Role of vitamin K-dependent proteins in the arterial vessel wall

Abstract

Vitamin K was discovered early last century at the same time as the vitamin K-antagonists. For many years the role of vitamin K was solely ascribed to coagulation and coagulation was thought to be involved only at the venous blood side. This view has dramatically changed with the discovery of vitamin K-dependent proteins outside the coagulation cascade and the role of coagulation factors at the arterial side. Vitamin K-dependent proteins are involved in the regulation of vascular smooth muscle cell migration, apoptosis, and calcification. Vascular calcification has become an important independent predictor of cardiovascular disease. Vitamin K-antagonists induce inactivity of inhibitors of vascular calcification, leading to accelerated calcification. The involvement of vitamin K-dependent proteins such as MGP in vascular calcification make that calcification is amendable for intervention with high intake of vitamin K. This review focuses on the effect of vitamin K-dependent proteins in vascular disease.

Full text

Hämostaseologie 4/2011
1 © Schattauer 2011Review
Role of vitamin K-dependent
proteins in the arterial vessel wall
M. L. L. Chatrou; C. P. Reutelingsperger; L. J. Schurgers
Department of Biochemistry, CARIM, Maastricht University, the Netherlands
Keywords
Vitamin K, vitamin K-antagonists, MGP,
vascular calcification
Summary
Vitamin K was discovered early last century at
the same time as the vitamin K-antagonists.
For many years the role of vitamin K was
solely ascribed to coagulation and coagu-
lation was thought to be involved only at the
venous blood side. This view has dramatically
changed with the discovery of vitamin K-de-
pendent proteins outside the coagulation cas-
cade and the role of coagulation factors at the
arterial side. Vitamin K-dependent proteins
are involved in the regulation of vascular
smooth muscle cell migration, apoptosis, and
calcification. Vascular calcification has be-
come an important independent predictor of
cardiovascular disease. Vitamin K-antagonists
induce inactivity of inhibitors of vascular cal-
cification, leading to accelerated calcification.
The involvement of vitamin K-dependent pro-
teins such as MGP in vascular calcification
make that calcification is amendable for inter-
vention with high intake of vitamin K. This re-
view focuses on the effect of vitamin K-de-
pendent proteins in vascular disease.
Correspondence to:
Leon J. Schurgers, PhD
Dept. of Biochemistry, Maastricht University
PO Box 616, 6200 MD Maastricht
The Netherlands
Tel. +31/43/388 16 80, Fax +31/43/388 41 59
E-mail: l.schurgers@maastrichtuniversity.nl
Schlüsselwörter
Vitamin K, Vitamin-K-Antagonisten, MGP,
vaskuläre Kalzifikation
Zusammenfassung
Vitamin K wurde im frühen vergangenen Jahr-
hundert zur gleichen Zeit wie die Vitamin-
K-Antagonisten entdeckt. Über Jahre wurde
die Rolle von Vitamin K ausschließlich der
Blutgerinnung zugeschrieben und man glaub-
te, dass sich die Koagulation nur auf der venö-
sen Blutseite abspiele. Mit der Entdeckung Vi-
tamin-K-abhängiger Proteine außerhalb der
Gerinnungskaskade und der Bedeutung von
Gerinnungsfaktoren auf der arteriellen Seite
hat sich diese Sicht grundlegend geändert. Vi-
tamin-K-abhängige Proteine sind an der Re-
gulation der Migration glatter Gefäßmuskel-
zellen sowie an der Apoptose und Kalzifikati-
on beteiligt. Vaskuläre Kalzifikation ist ein
wichtiger unabhängiger Prognosefaktor für
kardiovaskuläre Erkrankungen. Vitamin-K-
Antagonisten induzieren eine Inaktivität von
Inhibitoren der vaskulären Kalzifikation und
führen so zu beschleunigter Verkalkung. Die
Beteiligung Vitamin-K-abhängiger Proteine
wie MGP an der vaskulären Kalzifikation er-
öffnet eine Möglichkeit zur therapeutischen
Intervention durch die Einnahme hoher Dosen
Vitamin K. Das Thema dieses Reviews ist die
Wirkung Vitamin-K-abhängiger Proteine auf
vaskuläre Erkrankungen.
Die Rolle Vitamin-K-abhängiger Proteine in der
arteriellen Gefäßwand
Hämostaseologie 2011; 31: ■■
doi:10.5482/ha-1157
received: May 17, 2011
accepted: June 6, 2011
prepublished online: June 29, 2011
Vitamin K
Vitamin K is a fat-soluble vitamin and be-
longs to the family of vitamins A, D and E.
For most people, vitamin K is the least
known vitamin and even regarded as the
Cinderella (1). After the discovery of vit-
amin K in the early days of last century
clinicians and scientists believed that blood
coagulation was the only physiological pro-
cess in which vitamin K played a role. It was
the Danish researcher Dam who discovered
that chickens fed a fat-free diet suffered
from serious bleedings. It took some time
to isolate the micronutrient responsible for
this action, and it is now known as vitamin
K after the German word “Koagulation”.
In the 1970s it was discovered that vit-
amin K is a cofactor in the carboxylation
reaction. Simultaneously Stenflo et al. (2)
and Nelsestuen et al. (3) reported the dis-
covery of the unusual amino acid γ-carbo-
xyglutamate acid (Gla) in prothrombin as
the product of vitamin K action. These
groups independently identified the un-
equivocal role of vitamin K as a cofactor for
the post-translational carboxylation of glu-
tamate (Glu) residues. This carboxylation-
step is accomplished by an enzyme called
gamma glutamyl-carboxylase (GGCX) (4),
and requires a pro-peptide containing pro-
tein.
This process is driven by the oxidation
of reduced vitamin K into vitamin K-epox-
ide. The vitamin K-epoxide must be re-
cycled to vitamin K before it can be reused.
This reaction is catalyzed by the enzyme
vitamin K epoxide reductase (VKOR) (5,
6). In this way the efficiency of vitamin K is
very high:
One molecule vitamin K can assure some
500 carboxylation reactions.
Recently, the group of Oldenburg added a
new role for the VKOR enzyme in that the
subunit VKORC1L1 is responsible for
driving vitamin K-mediated intracellular
antioxidation pathways critical to cell sur-
vival (7).
Dietary vitamin K
Vitamin K is an essential dietary micro-
nutrient since man cannot synthesize it. Al-
though in our gut flora some bacteria pro-
duce large amounts of vitamin K2 (8) their
contribution to the vitamin K-status is
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Hämostaseologie 4/2011 © Schattauer 2011
2M. L. L. Chatrou; C. P. Reutelingsperger; L. J. Schurgers: Vitamin K-dependent proteins
questionable (9). The recommended daily
intake for vitamin K1 is 1 μg/kg body
weight and this is solely based on blood co-
agulation (10).
With the discovery of vitamin K-de-
pendent proteins in extra-hepatic tissues –
such as bone and vessel wall – this view
needs to be revised. Nutritional vitamin K
consists of two forms:
●
vitamin K1 and
●
vitamin K2 .
Vitamin K1 (also called phylloquinone) is
found in leafy green vegetables where it is
tightly bound to the chloroplast mem-
brane. This results in a poor absorption of
vitamin K1 from vegetables (11, 12) and
thus its contribution to the vitamin
K-status is overestimated.
Vitamin K2 (group name for the mena-
quinones) is found in fermented foods
such as cheese, sauerkraut and the Japanese
natto (12) which is derived from the bacte-
ria that are used for the fermentation pro-
cess. The absorption of vitamin K2 is much
better as compared to vi tamin K1 (13). The
difference between vitamin K1 and vitamin
K2 is related to the aliphatic side chain.
After being absorbed in the intestine vit-
amin K is transported by lipoproteins, as it
has no specific carrier protein. The differ-
ent lipophilicity of K1 and K2 may result in
substantial differences in plasma transport,
half life and delivery to target tissues (14)
(
씰
Tab. 1 ).
Vitamin K-antagonists (VKA)
Vitamin K-antagonists are 4-hydroxy -
coumarin derivatives. VKA were discover-
ed in the early 1920s as a malady of cattle
involving fatal bleeding showing up almost
simultaneously in the area of Wisconsin
(15). It turned out that if cattle ate spoiled
hay a loss in the clotting power of the blood
and as a resultant internal haemorrhage oc-
curred, which usually became fatal. It was
Campbell who isolated the crystalline di-
coumarol in 1939 (16).
Dicoumarol was launched as rat poison
in the early 1940s. After an unsuccessful
suicide attempt of an US soldier it became a
drug to lower the coagulation tendency of
blood. VKA have been given to patients for
more than six decades and besides an in-
creased bleeding tendency they are
relatively safe. By the year 2000 VKA were
the 11
th
most prescribed drug in the United
States (17).
Worldwide VKA form the mostly used drug
for the treatment and prevention of throm-
boembolic events, including atrial fibril-
lation, deep venous thrombosis and artifi-
cial heart valves.
In the USA, warfarin (named after the Wis-
consin Alumni Research Federation) is the
most used VKA whereas in Europe also
phenprocoumon and acenocoumarol are
used (
씰
Tab. 1 ).
The action of VKA is to block the VKOR
enzyme thereby rapidly exhausting vitamin
K tissue stores (18). VKA are used to pre-
vent thrombosis in patients at increased
risk for thrombosis. When patients are
over-anticoagulated with VKA this can be
reversed by a high amount of supplemental
vitamin K.
The liver has a very active antidotal
pathway for VKA, called the DT-diapho-
rase (19, 20). This enzyme is some 100-fold
less active in bone and vessel wall. There-
fore, VKA cause a pronounced vitamin
K-deficiency in extra-hepatic tissues (21,
22). With the knowledge of today – 16 vit-
amin K-dependent proteins are now
known, half of them synthesized by tissues
other than the liver – the use of VKA may
also induce unwanted side effects (
씰
Ta b.
2).
Vitamin K-dependent
proteins
It is now known that vitamin K-dependent
proteins constitute a family of 16 known
proteins with diverse functions, not only
involved in the haemostatic pathway
(
씰
Tab. 2). We will summarize the vitamin
K-dependent proteins, in particular those
involved in vascular disease.
Coagulation proteins
The clotting factors II, VII, IX and X are es-
sential for the coagulation cascade and are
γ-carboxylated in the liver to be function-
ally active. They are well balanced by the
anticoagulant factors protein C, protein S
and protein Z. These vitamin K-dependent
proteins are mainly synthesized and γ-glu-
tamylcarboxylated in the liver, with the ex-
ception of proteins S which is synthesized
some 45% by endothelial cells (23). Only
recently it has been realized that coagu-
lation factors also play an important role in
inflammation (24, 25). Minute amounts of
the coagulation proteins prothrombin and
FVII are synthesized de novo in the vessel
wall (24). The inhibition of thrombin by
melagatran (direct FIIa inhibitor) reduced
atherosclerotic plaque size and features of
plaque vulnerability (26).
Protein S acts in the coagulation cascade
as a cofactor of activated protein C (APC)
in the degradation of FVa and FVIIIa.
Tab. 1 Vitamin K and vitamin K-antagonists
name (trivial name) primary source half-life (t1/2) in hours
vitamin K phylloquinone leafy green vegetables ∼ 3
menaquinone meat, eggs ∼ 1.5
natto, cheese > 70
cheese, curd, sauerkraut > 70
vitamin K
antagonists
acenocoumarol (Sintrom) 10
dicoumarol (Warfarin) 50
phenprocoumon (Marcumar) 100
brodifacum (super coumarin) > 1500
-4
-7
-8, -9
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3 M. L. L. Chatrou; C. P. Reutelingsperger; L. J. Schurgers: Vitamin K-dependent proteins
Besides its coagulation inhibiting proper-
ties, protein S mediates a variety of regula-
tory phenomena including apoptosis and
phagocytosis (27).
Phagocytosis of apoptotic cells is thought
to limit the inflammatory response (28).
Protein S has been identified as a factor re-
sponsible for stimulation phagocytosis of
apoptotic cells by macrophages (27). Addi-
tionally, protein S regulates the expression
and function of scavenger receptor A
(SR-A) on macrophages resulting in dim-
inished uptake of acetylated low density li-
poprotein (AcLDL) (29, 30).
Recently, a new function of protein S
was discovered in vessel wall development.
Mice with protein S deficiency die in utero
(31). Mutants in which protein S was de-
leted specifically in hepatocytes – thought
to be the major source of circulating pro-
tein S – were viable as adults due to the en-
dothelial synthesized protein S. Protein S
deleted in endothelial cells revealed that en-
dothelial cells synthesize some 45% of the
blood-borne protein S (23). Interestingly,
the deficiency in the vessel wall resulted in
impaired angiogenesis, independent of
protein C (23). Mice with heterozygous
protein C deficiency not only exhibit severe
coagulation response to endotoxin but also
have significant differences in their inflam-
matory response (32). Additionally, APC
has been found to inhibit endotoxin-in-
duced production of TNF-α, IL-1β, IL-6,
and IL-8 by cultured monocytes/macro-
phages (33).
Extrahepatic vitamin K-dependent
proteins
Within the arterial vessel wall vitamin
K-dependent proteins are synthesized with
functions not related to blood coagulation.
Gas-6
Growth arrest specific gene 6 protein
(Gas-6) is a vitamin K-dependent protein
produced by vascular smooth muscle cells
(VSMCs) and involved in a pleiotropic of
physiologically processes (34). Gas-6 is as-
sociated with binding to its receptor Axl,
stimulating the anti-apoptotic protein
bcl-2 and inhibiting the pro-apoptotic pro-
tein caspase-3. Son et al. (35) showed that
Gas-6-Axl signaling protects VSMCs from
calcification by inhibiting apoptosis. It has
been shown that apoptotic bodies may
form a nidus for calcification (36).
MGP
The vitamin K-dependent matrix Gla-pro-
tein (MGP) is regarded as the strongest in-
hibitor of vascular calcification (VC) and
produced by many cells, including VSMCs.
MGP
●
promotes VSMC differentiation,
●
antagonizes BMP (BMP2 and BMP4)
signaling and
●
prevents osteochondrogenic lineage
reprogramming of VSMCs.
Both a high local and circulating inactive
MGP was associated with significantly
more VC and cardiovascular death (37, 38).
The role of MGP was elucidated in MGP
null mice (39). These mice were born
normally, but all died within eight weeks
after birth from ruptures of the large vessels
due to their massive calcification and loss of
elasticity. Rescue experiments in MGP null
mice demonstrated that MGP acts locally in
the vascular tissue as restoration of MGP
expression in arteries completely rescued
the arterial mineralization phenotype,
whereas hepatic MGP expression, resulting
in high systemic MGP levels, did not (40).
The crucial role of vitamin K in the in-
hibition of VC became clear from experi-
ments in which VKA was administered to
experimental animals (41). In this model –
in which VKA was given in the presence of
vitamin K to prevent bleedings – all extra-
hepatic vitamin K-dependent proteins, in-
cluding MGP, were synthesized in their in-
active uncarboxylated form, resulting in
vascular calcifications within 2–4 weeks.
Recently, we found that the VKA treat-
Tab. 2 Vitamin K-dependent proteins
VKD protein in tissue of γ-carboxylation (ref.) function (ref.)
coagulation
factor II
(prothrombin)
liver. limited extra-hepatic (24) coagulation, regulatory functions in
inflammation (24, 25, 26)
factor VII coagulation
factor IX liver
factor X
anti-coagulation
protein C liver (80) coagulation (25), anti-in flammatory
(32, 33)
protein S liver and endothelial cells (23) coagulation (25), anti-in flammatory,
phago cytosis and apoptosis (28–30)
Gla-rich protein regulator of mineralisation (83)
proline-rich
Gla proteins
unknown (84)
transmembrane
Gla proteins
periostin
most soft tissues
bone marrow mesenchymal
stromal cells, cadiomyocytes
unknown (85)
bone (86), myocardial (87)
protein Z liver (81) degradation of factor Xa (79)
other VKD proteins
MGP bone, cartilage, vascular tissue
and macrophages (38, 39, 57)
negative regulator of vascular
calcification (37–39)
Gas-6 VSMCs and endothelial cells TAM activating ligand (34)
osteocalcin EH: primarily osteoblasts (82) extra cellular matrix protein in bone
(78, 82)
TGF-α inducible
protein
most soft tissues ECM protein
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4 M. L. L. Chatrou; C. P. Reutelingsperger; L. J. Schurgers: Vitamin K-dependent proteins
ment also caused increased apoptosis in the
vascular media, which further supports the
relation between apoptosis and calcifi-
cation (36).
The role of vitamin K-dependent proteins
has also been studied in patient popu-
lations and it confirmed that treatment with
VKA induces excessive calcification of the
vascular arteries and aortic heart valves
(42–44).
Additionally, the duration of VKA treat-
ment seems to correlate with an increase in
vascular calcification.
Vascular calcification
VC is associated with increased cardiovas-
cular mortality and morbidity, and is re-
cognised as a strong and independent risk
factor for cardiovascular death (45–47).
The amount of VC, as measured and
quantified by multidetector computed to-
mography is an important predictor of
●
all-cause mortality,
●
vascular complications and
●
myocardial infarctions (45, 48, 49).
Patients with higher coronary artery cal-
cification scores were approximately ten
times more likely to have a cardiac event in
the next 3–5 years (50). Not only the pres-
ence of coronary artery calcification is pre-
dictive for cardiovascular outcome, also its
annual increase. It was shown that patients
with a calcification-progression over 15%
per year had a 17.2 fold increased risk of
myocardial infarction compared to pa-
tients without significant progression (51).
The amount of VC is even a stronger pre-
dictor than the Framingham risk score
(FRS) (52), a well accepted 10-year risk pre-
dictor for coronary vascular disease. Clini-
cally, VC causes stiffening of the vascular
arteries via elastic fiber and VSMC calcifi-
cation. The calcification may result in
●
decreased arterial compliance,
●
development of left ventricular hyper-
trophy and
●
decreased coronary perfusion leading to
an increased risk of fatal complications.
In spite of this, calcification of arteries has
been neglected and considered to be clini-
cally irrelevant. VC was regarded as an end-
stage passive process not amenable to
therapeutic intervention (53). However, re-
cent reports demonstrate that punctated
and spotty calcification in the athero-
sclerotic plaque influence stability
negatively and render the plaque vulner-
able to rupture (54, 55) (
씰
Fig. 1).
VC is now appreciated as a complex and
actively regulated process involving cells
and proteins acting as catalysts and
inhibitors (56, 57).
Recruitment of macrophages in the athero-
sclerotic plaque and consequently their se-
cretion of inflammatory cytokines may
serve as a signal for intimal calcification.
Indeed Nadra et al. showed that basic cal-
cium phosphate crystals are taken up by
macrophages in vitro (58). This was associ-
ated with the secretion of the pro-inflam-
matory cytokines TNF-α, IL-β and IL-8.
Furthermore, Pazár et al. showed that basic
calcium phosphate induced macrophage
IL-1β secretion through activation of the
NLRP3 inflammasome (59, 60). Also
VSMCs can execute phagocytosis of cal-
cium crystals. Ewence et al. showed that
when VSMCs phagocytose calcium crystals
it might destabilize atherosclerotic plaques
by initiating inflammation and by causing
VSMC death (61) (
씰
Fig. 2).
Detection of vascular calcification
VC ca n b e v isua li zed b y v ario us tech ni ques .
In the clinical setting, multidetector com-
puted tomography is often used and gener-
ates a quantitative calcium score, which is
used as a measure of atherosclerotic burden
(62, 63). VC is therefore a potent predictor
for cardiovascular events (64). Although
some research has linked the amount of
vascular calcium to a more stable plaque
phenotype (65) most studies identified
intimal calcification as predictor of a vul-
nerable plaque phenotype, in particular the
punctated “spotty calcification” (63, 66).
Indeed, finite element analysis implied that
macrocalcification in the plaque did not in-
crease plaque stress or rupture (55, 67)
Fig. 1 Detection of vascular calcification
a) in vivo micro-CT: calcified atherosclerotic plaque of an ApoE-/- mouse on western type diet for 6 months
b) histochemical staining (von Kossa) of intimal microcalcification of an ApoE-/- mouse on western type diet containing warfarin and vitamin K for 3 months
a) b)
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© Schattauer 2011 Hämostaseologie 4/2011
5 M. L. L. Chatrou; C. P. Reutelingsperger; L. J. Schurgers: Vitamin K-dependent proteins
whereas small calcified spots in the athero-
sclerotic cap increased stress, sufficient for
causing plaque rupture.
This resulted in studies screening for regu-
lating mechanisms of VC by multimodality
imaging. Derlin et al. (68), used a combination
of positron emitted tomography (PET) and
CT to asses the cardiovascular risk in patients.
The use of
18
F sodium fluoride for imaging
calcified atherosclerotic plaques showed a
more frequent uptake of Na
18
F in patients
with a high-risk profile. However, there was a
weaker correlation with risk factors compared
to the calcified plaque burden (68).
Imaging of VC in mice was done using
bisphosphonate near-infrared conjugated
probes. Bisphosphonates strongly bind to
calcified structures in coronary arteries
(69, 70). However, bisphosphonate also
strongly accumulates in bone, which results
in a high background signal. Therefore,
these studies are only applied ex vivo. New
specific probes imaging microcalcification
can provide a platform to study the earliest
events associated with VC at the molecular
and cellular level.
The use of circulating biomarkers such
as MGP for detecting or screening VC is an
attractive possibility. Vitamin K-dependent
proteins have been associated with the ear-
liest calcification areas in the plaque (71). It
was the uncarboxylated form of MGP that
strongly correlated with both medial and
intimal calcification (71, 72). By measuring
circulating MGP isoforms it was shown
that the majority of the healthy population
have sub-optimal levels of vascular vitamin
K (73, 74). Preliminary data suggest that
some MGP conformations are associated
with aspects of cardiovascular disease (37,
38, 75, 76). Patients with high VC scores
display high levels of inactive MGP, es-
pecially dialysis patients.
This creates possibilities for targeting VC
with vitamin K. Indeed high intake of vit-
amin K has been shown to regress preformed
medial calcifications in a rat model (77).
Recently, we conducted a first pilot
study in dialysis patients showing that vit-
amin K supplementation markedly re-
duced the level in plasma (78) of
●
uncarboxylated prothrombin (pivka-II),
●
uncarboxylated osteocalcin (ucOC),
●
inactive MGP (dp-ucMGP).
Fig. 2 Mechanism of vascular calcification
1) Adaptation of VSMCs from a contractile to a synthetic phenotype as a
result of multiple stress factors. VSMCs start loading calcium resulting in
calcification of the media.
2, 3) Additionally, modified lipoprotein binding to macrophage scavenger
receptors (2) are phagocytosed and accumulation results in foam cell
formation (3).
4) Foam cells secrete pro-inflammatory cytokines that amplify the local in-
flammatory response (4) resulting in an accelerated calcium loading and
vesicle release by VSMCs. These vesicles form a nidus for calcification. The in-
creased inflammatory profile results in osteogenic differentiation of VSMCs.
Additionally, VSMCs lose their calcifying inhibitors (such as MGP) resulting in
an acceleration of calcification.
5) As a result, VSMCs undergo apoptosis releasing more apoptotic bodies,
accelerating the calcification process.
6, 7) Macrophages phagocytose calcium crystals (7) which induces
activation of the NLRP-3 inflammasome.
8) Subsequently cytokines (such as TNF-α, IL-β and IL-8) are released. Also
VSMCs phagocytose calcium crystals, which leads to enhanced apoptosis of
VSMCs.
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6 M. L. L. Chatrou; C. P. Reutelingsperger; L. J. Schurgers: Vitamin K-dependent proteins
Conclusion
Effort must be directed towards retarding
or reversing the development of calcifi-
cation in the vasculature, especially in those
patients prone for vascular calcification
(i. e. chronic kidney disease, diabetes, car-
diovascular disease). In these patients the
treatment with vitamin K antagonists
should be reconsidered. Data suggest that
high vitamin K can induce regression of
VKA-induced vascular calcification (77).
Therefore, it is of importance to identify
patients with vascular disease and to evalu-
ate different strategies that are more effec-
tive in the prevention of
●
hypercoagulability as well as
●
vascular calcification.
New oral anticoagulants such as FIIa- and
FXa-inhibitors that specifically target one
protein in the coagulation cascade without
affecting vascular vitamin K-dependent
proteins may become the preferred choice.
Conflict of interest
The authors report no conflict of interests.
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