25 Uses and Function of Vitamin Κ

W A L T E R H . SEEGERS

I. Historical Sketch 687 II. Blood Coagulation Mechanisms 6 8 9

A. Introduction 6 8 9 B. Blood Coagulation Nomenclature 6 8 9 C. Three Basic Reactions of Blood Coagulation 690 D. Formation of Fibrin 691 E. Cross-Linking of Fibrin 692 F. Formation of Thrombin 6 9 2 G. Formation of Autoprothrombin C (Factor Xa) 6 9 6

III. Role of Vitamin K-Dependent Proteins 697 IV. Inactivation of Vitamin K-Dependent Proteins by

Antithrombin III 6 9 9 V. A Fifth Vitamin K-Dependent Protein 6 9 9

VI. Depression of Vitamin Κ Activity 701 VII. Vitamin Κ Function and the Structure of Prothrombin 702

VIII . Five Distinct Vitamin K-Dependent Molecules 707 IX. Some Possibilities and Needs for Investigation 708

References 7 1 0

I . H I S T O R I C A L S K E T C H

In a shor t while it will be a half cen tury since vitamin Κ was discovered by Dam (1929, 1935). It was o n e of the last vitamins to be found d u r i n g an era when vitamin studies were fashionable, a n d biochemistry was e m e r g i n g from the field of nut r i t ion . Subsequent ly , vitamins became r e g a r d e d as a special class of d r u g s . T h u s , in concer t with pharmacology , indust ry , a n d clinical applicat ions, a t tent ion became b road in scope. Vitamin Κ deficiency proved to be associated with p r o t h r o m b i n defi-

687

688 Walter Η . Seegers

ciency (Dam et al, 1936), bu t the original da ta were not especially con­vincing. Vi tamin Κ was isolated (Dam et al, 1939; Doisy et al, 1939) a n d was conveniently available for research purposes .

In the clinics the vitamin became especially impor t an t (Owen, 1974) for its usefulness in obstructive j aund ice , h e m o r r h a g i c disease of the newborn , poor absorpt ion syndromes , a n d liver diseases. A little later it was demons t r a t ed in clinical work that the h y p o p r o t h r o m b i n e m i a in­duced with dicumarol could be reversed with vitamin Κ (Overman et al, 1942; Davidson a n d MacDonald, 1943).

T h e backg round for the discovery of d icumaro l relates to nutr i t ion prob lems in agr icul ture . Spoiled sweet clover silage consumed by cows induced a b leeding tendency that was fatal in many he rds (Schofield, 1922, 1924). T h e p rob lem was not re la ted to infection, a n d se rum or def ibr inated blood temporar i ly s topped the h e m o r r h a g e . Th is work was conf i rmed (Roderick, 1929) a n d ex t ended (Roderick, 1931) to record that the p r o t h r o m b i n level was depressed in cattle with spoiled sweet clover disease.

T h e toxic agent was isolated a n d induced to crystallize (Campbell a n d Link, 1941). It was synthesized (S tahmann et al, 1941) a n d rela ted com­p o u n d s were also synthesized, tested, a n d in t roduced for clinical trials. T h e s e dramat ic events a re vividly described in the Harvey Lecture by Link (1944). Basic to the whole clinical en te rpr i se was the demons t ra t ion that withdrawal of Dicumarol was followed by a res torat ion of p ro ­th rombin concentra t ion a n d t he re was n o d a m a g e at the manufac tu r ing site which was known to be the liver.

T o learn about the effect of vitamin Κ on blood coagulat ion, it was essential to know more about these mechanisms. For that pu rpose , vitamin Κ a n d Dicumarol , considered as a family of c o m p o u n d s , served as convenient p r i m e modifiers for expe r imen t s in blood coagulation. Like the rap id proliferat ion of vitamins at one t ime, the discovery of new coagulat ion factors was equally exciting a n d occurred d u r i n g the mid-century per iod . It was essential to isolate each of the previously recog­nized ones , as well as the ones that very likely existed.

T h e main p rob lem with purification was the low concentra t ion of coagulat ion factors in blood, their lability, t he est imation of concentra­tion, and the state of technology in pro te in chemistry. T h e progressive conquest of these obstacles followed the in t roduct ion of ion exchange resins, polyacrylamide gel e lectrophoresis , gel filtration, amino acid analysis, new molecular weight de te rmina t ion me thods , a n d advances in general pro te in chemistry. Spectacular deve lopments re la ted to o u r knowledge of blood coagulation occur red d u r i n g the past 15 years. Very

25 . U s e s and Funct ion o f Vi tamin Κ 689

early in the purification of p r o t h r o m b i n , it was possible to show tha t vitamin Κ is not a prosthet ic g r o u p associated with the p r o t h r o m b i n molecule (Seegers, 1962).

T o d a y we know that the re a r e five prote ins in plasma that r equ i re vi tamin Κ for their synthesis by the liver. T h e s e a re qui te similar in s t ruc ture ; in fact, they a re so similar tha t my evidence for their origin in p r o t h r o m b i n was not easily set aside. I shall out l ine a useful a n d h o p e ­fully accurate way to study the n a t u r e of these mechanisms. T h e func­tion of the vitamin K - d e p e n d e n t prote ins can then be seen u p to the t ime of o u r p resen t knowledge.

In the synthesis of p r o t h r o m b i n , o n e of the functions of vi tamin Κ is at the postr ibosomal level (Suttie et al, 1974). T e n glutamic acid res idues a re conver ted to γ -carboxyglutamic acid a n d serve as calcium ion bind­ing sites (Stenflo et al., 1974; Magnusson et al., 1974; Nelsestuen et al, 1974). T h e o the r four vi tamin K - d e p e n d e n t prote ins also contain a cer­tain n u m b e r of γ -carboxyglutamic acid res idues .

I I . B L O O D C O A G U L A T I O N M E C H A N I S M S

A. Introduction

T h e need to u n d e r s t a n d the function of vi tamin Κ in te rms of b lood coagulat ion was expressed by Suttie (1969) when he stated, " . . . it has been possible for workers in teres ted in the mechanism of action of vitamin Κ to p roceed even t h o u g h details of the coagulat ion process a r e unresolved." I p r e s u m e by now h e has taken m u c h satisfaction f rom the recent deve lopments .

T h e l i tera ture on the subject of b lood coagulat ion is so vast that very few can keep u p with it and still have t ime to write themselves. I n o r d e r to keep the references at a sensible level a n d to avoid selective credit to cont r ibutors , I shall only indicate a few valuable sources, such as Biggs (1972), Bradshaw a n d Wessler (1975), Fearnley (1965), J o h n s o n (1971), Koch-Weser a n d Sellers (197la ,b) , Olson (1975), Quick (1942), Reich et al. (1975), Seegers (1962, 1967), Seegers et al. (1975), a n d Win t robe (1974).

B. Blood Coagulation Nomenclature

Each specialty in science has its own language a n d in the field of blood coagulat ion, plasma componen t s a r e r e fe r red to as factors a n d have

690 Walter Η . Seegers

been given R o m a n numera l s to serve as an equivalent to diverse com­m o n names . Platelet factors a re given Arabic n u m b e r s . T h e list below has been stable for several years. No te that the vitamin K - d e p e n d e n t factors a re I I , V I I , IX, X and XIV.

Factor I Fibrinogen Factor II Prothrombin

Factor III Thromboplastin, tissue factor Factor IV Calcium ions Factor V Ac-globulin, labile factor, proaccelerin

Factor VI Not verified Factor VII Cothromboplastin, stable factor, proconvertin, serum prothrombin

conversion accelerator (SPCA) Factor VIII Antihemophil ic factor A or globulin, platelet cofactor I, hemophil ia A

factor, thromboplastinogen Factor IX Antihemophil ia Β factor, autoprothrombin II, Christmas factor,

platelet cofactor II Factor X Autoprothrombin III, A u t o - I I I , Stuart-Prower factor,

prothrombokinase Factor XI Plasma thromboplastin antecedent (PTA), antihemophilic factor C

Factor XII Hageman factor, glass factor, contact factor Factor XIII Fibrinoligase, plasma transglutaminase, fibrin stabilizing factor

T h e above system has been e x p a n d e d to des ignate active enzymes as follows: Factor H a ( thrombin) ; Factor IXa; Factor Xa ( au top ro th rombin C, thrombokinase) and variants of the latter have been described. O t h e r active forms a re Factor XIa , Factor X H I a , a n d Factor X lVa . It is not correct to write Factor Va because n o enzyme activation occurs when th rombin potent iates the activity of Factor V, a n d the same seems to be t rue for Factor V i l l a as modified by th rombin .

C. Three Basic Reactions of Blood Coagulation

In this cybernetic system, the control l ing componen t s consist of posi­tive and negative feedback, control led chain reactions, mult iple enzyme involvement, a p p a r e n t stoichiometric reactions, a n d the re is integrat ion with o rgan function. It is convenient to consider inhibitors last, and to begin by dividing the blood coagulat ion system into th ree basic events as follows:

(1) the format ion of a u t o p r o t h r o m b i n C (Factor Xa); (2) the format ion of th rombin ; (3) the format ion of fibrin.

T h e s e reactions involve format ion of the clot a n d the activity of two enzymes. T h e events most likely occur in sequence a n d each successive reaction d e p e n d s u p o n the p reced ing one (Fig. 1).

2 5 . U s e s a n d Funct ion of Vi tamin Κ 691

VARIOUS CONDITIONS AND SUBSTANCES

H H — I (I) AUT0PR0THROMBIN Μ —• AUT0PR0THROMBIN C

(FACTOR X) 'PEPTIDES

(Z) PROTHROMBIN AUTOC

THROMBIN 'PEPTIDES

(3) FIBRINOGEN THROMBIN

FIBRIN 'PEPTIDES

Fig. 1 . T h r e e basic chemical reactions of blood coagulation. T h e first occurs under various conditions. T h e second is d u e to the specific formation of thrombin and fragments. In the third, the visible clot is formed.

D. Formation of Fibrin

Purified fibrinogen or the fibrinogen of plasma with a molecular weight nea r 340,000 has th ree polypept ide chains. T w o a re susceptible to limited proteolysis by th rombin . T h e process removes fibrinopeptide A and fibrinopeptide Β from each fibrinogen molecule. Th i s a m o u n t s to abou t 3 % of the large molecule. T h e fibrinopeptides a r e split f rom the Aa a n d Ββ chains, bu t no th ing is r emoved from t h e y chain. Each one of the th ree chains of fibrinogen is dupl ica ted in the fibrinogen molecule. T h e function of th rombin be ing cons idered can thus be r ep re sen ted as follows:

T h e fibrinogen, without fibrinopeptides, is r e fe r red to as a fibrin m o n o m e r a n d by a process of self-assembly, the m o n o m e r forms poly­mers . T h e molecules align end- to -end a n d side-to-side, a n d the fibrin has in te rconnect ing b ranches . T h e resul t ing gel is of variable s t rength a n d consistency d e p e n d i n g pr imari ly u p o n the concentra t ion of the pro te in a n d p H of the react ion. T h e ra te of fibrin format ion is d e p e n d e n t on p H , ionic s t rength of the m e d i u m , fibrinogen concentra t ion, t h rombin concentra t ion, calcium ion concent ra t ion , t e m p e r a t u r e , a n d many o the r variables.

T h r o m b i n (Αα ·Β0 · γ ) 2

(α φ · γ ) 2 + 2Α + 2Β

[(«·0·7Η

692 Walter Η . Seegers

Ε. Cross-Linking of Fibrin

Artificial fibrin obtained by clotting purif ied f ibrinogen with purif ied th rombin has several characteristics tha t dist inguish it from the t rue or genu ine fibrin obta ined from whole blood clots. T h e main p rope r ty generally fea tured is solubility in u r e a as a characteristic of fibrin f rom purif ied systems (fibrin-s) a n d insolubility for the fibrin (fibrin-i) f rom the natura l clot. T h e conversion of fibrin-5 to fibrin-i is b r o u g h t about by the activated cross-linking enzyme of plasma. It functions after it has been conver ted by t h rombin and /o r a u t o t h r o m b i n C to its active form (Factor X H I a ) . T h e enzyme forms pep t ide bonds between p re fe r r ed glutamic acid a n d lysine residues. T h e format ion of cross-linked fibrin can be indicated as follows:

Factor X I I I J T h r o m b i n

Factor X H I a ί(α'β'Ύ)2]η -[(<*-j3-y)E + N H 3

Plasma Factor X I I I , with a molecular weight of 340,000, is composed of two pairs of nonident ical po lypept ide chains A a n d B. T h e respective molecular weights a r e 75,000 a n d 88,000. T h e formula for the t e t r amer can be writ ten as A 2 B 2 . T h e A chains contain the catalytic function a n d a re the only chains found in the crystalline pro te in obtained from platelets. Activation occurs in two steps as follows:

T h r o m b i n C a 2 +

A 2 B 2 — — — — — — — — ι * - A 2 B 2 - * A 2

+ + 2 Pept ides B 2

T h e A 2 B 2 s t ruc ture is not active, bu t dissociation occurs in the presence of calcium ions. A n active center A ' — S H is u n m a s k e d to gene ra te the active pro te in while the Β chains then n o longer serve as carr iers .

F. Formation of Thrombin

Pro th rombin was the first one found to be vitamin K-dependen t for its synthesis. About half of the molecule is n e e d e d for the t h r o m b i n struc­tu re (Fig. 2). For many years, da ta completely suppo r t ed the view that the format ion of t h rombin is a process in which p r o t h r o m b i n is de­g raded . It was only recently, however , that functions for the n o n t h r o m -bin por t ion of p r o t h r o m b i n were found . It is the region where phos­phol ipids , calcium ions, and Ac-globul in b ind to form complexes , a n d

2 5 . U s e s a n d Funct ion o f V i tamin Κ 693

PROTHROMBIN P r o f r o g m e n t ϊ , ! , Α , Β Ι B 2

Ala f ^ |- .. ymmmmm Ser Prethrombin 1 Ser « τ \ | . ty/mum^m Ser

Prethrombin 2 Thr ι, |:. ζ Ser '—s-s—'

He

Thrombin Thr cpC Ser

Thrombin- E Thr r̂ > Lys Ser

Human ι j ^mm^mmmmm Fig. 2 . T h e prothrombin molecule, with a molecular weight near 73 ,000 , is drawn to scale as a single polypeptide chain. In experiments , prothrombin fragment 1 is removed by thrombin, yielding prethrombin 1. Prothrombin fragment 2 is removed by autoprothrom-bin C, yielding prethrombin 2. Alternatively, prothrombin fragment 1*2 can be removed by autoprothrombin C and later divided into prothrombin fragment 1 and prothrombin fragment 2 by thrombin. Autoprothrombin C breaks an Arg-I le bond in prethrombin 2 to develop thrombin activity. By a slow autolysis process, the Β1 chain of thrombin is lost and practically only thrombin esterase activity remains. T h e degradation of h u m a n prothrom­bin is the same, except that thrombin splits an A l peptide (13 amino acids) from the A chain of thrombin. This split involves an A r g - T h r bond. In the bovine material, the corresponding bond is Lys -Thr and is not attacked by thrombin.

thus m a k e the activation process efficient. T h e b ind ing sites in the p r o ­t h r o m b i n molecule a re pr imari ly p rov ided by the recently discovered γ-carboxyglutamic acid res idues at the N H 2 - t e r m i n a l region . Wi thout vi tamin K, an incomple te p r o t h r o m b i n is p r o d u c e d in which the corre­s p o n d i n g res idues a r e glutamic acid. A m o n g the degrada t ion f ragments of p r o t h r o m b i n p r o d u c e d d u r i n g activation a r e accelerators a n d in­hibitors.

PROTHROMBIN

0 40 60 HO TIME IN MINUTES

Fig. 3 . This illustrates the conversion of purified prothrombin (1,000 U/ml), in 25% sodium citrate solution, with only autoprothrombin C in various amounts (U/ml on each curve). T h e r e is a lag phase, followed by the generation of thrombin.

694 Walter Η . Seegers

I600Y ,0.3mg/ml

5 iZOO\ THROMBIN ZYMOGEN

^ 800 Ca ions Ac-globulin Lipids AUTOPROTHROMBIN C

S 400 THROMBIN + Peptide(s)

0*—> ' ' ' 1

0 5 10 15 20 ACTIVATION TIME (MIN.)

Fig. 4 . This illustrates the generation of thrombin from a system of purified pro­thrombin, purified Ac-globulin, purified autoprothrombin C, calcium ions, and crude cephalin. With all else kept constant, a decrease in phospholipid concentration is associated with a decrease in yield of thrombin. Similarly, with all else constant, any one of the reactants can be decreased to decrease the yield of thrombin. Compare with Fig. 3 to appreciate that the presence of lipids, Ac-globul in , and calcium ions introduces more rapid activation and mechanisms for control of rate at which thrombin generates.

For the conversion of p r o t h r o m b i n to classical t h rombin , only au to­p r o t h r o m b i n C (Factor Xa) is essential. Very favorable condit ions a re provided by a 2 5 % sodium citrate solution (Fig. 3). T h e powerful en­zyme is, nevertheless, not sufficiently active no r p resen t in high e n o u g h concent ra t ion u n d e r physiological condit ions to function adequately . T o compensa te for this and to in t roduce a means to control the enzyme, s u p p o r t is suppl ied from accessories; namely, plasma Ac-globul in (Fac­tor V), calcium ions, and platelet factor 3 (Fig. 4). A condensed equat ion for t h rombin format ion, r eco rded in two nomencla tu res , follows:

Calcium ions Platelet factor 3 Ac-globulin Autoprothrombin C

Prothrombin Thrombin + Peptides

C a 2 +

PF-3 Factor V Factor Xa

Factor II Factor I la -I - Peptides

T h e f ive-component system out l ined above yields all of the t h rombin possible from the selected a m o u n t of p r o t h r o m b i n . By reduc ing the

25 . U s e s and Funct ion of Vi tamin Κ 695

concent ra t ion of platelet factor 3 stepwise, the yield of t h r o m b i n can be r educed . Reduc ing the p r o t h r o m b i n , a u t o p r o t h r o m b i n C, Ac-globul in , or calcium ion concent ra t ion stepwise is associated with a corre­spondingly r educed yield of t h rombin . T h r o m b i n initially makes A c -globulin m o r e active bu t that is no t a prerequis i te for the function of Ac-globul in. O n the basis of this complex way to obtain t h rombin , five different deficiencies in t h r o m b i n format ion a re clearly possible: namely, a deficiency for p r o t h r o m b i n , for a u t o p r o t h r o m b i n I I I , for Ac-globulin, for platelet factor 3 , a n d for calcium ions. I n addi t ion, the a p p a r e n t stoichiometric n a t u r e of the react ion is the basis for buffer ing the genera t ion of t h rombin activity.

I m p o r t a n t facts for t h r o m b i n genera t ion can be summar ized (Fig. 5): P r o t h r o m b i n is a substrate which is d e g r a d e d by a u t o p r o t h r o m b i n C as enzyme. T h e enzyme can function by itself, bu t to control its function a n d to m a k e it m o r e effective than by itself, Ac-globul in a n d phos­pholipids f rom platelets a re involved. Lipids serve as a surface on which the molecular interactions take place. Calcium ions presumably enable the format ion of complexes involving calcium b ind ing sites on p ro ­th rombin (Henr iksen a n d Jackson , 1975). Lipids b ind directly to p r o ­t h r o m b i n in coopera t ion with calcium ions a n d γ-carboxyglutamic acid b ind ing sites. Ac-globul in b inds at the p ro f r agmen t 2 a rea in p r o t h r o m ­bin a n d p r e t h r o m b i n 1.

Calcium b ind ing sites of p r o t h r o m b i n a re at the N H 2 - t e r m i n a l e n d of p r o t h r o m b i n . T h e s e a re incomple te when the function of vitamin Κ is depressed . T h e s e a re also no t t he r e when p r e t h r o m b i n 1 is used as a

CALCIUM IONS -TISSUE EXTRACT—< |

C0THR0M80PLAST1NH (F-BL)

AUTOPROTHROMBIN I (H)

irOPROTHROMBM H ( F - I ) '-PLATELET COHICTO R (F -M) HUTELET FACTOR- 3 -CALCIUM ION S

ITHER WAY S

AUTOPROTHROMBIN C

^PLATELET FACTO R 3 .CALCIUM ION S

PRETHROMBIN and-PROTHROMBIN

^ » THROMBIN PEPTIDES 'PEPTIDES

Fig. 5. T h r e e basic reactions of blood coagulation, illustrating formation of thrombin by autoprothrombin C and accessories and the formation of autoprothrombin C by multi­ple ways.

696 Walter Η . Seegers

source of t h rombin . I gno red has been the fact tha t p r e t h r o m b i n 1 acti­vates perfectly well if sufficient p rocoagulan t material is used (Baker a n d Seegers, 1967). Unti l now the abnorma l p r o t h r o m b i n molecule isolated u n d e r vitamin K-deficient condit ions has not been s tudied adequate ly from the s tandpoin t of its activation with biological materials .

G. Formation of Autoprothrombin C (Factor Xa)

T h e format ion of a u t o p r o t h r o m b i n C occurs u n d e r a variety of condi­tions a n d with the suppo r t of several different substances. Based on their origin, at least four main classifications of substances can be m a d e ; namely, as follows:

1. Extrinsic g r o u p a. Calcium ions b. Tissue thromboplas t in c. Factor VI I (cothromboplast in)

2. Intr insic g r o u p a. Calcium ions b . Platelet cofactor (Factor V I I I ) c. Platelet factor 3 d. A u t o p r o t h r o m b i n II (Factor IXa)

3. Autocatalysis 4. Enzymes a n d enzymes f rom snake venom

a. Tryps in , papa in , cathepsin C b. Russell's viper, Echis carinatus, etc.

T h e autocatalytic format ion of a u t o p r o t h r o m b i n C from purif ied au­top ro th rombin I I I occurs most rapidly in s t rong salt solutions such as 2 5 % sodium citrate solution. It also occurs in physiological saline so­lutions.

T h e yield of a u t o p r o t h r o m b i n C by the extrinsic a n d intrinsic g roups of procoagulants is usually less than with snake venoms such as Russell's viper venom. As a consequence, the sera of most species contain a p p r e ­ciable amoun t s of residual or unact ivated a u t o p r o t h r o m b i n I I I . T h r o m ­boplastin of the extrinsic system is structurally a n d functionally different f rom PF-3 of the intrinsic system. W h e n the two systems a re in opera t ion at the same t ime, the result is a synergistic function.

T h e mechanics of a u t o p r o t h r o m b i n C format ion by the intrinsic sys­tem are similar to th rombin format ion. In both cases, an enzyme creates an active enzyme in the p resence of accessory substances. In the case of a u t o p r o t h r o m b i n C format ion, Factor IXa, platelet factor 3 , Factor V I I I , a n d calcium ions a re involved. A reduc t ion in concent ra t ion of a reactant f rom its o p t i m u m concentra t ion limits the yield of active enzyme. As

25 . U s e s and Funct ion o f Vi tamin Κ 697

d o n e previously for t h r o m b i n format ion , the r equ i r emen t s for intrinsic a u t o p r o t h r o m b i n C format ion (Fig. 5) a r e stated in two nomencla tures as follows:

Calcium ions Platelet factor 3 Antihemophil ic factor A Antihemophil ic factor Β

Autoprothrombin III • A u t o p r o t h r o m b i n C + Peptide(s)

Factor IV PF-3 Factor VIII Factor IXa

Factor X • Factor Xa + Peptide(s)

O n e view, which has become d o g m a , accounts for the format ion of Factor IXa. H a g e m a n factor becomes activated u p o n contact with certain surfaces, t hen p roduces Factor XIa f rom its p recursor . Factor XIa can t hen p r o d u c e Factor IXa. Factor XI seems to have a molecular weight nea r 160,000. Factor IXa is also p r o d u c e d by t h r o m b i n when the lat ter is in the o p t i m u m concentra t ion , a n d this may be an i m p o r t a n t way to account for coagulat ion in Factor X I I deficiency.

U n d e r condit ions of extrinsic a u t o p r o t h r o m b i n C format ion, only four componen t s a r e involved; namely, the substra te ( au top ro th rombin I I I ) , tissue thromboplas t in , Factor V I I a n d calcium ions. Presumably, Factor V I I is the enzyme. Tissue th romboplas t in consists of phos­phol ip id b o u n d to p ro te in . Equat ions a r e given in two nomenc la tu res :

Calcium ions Cothromboplastin Thromboplast in

Autoprothrombin III • A u t o p r o t h r o m b i n C + Peptide(s)

Factor IV Factor VII Factor III

Factor X »-Factor Xa + Peptide(s)

I I I . R O L E O F V I T A M I N K - D E P E N D E N T P R O T E I N S

T h e discussion of the n a t u r e of the blood coagulat ion mechanisms u p to this poin t has considered p rocoagu lan t effects. Ant icoagulant effects a r e equally impor t an t bu t need not be taken u p extensively to com­p r e h e n d the role of vitamin K - d e p e n d e n t prote ins a n d the effects of

698 Walter Η . Seegers

Kallikrein

Factor X I (ΡΤΔ ) •Facto r X l l o ^ X I I

Ca f tt

f (F-IXa)Auto-ll -i

Platelet Cofacto r Η (F-VIII )

Platelet F -3 - \ Calcium Ion s -J

Calcium Ion s - η Tissue Extrac t - Ί

Factor VII * Η

Factor XI a

i—Factor IX *

•Autoprothrombi n I I (Factor X )

*Prothrombin -

Peptide(s) CAc^lobuli n (F-V) -- Platele t Facto r 3 - Calciu m Ion s

C (")• . • = =

Autoprothrombi n 11- A (F-Xa Inhibitor )

Inactive Platelet Cofacto r

" Inactiv e Comple x

ANTITHROMBIN II I (Heparin Cofactor )

"Inactive Comple x

Inactive Ac-Globuli n

P* (t)(-

Fibrinogen- ^ FD P

ρ = Plasm m (Fibrinolysin )

* Vitami n K-dependen t

-Profragmen t I an d Prof ragment 2

Plasma Fibrinoligase

(F-ΧΙΙ Γ

35' \ t Activ e , I Cnlciur

(F-XIII ) W Ion s

Platelet F -2 ^Peptides

FIBRIN-S FDP -

Calcium

C K Ρ Solubl e Fibrin- i Polypeptide s

I SEEGERS - MURANQ I976~| (FDP )

Fig. 6 . Three basic reactions of blood coagulation. Same as Fig. 5, with additions to

illustrate inhibition of procoagulants, the role of platelets, the formation of Factor IXa, the

lysis of fibrinogen and fibrin by plasmin. Antithrombin III inactivates thrombin, autopro­

thrombin C, Factor IXa, and Factor VII . T h e place of the five vitamin K-dependent pro­

teins is marked. (Seegers and Murano, 1976.)

drugs . A d iagram of the procoagulan t system has been projected (Fig. 5) a n d repea ted to include the ant icoagulant effects extensively discussed in the l i terature , bu t not elaborated on in this p a p e r (Fig. 6).

T h e central prote in in these mechanisms is p r o t h r o m b i n , without which a clot cannot form unless bacteria, snake venoms, or o the r ex­t raneous materials come to bear on the situation. P r o t h r o m b i n activation is d e p e n d e n t u p o n a u t o p r o t h r o m b i n C which itself is der ived from the vitamin K-dependen t p roenzyme called Factor X. For the conversion of Factor X to Factor Xa, one mechanism involves Factor V I I and ano the r , Factor IX. Both of these are vitamin K - d e p e n d e n t prote ins . T h u s , four prote ins at the very center of the blood coagulat ion mechanisms a re requ i red . Removal of any one of t h e m f rom the blood is associated with a bleeding tendency. O n e en igma which remains is qui te evident. Why a re Factor VI I a n d Factor IX essential for p r o p e r hemostasis when each one , in purif ied systems, part icipates in the separate system that is qui te adequa te for the format ion of active Factor X?. T h e intrinsic a n d extrinsic

25 . U s e s a n d Funct ion o f Vi tamin Κ 699

systems a re r ep resen ted as r e d u n d a n t systems on the basis of excellent laboratory data , bu t in clinical cases they a re not .

I V . I N A C T I V A T I O N O F V I T A M I N K - D E P E N D E N T

P R O T E I N S B Y A N T I T H R O M B I N I I I

Ant i th rombin I I I is m o r e closely re la ted to the inhibit ion of active vitamin K - d e p e n d e n t prote ins t han any o the r inhibi tor . An t i t h rombin I I I is a p lasma pro te in a l ready known at the beg inn ing of this century a n d the re a re extensive l i te ra ture resources , as for example , Dombrose et al. (1971) a n d Bradshaw a n d Wessler (1975). Th i s plasma prote in has been obta ined in purif ied fo rm recently in several laboratories. Anti­t h rombin I I I neutral izes the active forms of all four vitamin K - d e p e n d e n t enzymes cons idered in the discussion thus far.

A mutua l deple t ion system is involved, in which the re is a one- to-one molecular neutral izat ion. In the case of t h rombin , the active serine center is r equ i red , as well as a rg in ine res idues of the inhibi tor . T h e mutua l inactivation process is accelerated by hepa r in , a n d for that func­tion, lysyl res idues of the inhibi tor probably serve as b ind ing sites for hepa r in . I n both cases of inhibit ion, the activity of an t i th rombin I I I is also d iminished because complexes form which r ep re sen t enzyme plus inhibi tor . An t i t h rombin I I I also reduces the activity of plasmin, bu t only limited informat ion about the mechan ism is available. T h e fact that a deficiency of an t i th rombin I I I is accompanied by a th rombos ing ten­dency is evidence that it is o n e of the most impor t an t inhibitors of the active vi tamin K-dependen t enzymes.

V . A F I F T H V I T A M I N K - D E P E N D E N T P R O T E I N

Attent ion to a fifth pro te in was pr imari ly d u e to work in this labora­tory. T h e names given to it a re Prote in C a n d a u t o p r o t h r o m b i n II -A. Ne i the r n a m e is entirely satisfactory. A u t o p r o t h r o m b i n I I -A is an in­hibi tor p r o d u c e d by th rombin . I n fact, it was discovered when purif ied p r o t h r o m b i n complex was activated by small a m o u n t s of purif ied th rombin . Ins tead of the expected genera t ion of t h rombin , an inhibi tor was p r o d u c e d ( M a m m e n et al., 1960). T e n years later, Marciniak (1970) concluded that it is not der ived f rom p r o t h r o m b i n . It is a competit ive inhibi tor of a u t o p r o t h r o m b i n C, a n d is composed of two polypept ide chains with tentatively arr ived at a p p a r e n t molecular mass of 40,000 a n d 22,000 dal tons ( M u r a n o et al, 1974).

Dur ing the 15 years that work on a u t o p r o t h r o m b i n II-A was p ro ­gressing in this laboratory, we were unab le to prove that it was distinct f rom o the r m e m b e r s of the p r o t h r o m b i n complex (vitamin

700 Walter Η . Seegers

K-dependen t proteins) . O u r a t tempts were nea r ing success when Stenflo (1976) isolated Prote in C a n d differentiated it f rom all the o thers . By using an ant ibody to Protein C, which Stenflo suppl ied a n d by applying o the r criteria, it was possible to p rove that a u t o p r o t h r o m b i n I I -A is an active form of Prote in C (Seegers et aL, 1976).

Stenflo (1976) surveyed the recent l i terature a n d p resen ted the evi­dence that all five vitamin K - d e p e n d e n t prote ins a re homologous (Fig. 7) a n d presumably this conclusion will gain fur ther exper imenta l suppor t . Th is implies that there was a c o m m o n ancestral gene a n d that all five prote ins a re the result of genetic d ivergence. Only p r o t h r o m b i n has a h igh molecular weight (73,000), as c o m p a r e d with the o thers (circa 55 ,000). Th i s is because p r o t h r o m b i n has in ternal molecular homology (Hewet t -Emmet t et aL, 1974) as a resul t of partial gene duplicat ion. Th i s genetic event must have occur red after the re were five vitamin K-dependen t prote ins . T o get an idea as to how long ago that occur red , consider the following: Chicken p r o t h r o m b i n , like bovine p r o t h r o m b i n , has a molecular weight near 73,000 (Walz et aL, 1974b), a n d let us assume that in ternal homology will be found , d u e to partial gene dupl icat ion. T h e n , we can go on to say that five pro te ins existed longer than 300 million years, because mammal s (cow) diverged from birds (chicken) 300 million years ago. It is not a wild use of the imaginat ion to suppose that vitamin Κ was requ i red in those times.

A n o t h e r interes t ing fact is tha t a u t o p r o t h r o m b i n I I -A serves to induce fibrinolysis in addi t ion to its ant icoagulant function. It is thus a u n i q u e pro te in , with a dual function. T h e molecular basis for its function in fibrinolysis is only to a minor d e g r e e d u e to activation of profibrinolysis (plasminogen). T h e major por t ion of the induced fibrinolysis is d u e to the suppress ion of inhibitors of fibrinolysis (Zolton a n d Seegers, 1973.)

Prothrombin

Factor I X Ty r

Factor X

Protein C

Factor VI I

Alo Asn|Lys|Gl y Ph e Le u Gl a Gl a

Leu Gl a Gl a Asn Se r Gl y

Ala As n Se r

Ala As n Se r

Ala As x

Lys

Phe Le u Gl a Gl a

Phe Le u Gl a Gl a

Gly Ph e Le u

Phe

Val Ar g

Val Ar g

Val

Lys

— Le u

Lys Gi n

Arg Pr o

Gly As n Le u

Gly As n Le u

Gly As n Le u

Gly As n Val

Stenflo J . B.C . 251 . 35 5 (1976 )

Fig. 7. Indication for homology of five vitamin K-dependent proteins. Modified from arrangement of Stenflo (1976). Gla = γ-carboxyglutamic acid.

25 . U s e s and Funct ion o f Vi tamin Κ 701

It r emains for fu ture work to d e t e r m i n e whe the r drastic reduc t ion of Prote in C concent ra t ion with oral ant icoagulants seriously impairs m u c h desi red fibrinolysis. T h e position of Prote in C in blood coagulat ion is inc luded on Fig. 6, a n d the following functions a r e known: (1) Inhibi tor of blood coagulat ion; (2) competi t ive inhibi tor of a u t o p r o t h r o m b i n C; (3) p r o m o t e s fibrinolysis by depress ing inhibi tors; (4) cofactor for epi­n e p h r i n e in platelet aggrega t ion ; a n d (5) inhibits activation of p r e t h r o m ­bin 1 by trypsin.

V I . D E P R E S S I O N O F V I T A M I N Κ A C T I V I T Y

T h e r e a re n u m e r o u s valuable pape r s , books, a n d reviews abou t vi tamin Κ deficiency a n d its depress ion with Dicumarol a n d related d rugs . T o illustrate the main points , I a m presen t ing a previously u n ­publ ished il lustration f rom the series of Mul le r -Berghaus a n d Seegers (1966). A 1.5 kg of rabbit was given 4 m g C o u m a d i n intravenously on 4 successive days (Fig. 8). T h e p r o t h r o m b i n concent ra t ion , as measu red by two-stage assay, d r o p p e d over a per iod of 4 days. T h e same assay was called the p r e t h r o m b i n assay when s u p p l e m e n t e d with pur i f ied au to ­p r o t h r o m b i n C a n d gave a h ighe r yield of t h r o m b i n . Th i s h ighe r yield became propor t iona te ly g rea te r as t he p r o t h r o m b i n concent ra t ion de ­creased. Someth ing built u p in the plasma tha t was resistant to the gen­era t ion of t h rombin , bu t fo rmed t h r o m b i n in the presence of m u c h a u t o p r o t h r o m b i n C.

DAYS HOUR S Fig. 8. A 1.6 kg rabbit was treated with 4 m g Coumadin on 4 successive days. T h e n a

single dose o f 15 m g vitamin K, was given on the fifth day. Histograms represent pre­thrombin 1 and prothrombin assays. T h e percentage of prethrombin 1 (prethrombin 1-prothrombin/prothrombin x 100), and the prothrombin time are represented by curves.

702 Walter Η . Seegers

A large dose of vitamin Κχ was given intravenously a n d within 2 h o u r s a response was no ted as follows: (a) the material responsive to the p re ­t h rombin assay decreased relative to the material responsive to the two-stage assay; (b) the p r o t h r o m b i n t ime decreased dramatically; a n d (c) the p r o t h r o m b i n concent ra t ion increased over a per iod of 24 hour s .

T h e expe r imen t gives a c r u d e indication of the half-life of p r o t h r o m ­bin. T h e rap id ra te of response to vi tamin K, a n d t he re is evidence that someth ing was in plasma that was difficult to convert to t h r o m b i n bu t did so in the presence of large a m o u n t s of a u t o p r o t h r o m b i n C. Th i s substance, as will now be discussed below, must have been , or at least included, an abnormal p r o t h r o m b i n molecule.

V I I . V I T A M I N Κ F U N C T I O N A N D T H E S T R U C T U R E O F

P R O T H R O M B I N

A series of contr ibut ions a p p e a r e d in several laboratories a n d some of these a re referenced (Carlisle et al., 1975; Davidson a n d MacDonald, 1943; Esmon et al, 1975a,b; Emson a n d Suttie, 1975; F e r n l u n d a n d Stenflo, 1975; Ganro t and Ni lehn, 1968; G a n r o t a n d Stenflo, 1970; G i t e l ^ a / . , 1973; He ldeb ran t et al, 1973; H e m k e r et al, 1963; J o h n s t o n a n d Olson, 1972; Josso et al, 1968; Malhotra a n d Car ter , 1971; Nelses-tuen a n d Suttie, 1972a,b, 1973; O l s e n ^ a / . , 1974; Shah a n d Suttie, 1971, 1972, 1974; Skotland et al, 1974; Stenflo, 1970, 1972, 1973, 1974, 1976; Stenflo a n d Ganro t , 1972, 1973; Stenflo et al, 1974; Suttie, 1969, 1970, 1973, W a l z ^ a / . , 1975).

F rom this work, one can conclude that vitamin Κ depr ivat ion is as­sociated with the p roduc t ion of an abnorma l p r o t h r o m b i n molecule. A previously unrecognized a m i n o acid was discovered in p r o t h r o m b i n (S tenf lo^a / . , 1974; Magnusson a/., 1974; Nelsestuen et al, 1974). Th is is γ -carboxyglutamic acid. T h e abnorma l p r o t h r o m b i n p r o d u c e d u n d e r condit ions of partial vitamin Κ depr ivat ion contains glutamic acid in place of the γ-carboxyglutamic acid. I n amino acid analysis of p ro ­th rombin , the latter is conver ted to glutamic acid u p o n hydrolysis a n d thus is not detected. T h e vitamin functions in the mechanics of in t roduc­ing the carboxyl g r o u p after the incomple te p r o t h r o m b i n is synthesized.

Beginn ing in 1967, Magnusson and his associates worked on de ter ­min ing the amino acid sequence of bovine p r o t h r o m b i n and the com­plete sequence has been p resen ted (Magnusson et al, 1975a,b). Inc luded was the posi t ioning of the disulfide bonds , as well as locations of carbo­hydra te (Figs. 9 a n d 10). In this laboratory we cont r ibu ted da ta on the n o n t h r o m b i n por t ion of t h rombin , as well as for the A chain of t h r o m b i n

ALA Ι

270 ALA \ 2 7 4 _

[ ILE-GLU-GLY-ARG.

Fig. 9. A m i n o acid sequence of nonthrombin portion of bovine prothrombin. T h e first 156 amino acid residues are called the prothrombin fragment 1 portion. T h e next 118 compose the prothrombin fragment 2. T h = thrombin and Xa = Factor Xa or autopro­thrombin C. Note ten γ-carboxyglutamic acid at positions 7 , 8 , 15, 1 7 , 2 0 , 2 1 , 2 6 , 2 7 , 3 0 , and 33 . Data of Reuterby et al. (1974), Hewett-Emmett et al. (1974, 1975), and Magnusson et al. (1974, 1975a,b).

Fig. 10. Amino acid sequence for thrombin portion of bovine prothrombin. Note A chain of 4 9 residues below, the B l chain up to residue 397 , the Ser, Asp, His active center, and the He to Asp salt bridge. Loss of Β1 includes active His and the resulting thrombin-E has only esterase activity. Β chain structure represents almost exclusively the data of Mag-nusson et al. (1975a,b), and all positions of disulfide bonds in bovine prothrombin origi­nated in that work. Table I gives structure of human prothrombin fragment 1.

2 5 . U s e s and Funct ion o f Vi tamin Κ 705

(Walz a n d Seegers , 1974; Walz et al, 1974a; Reute rby et al, 1974; H e w e t t - E m m e t t ^ a/., 1974, 1975). H e l d e b r a n t a n d associates (1973) also con t r ibu ted part ial sequences. We a re in the process of comple t ing the sequencing of the n o n t h r o m b i n por t ion of h u m a n p r o t h r o m b i n , a n d have p resen ted the a m i n o acid sequence of the A chain of h u m a n t h r o m b i n (Walz a n d Seegers, 1974).

T h e r e a re ten y-carboxyglutamic acid residues in bovine p r o t h r o m b i n located at positions 7, 8, 15, 17, 20, 2 1 , 26, 27 , 30 , a n d 3 3 . T h e s e a re also found in h u m a n p r o t h r o m b i n (Table I) . T h e s e positions have glutamic acid in the abnorma l bovine p r o t h r o m b i n isolated from plasma when vi tamin Κ activity is depressed . T h e γ-carboxyglutamic acid res idues a r e calcium b ind ing sites a n d function in phosphol ip id-b inding . A complex consisting of p r o t h r o m b i n , Ac-globul in , phosphol ip id , a u t o p r o t h r o m b i n C, a n d calcium ions then consti tutes the mix tu re ou t of which t h r o m b i n activity is genera ted .

If the bovine p r o t h r o m b i n molecule is d e g r a d e d to p r e t h r o m b i n 1 by th rombin , the p r o t h r o m b i n f r agment 1, with the γ -carboxyglu tamic acid res idues , is ou t of the react ions. Nevertheless , like p r o t h r o m b i n , the p r e t h r o m b i n 1 is conver ted to t h r o m b i n in a complex mix tu re consisting of p r e t h r o m b i n 1, Ac-globul in , phosphol ip id , a u t o p r o t h r o m b i n C, a n d calcium ions. T h e main difference be tween the genera t ion of t h r o m b i n f rom p r e t h r o m b i n 1 a n d p r o t h r o m b i n is the r equ i r emen t s in the la rger a m o u n t s of procoagulants for p r e t h r o m b i n (Baker a n d Seegers, 1967; Barthels a n d Seegers, 1969; Seegers al, 1972). P r e t h r o m b i n 1 requi res m u c h m o r e a u t o p r o t h r o m b i n C a n d Ac-globul in than p ro th rombin . If the purif ied p r o t h r o m b i n f r agment 1 is a d d e d to the p r e t h r o m b i n acti­vation mix tu re , it functions as an accelerator (Seegers et al, 1976). I t serves its function without be ing covalently b o u n d as it is in p r o t h r o m ­bin. I t is not correct to say (Magnusson et al, 1975a) that p r e t h r o m b i n 1 is not fu r ther activated to t h rombin . Unfor tuna te ly , t he re is really very little informat ion on the activation of the abnorma l Dicumarol - induced p r o t h r o m b i n . Malhol tra (1975) has publ ished an abstract a n d wrote to m e as follows: "I have had difficulty in convincing o thers tha t t h rombin can be gene ra t ed f rom Dicumarol - induced atypical p r o t h r o m b i n by biological accelerators. As a mat te r of fact, some of the reviewers t h o u g h t tha t bioactivities of atypical p ro te ins could be d u e to the pres­ence or no rma l p r o t h r o m b i n molecules in o u r purif ied p repara t ions . I n view of this p red icament , your recent letter is a grea t source of encour­a g e m e n t a n d confidence. I am , the re fore , very grateful to you." Pre­sumably, the o the r vitamin K - d e p e n d e n t proteins also have their func­tion impa i red bu t not completely lost d u r i n g vitamin Κ deficiency.

T A B L E I Comparison of A m i n o Ac id Sequences of H u m a n and B o v i n e Prothrombin Fragment 1 "

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

N H 2 - Ala -Asn- Thr- Phe -Leu- Gla -Gla- Val - Arg- Lys -Gly- Asn -Leu-Gla-N H 2 - Ala - Asn- Lys -Gly- Phe-Leu- Gla -Gla- Val -Arg- Lys -Gly- Asn-Leu-Gla-

16 17 18 19 20 21 22 23 24 25 26 27 28 2 9 30

H: Arg -Gla- Cys -Val- Gla -Gla- Thr -Cys- Ser -Tyr- Gla -Gla- Ala -Phe-Gla-B: Arg -Gla- Cys -Leu- Gla -Gla- Pro -Cys- Ser -Arg- Gla -Gla- Ala -Phe-Gla-

31 32 33 34 35 36 37 38 39 4 0 41 42 43 44 4 5

H: A l a - L e u - G l a -Ser- Ser -Gly- A l a - T h r - A s p -Val- P h e - T r p - A l a -Lys-Tyr-B: Ala -Leu- Gla -Ser- Leu Ser- Ala -Thr -Asp -Ala- P h e - T r p - Ala -Lys-Tyr-

4 6 47 48 4 9 50 51 52 53 54 55 56 57 58 59 60

H: Thr - Ala- Cys -Glu- Thr -Ala- Arg -Thr- Pro - Arg- Asp -Lys- Leu -Ala-Ala-B: Thr -Ala- Cys -Glu- Ser -Ala- Arg -Asn- Pro -Arg- Glu -Lys- Leu -Asn-Glu-

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

H: Cys -Leu- Glu -Gly- Asn -Cys- Ala -Glu- Gly -Leu- Gly -Thr- Asn -Tyr-Arg-B: Cys -Leu- Glu -Gly- Asn -Cys- Ala -Glu- Gly -Val- Gly -Met- Asn -Tyr- Arg-

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

H: Gly -Asn-Va l -Ser- He -Thr-Arg-Ser - Gly -He- G l u - C y s - G i n - L e u - T r p -B: Gly -Asn- Val -Ser- Val -Thr-Arg -Ser- Gly -He- Glu -Cys- Gin -Leu-Trp-

91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

H: Arg -Ser- Arg-Tyr- Pro -His- Lys -Pro- Glu -He- Asn -Ser- Thr-Thr-His -B: Arg -Ser- Arg-Tyr- Pro -His- Lys -Pro- Glu -He- Asn -Ser- Thr-Thr-His -

106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

Η: Pro -Gly- Ala - Asp- Leu -Gin- Glu - Asn- Phe -Cys- Arg - Asn- Pro - Asp-S^r-B: Pro -Gly- Ala - Asp- Leu -Arg- Glu - Asn- Phe -Cys- Arg - Asn- Pro - Asp-G/y-

121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

H: Ser -He- Thr -Gly- Pro -Trp- Cys -Tyr- Thr -Thr- Asp -Pro- T h r -Ala- Arg-B: Ser -He- T h r -Gly- Pro -Trp- Cys -Tyr- Thr -Thr- Ser -Pro- T h r -Leu- Arg-

136 137 138 139 140 141 142 143 144 145 146 147 148 149 150

H: Arg -Gin- Glu -Cys- Ser -Thr- Pro -Val- Cys -Gly- Gin -Asp- Gin -Val- Thr-B: Arg -Glu- Glu -Cys- Ser -Val- Pro -Val- Cys -Gly- Gin -Asp- Arg -Val-Thr-

( continued)

25 . U s e s and Funct ion o f Vi tamin Κ

T A B L E I, continued

707

151 152 153 154 155 156

H: Val -Met- Val -Thr- Pro -Arg-COOH B: Va l -G/w-Va l -lie- P r o - A r g - C O O H

n T h e numbering system corresponds to bovine (B) profragment 1. A single deletion occurs in human (H) profragment 1 at residue 4. Underl ined residues correspond to the differences between the two species. γ-Carboxyglutamic acid residues are designated Gla. Carbohydrate is attached to aspartic acid at positions # 7 7 and 101 of both the bovine and h u m a n fragments. Unpubl ished data from this laboratory by Daniel A. Walz, David Hewett-Emmett, and Walter H. Seegers.

T h e γ-carboxyglu tamic acids in h u m a n p r o t h r o m b i n f ragment 1 a re at exactly the same place as in the bovine species (Table I). Presumably, they function exactly as has been found with bovine material . In the h u m a n pro te in , t he r e a re 155 a m i n o acid res idues in place of 156, be­cause of a delet ion and substi tution at positions 3 a n d 4 . In o the r par ts of the molecule , t he re is r emarkab le similarity between h u m a n a n d bovine p r o t h r o m b i n f ragment 1.

V I I I . F I V E D I S T I N C T V I T A M I N K - D E P E N D E N T

M O L E C U L E S

At the midcen tury per iod , when p r o t h r o m b i n was known to r equ i re vi tamin Κ for its synthesis, four o the r activities became recognized as be ing vi tamin K-dependen t . T h e s e activities were all p resen t in my p ro ­t h r o m b i n p repa ra t ion , which a p p e a r e d to be h o m o g e n e o u s by the criteria available for testing at tha t t ime. I, the re fore , postula ted that these activities were der ived f rom a single p r o t h r o m b i n molecule (Seegers, 1962). Th i s was especially likely because t r ea tmen t of the p r o ­t h r o m b i n p repa ra t i on inactivated the p r o t h r o m b i n (it fo rmed p r e t h r o m -bin 1) a n d o the r activities a p p e a r e d . T o d a y , we know that activities were der ived f rom p r o t h r o m b i n f ragments a n d also f rom separa te molecules distinct f rom p r o t h r o m b i n , bu t hav ing homology in their amino acid sequence (Enfield et al, 1974, 1975; Fujikawa et al, 1973; McCoy et al, 1973; Ti tan i et al, 1972, 1975). T h e molecules d o not come f rom p r o ­th rombin , bu t we can postula te a c o m m o n ancestral g e n e which once coded for a pro te in . By genetic d ivergence , eventually five prote ins were p r o d u c e d .

T o see whe the r t he re would be five distinct pro te ins by the cri terion of immunology , we p r e p a r e d each o n e except Factor VI I in purif ied form

708 Walter Η . Seegers

T A B L E II B o v i n e Ant ibodies Tes ted against Antigens"*'

Antigens

Prothrombin + ± + ± — - + + Prethrombin 1 + - + + + Profragment 1 + ± + ± — - + + ± Profragment 2 + ± - - f + + Thrombin - ± - + - -Auto-III + -Auto-II-A - + + - + + -Protein C - + + - + + -Factor IX + + + -

n Immunology data by Houria I. Hassouna, and proteins prepared by Eduardo Novoa and Daniel A. Walz.

b ± , Partial identity; + , identity; - , no identity (cross reaction). c Plasma three times adsorbed with barium carbonate.

(Novoa et al, 1976). T h e s e p repara t ions were most likely of the best quality ever p r e p a r e d and were used to immunize rabbits. Not a single one of the antibodies recognized ano the r one of the vitamin K-dependen t proteins (Table I I ) . We feel that this generalizat ion could have included Factor V I I , if we h a d tested it, and because o the r reactions of the antibodies with plasma were consistent with that conclusion. T h e r e a re , thus , at least five immunological ly distinct vitamin K-dependen t proteins.

It is not the p u r p o s e to ex tend this discussion (Table I I ) , bu t it can be stated that p r o t h r o m b i n has at least four ant igenic d e t e r m i n a n t sites, one of these is probably covered because the ant ibody to t h r o m b i n does not recognize p r o t h r o m b i n no r does the ant ibody to p r o t h r o m b i n recognize th rombin . O n the o the r h a n d , p r o t h r o m b i n , p r e t h r o m b i n 1, p r o t h r o m ­bin f ragment 1, a n d p r o t h r o m b i n f ragment 2 show cross react ions. T h e s e can, in par t , be accounted for on the basis of in ternal homology in p r o t h r o m b i n (Fig. 6).

I X . S O M E P O S S I B I L I T I E S A N D N E E D S F O R

I N V E S T I G A T I O N

T h e p ro th rombin molecule has been in the vangua rd of the search for informat ion about the function of vitamin K. T h e r e a re many uncer ta in-

Pro

thro

mbi

n

Pre

thro

mbi

n 1

Pro

frag

men

t 1

Pro

frag

men

t 2

Th

rom

bin

Au

to-I

II

Au

to-I

I-A

Pro

tein

C

Fac

tor

IX

Pla

sma

Seru

m

Pla

smac

25 . U s e s a n d Funct ion o f Vi tamin Κ 709

ties abou t the activation of the Dicumaro l - induced p r o t h r o m b i n . I t is readily activated by Edits carinatus v enom, a n d that fact was p roper ly used as an exper imenta l convenience. Activation studies with clotting factors a re needed . How will it activate in 2 5 % sod ium citrate solution? If the molecule is devoid of γ -carboxyglu tamic acid residues, it probably can be conver ted to t h r o m b i n with a u t o p r o t h r o m b i n C a lone o r with the enzyme plus accessories. Pe rhaps a u t o p r o t h r o m b i n C without γ -carboxyglutamic acid res idues can also be isolated to find ou t how those residues function (Nelsestuen et al, 1974). T h e Dicumarol-induced p r o t h r o m b i n was usually isolated f rom animals given a dose that lowered the plasma concent ra t ion of p r o t h r o m b i n only partially. Pe rhaps h igher doses would be associated with reduc t ion of even the abnorma l p r o t h r o m b i n . In the case of some pre l iminary exper iments with dogs , p ro longed adminis t ra t ion of Coumadin in large amounts re­moved entirely the p r o t h r o m b i n complex as m e a s u r e d a n d detected by immunological techniques . T h e n a t u r e of species variation is not entirely decided by the fact that h u m a n a n d bovine p r o t h r o m b i n have the same γ-carboxyglu tamic acid res idues at the same position. T h e isolation of t he o the r four vi tamin K - d e p e n d e n t p ro te ins in a b n o r m a l fo rm should be possible, a n d their function would thus serve as models for informa­tion on modif ied enzymes a n d p roenzymes .

I have not discussed the role of vi tamin Κ in the incorpora t ion of ca rbohydra te . Th i s aspect has received m u c h at tent ion (Pereira a n d Cour i , 1971, 1972a,b).

A valuable old nut r i t ion expe r imen t f rom this laboratory is related to blood coagulat ion a n d has apparen t ly been forgot ten (Guest et al., 1947). Chickens were placed on a diet deficient in pteroylglutamic acid a n d developed an increase in ant ip lasmin activity. Th is increase was p re ­vented by feeding the crystalline vitamin. Th i s probably correlates with the fact tha t me tho t r exa te is an inhibi tor of folic acid a n d a well known neoplastic suppressant , used with m u c h success as a chemothe rapeu t i c agen t in the t r ea tmen t of neoplastic t rophoblast ic diseases (Her tz et al., 1958, 1961). I suggest that the the rapy is also associated with an in­creased inhibi tor titer which re ta rds fibrinolysis. In the opposi te direc­tion, the observations of Reich (1975) deal with facts related to increased fibrinolysis. H e finds that t u m o r cells have an increased p roduc t ion of p lasminogen activator in neoplasia. Similarly, we find tha t au top ro ­t h r o m b i n I I -A p romotes fibrinolysis by depress ing inhibitors of fib­rinolysis. Th i s opens the possibility tha t Dicumarol a n d related com­p o u n d s , by decreas ing Prote in C concent ra t ion , d o not favor fibrinolysis.

T h e function of vitamin Κ in the carboxylat ion of glutamic acid is a ided by the use of radioactive b icarbonate (Esmon et al., 1975a). It is only logical to suppose that enzymes a r e involved, a n d that the vitamin

710 Walter Η . Seegers

itself might be modified d u r i n g its function. T h e quest ion has been raised whe the r vi tamin Κ part icipates in the metabolism of no rma l b o n e deve lopment , on the basis tha t γ -ca rboxyglu tamate was identified in proteins isolated from mineral ized tissue (Hauschka^ /a / . , 1975). Prote in conta ining the a m i n o acid has also been found in pathologically mineral ized kidney stones (Lian a n d Pr ien , 1976). All this points in the direction of an increased interest in vi tamin K.

A C K N O W L E D G M E N T

This work was supported by research grant H L 03424-19 from the National Heart and Lung Institute, National Institutes of Health, U.S. Public Health Service. I am grateful to Dr. Houria I. Hassouna for her assistance with the references and discussion.

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