentrates. Whether factor (F)Va was present on the surface of these MPs was investigated. The activated protein C (APC)-catalyzed inactivation of MP-bound FVa was further determined. Also, inactivation of FVa at the surface of thrombin-activated PLTs and synthetic vesicles was determined. RESULTS: MPs in stored PLT products carry FVa at their surface. APC-catalyzed inactivation of MP-bound FVa resulted in 42 +/- 2 percent residual FVa activity after 20 minutes. The residual activity of FVa on thrombin-activated PLTs was 25 +/- 3 percent. Plasma-derived FVa was rapidly inactivated in the presence of synthetic vesicles, with 5 +/- 4 percent residual FVa activity. When synthetic vesicles were added to the inactivation mixture of MP- or thrombin-activated PLTs, a residual activity of 5 to 10 percent was found. Furthermore, addition of excess plasma-FVa to storage-induced MPs resulted in a residual activity of 26 +/- 2 percent. Moreover, the APC-resistant phenotype of MPs was confirmed in plasma in which thrombin generation was measured in the absence and presence of APC. Residual FVa activity in the presence of MPs, PLTs, or synthetic vesicles was 87 +/- 6, 65 +/- 3, and 8 +/- 19 percent, respectively. CONCLUSION: Together, these results suggest that the MP surface environment renders FVa resistant to APC. It is further concluded that the APC resistance of FVa at the surface of storage-induced MPs enhances their procoagulant nature.

Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation.

Platelets shed microparticles, which support haemostasis via adherence to the damaged vasculature and by promoting blood coagulation. We investigated mechanisms through which storage-induced microparticles might support blood coagulation. Flow cytometry was used to determine microparticle number, cellular origin and surface expression of tissue factor (TF), procoagulant phosphatidylserine (PtdSer) and glycoprotein (GP) Ib-alpha. The influence of microparticles on initiation and propagation of coagulation were examined in activated factor X (factor Xa; FXa) and thrombin generation assays and compared with that of synthetic phospholipids. About 75% of microparticles were platelet derived and their number significantly increased during storage of platelet concentrates. About 10% of the microparticles expressed functionally active TF, as measured in a FXa generation assay. However, TF-driven thrombin generation was only found in plasma in which tissue factor pathway inhibitor (TFPI) was neutralised, suggesting that microparticle-associated TF in platelet concentrates is of minor importance. Furthermore, 60% of all microparticles expressed PtdSer. In comparison with synthetic procoagulant phospholipids, the maximal rate of thrombin formation in TF-activated plasma was 15-fold higher when platelet-free plasma was titrated with microparticles. This difference could be attributed to the ability of microparticles to propagate thrombin generation by thrombin-activated FXI. Collectively, our findings indicate a role of microparticles in supporting haemostasis by enhancement of the propagation phase of blood coagulation.

Endogenous factor V synthesis in megakaryocytes contributes negligibly to the platelet factor V pool.

BACKGROUND AND OBJECTIVES: Coagulation factor V (FV) is distributed between two pools: 80% circulates in plasma and 20% is stored in platelets. The aim of the study was to determine the origin of platelet FV. DESIGN AND METHODS: We investigated a FV Leiden heterozygous patient who had received an allogeneic bone marrow transplant from a normal donor. The patient had been referred to our laboratory for his marked activated protein C (APC) resistance in the apparent absence of FV Leiden. Analysis of the DNA from a buccal swab showed that the patient was indeed a heterozygous carrier of FV Leiden. The difference in FV genotype between the hepatocytes (heterozygous FV Leiden) and the blood cells (homozygous normal) of the patient provided a good model to investigate the origin of platelet FV. Platelets were isolated from the patient and the bone marrow donor and activated with thrombin and ionomycin to release and activate FV. APC was then added and the inactivation of platelet FVa was followed over time with a highly sensitive prothrombinase-based assay. RESULTS: While the donor's platelet FVa showed a normal inactivation time course, the patient's platelet FVa was considerably resistant to APC. The kinetic pattern of APC-catalyzed inactivation of the patient's platelet FVa was indistinguishable from that of plasma FVa from a FV Leiden heterozygote. INTERPRETATION AND CONCLUSIONS: These data indicate that platelet FV is derived from plasma and that endogenous FV synthesis by megakaryocytes contributes negligibly to the platelet FV pool.