Vitamin K and Vitamin K-Dependent Proteins

Myrtle Thierry-Palmer. Cambridge World History of Food. Editor: Kenneth F Kiple & Kriemhild Conee Ornelas. Volume 1. Cambridge, UK: Cambridge University Press, 2000.

The term “vitamin K” was first introduced by Henrik Dam in 1935, following discovery of a fat-soluble substance that could prevent bleeding (Dam 1935, 1964). During the years 1928 to 1930, Dam conducted studies on the cholesterol metabolism of chicks at the University of Copenhagen. The chicks were being fed an artificial, practically sterol-free diet to which the then known vitamins A and D were added. He observed hemorrhages (bleeding) in different parts of the body in some of the chicks that had been on the diet for more than two or three weeks. In addition, blood that was examined from some of these chicks showed delayed coagulation or clotting (Dam 1964). The low amounts of cholesterol and fat in the diet were ruled out as the causes of the symptom.

Similar observations were made by W. D. McFarlane,W. R. Graham, Jr., and G. E. Hall (1931).W. F. Holst and E. R. Halbrook (1933) observed that the symptoms resembled scurvy, a disease caused by vitamin C deficiency, and could be prevented by addition of fresh cabbage to the diet. These investigators concluded that the protective agent in the cabbage was vitamin C. However, when pure vitamin C became available, it was injected into the chicks and failed to prevent the hemorrhagia (Dam 1964).

Large doses of vitamins A and D (fed in the form of fish-liver oils) and commercial carotene also did not prevent the hemorrhagia (Dam 1935). Cereals and seeds did prevent the symptom, and green leaves and hog liver were found to be potent sources of the anti-hemorrhagic factor. Research groups led by Dam and H. J. Almquist worked independently to show that the factor was a new fat-soluble vitamin. Dam’s report (Dam 1935) was followed in the same year by that of Almquist and E. L. R. Stokstad (1935). The factor was designated vitamin K (Dam 1935, 1964). According to Dam (1964: 10),”The letter K was the first one in the alphabet which had not, with more or less justification, been used to designate other vitamins, and it also happened to be the first letter in the word ‘koagulation’ according to the Scandinavian and German spelling.”

The elucidation of the structure of vitamin K was achieved in a relatively short time because of the number of large research groups involved. Several reviews are available on the isolation and characterization of vitamin K (Doisy, Binkley, and Thayer 1941; Dam 1942, 1964; Almquist 1979). In 1943, Dam received the Nobel Prize for Physiology or Medicine for his discovery of vitamin K. It is interesting to note that Almquist, who published later but in the same year as Dam, did not share the Nobel Prize. Dam did share the prize with E.A. Doisy, who was honored for the isolation, chemical synthesis, and structural identification of one of the forms of vitamin K.

The Structure of Vitamin K

Many compounds, all closely related, are now recognized as having antihemorrhagic activity. Common to all is the methylnaphthoquinone nucleus. This nucleus, known as menadione, has not been isolated from natural sources, but it has been synthesized and does possess biological activity. Attached to the methylnaphthoquinone nucleus are carbon chains that vary both in nature and length. Based on the nature of these carbon side chains, the natural K vitamins are subdivided into two groups: phylloquinone (vitamin K1) and the menaquinones (MK- n). Phylloquinone, the plant product, has a 20-carbon side chain with only one double bond. The menaquinones, synthesized by microorganisms, contain side chains with varying numbers of 5-carbon (isoprene) units but always with one double bond per 5-carbon unit. Chain lengths of the known menaquinones vary from 2 to 13 isoprene units. The n in MK- n refers to the number of 5-carbon units. A menaquinone with 4 isoprene units (20 carbons) in its side chain, for example, would be called menaquinone-4, abbreviated MK-4. Humans convert the synthetic menadione nucleus to menaquinone-4.

Nutritional Aspects of Vitamin K

Since the human body cannot synthesize the naphthoquinone entity, all the K vitamins in humans, at least the quinone nucleus of these compounds, are of extraneous origin. The K vitamins are synthesized by plants as phylloquinone and by bacteria in the intestines as the menaquinones.

Phylloquinone, the form of vitamin K synthesized by plants, is the major dietary form of vitamin K (Booth et al. 1993; Booth, Sadowski, and Pennington 1995; Shearer, Bach, and Kohlmeier 1996). It is found in plants and in the tissues of plant-eating animals and is the form of vitamin K that is added to infant formulas and given to infants at birth.

The best sources of phylloquinone are green leafy vegetables and certain oils, such as soybean, rape-seed, and olive oil (Shearer et al. 1996). Phylloquinone concentrations in plants, however, are affected by the stage of maturation and the geographic location of the plant. Further, different parts of the plant may differ in phylloquinone content. Outer leaves of the cabbage, for example, contain three to six times more phylloquinone than inner leaves (Shearer et al. 1980; Ferland and Sadowski 1992a). Although tea leaves and regular ground coffee contain high concentrations of phylloquinone, the brews are not a dietary source of the vitamin (Booth, Madabushi, et al. 1995). The phylloquinone content of oils declines slightly with heat and rapidly on exposure to daylight and fluorescent light when the oils are stored in transparent containers (Ferland and Sadowski 1992b; Shearer et al. 1996).

Recent evidence indicates that the intestinal absorption of phylloquinone from vegetables is poor and is improved by the simultaneous ingestion of fat (Vermeer et al. 1996). Bile acids, secreted into the intestines in response to fat in a meal, are necessary for the absorption of the fat-soluble K vitamins.There is a need for additional studies on the bioavailability of the K vitamins in foods to complement our knowledge of their vitamin K content (Vermeer et al. 1996).

Less is known about the vitamin K content of menaquinones (synthesized by bacteria) in foods. Livers of ruminant species, such as cows, have been found to have nutritionally significant quantities of menaquinones, but the concentrations of menaquinones in other animal organs were very low (Hirauchi et al. 1989; Shearer et al. 1996). Cheeses contained significant quantities of two different menaquinones, but very low quantities of menaquinones were found in milk and yoghurt (Shearer et al. 1996).

A topic currently in dispute is whether the menaquinones produced by bacterial action in the gut are substantially utilized by humans. Concentrations of various menaquinones have been found to be higher than that of phylloquinone in normal human livers (Usui et al. 1989) and, importantly, antibiotics have been known since the late 1940s and early 1950s to sometimes create a vitamin K deficiency. Nonetheless, the degree of importance of menaquinones in human nutrition has not yet been determined (Suttie 1995), and there are data to suggest that the long-chain menaquinones found in human liver are not as effective a form of the vitamin as phylloqui-none. Moreover, elimination of foods high in vitamin K from a normal diet can produce signs of vitamin K insufficiency, suggesting that bacterial synthesis of menaquinones only partially satisfies the human vita-min K requirement (Suttie 1995).

Documented cases of vitamin K deficiency in adults have been uncommon. The National Research Council of the National Academy of Sciences recommends a daily dietary allowance (RDA) of approximately 1 microgram (m g) vitamin K per kilogram (kg) of body weight (National Research Council 1989). Thus, the requirement for an individual weighing 150 pounds (68 kg) would be 68 m g phylloquinone. The average vitamin K intake of 10 male college students was determined to be 77 m g/day based on analysis of food composites (Suttie et al. 1988)—an average intake that corresponds to 1.04 m g vitamin K/kg body weight. Foods rich in vitamin K, such as spinach, broccoli, and Brussels sprouts, were not usually consumed in significant amounts by these students.This suggests that daily vitamin K intake may vary considerably depending on whether foods rich in vitamin K are consumed (Suttie et al. 1988). Table IV.A.6.2 makes it clear that 100 grams (g) of spinach (380 m g phylloquinone per 100 g) would contribute much to the recommended daily dietary intake of vitamin K, even if phylloquinone is poorly absorbed from this food.

Vitamin K deficiency can result from inadequate intestinal absorption. Because vitamin K is a fat-soluble vitamin, bile acids and pancreatic juice are needed for its absorption. Thus, causes of poor intestinal absorption can be insufficient production of bile acids, inadequate release of bile acids (for example, obstruction of the bile ducts by gallstones), or pancreatic insufficiency (Report of the Committee on Nutrition 1961; Suttie 1991). Levels of all the K vitamins were reduced in patients with chronic hepatitis and liver cirrhosis (Usui et al. 1989). In addition, secondary vitamin K deficiency has been observed in human subjects taking megadoses of vitamin E (Korsan-Bengtsen, Elmfeldt, and Holm 1974). Vitamin E supplementation is currently on the rise because of its role as an anti-oxidant. However, the effect of large doses of vitamin E on vitamin K status must always be borne in mind.

An upper limit for vitamin K intake has not been set, but 20 μg/100 kilocalories has been suggested by J. W. Suttie and R. E. Olson (Olson 1989). Menadione, which is not a natural form of vitamin K, can cause severe toxic reactions in infants if administered in large doses. Phylloquinone, however, has been given without adverse effects to infants in a single intramuscular dose that is 100 times their RDA. Adult dietary intakes of 10 to 15 times the RDA also cause no adverse effects (Olson 1989).

Function of Vitamin K

Vitamin K is involved in the synthesis of four clotting factors, one procoagulant protein, two anticoagulant proteins, and two bone proteins. The vitamin K–dependent clotting factors are integral to blood coagulation, such that an untreated vitamin K deficiency results in death due to bleeding. There also must be control of coagulation, and the body, therefore, has anticoagulant systems. Two of the known anticoagulant proteins are vitamin K–dependent.

The vitamin K–dependent bone proteins (in contrast to the vitamin K–dependent clotting proteins) have only recently been discovered, and the physiological role of these proteins in bone has not yet been determined.

Mechanism of Action of Vitamin K

Vitamin K is a cofactor for an enzyme (vitamin K–dependent carboxylase) that carboxylates specific glutamic acid residues of proteins, converting these amino acid residues to gamma-carboxyglutamic acid residues. This modification occurs after the protein has been synthesized. The gamma-carboxyglutamyl residues are abbreviated Gla.

Although it was established in the early 1950s that vitamin K was necessary for the synthesis of the clotting proteins prothrombin (factor II) and factors VII, IX, and X, the vitamin K–dependent carboxylation reaction was not discovered until 1974. Investigators at that time demonstrated the presence of a new amino acid, gamma-carboxyglutamic acid, in prothrombin (Nelsestuen, Zytkovicz, and Howard 1974; Stenflo et al. 1974). The nature of the carboxylase reaction was soon delineated (Shah and Suttie 1974; Esmon, Sadowski, and Suttie 1975; Suttie 1985).A reduced form of vitamin K (vitamin K hydroquinone) is converted to vitamin K epoxide as the glutamic acid residues of the specific protein are converted to gamma-carboxyglutamic acid residues. At physiological concentrations, vitamin K must be regenerated and reutilized and is recycled. Two reactions take place sequentially to convert vitamin K epoxide to its reduced form (vita-min K hydroquinone) for continued carboxylation. These two reactions are prevented by coumarin anticoagulants, synthetic compounds such as warfarin, which are used medically to counteract excessive clotting.

The vitamin K–dependent (Gla-containing) proteins that have been well characterized include prothrombin and the clotting factors VII, IX, and X (plasma proteins synthesized by the liver); protein Z, a procoagulant plasma protein; protein C and protein S, lasma anticoagulants synthesized by the liver; osteocalcin, also called bone Gla protein; and matrix Gla protein, a bone-matrix protein.

Vitamin K–Dependent Proteins

The clotting proteins. When a cut or injury occurs, platelets (cells in blood) converge on the injury to form a plug, and a clot forms on the platelet plug. This action stops blood loss and prevents the injured person from bleeding to death. A clot is formed by a series of transformations involving more than ten different proteins. In the final stage, fibrinogen, a soluble blood protein, is cleaved by thrombin and converted to fibrin. Fibrin monomers are then cross-linked to form the insoluble or hard clot. Hundreds of papers have been published on the blood clotting cascade; reviews by C. M. Jackson and Y. Nemerson (1980) and by B. Furie and B. C. Furie (1992) provide additional information.

The proteins of the clotting cascade that are vita-min K–dependent are prothrombin (factor II), factor VII, factor IX, and factor X.These proteins, which are central to the blood clotting cascade, are carboxylated after synthesis in a reaction requiring vitamin K (Uotila 1990). The Gla residues make the proteins more negative and endow them with an increased ability to bind positively charged calcium ions. Calcium ions serve as bridges between these proteins and the negatively charged phospholipids of the platelet membrane. The proteins are brought in close proximity to each other on the platelet membrane, augmenting their activation. In the absence of vitamin K, carboxylation of prothrombin and factors VII, IX, and X does not occur. Activation of these factors proceeds so slowly that bleeding may result.

At the time of identification of vitamin K as the antihemorrhagic factor, prothrombin and fibrinogen were the only proteins in the clotting cascade characterized as involved in the formation of the fibrin clot. The laboratories of Dam (Dam, Schønheyder, and Tage-Hansen 1936; Dam 1964) and A. J. Quick (Quick 1937) independently demonstrated that the activity of prothrombin was decreased in the plasma of chicks fed hemorrhagic diets. The choice of the chick as the experimental animal in these early studies was fortuitous, since chicks develop the symptoms of vitamin K deficiency more readily than other experimental animals (Suttie 1991). Factors VII, IX, and X, the other vitamin K–dependent clotting factors, were not established as essential plasma proteins and vitamin K–dependent proteins until the 1950s.

Human protein Z, another vitamin K–dependent protein, was purified and first described in 1984 (Broze and Miletich 1984). It is synthesized by the liver and promotes the association of thrombin (activated prothrombin) with phospholipid surfaces, a process necessary for clotting. Protein Z deficiency has recently been described as a new type of bleeding tendency (Kemkes-Matthes and Matthes 1995).

The anticoagulants. For several decades prior to the discovery of protein C (Stenflo 1976), the only known vitamin K–dependent plasma proteins were the clotting proteins II, VII, IX, and X. Protein S was subsequently isolated and purified (Di Scipio et al. 1977). Protein C and protein S are now known to be components of a very important anticoagulant system in plasma. Protein C, when activated and bound to protein S, is able to cleave, and thereby inactivate, two of the activated clotting factors, factors Va and VIIIa (Walker 1980; Stenflo 1984; Esmon 1987).The presence of both coagulant and anticoagulant systems in plasma allows for control of clotting.

Hereditary protein C deficiency and protein S deficiency have been discovered (Griffin et al. 1981; Pabinger 1986; Miletich, Sherman, and Broze 1987; Broekmans and Conrad 1988; Bertina 1989; Preissner 1990; Rick 1990).The clinical manifestation of protein S or protein C deficiency is thrombosis (excessive clotting).

The bone proteins. Until the discovery of osteocalcin (bone Gla protein, BGP) in the 1970s, vitamin K was assumed to function only in coagulation. The Gla residues of osteocalcin, a major protein of bone, provide a point of interaction between the protein and bone mineral (Hauschka, Lian, and Gallop 1975; Poser and Price 1979). Matrix Gla protein was isolated and identified in 1983 (Price, Urist, and Otawara 1983). The synthesis of osteocalcin and matrix Gla protein is regulated by the hormonal form of vitamin D (1,25-dihydroxyvitamin D) (Price 1988).Two vitamins, vita-mins K and D, are thus involved in the synthesis of osteocalcin and matrix Gla protein.

A fetal warfarin syndrome, first reported in 1975, has been found in infants born to mothers on warfarin anticoagulant therapy during pregnancy (Becker et al. 1975; Pettifor and Benson 1975; Shaul, Emery, and Hall 1975). Some of the abnormalities identified have been associated with bone formation. Although the functions of osteocalcin and matrix Gla protein are not yet established, defective carboxylation of these two proteins in the presence of warfarin may be a cause of the bone abnormalities associated with fetal warfarin syndrome (Price et al. 1983; Pauli et al. 1987; Price 1988).

In recent years, several studies have implicated vita-min K insufficiency in the pathogenesis of osteoporosis (Binkley and Suttie 1995;Vermeer, Jie, and Knapen 1995; Shearer et al. 1996; Vermeer et al. 1996). Osteoporosis is an age-related disorder characterized by inadequate skeletal strength due to bone loss (decreased bone density) and a predisposition to bone fractures. Peak bone mass is achieved between ages 25 and 35, and bone loss is a natural process that begins thereafter. The highest rate of bone loss for women, however, occurs within the first three to six years after the onset of menopause.Women are at greater risk for osteoporosis than men because of a lower peak bone density and the rapid postmenopausal loss of bone, related, in part, to reduced estrogen levels.

Low concentrations of vitamin K have been found in the blood of patients with bone fractures.When the vitamin K status is insufficient, vitamin K–dependent proteins tend to be undercarboxylated. Osteocalcin is more sensitive to a low vitamin K intake than the coagulation proteins, and, thus, undercarboxylated osteocalcin is the most sensitive known marker of vitamin K insufficiency. Undercarboxylated osteocalcin has been reported to be increased in postmenopausal women and to be highest in those individuals with the lowest hip bone density and greatest hip fracture risk. Vitamin K supplementation has been shown to decrease bone loss, and clinical trials have been initiated to test the effect of long-term vitamin K supplementation on bone mass. Measurements of undercarboxylated osteocalcin suggest that vitamin K insufficiency, formerly characterized by the status of the coagulation system, may be more common than previously thought (Binkley and Suttie 1995;Vermeer et al. 1996).

Other proteins. Gla-containing proteins have also been purified from kidney (Griep and Friedman 1980), urine (Nakagawa et al. 1983), liver mitochondria (Gardemann and Domagk 1983), and human spermatozoa (Soute et al. 1985).The function of these proteins is not presently known.

Abnormal Carboxylation

Hereditary deficiency of vitamin K–dependent clotting proteins (factors II,VII, IX, and X) can result in a rare bleeding disorder that has so far been diagnosed in only seven patients. The first reports of this disorder were in 1966 (Fischer and Zweymuller 1966; McMillan and Roberts 1966), and data (Brenner et al. 1990) suggest that the defect is abnormal carboxylation (due to a defective liver vitamin K–dependent carboxylase enzyme). Deficiencies in the plasma anticoagulants protein C and protein S have also been observed (Brenner et al. 1990).

Hemorrhagic Diseases of Infants

Although vitamin K deficiency symptoms are not very prevalent in adults, hemorrhagic diseases related to vitamin K deficiency are observed in infants worldwide. Three patterns of vitamin K–deficiency hemorrhage—early hemorrhagic disease of the newborn (HDN), classic hemorrhagic disease of the newborn, and late hemorrhagic disease—have been described. The frequency of these diseases, particularly that of late hemorrhagic disease, has increased during the last decade (Lane and Hathaway 1985; Kries, Shearer, and Göbel 1988; Greer 1995).

Early Hemorrhagic Disease

Early hemorrhagic disease of the newborn is characterized by severe, and sometimes life-threatening, hemorrhage at the time of delivery or during the first 24 hours after birth (Lane and Hathaway 1985; Kries et al. 1988). The bleeding varies from skin bruising, cephalohematoma, or umbilical bleeding to widespread and fatal intracranial, intra-abdominal, intrathoracic, and gastrointestinal bleeding (Lane and Hathaway 1985).The disease is often seen in infants whose mothers have taken drugs during pregnancy that affect vitamin K metabolism, such as therapeutic doses of warfarin. Idiopathic cases (no known cause) have also been reported.

Classic HDN

C. W. Townsend (1894) was the first person to use the term “haemorrhagic disease of the newborn.” He described 50 infants who began bleeding on the second or third day of life, most commonly from the gastrointestinal tract, and speculated that the disease was of infectious origin. Dam and colleagues (1952) studied 33,000 infants and concluded that low levels of the clotting factor prothrombin in newborns was secondary to a vitamin K deficiency and occurred primarily in breast-fed infants. The researchers showed that this problem could be prevented by administration of vitamin K to mothers prior to delivery and to the infant shortly after delivery.

Normal full-term infants have reduced blood concentrations of the vitamin K–dependent clotting factors II, VII, IX, and X. Further, the levels of these factors decline during the first few days of life. Hemorrhagic disease of the newborn, often called early vitamin K deficiency, occurs from 1 to 7 days after birth. Breast-fed infants are at risk because of the low vitamin K content of breast milk. Bleeding occurs in the gastrointestinal tract, skin, and nose and at circumcision.The disease is prevented by vitamin K prophylaxis at birth (Lane and Hathaway 1985; Kries et al. 1988; Greer 1995), as indicated in 1952 (Dam et al. 1952).

Late Hemorrhagic Disease

Late hemorrhagic disease (late neonatal vitamin K deficiency) strikes at some infants (1 to 12 months of age) who are predominantly breast-fed and who do not receive vitamin K supplementation (Lane and Hathaway 1985; Hanawa et al. 1988; Kries et al. 1988). It is characterized by intracranial, skin, and gastrointestinal bleeding.

That late hemorrhagic disease of the neonate has been observed primarily in breast-fed infants may result from the low vitamin K content of human milk (Lane and Hathaway 1985; Kries et al. 1988; Canfield et al. 1990). However, even if administered at birth, vitamin K may not always prevent deficiency in older infants because of their metabolism rates and consequent elimination of the vitamin. In Japan, from January 1981 to June 1985, 543 cases of vitamin K deficiency were reported in infants over 2 weeks of age (Hanawa et al. 1988). Of these, 427 were diagnosed as having idiopathic vitamin K deficiency, and 387 (90 percent) of this group had been entirely breast-fed (Hanawa et al. 1988). The concentrations of phylloquinone in human milk (mean 2.1 μg/liter) have been found to be significantly lower than those found in cows’ milk (mean 4.9 μg/liter) and in unsupplemented infant formulas containing only fat from cows’ milk (mean 4.2 μg/liter). Supplemented infant formulas, by contrast, had higher levels of phylloquinone than cows’ milk; two of those studied had levels of 75.1 μg/liter and 101.8 μg/liter (Harroon et al. 1982; Kries et al. 1987).

It has been proposed that the late neonatal vitamin K deficiency that occurs in predominantly breast-fed infants may not result solely from lower vitamin K intake but from its combination with other factors, including subclinical liver dysfunction (Hanawa et al. 1988; Kries and Göbel 1988; Matsuda et al. 1989). In fact, I. Matsuda and colleagues (1989) have suggested that the higher content of vitamin K in formulas, unlike the lower content of vitamin K in human milk, can actually mask such defects.

In another study, phylloquinone levels and coagulation factors were measured in healthy term newborns until 4 weeks after birth (Pietersma-de Bruyn et al. 1990). Although breast-fed infants had lower serum phylloquinone levels than formula-fed infants, the levels of the vitamin K–dependent clotting factors II and X were comparable in the two groups. Thus, the authors of this study concluded that breast milk contains sufficient vitamin K for optimal carboxylation of the clotting factors.They have also suggested that the vitamin K supplementation of infant formulas results in higher than normal serum levels of vitamin K in infants without a concomitant rise in the levels of the vitamin K–dependent clotting proteins (Pietersma-de Bruyn et al. 1990).

Vitamin K Prophylaxis

Prophylactic use of vitamin K has been recommended for all newborns by the American Academy of Pediatrics since 1961. Vitamin K prophylaxis is standard for all infants in some geographical regions but is, or has been, “selective” (given only to at-risk infants) in others. However, in some regions where the “selective policy” was in effect, there was a resurgence (in the 1980s and 1990s) of hemorrhagic disease (Tulchinsky et al. 1993; Greer 1995).

The mode of administration (oral versus intramuscular) of vitamin K also became an issue of controversy in the 1990s because of reports from one research group (unconfirmed by two other groups) suggesting an association between intramuscular administration of vitamin K to the neonate and the subsequent development of childhood cancers. But in several countries (for example, Sweden, Australia, and Germany) where use of oral vitamin K prophylaxis (compared with intramuscular) has increased, there has been an increased incidence of late hemorrhagic disease. Oral prophylaxis prevents classic hemorrhagic disease but is less effective in preventing late hemorrhagic disease. Prevention of the latter requires repeated oral doses of vitamin K during the first 2 months of life for exclusively breast-fed infants or other infants at risk (for example, infants with liver disease), all of which presents problems with compliance (Greer 1995;Thorp et al. 1995).

The Vitamin K Ad Hoc Task Force of the American Academy of Pediatrics (1993) has recommended that to prevent hemorrhagic disease, phylloquinone be administered at birth to all newborns as a single, intramuscular dose of 0.5 to 1 milligram. The Task Force has also recommended that research be done to judge the efficacy, safety, and bioavailability of oral formulations of vitamin K. Oral supplements of vitamin K to nursing mothers might become an alternative method of prophylaxis to prevent late hemorrhagic disease (Greer 1995). Physicians could also advise pregnant and nursing mothers to increase their intake of green, leafy vegetables.

The Oral Anticoagulants

The discovery of the first coumarin anticoagulant, dicoumarol, has been interestingly described by K. P. Link (1959). During a blizzard on a Saturday afternoon in February 1933, a Wisconsin farmer appeared at the University of Wisconsin Biochemistry Building with a dead cow, a milk can containing blood without clotting capacity, and about 100 pounds of spoiled sweet clover hay. The farmer’s cows were suffering from “sweet clover disease,” a malady caused by feeding cattle improperly cured hay made from sweet clover. If the type of hay was not changed and if the cows were not transfused, they developed a prothrombin deficit and bled to death. Link and his colleagues began to work on the isolation and identification of the hemorrhagic agent, and in 1939, it was identified as dicoumarol, a coumarin derivative.As a result of the spoilage, coumarin, a natural component of sweet clover, had been converted to dicoumarol (Link 1959).

Since the discovery of dicoumarol, other coumarin derivatives have been synthesized (Link 1959). The most famous of the coumarin derivatives is warfarin, named for the University of Wisconsin Alumni Research Foundation (WARF), which received a patent for the compound. The coumarin compounds interfere with the metabolism of vitamin K, preventing conversion of vitamin K epoxide to vitamin K and also preventing reduction of vitamin K to vitamin K hydroquinone. This interference causes a buildup of vitamin K epoxide, and when physiological levels of vitamin K are present, the inability of the epoxide to be converted to the vita-min creates a relative vitamin K deficiency. The vita-min K–dependent proteins, including the clotting proteins II,VII, IX, and X, are, therefore, not carboxylated—or only partially carboxylated—and remain inactive (Suttie 1990).

The coumarin compounds, particularly warfarin, have been used as oral anticoagulants in the long-term treatment of patients prone to thrombosis (clot formation). The formation of a clot in a coronary artery narrowed by atherosclerosis is a causative factor in the development of acute myocardial infarction (heart attack), and warfarin is used after acute myocardial infarction to prevent further thrombus (clot) formation and reinfarction.

A recent study of the effect of warfarin on mortality and reinfarction after myocardial infarction concluded that “long-term therapy with warfarin has an important beneficial effect after myocardial infarction” (Smith, Arnesen, and Holme 1990: 147). This study involved 607 patients treated with warfarin and 607 patients treated with a placebo. Compared with the placebo group, warfarin therapy reduced the number of deaths by 24 percent, the number of rein-farctions by 34 percent, and the number of cerebrovascular accidents (strokes) by 55 percent.

In anticoagulant therapy, a dose of the anticoagulant is selected that achieves effective anticoagulation and minimizes bleeding complications (Second Report of the Sixty Plus Reinfarction Study Research Group 1982). The advantage of using warfarin for long-term therapy is that an overdosage is corrected by the administration of a large dose of vitamin K. Large doses of vitamin K are converted to vitamin K hydroquinone by enzymes that are not inhibited by warfarin (Link 1959; Suttie 1990). Correspondingly, persons undergoing warfarin anticoagulant therapy should avoid a diet that is high in vitamin K by limiting, among other things, the intake of green, leafy vegetables (Pedersen et al. 1991).

A Note on Hemophilia

Hemophilia is a bleeding disorder caused by a deficiency in one or, more rarely, two of the clotting proteins (De Angelis et al. 1990). The majority (85 percent) of hemophilia patients are deficient in factor VIII (hemophilia A), with 10 to 12 percent deficient in factor IX (hemophilia B). Rarer forms of the disease result from deficiency in other clotting factors. The hemophilia patient must receive injections of the missing protein in order to maintain proper coagulation. Hemophilia is a genetic disease, most commonly transmitted as a sex-linked recessive trait, and is not related to vitamin K status (Furie and Furie 1988).