congenital deficiency of blood clotting factors ii, vii ......factors vii, ix, and x. studies on...

776 Blood, Vol. 53, No. 4 (April), 1979

Congenital Deficiency of Blood Clotting FactorsII, VII, IX, and X

By Kuo-San Chung. Annie Bezeaud, Jonathan C. Goldsmith, Campbell W. McMillan,

Doris M#{233}nach#{233},and Harold R. Roberts

A patient congenitally deficient in factorsII. VII. IX. and X has been further investi-gated after a follow-up of 1 5 yr. At birth,these factors. when determined by clottingassays, were undetectable. Following ther-apy with vitamin K1. the clotting activity ofthese factors rose but never exceeded1 8 % of normal. Immunologic assaysrevealed much higher levels of thesefactors than did clotting assays, thussuggesting that the vitamin-K-dependentfactors were present in abnormal forms.Two-dimensional crossed immunoelectro-

phoresis showed that at least two forms ofprothrombin were present in the patient’s

plasma. One form was similar to normalprothrombin; the other had the same

mobility as acarboxyprothrombin. In addi-

tion. the majority of this fast-migrating

peak was not adsorbable onto insolublebarium salts. These observations sug-

gested that some molecules of the

patient’s prothrombin lacked the normalcomplement of gamma-carboxyglutamic

acid residues. This observation was con-

firmed by a specific assay for gamma-

carboxyglutamate. Since malabsorption ofvitamin K, warfarin intoxication, and

hepatic dysfunction were excluded as

causes of this patient’s syndrome, this rare

congenital abnormality could represent

either a defective gamma-carboxylation

mechanism within the hepatocyte or faulty

vitamin K transport.

I T HAS BEEN KNOWN for many years that vitamin K is necessary for the

complete synthesis of prothrombin (factor II) and factors VII, IX, and X.’

Vitamin K is also necessary for the production of protein C in the bovine species

and protein S in man.2’3 Recent investigations have shown that vitamin K acts at a

postribosomal level in the hepatocyte to modify precursor forms of these factors.4

Thus, vitamin K, in the presence of a carboxylating enzyme, hepatic microsomes,

02, and CO2. converts glutamic acid residues on the amino-terminal regions of

precursor forms of prothrombin and factors VII, IX, and X to gamma-carboxyglu-

tamic acid residues.5 These gamma-carboxyglutamic acid residues are necessary

for calcium-dependent phospholipid binding by the vitamin- K-dependent clotting

factors and are prerequisites for normal blood coagulation.6

Whereas defective gamma-carboxylation of glutamic acid residues occurs

following therapy with coumarin drugs,’ a congenital abnormality resulting in

defective gamma-carboxylation of the vitamin-K-dependent factors has hitherto

not been reported.

We have reevaluated a unique I 5-yr-old female who has congenital deficiency of

From the Departments of Medicine. Pediatrics. and Pathology. School of Medicine, Universit�’ of

North Carolina at Chapel Hill, NC., and the Service Central d’Immunologie et H#{234}matologie, Hbpital

Beaujon, Clichy, France.

Submitted October 8, 1978; accepted December 18. 1978.

Supported by NIH Grants HL-06350. HL-21975. HL-07149, and HL-20319 from the National

Heart, Lung. and Blood Institute. Dr. Goldsmith is a Fellow ofthe National Hemophilia Foundation.

Address reprint requests to Dr. K. S. Chung. Department of Medicine, University of North Carolina

School of Medicine, 415 Burnett-Womack Clinical Sciences Building 229-H, Chapel Hill, NC.

275 /4.(C) /979 by Grune & Stratton, Inc. JSSN 0006-4971/79/5304-0024$02.00/0

For personal use only.on October 3, 2017. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

FACTORS II, VII, IX, AND X 777

the vitamin-K-dependent blood clotting factors.7 She was initially seen at the age of

3 mo with multiple bruises and a history of numerous hemorrhagic episodes.

Vitamin K deficiency due to malabsorption, liver disease, and warfarin intoxication

was excluded, both at the time of initial presentation and on several subsequent

occasions. Based on current studies, it appears that this patient may have a defect

in the gamma-carboxylation mechanism that results in the production of immuno-

logically recognizable factors II, VII, IX, and X that lack the full complement of

gamma-carboxyglutamic acid residues and therefore lack coagulant activity.

MATERIALS AND METHODS

Patient

The patient has been previously described.7 When she was initially seen it was determined by assay of

her plasma for bishydroxycoumarin and warfarin that she had received no anticoagulant drugs.

Subsequent assays for these drugs have also been negative. At the age of 6 mo the patient was observed

for 2 wk in the hospital, at which time it was ascertained that she was not receiving vitamin K

antagonists surreptitiously. In addition, studies at that time revealed no evidence for malabsorption or

liver disease.7 Subsequent to the initial report, the patient has been followed periodically for I 5 yr.

During this time she has required daily vitamin K supplements. Initially vitamin K was administered

parenterally, but subsequently oral doses have been shown to be efficacious. However, even with massive

intravenous doses of vitamin K1, the patient’s K-dependent factors never returned to normal, although

the prothrombin time decreased from more than 100 sec to 20-25 sec. Withdrawal of vitamin K1

resulted in prompt return of bleeding and a marked decrease in the clotting activities of prothrombin and

factors VII, IX, and X. Studies on parents ofthe patient showed normal levels offactors II, VII, IX, and

x.

Plasma

Normal human blood was collected from 10 normal donors into 3.2% trisodium citrate (8 parts blood

to I part citrate). After centrifugation the plasma was pooled and stored at -70#{176}C. The patient’s

plasma was prepared in the same manner.

Reagents

Sodium chloride, sodium citrate, kaolin, calcium lactate, diethylbarbituric acid, tris(hydroxyme-

thyl)aminomethane, sodium azide, potassium hydroxide, benzene, monobasic and dibasic sodium

phosphate, and barium chloride were purchased from Fisher Scientific, Fair Lawn, N.J. Imidazole,

acrylamide, and cyanogen bromide were purchased from Eastman Kodak, Rochester, N.Y. Calcium

chloride, methanol, glacial acetic acid, and formic acid were obtained from Mallinckrodt, St. Louis, Mo.

Sephadex G-100 and Sepharose 4B were obtained from Pharmacia Fine Chemicals, Piscataway, N.J.,

and Uppsala, Sweden. Alumina C--y gel was purchased from Calbiochem, La Jolla, Calif. Borosilicate

glass clotting tubes (10 X 75 mm) were purchased from Arthur H. Thomas, Philadelphia, Pa. Agarose

and Coomassie brilliant blue were from Bio-Rad Laboratories, Richmond, Calif. Simplastin was

purchased from General Diagnostics, Morris Plains, N.J. Thrombofax was from Ortho Diagnostics,

Raritan, Ni. Dansyl chloride and the venom from Echis carinatus were obtained from Sigma

Chemical, St. Louis, Mo. Staphylocoagulase was purchased from Diagonostica Stago, Asnieres, France.

Canine factor V and fibrinogen were obtained from platelet-poor canine oxalated plasma that was

exhaustively treated with barium sulfate and then raised to 25% saturation with (NH4)2S04 (to obtain

fibrinogen-rich material) and then to 33% (to obtain factor-V-rich material).

Normal Human Prothrombin

Highly purified normal human prothrombin was a by-product ofour factor IX purification procedure

employing heparin agarose chromatography as the last step.t’9

Heterologous Antibodies to Normal Human Prothrombin

Antibodies to normal human prothrombin were raised in two rabbits by four weekly injections of 0.25

mg of pure prothrombin and an equal volume of complete Freund’s adjuvant. The rabbits were then bled




778 CHUNG ET AL.

at weekly intervals, beginning 1 wk after completion of the immunization. The rabbit serum was heated

at 56#{176}Cfor 30 mm and adsorbed with one-tenth volume of alumina C-’y gel. After centrifugation, the

supernatant was dialyzed against 0.0l-M sodium phosphate buffer (pH 7.0), and the dialysate was

applied to a prothrombin-Sepharose affinity column that was prepared by coupling normal prothrombin

to cyanogen-bromide-activated Sepharose 4B.’#{176}Antiprothrombin antibodies were eluted with 0.l-M

glycine-HCI (pH 2.8), then immediately brought to pH 7.0 by the dropwise addition ofdilute NaOH.

Purification ofthe Patient ‘s Prothrombin

Because there were only limited quantities of the patient’s plasma, the patient’s prothrombin was

prepared from small amounts of citrated plasma. Ten milliliters of the patient’s plasma was stirred with

1 ml of l-M BaCl2 and centrifuged. The supernatant containing nonadsorbable prothrombin was

dialyzed against 0.01-M sodium phosphate buffer (pH 7.0) and applied to an antiprothrombin antibody

affinity column ( I .5 X 4.0 cm) that was prepared by coupling the purified antibody to cyanogen-

bromide-activated Sepharose 4B. The prothrombin was eluted with 0.l-M glycine-HCI buffer (pH 2.8)

and immediately raised to pH 7.0 with dilute NaOl-1. The eluate was then concentrated and subjected to

gel filtration on a Sephadex G-I00 column (0.35 x 8.5 cm). The purification was monitored

immunologically by the Ouchterlony double-diffusion technique.#{176} The results showed a single precipitin

line when the preparation was tested against an antiprothrombin antibody, against anti-whole-

human-plasma, and against antiserum to the barium citrate eluate of normal human plasma.

Determination ofGamma-Carboxyglutamic Acid in Patient

and Normal Prothrombin

Gamma-carboxyglutamic acid residues were assessed in the purified prothrombin from both normal

plasma and the patient’s plasma using thin-layer polyamide chromatography.’2 Purified prothrombin

was hydrolyzed for 24 hr at 100#{176}Cin 2-M KOHl3 The resulting amino acids were labeled with dansyl

chloride and subjected to two-dimensional chromatography on thin-layer polyamide sheets using

H,0-formic acid (100:1.5 v/v) in the first dimension and benzene-acetic acid (9:1 v/v) in the second

dimension. Labeled amino acids could be detected under short-wave ultraviolet light (A = 254 nm).t2 A

synthetic monomeric gamma-carboxyglutamic acid was prepared according to the methods of Boggs et

al.’4 It was then dansylated, subjected to chromatography as described previously, and used as a control

to detect a similar amino acid in normal prothrombin and in the patient’s prothrombin.

Coagulation Assays

Blood clotting factors VII, IX, and X were assayed in one-stage tests using as substrate platelet-poor

plasma obtained from patients congenitally deficient in the respective factors.�#{176} Prothrombin coagu-

lant activity was measured with the one-stage method,’t the two-stage method,’9 staphylocoagulase,2#{176}

and venom from E. carinalus. For the latter method, normal plasma and the patient’s plasma diluted

1 :7, 1 : I 4, 1 :2 1 , and I :28 in imidazole-HCI buffer (pH 7.2) were tested. Two milligrams of E. carinatus

venom were dissolved in I ml of normal saline; 10 MI of this solution were added to 0.2 ml of the plasma

dilutions and incubated for 10 mm at 37#{176}C.Two-tenths milliliter ofcanine fibrinogen was then added, a

stop watch was started simultaneously, and the tubes were tilted and observed for the appearance of a

clot.

Immunologic Procedures

Antibody neutralization was monitored in clotting assays as follows: Factor IX neutralization assays

were conducted by a previously described method2’ with a well-characterized specific human inhibitor of

factor IX.22 Factor X neutralization was a modification of the factor IX neutralization assay using a

heterologous antibody to factor X. Prothrombin neutralization was conducted using a heterologous

antibody to factor II in a two-stage assay to detect residual prothrombin activity.

Radial immunodiffusion was performed in 1% agarose in 0.I-M phosphate-buffered saline at pH

7�23 Electroimmunoassays were performed with agarose containing a specific heterologous antibody to

prothrombin.24 Crossed immunoelectrophoresis was conducted in 0.075-M tris-barbital buffer (pH 8.6)

containing 2-mM calcium lactate at 4#{176}Cin both directions.2126

RESULTS

Clotting factors not dependent on vitamin K for synthesis have been normal

throughout the patient’s life, as shown in Table I . Factor V was initially 57% of





Table 1 . Speci fic Clotting Factor Assays (Non-Vitamin-K-Dependent)

Patient Control

Fibrinogen (mg/mI)

FactorV(%)

Factor VIII (%)

FactorXl(%)

FactorXll(%)

3.27 2.95

57-100 100

108 100

100 100

100 100

Table 2. Patient: Vitamin-K-Dependent Clotting Factors

Before After

Vitamin K, Vitamin K,

Treatment Treatment)% of normal) )% of normal)

Factorll

FactorVll

FactorlX

FactorX

<1 7.0

<1 6.9

<1 18.2

<1 7.0

normal, but has subsequently been 100% of normal. The patient’s vitamin-

K-dependent clotting factor levels are shown in Table 2. Note that factors II, VII,

Ix, and X were undetectable before the patient was treated with vitamin K.

Administration of large doses of oral vitamin K, resulted in an increase in these

factors not exceeding 18% of normal. However, despite massive doses of parenteral

vitamin K,, the patient’s vitamin-K-dependent factors never reached normal levels.

To determine if the vitamin-K-dependent factors were actually as low as

indicated by clotting assays, factors II, IX, and X were measured by immunologic

assays using antibody neutralization tests. The results shown in Table 3 indicate

that prothrombin is present in a concentration 57% of normal, factor IX in a

Table 3. Patient: Comparison of Clotting and Immunologic Activities

of Vitamin-K-Dependent Factors

Antibody

ClottingActivity

(% of normal)

NeutralizationActivity

(% of normal)

Factor II 7.0 57

FactorVll 6.9 -a

FactorlX 18.2 100

Factor X 7.0 55

Appropriate monospecific antibody not available.

Table 4. Prothrombin Determinations

(Levels in Percent of Normal)

Patient’s Plasma

Warfarin PlasmaNative Adsorbed

One-stage assay 3.5 1 -Two-stage assay 7 0 57

E. carinatus 47 1 8.2 75

Staphylocoagulase 40 - -

Anti-Il neutralization 56.8 55 89

Electroimmunoassay 58.4 45 85

Radial immunodiffusion 51 39 81.5




.. .

. -: -#{176}�

B �e�

A.

I

j

c

780 CHUNG ET AL.

concentration 100% of normal, and factor X in a concentration 55% of normal.

Factor VII was not measured immunologically because an appropriate antibody

was not available for this purpose. These data strongly suggested that altered forms

of the vitamin-K-dependent factors were present in the patient’s plasma, forms that

retained immunologic activity but lacked coagulant activity.

To explore this possibility further, the prothrombin content of the patient’s

plasma was measured by several techniques and compared with that of normal

pooled human plasma and of plasma obtained from a patient on warfarin therapy.

Alp ., . . - � 4�#{149}C�.

:�‘� #{149}�,

.i .�‘�:“ � : #{231}..‘�‘: �

,�. :

I

., *4� � � �t

�1

:. �

): � “ � ‘k’.,.

.-.---,

-9

Fig. 1 . Crossed immunoelectrophoresis: (A) patient’s plasma. (B) plasma from a patient onwarfarin therapy. (C) normal pooled plasma. The first dimension was run for 3.5 hr at 1 Ov/cm. andthe second dimension was run overnight at 20 v/cm. Both dimensions were run in 1 % agarosecontaining 2-mM calcium lactate. In the second dimension the agarose contained a specific antibody

to prothrombin.




- . ....- .� �-


As shown in Table 4, when using physiologic activators, i.e., one- and two-stage

clotting assays, the prothrombin clotting activity was found to be very low (less

than 10% of normal). In contrast, when using nonphysiologic activators such as E.

carinatus venom and staphylocoagulase, the levels of prothrombin were found to be

47% and 40% of normal, respectively. Immunologic techniques using a specific

heterologous antiprothrombin antibody gave results similar to those obtained using

nonphysiologic activators. Similar results were obtained when assaying plasma

from a patient receiving long-term anticoagulant therapy with warfarin. After

adsorption of the patient’s plasma by barium citrate, the supernatant exhibited

prothrombin activity when using nonphysiologic activators, and these results were

corroborated by immunologic assays.

When subjected to crossed immunoelectrophoresis (Fig. 1 ), the patient’s plasma

exhibited at least two peaks of prothrombin (Fig. la). The smaller of the two peaks

had the same electrophoretic mobility as normal prothrombin (Fig. ic), whereas

the major peak had the same electrophoretic mobility as the acarboxyprothrombin

found in plasma from a patient on warfarin therapy (Fig. ib). These data suggest

that most of the patient’s prothrombin existed in the acarboxy form.

The patient’s plasma was then adsorbed with barium citrate, which usually

removes all normal prothrombin, but not acarboxyprothrombin, in warfarin plas-

ma. Resulting supernatant plasma was tested by crossed immunoelectrophoresis for

residual prothrombin (Fig. 2). The peak corresponding to normal prothrombin was

A�� ..

� . #{149}:-‘‘

. .. �-:-t.�� .,

�4I�

Fig. 2. Crossed immuno-

electrophoresis: (A) patients

plasma after adsorption by bar-ium citrate. (B) normal pooled

plasma control. The conditions

are the same as those de-

scribed in Fig. 1.




782 CHUNG ET AL.

removed by barium citrate, whereas most of the peak with the same electrophoretic

mobility as acarboxyprothrombin remained. The barium citrate cake resulting

from adsorption of the patient’s native plasma was eluted and then subjected to

crossed immunoelectrophoresis (Fig. 3). A peak of prothrombin with the same

electrophoretic mobility as normal prothrombin could be found, but in addition a

faster-migrating peak was also found, thus indicating that at least some of the

abnormal prothrombin was adsorbed by barium salts.

A more direct method was used to determine the content of gamma-carboxyglu-

tamic acid residues in the patient’s purified nonadsorbable prothrombin. Alkaline

hydrolysis and two-dimensional thin-layer chromatography of the patient’s

prothrombin and normal prothrombin were performed. The dansylated amino acids

from the patient’s prothrombin and normal prothrombin were compared to each

other and to a standard synthetic monomeric gamma-carboxyglutamic acid. The

migration of the synthetic monomeric gamma-carboxyglutamic acid in this experi-

ment is shown in Fig. 4c. A fluorescent spot corresponding to the gamma-

carboxyglutamic acid control was found in normal prothrombin even when normal

prothrombin was diluted to about the same antigenic concentration as the patient’s

prothrombin (Figs. 4b and 4d). However, when the patient’s prothrombin was

chromatographed in this system (Fig. 4a), no fluorescent spot corresponding to

. . - S .

-� � . � �

�; - � . . ‘ . -.. . ��:-r� #{149}��-�Y

� A�’�”t� S ‘ � � � 1�

�

�- �

� .� ;.�_�_;� #{149};;t6��,� ‘. .. . � __________

C - . �. 1�

� . -:�.. �

� � .. ------;---,, �

B � :‘�

Fig. 3. Crossed immunoelectrophoresis: (A) eluate from barium citrate cake resulted from

adsorption of the patient’s native plasma, (B) normal pooled plasma control. The conditions are thesame as those described in Fig. 1 and 2.





Fig. 4. Thin-layer chromatography of dansylated amino acids from alkaline hydrolysis of: (A)

patient�s purified nonadsorbable prothrombin. (B) normal purified prothrombin. (C) syntheticmonomeric gamma-carboxyglutamic aci ontrol. (D) normal purified prothrombin diluted to approxi-

mately the same antigenic concentrati the patient’s prothrombin. Arrows denote the location ofgamma-carboxyglutamic acid.

gamma-carboxyglutamic acid was seen. This technique is sensitive to a prothrom-

bin gamma-carboxyglutamic acid content 1% of normal.

DISCUSSION

A unique patient with congenital deficiency of factors II, VII, IX, and X has

been evaluated. Even after therapy with massive doses of vitamin K,, either

parenterally or orally, her plasma showed greatly reduced levels of the vitamin-

K-dependent factors when measured by clotting assays. However, when immuno-

logic techniques were employed to measure these factors, much greater quantities

of the vitamin-K-dependent clotting proteins were found. Since vitamin K

deficiency due to malabsorption, liver disease, and warfarin ingestion had been

excluded by appropriate biochemical and spectrophotometric tests, the patient’s

partial response to parenteral and oral vitamin K, suggested a defect in vitamin K




784 CHUNG ET AL.

metabolism. Defective vitamin K metabolism could lead to the production of blood

clotting factors deficient in gamma-carboxyglutamic acid. Gamma-caroboxyglu-

tamic acid residues are normally found on the amino-terminal end of molecules of

the vitamin-K-dependent factors and are necessary for these factors to bind not

only to calcium but also to insoluble barium salts. Calcium binding is necessary for

the subsequent binding of these factors to phospholipids or specific receptors on the

platelet surface. In the absence of calcium-dependent phospholipid binding, the

vitamin-K-dependent factors do not participate normally in blood coagulation.

The patient’s prothrombin after vitamin K treatment was found to be 3.5% and

7% of normal when measured using clotting assays dependent on calcium binding.

However, when the patient’s prothrombin was assessed by nonphysiologic activa-

tors, which do not depend on the presence of gamma-carboxyglutamic acid residues

for the generation of thrombin, the level of prothrombin was found to be 40% and

47% of normal. Similarly, immunologic assays of prothrombin, factor IX, and

factor X were 57%, 100%, and 55% of normal. These data strongly suggest that

factors II, IX, and X in the patient’s plasma lacked the full complement of

gamma-carboxyglutamic acid residues. This hypothesis is supported by the finding

that on crossed immunoelectrophoresis in the presence of calcium ions the patient

had at least two peaks of prothrombin, one corresponding in mobility to normal

prothrombin and the other corresponding to a more rapidly migrating peak found in

the plasma of patients treated with warfarin. The rapid peak in the patient’s plasma

is essentially not adsorbable onto barium salts, again suggesting that the patient’s

prothrombin contained a decreased complement of gamma-carboxyglutamic resi-

dues. This hypothesis was substantiated with the observation that amino acids from

the patient’s purified nonadsorbable prothrombin contained no detectable gamma-

carboxyglutamic acid residues when tested by a qualitative technique employing

thin-layer chromatography of dansylated amino acids.

In addition to normal prothrombin and acarboxyprothrombin, the patient’s

plasma contains another form of prothrombin that behaves on crossed immunoelec-

trophoresis, in the presence of calcium ons, as a fast-migrating peak but has the

ability to be adsorbed onto insoluble � ‘im salts. These results suggest that the

patient’s prothrombin is heterogenous ‘� respect to the content of gamma-

carboxyglutamic acid residues. This hypoth :� is supported by the findings of

Esnouf and Prowse,27 who reported different ue,, � �f carboxylation of prothrom-

bin molecules obtained from patients on warfarin t. py.

Since even with immunologic techniques the patt5 ��t’s plasma contained only

about 50% of the prothrombin found in normal plasma, the question arises why

prothrombin deficient in gamma-carboxyglutamic acid residues is not present in

amounts approaching 100% of normal. The answer to this question is not known,

but it is possible that acarboxyprothrombin has a shorter biologic half-life than

normal prothrombin, as has been suggested by Lavergne and Josso.28 Thompson29

has suggested a similar explanation for decreased immunologic levels of acarboxy

factor IX found in patients on warfarin therapy.

Gamma-carboxyglutamic acid as a constituent of the vitamin-K-dependent

clotting factors has been described by Stenflo,3#{176}Nelsestuen et al.,3’ and Magnusson

et al.32 Later, Hauschka et al.33’34 described the presence of these same residues in

bone and in the kidney. Their results suggest that the gamma-carboxyglutamic acid

in proteins other than the blood clotting factors is also apparently vitamin-

K-dependent. It should be noted, however, that our patient had normal renal





function and normal skeletal development, even though her vitamin-K-dependent

clotting factors had been decreased since birth and during the subsequent I 5 yr of

follow-up. Thus if the patient’s defective vitamin K metabolism had affected her

skeletal or renal development, the defects were either obscured by vitamin K

therapy or not detected in these tissues, our end points being currently too crude. It

is also possible that defects in the renal or skeletal system will be manifest only after

the patient gets older.

Although vitamin K deficiency per se and vitamin K antagonism have been

excluded in this patient, the precise defect in vitamin K metabolism is yet to be

clarified. There could be an abnormality in transport of vitamin K in the blood.

However, a carrier protein for vitamin K has not been identified. A second

possibility is that vitamin K may not be handled normally in the liver. Suttie et al.’

as well as others,35’36 have suggested that vitamin K,, when it enters the hepatocyte,

is obligatorily converted to vitamin K, epoxide by an epoxidase system. Vitamin K,

epoxide is then converted to reduced vitamin K, by a specific reductase system in

the hepatocyte before it can act as a co-factor in the carboxylating mechanism. We

have been unable to exclude in this patient defects of the epoxidase or reductase

systems. Another possibility is that vitamin K is metabolized normally within the

hepatocyte but that it does not function as a co-factor for gamma-carboxylation

because of a defective carboxylating mechanism. Suttie et al.’ have demonstrated

that the gamma-carboxylation of glutamic acid residues in the hepatocyte requires

hepatic microsomes, reduced vitamin K,, a carboxylating enzyme, 02, and CO2. It

seems unlikely that the patient’s hepatic microsomes are defective, but it is possible

that the patient has a congenitally defective carboxylating mechanism for the

vitamin-K-dependent clotting factors. Evaluation of the patient’s own hepatic

microsomes would be necessary to test this hypothesis, but this experiment is not

possible at the present time.

Nevertheless, it is clear that this unique patient has congenital deficiency in the

clotting activity of factors II, VII, IX, and X, and to our knowledge she is the only

such patient reported with this defect. We have not yet tested this patient for the

presence of protein 5, which is anoth. :jtamin..K�dependent factor found in

humans. The function of this protei’ not yet known. The current studies

demonstrate that the defect in thic dtient is due to synthesis of clotting factors

lacking the normal compleme �n a’� gamma-carboxyglutamic acid residues. Since

carboxylation of glutamic a �sidues is dependent on vitamin K, it follows that

there is some type of interft �.nce in metabolism of vitamin K, either by defective

transport in the blood or by defective intrahepatic epoxidase, reductase, or

carboxylase systems. While the latter hypothesis may be more attractive in view of

recent knowledge, it is not yet possible to distinguish among these possibilities.

ACKNOWLEDGMENT

The authors acknowledge the helpful experiments of Su Chung and the technical assistance of P. B.

Soule. We wish to thank Dr. R. G. Hiskey for supplying synthetic monomeric gamma-carboxyglutamic

acid. We express our appreciation to T. H. Duncan for superb secretarial assistance.

REFERENCES

l . Suttie iW: Oral anticoagulant therapy: The protein purification from bovine plasma and

biosynthetic basis. Semin Hematol 14:365, 1977 preliminary characterization. i Biol Chem

2. Stenflo J: A new vitamin K-dependent 251:355, 1976




786 CHUNG ET AL.

3. Di Scipio RG, Hermodson MA, Yates SG,

Davie EW: A comparison of human prothrombin,

factor IX (Christmas factor), factor X (Stuart

factor), and protein S. Biochemistry 16:698, 1977

4. Suttie JW: Mechanism of action of vitamin

K: Demonstration of a liver precursor of

prothrombin.Science 179:192, 1973

5. Jones JP, Gardner EJ, Cooper TG, Olson

RE: Vitamin K-dependent carboxylation of

peptide-bound glutamate. J Biol Chem 252:7738,

I 977

6. Esmon CT, Suttie iW, Jackson CM: The

functional significance of vitamin K action.

Difference in phospholipid binding between

normal and abnormal prothrombin. J Biol Chem

250:6125, 1975

7. McMillan CW, Roberts HR: Congenital

combined deficiency of coagulation factors II,

VII, IX and X. N EngI i Med 274:1313, 1966

8. Chung KS, Reisner HM, Roberts HR:

Purification and characterization of human blood

clotting factor IX. Circulation 53-54 [Suppl Ill:

464, 1976 (abstract)

9. Chung KS, Madar DA, Goldsmith JC,

Kingdon HS, Roberts HR: Purification and char-

acterization of an abnormal factor IX (Christmas

factor) molecule: Factor IX Chapel Hill. J Clin

Invest 62:1078, 1978

10. Cuatrecasas P. Protein purification by

affinity chromatography. J Biol Chem 245:3059,

I970

1 1. Ouchterlony 0: Handbook of lmmunodif-

fusion and lmmunoelectrophoresis. Ann Arbor,

Mich. Arbor Science Publishers, 1968

12. Woods KR, Wang KT: Separation of

dansyl-amino acids by polyamide layer chroma-

tography. Biochim BiophysActa 133:369, 1967

13. Hauschka PV, Lian JB, Gallop PM: Direct

identification of the calcium-binding amino acid,

‘y-carboxyglutamate, in mineralized tissue. Proc

Natl Acad Sci USA 72:3925, 1975

14. Boggs NT, Gawley RE, Koehler KA,

Hiskey RG: Synthesis of Dl--y-carboxyglutamic

acid derivatives. i Org Chem 40:3850, I 975

15. Biggs R: Human Blood Coagulation,

Haemostasis and Thrombosis (ed 2). Oxford,

Blackwell Scientific, 1976, p 704

16. Barrow EM, Bullock WR, Graham JB: A

study of the carrier state for plasma thromboplas-

tin component (PTC, Christmas factor) deficien-

cy, utilizing a new assay procedure. i Lab Clin

Med 55:936, 1960

17. Hougie C, Barrow EM, Graham JB: Stuart

clotting defect. I. Segregation of an hereditary

hemorrhagic state from the heterogeneous group

heretofore called �‘stable factor” (SPCA, procon-

vertin, factor VII), deficiency. J Clin Invest

36:485, 1957

18. Soulier iP, Larrieu Mi: Etude analytique

des temps de Quick allong#{233}s. Dosages de

prothrombine, de proconvertine et de proacc#{233}l#{233}r-

inc. Vox Sang 23:459, 1952

19. Smith HP, Warner ED, Brinkhous KM:

Prothrombin deficiency and the bleeding tendency

in liver injury (chloroform intoxication). J Exp

Med 66:801, 1937

20. Soulier JP, Prou-Wartelle 0: Etude com-

parative des taux de cofacteur de Ia staphyloco-

agulase (CRF) et des taux de facteur II (pro-

thrombine) dans diverses conditions. Nouv Rev Fr

Hematol 6:623, 1966

21. Roberts HR. Grizzle iE, McLester WD,

Penick GD: Genetic variants of hemophilia B:

Detection by means of a specific PTC inhibitor. J

Clin Invest 47:360, 1968

22. Pike IM, Yount Wi, Puritz EM, Roberts

HR: Immunochemical characterization of a

monoclonal lgG4 human antibody to factor IX.

Blood4O:l, 1972

23. Fahey JL, McKelvey EM: Quantitative

determination of serum immunoglobulins in anti-

body agar plates. i Immunol 94:84, 1965

24. Laurell CB: Quantitative estimation of

proteins by electrophoresis in agarose gel contain-

ing antibodies. Anal Biochem 15:45, 1966

25. Weeke B: Crossed immunoelectrophoresis.

Scand i Immunol I [Suppl 2]:47, 1973

26. Ganrot P0: Crossed immunoelectrophore-

sis. Scand J Clin Lab Invest 29(Suppl l24J:39,

1972

27. Esnouf MP, Prowse CV: The gamma-

carboxyglutamic acid content of human and

bovine prothrombin following warfarin treatment.

Biochim Biophys Acta 490:47 1 , I 977

28. Lavergne iM, iosso F: Metabolism of

PIVKA II in man, in Hemker HC, Veltkamp JJ

(eds): Prothrombin and Related Coagulation

Factors. Leiden, Leiden University Press, 1975, p

I 83

29. Thompson AR: Factor IX antigen by

radioimmunoassay-abnormal factor IX protein

in patients on warfarin therapy and with hemo-

philia B. i Clin Invest 59:900, 1977

30. Stenflo i: Vitamin K and the biosynthesis

of prothrombin. II. Structural comparison of an

amino terminal fragment from normal and from

dicoumarol-induced bovine prothrombin. J Biol

Chem 248:6325, 1973

31 . Nelsestuen GL, Zytkovicz TH, Howard

JB: The mode of action of vitamin K. Identifica-

tion of ‘y-carboxyglutamic acid as a component of

prothrombin. i Biol Chem 249:6347, 1974

32. Magnusson 5, Sottrup-iensen L, Petersen

TE, Morris HR. Dell A: Primary structure of the

vitamin K-dependent part of prothrombin. FEBS

Lett44:l89, 1974




FACTORS II. VII, IX, AND X 787

33. Hauschka PV, Reid ML, Lian iB, Fried-

man PA, Gallop PM: Probable vitamin K-depen-

dence of ‘y-carboxyglutamate formation in bone.

Fed Proc 35:1354, 1976

34. Hauschka PV, Friedman PA, Traverso JP,

Gallop PM: Vitamin K-dependent ‘y-carboxyglu-

tamic acid formation by kidney microsomes in

vitro. Biochem Biophys Res Commun 71:1207,

I 976

35. Willingham AK, Matschiner iT: Changes

in phylloquinone epoxidase activity related to

prothrombin synthesis and microsomal clotting

activity in the rat. Biochem i 149:435, 1974

36. Bell BG, Caldwell PT, Holm EET:

Coumarins and the vitamin K-K epoxide cycle.

Lack of resistance to coumatetralyl in warfarin-

resistant rats. Biochem Pharmacol 25:1067, 1976




1979 53: 776-787

KS Chung, A Bezeaud, JC Goldsmith, CW McMillan, D Menache and HR Roberts Congenital deficiency of blood clotting factors II, VII, IX, and X

http://www.bloodjournal.org/content/53/4/776.full.htmlUpdated information and services can be found at:

Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of


http://www.bloodjournal.org/content/53/4/776.full.html

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





congenital deficiency of blood clotting factors ii, vii ......factors vii, ix, and x. studies on...

Documents