66

THE A C T I O N OF VITAMIN K A N D C O U M A R I N
ANTICOAGULANTS
LIV H E L G E L A N D

Biokjemisk Institutt
University of Oslo
Blindern, N o r w a y

Introduction In 1929 an antihaemorrhagic factor was discovered by the Danish biochemist Henrik
Dam. 1 The dietary factor, which was shown to be required for normal haemostasis in the
chick, was a new fat-soluble vitamin, and in 1935 the name vitamin K (for Koagulation)
was introduced. Some years later the factor was isolated as a yellow oil from alfalfa and
characterized as a phylloquinone, 2-methyl-3-phytyl-l,4-naphthoquinone (Fig 1) by Doisy
and his collaborators. The compound, designated vitamin Kx, was found to be present in
green plants, hemp seed, cereals, and liver. At the same time the menaquinone (vitamin Kz)
was isolated as a crystalline product (Fig 1) from putrefied fish meal. An early observation
was that the coagulation factor, prothrombin, was present at a reduced level in the plasma
of vitamin K-deficient chicks. As further details of the clotting system were revealed in the
1950s, vitamin K was also shown to be essential for the synthesis of the blood coagulation
factors VII, IX and X.
0 O 0

I II III

FNu~l Vitamin K compounds: (I) Vitamin Ka (phylloquinone;2-methyl-3-phytyl-l,4-naphthoquinone),

(II) Vitamin K z (menaquinone-7;2-methyl-3-farnesylgeranylgeranyl- l, 4-naphthoquinone) a number
of vitamin K 2 analogs are known which differ in the length of the side chain), (III) Vitamin K 3
(menadione;2-methyl-l,4-naphthoquinone). The vitamin K group is defined as being made up of 2-
m ethyl-l,4-naphthoquinone and all derivatives of this compound which exhibit an antihaemorrhagic
activity in animals fed a vitamin K-deficient diet.

Analogues of vitamin K Structural analogues of vitamin K have not only been useful as clinical anticoagulants and
rodenticides, but also represent valuable tools in studying the mode of action of vitamin K.
The first coumarin anticoagulant to be discovered was dicoumarol, 3,3'-methylenebis-(4-
hydroxycoumarin (Fig 2), which was isolated from spoiled sweet clover hay and identified
as the causative agent of a fatal haemorrhagic disease of cattle by Link's group during the
years 1933-41. Since then, a large number of substituted 4-hydroxycoumarins have been
synthesized, among which Warfarin (Fig 2) is well known and has been successfully used in
anticoagulant therapy and also as a rodenticide. The administration of Warfarin or
dicumarol to experimental animals or man causes a decrease of the vitamin K-dependent
coagulation factors of plasma thus reducing the clot-forming tendency in cases of threatened
thrombosis.
0

I II

F~u~ 2 Coumarin anticoagulants: (I) Dicumarol; 3,3"-methyl-bis-(4-hydroxycoumarin); (II) Warfarin; 3-(0,

acetonylbenzyl)-4-hydroxycoumarin

Function An established function of vitamin K in higher organisms is related to blood clotting. 2, 3

The coagulation of blood involves a complex cascade reaction sequence, in which there is a
sequential conversion of zymogens to the corresponding active forms. In the penultimate
stage of the process, prothrombin is the proenzyme for the proteolytic enzyme thrombin
which subsequently converts soluble fibrinogen into insoluble fibrin (Fig 3). The numerous
steps yield a large amplification which ensures rapid clotting of the blood. More than ten
different proteins are involved in the reaction sequence, several of them being proteolytic
enzymes. These coagulation proteins participate in two closely closely related clotting
mechanisms, the intrinsic pathway which utilizes factors in the blood to generate thrombim

BIOCHEMICAL EDUCATION 8(3) 1980

 67

and the extrinsic pathway in which plasma factors as well as tissue components are involved
(Fig 3). In the test tube the two pathways can be studied separately, but in vivo, the
intrinsic and extrinsic pathways apparently function simultaneously as both pathways are
needed for proper clotting. Since vitamin K is required for the biosynthesis of the
functionally active form of prothrombin and for factors VII, IX and X, the vitamin
interferes with both the extrinsic and intrinsic coagulation system.
Intrinsic p a t h w a y

1 Extrinsic pathway

IX
1 " I Xa

TiSSUe-

l
'IIII factor
PL
Ca 2+

x ,xa, x

Prothrombin • Thrombin

Fibrinogen
1 i Fibrin

F~um3 Simplified scheme of the coagulation mechanism with the four vitamin K-dependent clotting factors
underlined. PL refers to phospholipid; V, proaccelerin; VII, proconvertin; VIII, antihaemophilic
factor A; IX, Christmas factor or antihaemophilicfactor B; X, Stuart factor. The subscript 'a' means
that thefactor is in the activeform.
Role o f calcium The requirement of Ca~+ ions for blood clotting as well as the use of Caz+ binding agents
such as citrate, oxalate and EDTA (ethylenediaminetetraacetate) to prevent clotting of
blood samples have been known for many years. Caz+ ions were later shown to play an
essential role in several of the steps in the coagulation pathways. Caz+ binding is a
characteristic property of the vitamin K-dependent clotting factors, and vitamin K is
essential for the formation of specific calcium binding sites in the molecules. Our
knowledge of the structure and biochemistry of the vitamin K-dependent coagulation
factors has been derived in the main from studies of prothrombin which possesses a central
position in blood coagulation (Fig 3). Prothrombin and the other vitamin K-dependent
clotting factors are produced in the liver. By administration of vitamin K antagonists such
as Warfarin or dicoumarol, or by vitamin K deficiency, biologically-inactive prothrombin
results. This was discovered originally in human plasma and given the name PIVKA
(Protein Induced by Vitamin K Absence and Antagonists). Evidence that the protein was a
prothrombin precursor was obtained by immunochemical methods. The existence of a
similar protein, which in many ways resembled prothrombin, was later demonstrated in
bovine plasma. Such inactive prothrombin molecules, in contrast to normal prothrombin
do not bind Caz+ which is necessary for the physiological conversion to thrombin. Further-
more, inactive prothrombin is not adsorbed by insoluble barium salts, a characteristic
property of the vitamin K-binding dotting factors. The main antigenic determinants were,
however, found to be the same in normal and inactive prothrombin. Inactive forms of the
other vitamin K-dependent factors have also been described. In 1973 Suttie demonstrated
the presence of a prothrombin precursor in the livers of Warfarin-treated rats. This
observation was based on the finding that the prothrombin precursor could be converted to
thrombin by Echis carinatus venom. It became evident that the prothrombin precursor
accumulates in rat liver, whereas in cattle and man, the precursor is secreted into the
plasma.

y-carboxy-glutamic acid The production of inactive prothrombin in different species by vitamin K deficiency or by
the use of antagonists suggested that vitamin K was involved in a post-translational
conversion of prothrombin precursor to biologically-active prothrombin and not in
regulating the de novo synthesis of prothrombin as was proposed earlier. In 1974 Stenflo
discovered a previously unknown amino acid, y-carboxyglutamic acid, in bovine
prothrombin. Subsequently, y-carboxyglutamic acid residues were found in ten different
positions; 7, 8, 15, 17, 20, 21, 26, 27, 30, and 33, all in the non-thrombin part of the
molecule (Fig 4), whereas inactive prothrombin had glutamic acid residues at these
positions. Due to the lability of y-carboxyglutamic acid, which readily decarboxylates and
gives glutamic acid in acid solution, y-carboxyglutamic acid was not recognized as a
constituent of proteins. The amino acid has now been found in all four vitamin K-
dependent dotting factors and also in several other proteins which will be described3 The
y-carboxyglutamic acid residues are responsible for the CaZ+-binding properties of

BIOCHEMICAL EDUCATION 8(3) 1980

68

CH CH
I I
NH2- ~
t -CH
75 18S

Prothrombin

Figure 4 Prothromtn'n, mol wt 70,000. CH, carbohydrate; hatched, carboxylation; solid black, thrombin

prothrombin. The prothrombin molecule is bound to phospholipid during the activation,

ie the physiological conversion to thrombin, and this binding is mediated by Ca2+ ions.
y-carboxyglutamic acid in vitamin K-dependent proteins results from a post-translational
vitamin K-dependent carboxylation of glutamic acid residues (Fig 5). The molecular
mechanism of vitamin K action has been studied in a rat liver microsomal system especially
by Suttie et al. 3 Prothrombin production was dependent on vitamin K, 0 2, CO 2 and
prothrombrin precursor present in the microsome fraction isolated from vitanfin K-
deficient rats. Furthermore the carboxylase activity was stimulated by an energy source such
as ATP and by factors present in the post-microsomal supernatant. The latter can be omitted
if vitamin K is replaced by the reduced form of the vitamin. Vitamin K can be reduced

COO- OO14C COO-

J \/
CH 2 CH
I I
14C02 02

Gtu in peptide Vitamin KH 2 y - c a r b o x y Glu in

peptide

Fig ure 5 Post-translationalformation of y-carboxyglutamic acid by vitamin K-dependent carboxylation

enzymically with NADH or NADPH. Warfarin inhibits the carboxylase reaction and the
inhibition can be overcome by high concentrations of vitamin K. The vitamin K-dependent
carboxylase, which is bound to the microsomal membrane, has been solubilized in various
detergents and the solubilized preparation differs in some respects from the membrane-
associated enzyme system, although the basic requirement for 0 2, CO 2 and reduced
vitamin K is retained. The solubilized enzyme system is not stimulated by ATP. Further-
more, Warfarin has no effect on solubilized carboxylase, whereas another vitamin K
antagonist, the 2-chloro analogue of vitamin K (2-chloro-3-phytyl-l,4-naphthoquinone) is
still a powerful inhibitor. This demonstrates that the coumarin anticoagulants such as
Warfarin do not act as competitive inhibitors of vitamin K. It thus appears that the
"coumarins act indirectly to antagonize the vitamin. Vitamin K 2,3-epoxide has been
revealed as a major metabolite of vitamin K (Fig 6), and by Warfarin-treatment large
amounts of the 2,3-epoxide accumulate in liver. It has been suggested that Warfarin exerts
its anticoagulant effect by blocking the regeneration of vitamin K from its 2,3-epoxide
metabolites. The enzyme which converts the epoxide back to vitamin K, vitamin K
epoxide reductase, is inhibited by Warfarin and by other coumarin anticoagulants. The
Warfarin resistance in some strains of wild rats is probably due to a mutation that has
altered this enzyme so that it is no longer inhibited by Warfarin. The epoxidation of

0 ~ ° a' d~ ' N ' A 0

o o
Vitamin K . . . . Vitamin t(
epoxicle
Warfarin

Figure 6 The role of Warfarin in the metabolism of vitamin K

vitamin K (Fig 6) may possibly be linked to the vitamin K-dependent carboxylation

reaction. Furthermore, it is suggested that vitamin K may function as a peroxide carrier and
that the carboxylase uses peroxide to generate oxide ion which could act by abstracting a
proton from the y-methylene group of the appropriate glutamic acid residue.
Carboxylation could then be affected. The finding that synthetic peptides which are
analogous to the N-terminal Glu-containing regions of the prothrombin precursor could
serve as substrates for the carboxylase is obviously of great value in efforts to purify the
carboxylase and elucidate the mode of action of vitamin K.

BIOCHEMICAL EDUCATION 8(3) 1980


Vitamin K-dependent proteins
References
BIOCHEMICAL EDUCATION 8(3) 1980
69
With the important discovery in 1975 of a low-molecular weight protein in bone which
contained y-carboxyglutamic acid, it was realized that vitamin K-dependent proteins might
be more widely distributed than expected. This has clearly proved to be the case, and
y-carboxyglutamic acid residues have now been identified in other plasma proteins as well
as in proteins from various tissues; renal tissue, placenta, lung and spleen, and in
ribosomes? and proteins associated with ectopic calcification. Vitamin K-dependent car-
boxylating systems have also been found in some of the tissues containing y-carboxy-
glutamic acid. These systems appear to be quite similar to that of rat liver microsomes
which carboxylate the prothrombin precursor. The physiological function of these Gla-
containing proteins has not yet been established. Possibly they may be involved in calcium
metabolism, transport and deposition. It must be pointed out, however, that many Ca-
binding proteins do not contain y-carboxyglutamic acid. The Gla-containing protein of
bone, also called osteocalcin, is proposed to play a role in the regulation of the mineraliza-
tion process in bone. Osteocalcin, a small protein, mol wt 570(06500, which contains 3-4
Gla residues depending on the species, is the most abundant non-collagenous protein in
bone. The protein appears to be a constituent of all osseous tissue and binds tightly to
hydroxyapatite, the major mineral component of bone. The formation of y-carboxy-
glutamic acid in osteocalcin, which takes place in bone cells, is a vitamin K-dependent
reaction, and the production of the Gla-containing protein is inhibited by coumarin anti-
coagulants.6 More recently, a calcium-binding protein from chick chorioallantoic
membrane has been described.7 The protein which has a molecular weight of 100,000, is
composed of four subunits of identical size, containing 2-10 Gla residues. This Gla-
containing protein seems to be of importance in the transport of calcium by the chorio-
aUantoic membrane from the egg shell to the embryo. Thus vitamin K may be involved in
calcium transport by regulating the functional state of this calcium-transporting protein.
Human placenta has also been shown to contain a vitamin K-dependent carboxylating
system.e Furthermore, foetal bone abnormalities have been reported to be correlated with
ingestion of coumarin anticoagulants in the first trimester of pregnancy. The discovery of a
vitamin K-dependent protein in kidney, a tissue which plays an important role in calcium
homeostatis, is also of great interest. The function of this Gla-containing protein is not
known, but it may be involved in resorption of calcium in the renal tube. Gla-containing
proteins have also been detected in calcified lesions formed during pathological mineraliza-
tion. Furthermore, Gla-containing proteins in kidney stones and in urine have been
reported. Normally, most of the y-carboxyglutamic acid in urine exists as the free amino
acid. Determination of free and protein bound y-carboxyglutamic acid in the urine may
possibly be of diagnostic value in diseases related to calcium metabolism and vitamin K
deficiency. Further studies of the Gla-containing proteins will establish the role of vitamin
K in normal and pathological mineralization. 9
tOlson, R E (1979) TIBS 4 (No 5), 18-20
ZDavie, E W and Fujikawa, K (1975) Ann Rev Biochem 44,799-829
~Suttie, J W (1978) in The.fat-soluble vitamins, Vol 2, DeLuca, H F (ed). Plenum Press, New York and London.
211-277
4Hauschka, P V and Reid, M L (1978)JBiol Chera 253, 9063--9068
SVanBnskirk, J J and Kitsch, W M (1978) BiochemBiolshysRes Commun 82, 1329--1331
s Nishimoto, S K and Price, P A (1979)J Biol Chera 254, 437-441
7Tuan, R S, Scott, W A and Cohn, Z A (1978)J Cell Bio177, 752-761
s Friedman, P A, Hauschka, P V, Shia, M A, and Wallace, J K (1979) Biochim BiolohysActa 583,261-265
9Hauschka, P V, Lian, J B and Gallop, P M (1978) TIBS 3 (No 4), 75-78