The riddle of vitamin K1 deficit in the newborn

The Riddle of Vitamin K1 Deficit in the Newborn Lyonel G. Israels, Esther D. Israels, and Satya P. Saxena

Vitamin K in the fetus and newborn is maintained at levels less than that necessary to achieve full y-carboxylation o f the K-dependent proteins, including those required for hemostasis. As the infant matures and even into adulthood, there is no significant storage pool for this vitamin, and a K1deficient state can be produced by placing an adult on a K-deficient diet for 7 to 10 days. Questions arise as to why the level of vitamin K is so rigidly controlled and why the placental gradient in humans and other mammals maintains the fetus in a K-"deficient" state. The evidence is reviewed that suggests that K-dependent proteins are llgands for receptor tyrosine kinases, which, in the rapidly proliferating cell milieu of the fetus, control growth regulation. Increased stimuli may result in growth dysregulation whereas conversely, the further depletion of vitamin K-dependent proteins, as in warfarin toxicity, depletes the required stimuli for normal embryogenesis. These findings argue for the need for tightly controlled levels of vitamin K consistent with normal embryogenesis.

Copyright 9

1 9 9 7 by W.B. Saunders

Company

uring embryogenesis and into the postnatal period, the vitamin K1 level in the h u m a n fetus and newborn is markedly lower than in the adult. This low Ka milieu may be of significant benefit to the fetus: offsetting the fact that it does place the newborn at hemorrhagic risk. In this article, we examine some of the physiological processes in which KI may participate and seek an explanation for the "K-deficient" state of the fetus and newborn.

D

Introduction Vitamin K1 (2 methyl-3-phytyl-l,4 naphthoquinone) is the essential cofactor for the posttranslational y-carboxylation of a series of glutamic acid (Gla) residues in juxtaposition to the N-terminus of the vitamin K-dependent proteins. 1-3 These Gla residues facilitate the binding of vitamin K-dependent proteins to phospholipid From the Departments of Medicine and Pediatrics, Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba Canada. Supported in part by grants from the Manitoba Medical Seroices Foundation (S.P.S. and L.G.I.) and the Children's Hospital Research Foundation, Winnipeg (S.P.S.). S.P.S. is a recipient of a Heart and Stroke Foundation of Canada Scholarship. Address reprint requests to Lyonel G. Israels, MD, Departments of Medicine and Pediatrics, Manitoba Institute of CellBiology, University of Manitoba, 100 Olivia Street, Winnipeg, Manitoba, R3E OV9 Canada. Copyright 9 1997 by W.B. Saunders Company O146-0005/97/2101-0012505. 00/0

90

m e m b r a n e in the presence of calcium.4 The vitamin K level in the mammalian fetus is tightly regulated by a maternal/fetal placental gradient5; the median vitamin KI concentration in h u m a n cord plasma is 16 p g / m L c o m p a r e d with a maternal median plasma level of 470 p g / m L : a transplacental gradient greater than for any other fat-soluble vitamin. 6 The lower vitamin K1 concentration in the fetus and newborn is refleeted in reduced y-carboxylation of coagulation factors II, VII, IX, and X; proteins C and S; bone protein matrix-Gla protein; osteocalcin; and other Ca 2§ binding proteins whose functions are less well-defined. Although delayed osteocalcin production without rigid skeletal formarion may be of benefit to the fetus, the low levels of the coagulation factors with the attendant hemorrhagic risk is difficult to explain.

Vitamin K1 Levels and P o o l Size in Infants and Adults To prevent hemorrhagic disease due to the low levels of the vitamin K-dependent coagulation factors, newborns usually receive vitamin K1 at birth. 7'8 T h e administration of vitamin K1 to the neonate results in a three to four log increase in hepatic K1 concentration. Guillaumont et al measured hepatic K1 levels in 18 infants who died in the neonatal period. Their mean gestational age was about 34 weeks and mean weight about 2,000 g. In those infants who had not re-

Seminars in Perinatology, Vol 21, No 1 (February), 1997: pp 90-96

Vitamin Kl Deficit in the Newborn

ceived K1, the total hepatic content of K~ ranged from 0.1 to 0.9 #g. Those who had received 2 mg of K1, either orally or intravenously within 24 to 48 hours of birth, had levels between 405 and 2,824 #g.9 Although in the adult the plasma levels of K1 are higher, the storage pool of KI is surprisingly small. The hepatic storage pool of K1 measured in adult liver by Shearer et al 6 ranged from 1.7 to 38.3 #g (median, 7.8 #g). In vitamin K-depleted elderly adults, Frick et al 1~ estimated the daily requirement to maintain a normal p r o t h r o m b i n time to be about 0.5 # g / k g / d or 35 #g per 24 h o u r s - - a n a m o u n t that approximates the size of the hepatic pool. In healthy young adults, restriction o f the total vitamin K~ intake to 10 # g / d resulted in a rapid decrease in the plasma K1 from control levels of over 1.0 n m o l / L to 0.2 n m o l / L within 7 daysJ 1 This was accompanied by a decrease in urinary Gla protein and an increase in descarboxy-prothrombin (PIVKA II). Vitamin K1 deficit is not u n c o m m o n in the elderly and has been suggested to be a significant factor in osteoporosis and hip fractures. Because there is no major storage pool, humans are dep e n d e n t on a continual dietary supply of Ki. Is there a possible disadvantage to higher tissue levels of K~ in the adult as well as the fetus?

The Risks Attending Rapid Cell Turnover in Fetal Growth In considering the various risks to the fetus during a period of rapid growth, the high mitotic rate represents a potential for mutagenic and teratogenic events. Vitamin K~ is not mutagenic in the Ames assay 12 although, in some lymphocyte systems, it does produce small but significant increases in sister chromatid exchange, is Benzo(a)pyrene (BP) is a classical procarcinogen that produces pleomorphic small cell tumours when given by intraperitoneal injection in mice. This polycyclic aromatic hydrocarbon (PAH) is metabolized to its carcinogenic products by arylhydrocarbon hydroxylase (AHH) via a cytochrome p450-dependent mixed function oxidase system. Earlier studies in this laboratory found the following: (1) in the chick embryo, vitamin Ka increases the induction of A H H when given together with BP as c o m p a r e d with BP alonel4; (2) in vitro, vitamin K~ increases the production of BP metabolites and the derived proxi-

91

mate carcinogens by rat liver microsomes; (3) dietary-induced K1 deficiency reduces B P / D N A adducts in the liver o f mice given intravenous BpI~; (4) mice maintained on a K-deficient diet for a 2-week period before and after the injection of BP develop tumors at a slower rate and survive significantly longer than the BP-injected mice fed a normal Kl-replete diet; (5) the co-administration of warfarin with BP reduces the t u m o r incidence and mortality to that of mice on the Kl-deficient diet; (6) conversely, the concomitant supplementation of intraperitoneal K1 with BP accelerates t u m o r growth. 16 Tobacco is a primary source o f exposure to PAHs for humans. Microsomes obtained from the placentas of women who smoke during pregnancy have A H H levels up to 15 times greater than the placentas of nonsmokers. 17 Despite these observations, there is no consistent epidemiological evidence of an increased incidence of childhood cancers associated with maternal smoking during pregnancyJ s'19 The endothelial cells cultured from the umbilical veins o f placentas (fetal in origin) from mothers who smoke have the same low A H H activity as from nonsmokers. A H H levels in various tissues, including liver, measured in medically aborted h u m a n fetuses are no different in smoking and nonsmoking womenfl ~ However, A H H activity is readily inducible on in vitro culture of fetal endothelial cells with 3-methylcholanthrene~7; that is, although A H H is inducible, it is not elevated in fetuses of mothers who smoke. W h e t h e r the placenta allows PAHs or their metabolites to pass is not established in humans, but it does occur in pregnant mice; in these mice, B P / D N A adducts are present in the fetus but are m u c h lower than in the corresponding maternal organsfl I In humans, although most of the PAH may be intercepted and metabolized by the placental barrier, it is unlikely that the placenta is completely impervious. Why there is no induction o f A H H in the fetuses of mothers who smoke is unknown. Perhaps the low K environment of the fetus is a secondary mechanism for protecting the fetus from these and o t h e r xenobiotics not intercepted by the placenta.

Vitamin KI as a Regulator of Embryogenesis Protein tyrosine phosphorylation has a pivotal role in the precise regulation of cell division,

92

Israels, Israels, and Saxena

differentiation, and migration required for normal embryogenesis. The overall level of tyrosine phosphorylation is high in most embryonic tissues during the early stages of development, decreases significantly in late embryogenesis, and is low or undetectable in the same tissues in the adult. 22 The identification of several receptor and non-receptor kinases in embryonic tissues 23'24 associated with major sequential changes in the levels of protein-tyrosine phosphorylation22 and tyrosine kinase activity25 during embryonic development, suggests that such posttranslational modification of target proteins is key to the regulation of orderly embryogenesis. Recently, two vitamin K-dependent proteins, Gas 6 26 and the coagulation inhibitor Protein S,27 respectively, have been identified as ligands for the Tyro 7 (alternatively called Axl, UFO, or Ark) and Tyro 3 (alternatively called Sky, Rse, Brt, or Tif) families of receptor tyrosine kinases (RTKs). 2s'29 The extracellular domain of the RTK family Tyro 3, 7 and Tyro 12 (alternatively called c-Eyk, c-mer) consists of a combination of fibronectin type III and immunoglobulin motifs common to: extracellular matrix proteins, neural cell adhesion molecules, and cell-surface receptors with tyrosine kinase or phosphatase activity.3~ It is believed that these Tyro 3, 7, and 12 RTKs function as both cell adhesion proteins and components of signal transduction pathways. The role of vitamin K-dependent receptorligand systems in cellular processes is as yet unclear, but studies showing transforming activity of Axl (Tyro 7) in NIH 3T3 cells 32'33 and the mitogenic potential of Protein S in smooth muscle cells ~4 suggest a role in growth regulation. The involvement of vitamin K metabolism and function in two well-characterized birth defects, warfarin embryopathy35 and vitamin K epoxide reductase deficiency,36 suggests that developmental signals from K-dependent pathways may be required for normal embryogenesis. Because c-Eyk, a chicken counterpart of the Tyro 12 family, exhibits a broad spatial and temporal expression during developmentY we explored the possible role (s) of a vitamin K-dependent regulatory pathway(s) in embryogenesis. An easily accessible in vivo system to study cell signaling pathways during development is the chick embryo. Well-defined organ development is present by day 10. Hatch occurs on day 21. In the following studies, we injected the air space

of eggs on either day 10 or day 16 with vitamin K1 or warfarin and, after 48 hours, monitored the changes in protein-tyrosine phosphorylation in embryonic tissues by Western blot using 4G10, a monoclonal anti-phosphotyrosine (PY) antibody. Supplementation of vitamin K1 both at an early stage (day 10) or later stage (day 16), significantly increased the tyrosine phosphorylation of many proteins (Fig 1A, 1B), ineluding two intracellular proteins associated with cell attachment, namely focal adhesion kinase (pp125 FAK)and paxillin; pp60 src, a non-receptor protein tyrosine kinase; and c-Eyk, an RTK chicken counterpart of the Tyro 12 family38 (Fig 2). Based on body weight, these effects were observed at doses of vitamin K~ that ranged from three logs below and up to a dose equivalent by weight to that given a normal full-term newborn (0.5 mg). Warfarin, an inhibitor of vitamin K1 epoxide reductase, inhibited the effects of low dose vitamin K1 on tyrosine phosphorylation. This was completely reversible at higher K1 dosage sufficient to by-pass the warfarin metabolic block 35 (Fig 1C). The observation, that vitamin K1 supplementation induces tyrosine phosphorylation of proteins associated with cell attachment, is of particular interest because during the earlier stages of development cell-cell and cell-extracellular matrix junctions are continually remodeled to a e c o modate rapid cell proliferation, growth, and differentiation. A link between the extracellular matrix and the actin cytoskeleton is made at focal adhesion sites. 39 These focal adhesions not only provide an anchor to which the cytoskeleton can apply stress, but are also involved in transducing extracellular information into the cell. 4~ It is known that protein expression and tyrosine phosphorylation of pp125 FAKand its potential substrate paxillin are under developmental control. 42'43The concomitant increase in the tyrosine phosphorylation of pp125 FAKas well as paxillin, an in vivo substrate of both pp125 FAK and pp60S%44 is consistent with the propagation of growth regulatory signals in a vitamin Kl-induced cascade, and suggests that alterations in the levels of vitamin K1 during embryogenesis may result in dysregulation of cell-cell or cellmatrix adhesion and other growth regulatory pathways. It is not clear from our studies whether increased tyrosine phosphorylation of pp125 yak in

V i t a m i n K1 Deficit i n the N e w b o r n

A

B 9d

m

93

C K l 0.4511g+

~. I

,91 "11

4S

K t 4.Slag + II

!

1

1" -i ~

~

,,jr,,lllr-~ v

31"1.

Figure 1. Vitamin K1 induces protein tyrosine phosphorylation during early and late embryogenesis. Eggs were injected with vitamin K~ on day 10 or day 16, and brain tissue was obtained 48 hours later. Control designates uninjected eggs; acetone is the vehicle used to suspend vitamin K~ and warfarin. Proteins were separated by SDS-PAGE (7.5%), transferred to nitrocellulose, and blots probed with an anti-phosphotyrosine (anti-IN) antibody. (A) Anti-PY immunoblot from day 12 brain; (B) anti-PY immunoblot from day 18 brain; (C) same as (A) except that varying doses of warfarin were injected together with vitamin K1 0.45 #g or 4.5 #g. The positions of tyrosine phosphorylated proteins exhibiting changes are indicated. These were identified as pp125FAK (120130 kd), c-Eyk (105-110 kd), paxillin (67-70 kd), and pp60 src (55-60 kd). The topmost band (140 to 150 kd) has not been identified. (Reprinted from TheJournal of Clinical Investigation, 1997, vo199, pp 602-607, by copyright permission of The American Society for Clinical Investigation.)

the presence of vitamin K~ is a result of its autophosphorylation at tyrosine (Tyr)-397, a modification that has been shown to activate other tyrosine kinases, or is caused by the activity of other tyrosine kinase (s) regulated either by a vitamin K-dependent receptor-ligand system or by pp125 F~ itself. Recent studies showed that the autophosphorylation of pp125 r~: generates an S H - 2 - m e d i a t e d interaction with a m e m b e r of the Src family. 45 This interaction enzymatically activates the Src family kinase which, in turn, phosphorylates Tyr-407, Tyr-576, and Tyr-577 of pp125 F~a~to fully activate this kinase. 46 Consistent with these findings, anti-Src i m m u n e complexes isolated from embryos pretreated with vitamin K1 showed increased tyrosine phosphorylation and kinase activity as measured by the phosphorylation of a synthetic peptide. Although evidence for an association between the Src family tyrosine

kinases and the tyrosine phosphorylated Sky (Tyro 3) has been presented, 47 it is not clear whether K-dependent receptor-ligand systems can directly activate Src family kinases and the tyrosine phosphorylation/activity o f pp125 FAK. These effects by vitamin KI on protein tyrosine phosphorylation occur in concert with the increased tyrosine phosphorylation o f c-Eyk, the chicken counterpart of the Tyro 12 family, s7 T h e ligands for c-Eyk or related RTKs involved in mediating the effects of vitamin K~ during chick embryogenesis are as yet unknown. Recent evidence supporting the involvement o f vitamin K for full activity o f Gas6, a ligand for Tyro 7 RTKs, has been presented. 4s-51 The binding of Gas6 to its receptors in HOS cells is largely d e p e n d e n t on the presence of divalent cations; chelation o f Ca 2§ results in loss o f the ability o f r e c o m b i n a n t Gas6 to bind and stimulate Sky in B31 cells. 49's~

94

Israels, Israels, and Saxena

~ VITAMIN KI i"" ~

~

VITAMIN KI,

i

i.p. anti-FAK i.b. anti-PY

i.p. anti-FAK i.b. anti-FAK

FAK .a ~ VITAMINKI

VITAMIN K 1

I

!

Paxillin

i.p. anti-Paxillin i.b. anti-PY

i.p. anti-Paxill|n i.b. anti-Paxillin

ig

i.p. anti-Eyk i.b. anti-PY

c-Eyk

i.p. anti-Eyk i.b. anti-Eyk

Figure 2. Increased tyrosine phosphorylation of pp125 yAK,paxillin and c-Eyk in the brain of vitamin Krpretreated day 12 embryos, pp125 FA~, paxillin, and c-Eyk were immunoprecipitated (i.p.) from detergent-soluble lysates of brain after injection of vitamin K1 on day 10. Proteins were resolved by SDS-PAGE (7.5%), transferred to nitrocellulose, and analyzed by anti-PY immunoblotting (ib) to monitor vitamin K,-induced changes in tyrosine phosphorylation (left panel). The blots were stripped and reprobed with anti-FAK, anti-paxillin, or anti-c-Eyk antibodies for protein expression (right panel). The blots indicate that there was no change in total protein expression, whereas tyrosine phosphorylation was markedly increased by K1. Ig represents an IgG heavy chain. (Reprinted from The Journal of Clinical Investigation, 1997, vol 99, pp 602-607, by copyright permission of The American Society for Clinical Investigation.) These studies suggest that, like other K-dependent proteins, binding of Ca 2+ through Gla residues is important to the full activity of Gas6. Although deletion variants o f Gas6, lacking both Gla domain and epidermal growth factor-like repeats, are capable of binding to the extracellular domains of both Rse and Axl, the presence of Gla residues may facilitate the Ca2+-dependent binding of these proteins to negatively charged m e m b r a n e phospholipids and may also function to p r o m o t e the establishment of a local concentration gradient of the ligand on the surface of receptor-bearing target cells. 52 Consistent with these reports, inhibition of vitamin K effect by warfarin in the present in vivo studies suggests that vitamin K-dependent y-carboxylation of proteins/ligands may be required for the vitamin K1 effects in the embryo.

Previous work from these laboratories relating to the possible regulatory effects of vitamin K1 has recently been summarized. 5~ It is of more than passing interest that vitamin K1 is maintained at low concentration in the h u m a n fetus and increases slowly to adult levels after birth in breast-fed babies u n s u p p l e m e n t e d with vitamin K. In most western countries, vitamin K, is now administered orally or intramuscularly to prevent hemorrhagic disease due to low levels of the vitamin K-dependent coagulation factors at the time of birth. The inhibition of protein-tyrosine phosphorylation by warfarin is consistent with its known toxicity to the h u m a n fetus, 35 because warfarin crosses the placenta and results in fetal death or skeletal anomalies similar to those described in congenital vitamin K epoxide dehydratase deficiency) 6 T h e present studies

Vitamin K1 Deficit in the Newborn

suggest that vitamin Ka is an important element in embryonic development and may explain, at least in part, the advantage of limiting its concentration in the mammalian and human fetus by a tightly regulated maternal/fetal placental gradient. This demonstration of a novel role of vitamin K1 in the tyrosine phosphorylation cascade involving cytoskeletal proteins argues for the advantage of preset margins for vitamin K1, consistent with normal embryogenesis. The transforming and mitogenic potential, in vitro, of the vitamin K-dependent ligand systems Axl and Protein S, and the K~ upregulation of a protein tyrosine cascade in the in vivo chick embryo system, support a role for KI as a potential signal for mitogenesis, cell dysregulation, or transformation. A study showing that adenovirus transformed BHK cells increase y-carboxylation and secretion of a K-dependent protein 54 suggests that such an increase in the production of K-dependent proteins, as potential ligands for one or more RTKs could, in turn, upregulate tyrosine phosphorylation and contribute to cell transformation. Our previous studies with a known carcinogen indicate that there is augmentation of DNA adduct formation, increased procarcinogen metabolism, and tumorogenesis in the presence of K~. We propose that upregulation of signaling by vitamin K~ may provide more fertile ground for mutagenesis in the presence of a chemical or viral signal, and that the relatively small KI pool in the adult and the greatly reduced levels in the fetus are potentially protective.

Acknowledgment It is a special j o y for m e to have the o p p o r t u n i t y to h o n o r Alvin Zipursky. A s u p e r b t e a c h e r a n d clinician, his studies have also materially a d v a n c e d o u r knowle d g e o f n e o n a t a l hematology. His e n t h u s i a s m today for new ideas a n d his d e d i c a t i o n to his art are as great as w h e n we first m e t 40 years ago. May it c o n t i n u e .

References 1. Shearer MJ: Vitamin K metabolism and nutriture. Blood Rev 6:92-104, 1992 2. Olson RE: The function and metabolism of vitamin K. Annu Rev Nutr 4:281-337, 1984 3. Dowd P, Ham SW, Naganathan S, et al: The mechanism of action of vitamin K. Annu Rev Nutr 15:419440, 1995

95

4. Furie B, Furie BC: Molecular basis of vitamin K-dependent y-carboxylation. Blood 75:1753-1762, 1990 5. Shearer MJ, Rahim S, Barkhan P, et al: Plasma vitamin KI in mothers and their newborn babies. Lancet ii:460463, 1982 6. Shearer MJ, McCarthy Fr, Crampton OE, et al: The assessment of vitamin K status from tissue measurements, in Suttie JW (ed): Current Advances in Vitamin K Research. New York, NY, Elsevier Science, 1988, pp 437452 7. von Kries R, Greer FR, Suttie JE: Assessment of vitamin K status of the newborn infants. J Pediatr Gastroenterol Nutr 16:231-237, 1993 8. von Kries R, Shearer MJ, Gobel U, et al: Vitamin K in infancy. EurJ Pediatr 147:106-112, 1988 9. Guillaumont M, Sann L, Leclercq M, et al: Changes in hepatic vitamin K1 levels after prophylactic administration to the newborn. J Pediatr Gastroenterol Nutr 16:1014, 1993 10. Frick PG, Riedler G, Brogli H: Dose response and minimal daily requirement for vitamin K in man.J AppI Physiol 23:387-389, 1967 11. Ferland G, Sadowski JA, O'Brien ME: Dietary induced subclinical vitamin K deficiency in normal human subjects. J Clin Invest 91:1761-1768, 1993 12. Tikkanen L, Matsushima T, Natori S, et al: Mutagenicity of natural naphthoquinones and benzoquinones in the salmonella/microsome test. Mutat Res 124:25-34, 1983 13. Israels LG, Friesen E, Jansen A, et al: Vitamin K~ increases sister chromatid exchange in vitro in human leukocytes and in vivo in fetal sheep cells: A possible role for "vitamin K deficiency" in the fetus. Pediatr Res 22:405-408, 1987 14. Dogra SC, Israels LG: Vitamin K~ amplification ofbenzo(a)pyrene metabolism in chick embryo. Int J Biochem 19:471473, 1987 15. Israels LJ, Ollmann DJ, Israels ED: Vitamin Kl as a modulator of benzo(a)pyrene metabolism as measured by in vitro metabolite formation and in vivo DNA-adduct formation. IntJ Biochem 17:1263-1266, 1985 16. Israels LJ, Walls GA, Ollmann DJ, et al: Vitamin Kl as a regulator of benzo(a)pyrene metabolism, mutagenesis, and carcinogenesis. J Clin Invest 71 :1130-1140, 1983 17. Manchester DK, Parker NB, Bowman CM: Maternal smoking increases xenobiotic metabolism in placenta but not umbilical vein endothelium. Pediatr Res 18:10711075, 1984 18. Neutel CI, Buck C: Effect of smoking during pregnancy on the risk of cancer in children. J Natl Cancer Inst 47:59-63, 1971 19. Pershan G, Ericson A, Otterblad-Olausson P: Maternal smoking in pregnancy: does it increases the risk of childhood cancer? IntJ Epidemiol 21:1-5, 1992 20. Rifkind AB, Tseng L, Hirsch MB, et al: Aryl hydrocarbon hydroxylase activity and microsomal cytochrome content of human fetal tissues. Cancer Res 38:1572-1577, 1978 21. Lu LW, Disher RM, Reddy MV, et al: 32P-postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4-aminobiphenyl and benzo(a)pyrene. Cancer Res 46:3046-3054, 1986 22. Maher PA, Pasquale EB: Tyrosine phosphorylated proteins in different tissues during chick embryo development. J Cell Biol 106:1747-1755, 1988

96

Israels, Israels, and Saxena

23. Pasquale EB, Singer SJ: Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library. Proc Natl Acad Sci USA 86:5449-5453, 1989 24. Adamson ED: Oncogenes in development. Development 99:449-471, 1987 25. Maher PA: Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development. J Cell Biol 112:955-963, 1991 26. Manfiotetti G, Brancolini C, Avanzi G, et al: The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregnlator in the blood coagulation cascade. Mol Cell Biol 13:4976-4985, 1993 27. Dahlback B: Protein S and C4B-binding protein: components involved in the regulation of the protein C anticoagnlant system. Thromb Haemost 66:49-61, 1991 28. Lai C, Lemke G: An extended family of protein-tyrosine kinase genes differentially expressed in the vertebrate nervous system. Neuron 6:691-704, 1991 29. Stitt TN, Conn G, Gore M, et al: The anticoagnlation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 80:661-670, 1995 30. Edelman GM, Crossin KL: Cell adhesion molecules: Implications for a molecular histology. Annu Rev Biochem 60:155-190, 1991 31. Krueger NX, Saito H: A human transmembrane proteintyrosine phosphatase, PTP (, is expressed in brain and has an N-terminal receptor domain homologous to carbonic anhydrase. Proc N a t Acad Sci USA 89:7417-7421, 1992 32. O'Bryan JP, Frye RA, Cogswell PC, et al: axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 11:5016-5031, 1991 33. McCloskey P, Pierce J, Koski RA, et al: Activation of the Axl receptor tyrosine kinase induces mitogenesis and transformation in 32D cells. Cell Growth Diff 5:11051117, 1994 34. Gasic GP, Arenas CP, Gasic TB, et al: Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells. Proc Natl Acad Sci USA 89:231%2320, 1992 35. Hall JG, Panli RM, Wilson KM: Maternal and fetal sequelae of anticoagulation during pregnancy. Am J Med 68:122-140, 1980 36. Pauli RM, Lian JB, Mosher DF, et al: Association of congenital deficiency of multiple vitamin K-dependent coagulation factors and the phenotype of the warfarin embryopathy: Clues to the mechanism of teratogenicity of coumarin derivative. A m J Hum Genet 41:566-583, 1987 37. Jia R, Hanafusa H: The proto-oncogene of v-eyk (v-ryk) is a novel receptor-type protein kinase with extraceUular Ig/FN-III domains. J Biol Chem 269:1839-1844, 1994

38. Saxena SP, Fan T, Li M, et al: A novel role for vitamin K1 in a tyrosine phosphorylation cascade during chick embryogenesis. J Clin Invest 99:602-607, 1997 39. Burridge K, Fath K, Kelley T, et al: Focal adhesions: Transmembranejunctions between the extracellular matrix and the cytoskeleton. Ann Rev Cell Biol 4:487-525, 1988 40. Juliano RL, Haskill S: Signal transduction from the extracellular matrix. J Cell Biol 120:577-585, 1993 41. Luna EJ, Hitt AL: Cytoskeleton-plasma membrane interactions. Science 258:955-964, 1992 42. Turner CE, Schaller MD, ParsonsJT: Tyrosine phosphorylation of the focal adhesion kinase pp125 FAK during development: relation to paxillin.J Cell Sci 105:637-645, 1993 43. Turner CE: Paxillin is a major phosphotyrosine-conmining protein during embryonic development. J Cell Biol 115:201-207, 1991 44. Turner CE: Paxillin: A cytoskeletal target for tyrosine kinases. Bioessays 16:47-52, 1994 45. Schaller MD, Hildebrand JD, Shannon JD, et al: Autophosphorylation of the focal adhesion kinase, pp125 F~, directs SH2-dependent binding of pp60 src. Mol Cell Biol 14:1680-1688, 1994 46. Calalb MB, Polte TR, Hanks SK: Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: A role for Src family of kinases. Mol Cell Biol 14:954-963, 1995 47. Toshima J, Ohashi K, Iwashita S: Autophosphorylation activity and association with Src family kinase of Sky receptor tyrosine kinase. Biochem Biophy Res Commun 209:656-663, 1995 48. Varnum BC, Young C, Elliott G, et al: Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene6. Nature 373:623-626, 1995 49. Nakano T, KishinoJ, Arita H: Characterization of a highaffinity and specific binding site for Gas6. FEBS Letters 387:75-77, 1996 50. Ohashi K, Nagata K, ToshimaJ, et al: Stimulation of Sky receptor tyrosine kinase by the product of growth arrestspecific gene 6. J Biol Chem 270:22681-22684, 1995 51. Nakano T, Kawamoto K, Kigashino K, et al: Prevention of growth arrest-induced cell death of vascular smooth muscle cells by a product of growth arrest-specific gene, gas6. FEBS Letters 387:78-80, 1996 52. Mark MR, Chen J, Hammonds RG, et al: Characterization of Gas6, a member of the superfamily of G Domaincontaining proteins, as a ligand for Rse and Axl. J Biol Chem 271:9785-9789, 1996 53. Israels LG, Israels ED: Observations on vitamin K deficiency in the fetus and newborn. Has nature made a mistake? Sere Thromb Haemost 21:364-370, 1995 54. Berg DT, McClure DB, WaUsJD, et al: Viral transformation increases vitamin K-dependent gamma-carboxylation of glutamate. Exp Cell Res 192:32-40, 1991