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CHARACTERIZATION OF FACTOR VIII INHIBITOR
BY-PASSING ACTIVITY OF FACTOR IX CONCENTRATES
BY

Karen L. Hess

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1983

U/&fuwfip

ABSTRACT
CHARACTERIZATION OF FACTOR VIII INHIBITOR BY-PASSING
ACTIVITY OF FACTOR IX CONCENTRATES
BY

Karen L. Hess

Hemophilia A is a familial bleeding disorder associated
with an abnormal Factor VIII molecule. Bleeding episodes
are treated by infusion of antihemophiliac globulin
concentrate or cryoprecipitate. Between 5-18% of
hemophiliacs develop an antibody to Factor VIII which makes
such treatments ineffective. Infusion of Factor IX
concentrates may provide a possible solution to this
problem.

This study was designed to examine the mechanism
responsible for Factor VIII inhibitor by-passing activity
(FEIBA) of Factor IX concentrate. A nmdified activated
partial thromboplastin time was designed to assay for FEIBA.
The procoagulant and coagulant proteins used in the assay
were obtained from the prothrombin complex by biochemical
fractionation. In our ;_'L_n xii-£53 system, FEIBA proved
dependent on the interaction of Factor X, 11a and V.

Further attempts were nmde tx> correlate anddolytic
activity of FEIBA with the clotting activity utilizing a
synthetic chromogenic peptide substrate,
[H-D-Phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide

dihydrochloride].

ACKNOWLEDGMENTS

I wish to express my appreciation to the following people:

Garson H. Tishkoff, M.D., PhD, my major professor, who
awakened my interest in the subject matter, encouraged my
initiative, and guided my studies.

Martha Thomas, M.S., MT (ASCP), my academic advisor,
for her guidance in my studies.

Mary Boss, M.S., MT (ASCP), for her willingness to
serve as a member on my committee.

Janice Harte, M.S., Nancy Shih, M.S., and Douglas
Estry, M.S., MT (ASCP), for their friendship and assistance.

To the Great Lakes Regional Red Cross Center in
Lansing, Michigan for their cooperation in extending their
full use of facilities (plasma) and assistance of their

personnel.

ii

TABLE OF CONTENTS

Page
INTRODUCTION 0 0 O O O O O O O O O O O O O O O O O O 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . 3

MATERIALS AND METHODS . . . . . . . . . . . . . . . 21

Chromatographic Material . . . . . . . . . . . 21
Equipment . . . . . . . . . . . . . . . . . . . 21
Coagulation Reagents . . . . . . . . . . . . . 22
Chemicals . . . . . . . . . . . . . . . . . . . 22
Protein Determinations . . . . . . . . . . . . 23
Conductivity Measurements . . . . . . . . . . . 23
Coagulation Assays . . . . . . . . . . . . 23
Preparation of Dialysis Tubing . . . . . . . . 24
Antithrombin III . . . . . . . . . . . . . . . 24
Human Plasma . . . . . . . . . . . . . . . . . 25
Polyacrylamide Gel Electrophoresis . . . . . . 25

Factor VIII Inhibitor By-Pass Activity
(FEIBA assay) . . . . . . . . . . . . . . . . 26

Chromogenic Substrate Assay . . . . . . . . . . 26
Preparation of Chromatographic Material
Blue Dextran Resin . . . . . . . . . . . . 28

Sephadex G-200 . . . . . . . . . . . . . 28
DEAR-52 . . . . . . . . . . . . . . . . . 28
Heparin Sepharose . . . . . . . . . . . . 28
Hydroxyapatite . . . . . . . . . . . . . . 29
Thrombin Sepharose . . . . . . . . . . . . 29

Preparation of Thrombin . . . . . . . . . . 30
Preparation of ATIII- -Sepharose . . . . . . . . 31
Purification of Factor X . . . . . . . . . . . 32

Vician-Tishkoff's method . . . . . . . . . 32

Kosow' s method . . . . . . . . . . . . . . 35
Isoelectric focusing . . . . . . . . . . . 37
Reduction and Alkylation of Factor X . . . . . 37
Activation of Factor X . . . . . . . . . . . . 38
Antisera Production . . . . . . . . . . . . . . 39

RESULTS 0 O O O O O O O O O O O O O O O O O O O O O 40

Purification of Human Factor X . . . . . . . . 40
Vician-Tishkoff‘s method . . . . . . . . 4O
Kosow's method . . . . . . . . . . . . . . 47

iii

Activation of Prothrombin to Thrombin
Clotting Studies . . . . . . . . . .
Molecular Studies . . . . . . . . . .
Reduction and Alkylation of Factor X
SDS Gel Electrophoresis . . . . . . .
Chromogenic Substrate Assays . . . .
Ouchterlony Diffusion . . . . . . . .
Isoelectric Focusing . . . . . . . .

DISCUSSION . . . . . . . . . . . . . . . .

CONCLUSION O O O O O O O O O O O O O O O 0

LITERATURE CITED . . . . . . . . . . . . .

VI

F1

1

A

iv

Page

51
56
66
70
71
75
75
76

77
84
86

97

Table

10.

ll.

12.

13.

LIST OF TABLES

A summary of the major and minor
procoagulants and coagulants in
Factor IX concentrates . . . . . . . . . .

Human Factor X Purification (Vician-
Tishkoff method): A summary of several
preparations . . . . . . . . . . . . . .

Congenitally deficient plasma and the
interaction of X+IIa in the FEIBA assay. .

Preincubation of X+IIa for various time
intervals, with and without the inhibitor
substrate, and proceeding with a "l-minute"
FEIBA . . . . . . . . . . . . . . . . . .

Preincubation of X+IIa without the substrate
and continuing with a "40-minute" FEIBA .

The effect of Factor IX in the FEIBA system

The effect of human serum in the generation
Of FEIBA O O O O O O O O O O O O O O O O O

FEIBA results on X+IIa fractionated off
Sephadex G-100 . . . . . . . . . . . . . .

FEIBA results of the interaction of X with
immobilized thrombin . . . . . . . . . . .

SDS gel results on the interaction of X
with immobilized thrombin . . . . . . . .

FEIBA results of X+IIa incubated with
AT-III sepharose o o o o o o o o o o o o

FEIBA results of reduced and alkylated
FaCtOr x O O O O O O O O O O O O O O O O O

Apparent molecular weights of bands
appearing on SDS gels . . . . . . . . . .

Page

14

46

63

64

65

67

68

69

70

71

74

Figure

l.

10.

ll.

12.

13.

14.

15.

LIST OF FIGURES

Cascade scheme for blood coagulation . . . .
Schematic illustration of bovine prothrombin
The conversion of prothrombin to thrombin.

Schematic representation of Factor X . . . .

Procoagulant profile of prothrombin complex
eluted from Sephadex G—200 . . . . . . . . .

Procoagulant profile of Auto Factor IX
(HYLAND) chromatographed on Sephadex G-200

Pooled prothrombin complex chromatographed
on Blue Dextran resin . . . . . . . . . .

Chromatography pattern of protein off of an
ion exchange resin . . . . . . . . . . . .

Sodium dodecyl sulfate polyarcylamide gel
electrophoresis of purified Factor X
(Vician-Tishkoff's method) . . . . . . . .

Elution pattern of prothrombin complex from
DEAE—ASO o o o o o o o o o o o o o o o o o 0

Protein profile of Factor X from heparin
agarose column . . . . . . . . . . . . . .

Factor X pattern off of Hydroxyapatite
COlumn O O O O O O I O O O O O O O I O O 0

Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of purified Factor X
(Kosow's method) . . . . . . . . . . . . .

Australian Taipan Venom chromatograph on
DESZ O O O O O O O O O O O O O I O O O O O O

Thrombin chromatographed on Bio-Rex 7O . .

vi

Page

12

41

42

43

45

48

49

50

52

53

54

55

Figure Page
16. Generation of FEIBA with Factor X + II . . . 57

17. Calculating optimum amounts of prothrombin
necessary for FEIBA generation . . . . . . . 57

18. Calculating optimum amounts of Factor X

necessary for FEIBA generation . . . . . . . 58
19. FEIBA assay with depleted Factor V substrate 58
20. Impaired FEIBA generation . . . . . . . . . 60
21. Significant FEIBA generation with X + IIa. . 60

22. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of Factor X intermediate . . 72

vii

INTRODUCTION

Classic hemophilia, or hemophilia A, is an inherited,
sex-linked bleeding disorder. It exhibits decreased clot-
ting activity due to a Ideficiency’ of a specific plasma
protein, Factor VIII, which is necessary for normal hemo-
stasis. Clinical manifestations of the disorder include
deep tissue bleeding and the residual effects of bleeding.
Current treatment of a hemorrhagic episode includes infusion
of cryoprecipitate, a by-product of whole blood fraction-
ation, or infusion of lyophilized Factor VIII concentrate,
prepared from the plasma of blood donors. However, 7-18% of
individuals with hemophilia A develop an inhibitor (an
antibody which acts specifically against Factor VIII
clotting activity) which makes the standard form of
treatment ineffective.

The most efficacious treatment of a bleeding episode in
the hemophiliac with an inhibitor, is the infusion of
prothrombin complex concentrates. The exact mechanism of
the coagulant effect of these concentrates is unknown.

It is the purpose of this thesis to characterize the
mechanism. of the jprothrombin concentrates. In order to

approach this problem it was necessary to: l) isolate and

2
purify procoagulant and coagulant proteins; 2) devise an
assay for evaluating Factor VIII inhibitor by-passing
activity (FEIBA); and 3) utilize in 22332 clotting studies,
chromatographic techniques and synthetic substrate assays to

analyze the precise mechanism.

The Clotting Factors

 

Observations of an enzymatic activity involved in
clotting date back to 1872 when Schmidt (1) used the term
"fibrin ferment" or "thrombin" to describe the material
precipitated from serum, not from fresh blood. Morowitz
(2), in his classic theory on blood coagulation in 1905,
describes thrombokinase and thrombin as two active enzymes
in the blood clotting scheme. During the next fifty years,
the discovery of Factor V (3), Factor VII (4), and Factor X
(5) and the discovery of their role in the conversion of
prothrombin to thrombin lent support to Morowitz's theory.
Prothrombin, which was merely a hypothetical substance in
the early 1900's, was investigated extensively and.tn/ the
early 1960's Seegers (6) and others (7-9) had elucidated the
biochemistry of prothrombin. The study of the coagulation
protein interactions led to a hypothetical scheme, proposed
by Macfarlane (10), of the pathophysiology of the clotting
activity from surface contact to fibrin formation. Davie
and Ratnoff (11) also proposed at the same time as
Macfarlane the sequential activation of the clotting
proteins in a Ichain-like manner. This is known as the

"cascade" or "waterfall" hypothesis.

Figure 1 shows a modified cascade scheme which also
includes an alternate pathway referred to as the extrinsic

pathway, as well as feedback reactions.

l[—INTRINSIC SYSTEM

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

 

 

 

‘ I :FACTOR n!
FACTOR m—bl Surface :
FACTOR Inc
Contact l
m‘KIncnoggn ; P'Osmmoqen : Plasmmogen
. :guolhhtem—) Kollnlucm . 2 P'OOCHVOIOI ACHVO‘O!
....................... B'Odykmm) ' -

 

Plosmanoqon-iePlogmm

 

FACTOR 31—3 FACTOR x10

  
  

 

 

 

EXTRINSIC FACTOR II c" FACTOR IIO+FACTOR mt- ----- -.
SYSIEE-\-§ --____ _____ _ PL Co
. FACTOR I ‘FAC TOR Io + -'

 

FACTOR m + £0 FACTOR IG--

TISSUE FACTOR \
musseu's Co PL C°

VIPER VENOM) \ PROTHROMBINASE

PROTHROMam-c-LNHROMOIN

 

__-____L_-_-

 

FIBRINOGEN—éFIBRIN
CO
FACTOR III —I:-) FACTOR mo

 

'-—-------

 

L--

 

FIGURE 1. A Modified Cascade Scheme For Blood Coagulation.
(12).

There is another theory of blood coagulation proposed
by Seegers (13) which involves specific terminology relating
the clotting proteins to the prothrombin molecule
(prothrombin derivatives). At the present time, with
modification of both theories and with more extensive

characterization of the clotting factors, a more current

scheme of the protein - protein interactions can be

postulated.

Prothrombin and Thrombin

The complete biochemistry of bovine prothrombin and its
activation is now ‘well known. Bovine prothrombin is a
glycoprotein with a single polypeptide chain (molecular
weight 70,000 daltons). The complete amino acid sequence of
bovine prothrombin is shown schematically in Figure 2. The
prothrombin molecule can be divided into three main parts:
1) Prothrombin Fragment l which is one activation product;
2) Prothrombin Fragment 2, another activation product; 3)
and the remaining portion which constitutes thrombin after

the appropriate activation.

Ihvombm'Fon-mng Reguon o. onvhmmbm —H

(MD ®(323) ©(274)

'me—fl E233"? 1...... T

'S v S
S
1 )3 onthtombm
Ftogmoni 2
lemon

Sc:

 

 

(SSlz

‘5“5 CHO
A HIM W\N\W\\\W‘k\\\\“\\\\‘ A ' °® S . ' _ 1L.

 

©(l56)
H—onlhvombm fragment I Roman ———'|

FIGURE 2. Schematic Illustration Of Bovine Prothrombin (14)

Human_ prothrombin has also .been fully characterized
(15) and the amino acid sequence is known. Human
prothrombin is also a single chain polypeptide with a
molecular' weight of 72,000 daltons. Recent. purification
procedures of human prothrombin are included in the work of
DiScipio, et al (16). There are some differences in the
amino-terminal region of the bovine prothrombin molecule as
compared to the human prothrombin molecule. The human
prothrombin molecule has a threonine residue in position 3
where the bovine prothrombin has a lysine residue. In
position 4, a glycine is present in the bovine species; the
human species omits a residue at this position. The purpose

for the omission is to allow greater homology with other

human vitamin 1( dependent proteins. Although ndnor
differences are present among the various clotting proteins,
their similarities are quite significant.

The conversion of prothrombin to thrombin is a rapid
process dependent on the interaction of Factor Xa, Factor V
(Va), lipid surfaces and calciunl ions. Factor Xa is aa
proteolytic enzyme responsible for the cleavage of two
peptide bonds in the prothrombin molecule. Factor V, a
labile plasma component, binds II, Xa and phospholipid. The
lipid surface increases the association between Factor xa
and prothrombin and is :made available from jplatelet or
damaged cell membranes. Calcium ions are necessary for the
binding of Factor Xa and prothrombin binding to the lipid
surface. Figure 3 schematically illustrates the conversion

of prothrombin to thrombin (17).

[Xo,Va,PL,C02']__I (’7

Prothlombm I Bw'hrombin2 A Thvombm
7 Fmgmenl 1-2

 

 

[Xo,Vo,Pl.Coz°]_l

Wyflfl/ézfi-

   

    

? Fragment L?
FIGURE 3. The Conversion Of Prothrombin To Thrombin (17)

When prothrombin is activated in the presence of
antithrombin III, an additional pathway is reported (18) in

which the zymogen is converted to a pwecursor (P3) with a

molecular weight of 37,000 and fragment F This takes

A B'
place before thrombin generation.

A slower activation of prothrombin occurs when high
concentrations of Factor Xa are reacted with Factor II in
the presence of 25% sodium citrate (19).

Since the zymogen itself is devoid of biological
activity, assay methods are based on the measurement of
thrombin formation. The first assay was a one stage
procedure developed by Quick (20) in 1935 and modified by
Link and Shapiro in 1949. The two stage assay first used by
Warner, Brinkhaus, and Smith (21) in 1936 involves the
addition of tissue extracts and calcium ions to Factors V,
VII, X and prothrombin. This results in optimal thrombin
formation. At specified intervals, aliquots of the reaction
mixture are removed and added to a standard amount of
fibrinogen. Clotting times are recorded. Clotting times of
fibrinogen are plotted against the time at which the samples
were removed. Clotting times are converted into thrombin
units according to appropriate calibration curves. One unit
of prothrombin yields one unit of thrombin.

Thrombin

Thrombin is a serine protease that catalyzes the
cleavage of specific arginyl and lysyl bonds. It is present
in the blood as a result of the proteolytic conversion of
prothrombin. Thrombin is less negatively charged than

prothrombin and was first obtained as a highly purified

protein in 1955 (22). The molecular weight of bovine
thrombin is reported as 33,700 daltons (23) as well as
40,000 daltons (24). The amino acid sequence of bovine and
human thrombin has been characterized (25, 26). Very few
differences exist between the two species.

Thrombin is responsible for the following actions: 1)
conversion of fibrinogen into fibrin; 2) activation of
Factor XIII; 3) initiation of platelet secretion and aggre—
gation; 4) reactions with plasma protease inhibitors and 5)
activation of Factor V and Protein C.

Thrombin can be separated into three components:
alpha, beta and gamma thrombin. Human alpha thrombin is a
double chain polypeptide with a molecular weight of 36,600
daltons. It is the form primarily responsible for clotting
activity. Alpha thrombin also possesses esterase activity.
Beta and gamma thrombin do not exhibit significant clotting
activity. All three forms are inhibited by alpha-tosyl-L-
lysylchloro-methyl ketone.

Thrombin, whether human or bovine, can undergo
autolytic degradation. The degraded species lack clotting
activity but exhibit the esterase activity.

Thrombin. :may be assayed enzymatically utilizing
chromogenic peptide substrates. 82160 (Ortho) is sensitive
for the determination of thrombin, but is also sensitive to
other proteases such as trypsin, papain, and brinase. A

more newly developed synthetic peptide is [H-D-phenylalanyl-

10

L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride], or
82238 which is more sensitive and more soluble than 82160.
52238 is a small peptide with the chromophore attached to
the N terminal amino acid. The rate of p-nitroanaline
formation is prOportional to the enzymatic activity of
thrombin.
Factor X

Factor X, also known as Stuart Prower factor, is a
plasma glycoprotein.enui a major proenzyme activated during
blood coagulation. Factor X, along with Factors II, VII and
IX, are dependent upon vitamin K for their biosynthesis.
These coagulant proteins can be absorbed out of the plasma
by barium and aluminum salts. Seegers (27) refers to Factor
X as autoprothrombin III. Aronson‘s (28) findings support
evidence of a separate origin for Factor X due to the
presence of only 1/3 similar amino acid sequences for Factor
X as compared. with prothrombin. The remaining 2/3 are
unique for each molecular species. Later studies (29)
report similarities between. Factcm' X and. prothrombin as
follows: 1) both require Ca++ and phospholipid for
physiological activation; 2) both have similar
amino—terminal amino acid sequences; 3) both are serine
esterases with common amino acids at the active site and 4)
both contain similar vitamin K dependent calcium binding

regions.

ll

Bovine Factor )( has been successfully purified using
various isolation procedures. (30, 31, 32, 33) It is a
glycoprotein (molecular weight 55,000 daltons) composed of
two polypeptide chains held together by disulfide bonds. It
is also reported to exist as a single polypeptide chain
(34). Jackson (35) isolated two glycoprotein variants of
bovine Factor X, which differed in carbohydrate composition.
Both, however, contained two polypeptide chains. The amino
acid composition of the heavy chains of the Factor X
molecule is similar to that of thrombin with the exception
of three amino acids: lysine, threonine and alanine.

Purification of human Factor X has been more difficult
to achieve. Procedures have included preparative
isoelectric focusing (36), purification utilizing DEAE
cellulose (37) and hydroxyapatite (38). A unique approach
has been through isolation of Factor X utilizing blue
dextran agarose (BDA) affinity chromatography (39). This
purification process yields a homogeneous Factor X
(molecular weight 69,000 daltons). Like bovine Factor X, it
is composed of a.tmavy (50,000 daltons) and light (13,000
daltons) chain. DiScipio (40) has characterized human Factor
X. He reported a molecular weight of 60,000 daltons.
Schematically, Factor X is illustrated in Figure 4. The
heavy chain of Factor X (solid black line) contains the

active site serine, aspartate and histidine residues.

12

fouo: X Achvonon Pcphd.
Heavy Polypophdc Chom 0’ 5000: X0 vuf I ”0"0' ‘

(bov human! 3)

(H0
PAan'Hu—uogm fin?)

 

 

 

 

 

k‘lnghl Polypophde Chem a! foam X

FIGURE 4. Schematic Representation of Factor X (41)

Kosow (42), who purified human Factor X by a
combination (n3 DEAE-sephadex, Heparin Sepharose, and
hydroxyapatite, achieved a final human Factor X product with
a molecular weight of 75,000 daltons.

Factor X can be activated to a serine protease, Xa, via
the extrinsic pathway by tissue factor and Factor VII
(43,44), or through the intrinsic pathway by Factor IXa and
Factor VIII in complex with phospholipid and calcium (45).
Factor X can also be activated by trypsin (46, 47), papain
(48), or a protease from Russells viper venom (49, 50, 51).
Formation of Factor Xa involves cleavage of a specific
arginyl - isoleucine peptide bond in the heavy chain of the
precursor protein (52). The esterase and coagulant
activities of activated Factor X are variably inhibited by
diisopropylfluorophosphate (53). Activated Factor X is also

inhibited by antithrombin III in a reversible reaction (54)

13

following second order kinetics. In the absence of heparin,
the rate of inhibition is slow.

The qualitative clotting assay for Factor X involves a
modified one stage prothrombin assay' in. which Russell's
viper venom is added to Factor VII-X deficient plasma.
Russell's viper venom is sensitive to extraneous Factor X
but not to Factor VIII. The appropriate lipid and calcium
chloride are added to complete the assay.

Factor IX Concentrates

 

Factor IX concentrates, also referred to as prothrombin
complex concentrates (PCC), have been used since 1959 for
the treatment of hemorrhagic disorders associated with
Christmas disease (55) and liver disease (56). Clinical use
of the concentrates was further investigated by Tullis 23 21
(57) who reported therapeutic effectiveness in patients with
acquired or hereditary deficiencies of Factors II, VII and
X.

The only commercial products available from 1960 to
1968 were made by the French National Transfusion Center or
by British laboratories (58). The first commercial
concentrate produced in the United States was developed by
Cutter Laboratories of Berkeley, California in the late
sixties. This concentrate, Hemoplex, which also contained
Factors II, VII and X was first reviewed by Hoag 33 a1 (59).

Since that time, additional commercial concentrates

have been developed. A study by Menache (60) reported 23

14

different Factor IX 'concentrates produced by eighteen
laboratories. These concentrates vary in several ways
including starting' material, volume ‘used. to prepare one
batch, adsorbent used, and the presence or absence of

heparin in the final product.

TabLe 1: A summary of the major and minor procoagulants and
coagulants in Factor IX concentrates.

 

 

Major Minor Miscellaneous
Factor 11 11a Antithrombin III
Factor X Xa Phospholipid
Factor IX IXa
Factor VII VIIa
Protein C (?) XII

XI

VIII
V

 

The Use of Factor IX Concentrates to Treat
Immune Inhibitor in Classic Hemophilia

 

 

The life expectancy of a patient with classic hemophilia
has been extended by the use of Factor VIII concentrates.
However, development of an inhibitor to Factor VIII makes the
standard form (ME treatment ineffective during bleeding
episodes. Strauss (61) estimated that up to 21% of Type A
hemophiliacs develop an antibody to Factor VIII, while other
researchers (62) have reported the appearance of an inhibitor
in approximately 7% of hemophiliacs. The inhibitors in
hemophilia A are IgG antibodies, mainly monoclonal with kappa
light chains. They are species specific and act specifically

against Factor VIII procoagulant activity. It is unknown why

15

some hemophiliacs develop an antibody and others do not. It
may be related to higher exposure to Factor VIII or merely
reflect the tendency of some patients to produce antibodies
more readily.

Management. of patients 'with inhibitors during' bleeding
episodes has involved plasmapheresis (63) (64), immuno-
suppressive therapy (65) (66) and administration of bovine and
porcine Factor VIII (67). Plasmapheresis has been ineffective
in completely removing or diluting the circulating antibody and
provides only short term improvement of the bleeding episode.
There is conflicting data as to the suppression of antibody
titers of immunosuppressive drugs such. as cyclophosphamide,
chlorambucil and 6-mercaptopurine. Bovine and porcine Factor
VIII have been used with limited success. The higher level of
Factor VIII in animal species enables use of a smaller
therapeutic volume. However, allergic reactions to the animal
concentrates are serious <disadvantages. Another ‘therapeutic
approach has been neutralization of the inhibitor by continuous
infusion of large amounts of Factor VIII. This has not been
uniformly effective.

For the treatment of hemophiliacs with an inhibitor, the
most dramatic response has been the use of prothrombin complex
concentrates. Fekete et a1. (68) first reported a unique
therapeutic approach using an "activated prothrombin complex"
product (produced by Hyland Laboratories, Chicago, Illinois).

From 1972 to 1976 there were five other reports of the

l6

efficacious use of Factor IX concentrates for hemophiliacs with
an immune inhibitor (69), (70), (71), (72), (73). All
investigators reported control of the hemorrhagic episode after
infusion of the concentrate. Both activated Factor IX
concentrates (auto Factor IX) which contain trace amounts of
Factors Xa, IXa and other coagulants, and standard concentrates
such as Proplex (Hyland Laboratories) were used. More recent
studies (74), (75), (76), (77) have used a new concentrate,
FEIBA (Factor Eight Inhibitor By-Passing Activity). This
product does run: contain any Factor VII. FEIBA concentrates
(Fraction. R. made by Immuno .AG, ‘Vienna, Austria) have been
successful in controlling hemorrhagic complications of the
disorder and are widely used in Europe.

The non-activated products which are currently available
in the United States are Konyne and Proplex. These, however,
for reasons unknown, have been less effective since 1976 for
the management of the bleeding episode. The activated product
available in the United States in current use is Autoplex

(Hyland Laboratories).

Complications of Therapy With
Prothrombin Concentrates

Potential complications (If post-infusion prothrombin
complex concentrates have Ibeen. recorded. The .reported
incidence of viral hepatitis is high after infusion of Factor
IX concentrates, but varies from country to country (78), (79),

(80). The risk of hepatitis B virus transmission increased

17

when Factor IX concentrates were manufactured from paid
commercial donors as opposed to volunteer donors. It has been
recommended (81) that even though present screening techniques
are not 100% effective in detecting blood or plasma
contaminated with hepatitis B virus, all manufacturers should
perform third generation testing. These tests include
radioimmunoassay, reversed passive hemagglutination and the
agglutination flocculation test.

Another complication has been an increase in the Factor
VIII inhibitor concentration after infusion (82), (83), (84).

A third concern regarding the use of the concentrates is
their thrombogenic properties. Menache (85) first noted the
potential thrombogenicity of these concentrates. The greatest
number of thrombotic episodes has occurred after infusion of
concentrates into patients with liver disease. This may be due
to decreased clearance by the damaged liver of activated
factors present in the concentrates and decreased levels of
antithrombin III. Fatal thromboembolism has been reported
(86). Consequently, several investigators have strongly
discouraged the use of prothrombin complex products in patients
with liver disease.

The incidence (ME thrombotic complications resulting from
the use of prothrombin concentrates has been low. Studies have
shown (87) that several commercial preparations do contain
substantial amounts of potentially thrombogenic enzymes and are

able to produce thrombi in animal models. Thromboembolic

18

complications have been recorded with non-activated products as
well as FEIBA (Immuno). Some researchers (88) (89) have
suggested the addition of heparin to the reconstituted material
to reduce thrombogenicity.

The mechanism of the hypercoagulable state after infusion
of the complex is not completely understood. It may be due to
the presence of Factor Xa or IXa, or the concentrates may be
thrombogenic under certain conditions i2 ytyg (90). It may be
important to infuse a concentrate immediately after
reconstitution since Elodi (91) reported generation of Factor
IXa during incubation of the concentrate at room temperature
for 20 minutes.

£3 ytttg screening techniques have been proposed to test
neW' batches of Factor IX concentrates for thrombogenicity.
Blatt gt gt (92) suggested conduction a "pre-infusion stability
check". This involves the addition of plasma to reconstituted
concentrate at room temperature. If no clot forms after five
minutes, the concentrate is assumed safe for administration.
Kingdon gt gt (93) introduced a test in. which Factor IX
concentrates were added to a nonactivated partial
thromboplastin time system! (NAPTT). Shorter reaction times
indicated possible thrombogenicity. Cash ‘gt_ gt (94) found
major discrepancies between the NAPTT and the thromboplastin

generation test (TGt The TGt represents the incubation

50" 50

time required to give a fibrinogen clotting time of 50 seconds.

Products which had a short TGt50 were those in which thrombin

19

generation occurred more readily. Cash gt a_l_ (95) concluded
that the TGt50 correlated more closely than the NAPTT with EB
vivo systems, although it could not be recommended as the only

monitor for thrombogenicity testing.

Mechanisms Responsible for By-Passing
Factor VIII Inhibitor

 

 

There are many theories attempting to explain the
mechanism of the coagulant effect of the prothrombin
concentrates in the hemophiliac with an inhibitor. Kurczynski
(96) suggested the presence of Factor IXa, Xa, or the mass
effect of various factors on coagulation are responsible for
clot promoting activity. Elsinger (97) reported that
by—passing of the inhibitor is due to presence of Factors VII,
IX, and X. Elsinger gave evidence that the presence of Factors
VII, IX, and X are responsible for by-passing the Factor X
activity although Factor \7 is not by-passed. In his
experiments with the reaction of fraction FEIBA (active portion
of preparat1on) and soybean trypsin inhibitor, he gave evidence
to support the theory that the active component is not Factor
Xa itself.

Vermylen (98) proposed the enhancement of Factor X
activator activity of platelet, independent of the intrinsic or
extrinsic cycle, is the by-pass mechanism. He found that
platelets, prepared from plasma pre-incubated with fraction
FEIBA, have increased coagulant activity. This can be
demonstrated by decreasing the latent period of aggregation.

This theory proved correct in patients who were infused with

20

FEIBA (Immuno). It was discovered that three hours post
infusion there was a dramatic increase in the coagulant
activity of the patients' washed platelets.

Tishkoff (99) in 1976 postulated that the active protease
in the concentrates may be a binary complex of Factor X and
thrombin based on molecular size measurements. Factor V is
also a necessary component of this reaction. At that time, he
suggested that this mechanism was an alternate extrinsic
pathway for the generation of prothrombokinase. Factor X and
thrombin, when reacted in a modified APTT, have the ability to
by-pass the Factor VIII inhibitor substrate plasma to shorten
the APTT.

CURRENT STATUS OF PROTHROMBIN COMPLEX

CONCENTRATES IN MANAGEMENT OF BLEEDING
EPISODES IN INHIBITOR PATIENTS

 

 

 

The commercially available products used today in the
United States are KonyneR and ProplexR. AutoProplex
(HYLAND) is used with a limited basis. Several problems
remain concerning the clinical use of the prothrombin
concentrates. Because the exact mechanism is unknown, there
is no standardization of the commercial concentrates. This
accounts for a wide degree of variation in the products.
There is still the potential for thrombogenic complications
as well as anamnestic response in some patients. Finally,
there have been in the past few years conflicting reports as
to the efficacy of the concentrates in managing a bleeding

episode.

Materials and Methods

 

Chromatographic Materials

 

Sephadex G-100, G-200, BioGel A-15M, DEAE-A50, and all
column. «chromatographic equipment. 'were purchased from
Pharmacia Five Chemicals, Piscataway, New Jersey. Whatman
DEAE Cellulose was obtained from H. Reeve Angel and Co.,
Clifton, New Jersey. Dowex 50X8 (20-50 mesh, Na form) from
J.T. Baker Chemical Co., Philipsburg, New Jersey.
Hydroxyapatite, Bio—Rex 70 (110-200 mesh), and Chelex resin
(100—200 mesh, Na form) were purchased from Bio Rad

Laboratories, Richmond, California.

Equipment

 

Major equipment used included a Beckman Du
Spectrophotometer, Fullerton, California, with Gilford light
source and cuvette position, Oberlin, Ohio; Radiometer pH
meter, Copenhagen, Denmark; LKB fraction collector,
Rockville, Maryland; Amicon concentrating ultrafilter
system, Lexington, Massachusetts; Bio Rad ultrafilter
beaker; and Sorvall centrifuge, DuPont Instruments, Newtown,

Connecticut.

21

Coagulation Reagents.

 

Bovine plasma deficient in Factors VII and X, crude
Rusell's viper venom in cephalin, human Factor V and
heparin, grade 1, sodium salt from porcine intestinal mucosa
were all obtained from Sigma Chemical Co., St. Louis,
Missouri. Activated thromboplastin and activated
cephaloplastin (ACTIN) were purchased from Dade, Miami,
Florida. Human fibrinogen, grade L, was obtained from
ABKABI, Stockholm, Sweden. The fibrinogen was dialyzed to
reverse excess citrate which interfered with the two-stage

rothrombin assay. The fibrinogen (1.59 dissolved in 66 ml
of distilled H20) was dialyzed overnight at 4C against 4
liters of buffer consisting of 35.35g NaCl, 2.87g sodium
citrate, 17.74g sodium acetate. It was subsequently frozen
at -80C in 10 m1 aliquots. Factor VIII Inhibitor plasma was
donated by Dr. J. Penner, then of the University of

Michigan, Ann Arbor, Michigan.

 

Chemicals
Acrylamide, methylenebisacryalamide, N,N,N',N' - tetra-
methylethylene - diamine (TEMED), ammonium persulfate,

sodium dodecyl sulfate, dithioerythritol, and all
electrophoretic equipment was purchased from Bio Rad
Laboratories. Ethanolamine, benzamidine-hydrochloride (HCl)
was obtained from Eastman Organic Chemicals, Rochester, New

York. Blue dextran 2000 was obtained from Pharmacia Fine

22

23

Chemicals. Cyanogen bromide and polybrene were purchased
from Aldrich Chemical Co., Milwaukee, Wisconsin. Australian
Taipan venom and Soybean trypsin inhibitor was purchased
from Sigma Chemical Company. Kabi chromogenic substrates
$2160 and 82238 were purchased through Ortho Diagnostics,
Raritan, New Jersey. All other chemicals were reagent grade

and all solutions prepared with distilled, deionized water.

Protein Determinations

 

Protein was monitored by absorbance readings at 280 nm.
Protein concentrations were then calculated according to the

formula: (OD at 280 nm)(0.795) + 0.006 = mg protein per ml.

Conductivity Determinations

 

A radiometer type CDM 2e conductivity meter was
utilized to measure conductivity. The readings of unknown
samples were compared to standard curves that were

constructed using buffers of known ionic strengths.

Coagulation Assays

 

Prothrombin and thrombin were assayed by modifying the
2 stage prothrombin procedure of Ware and Seegers (100) by
replacing bovine fibrinogen with human fibrinogen. The
results are expressed in Iowa units. One unit of
prothrombin yields one unit of thrombin. Normal plasma

values assayed 250 units of prothrombin per ml.

24

Factor X and Xa were assayed according to the method of
Bachman (101). Factor X was assayed using bovine plasma
deficient in Factors VII-X and Russell's viper venom. One
unit of Factor X activity is equivalent to the activity in
one ml of normal human plasma. Factor Xa was assayed by
substituting rabbit brain cephalin for Russell's viper venom
and comparing the clotting times obtained to the Factor X
standard curve.

Factor VIII was assayed according to a modified one
stage Quick (102) procedure using human Factor VIII
deficient plasma donated by Dr. J. Penner.

Factor IX was assayed according to the method of
Proctor and Rappaport (103).

Standard curves were prepared for all clotting assays
utilizing a fresh plasma pool from male donors. The

anticoagulant was 3.8% sodium citrate.

Preparation of dialysis tubing

 

Dialysis tubing was treated according to Methods of

Enzymology (104).

Antithrombin III

 

The partially purified human antithrombin III was
obtained from the National Red Cross Research Laboratory,
Bethesda, Maryland. It was further purified on a Sephadex

G-100 column before using it in the assay procedures.

Human Plasma

 

Plasma was obtained from the Great Lakes Regional Red
Cross Services, Lansing, Nuchigan. Three inhibitors were
added to the fresh plasma as follows: 0.157g/1iter
Benzamidine - HCl; 50 mg/liter polybrene; and 20 mg/liter of
soybean trypsin inhibitor. The plasma was then centrifuged
for 30 minutes, at 10,000 rpms at 4C. The supernatant was
collected in plastic transfer bags and frozen at -80C
overnight. It was then slowly thawed at 4C and centrifuged
at 8,000 rpm for 15 minutes and the supernatant
(cryo—precipitate poor plasma) finally stored in plastic

containers at -80C.

Polyacrylamide Gel Electrophoresis

 

Sodium dodecyl sulfate gels (SDS) were prepared
according to the method of Fairbanks (105).

Molecular weight markers purchased from Schwartzman,
Orangebury, New York, were run in the same manner as the
protein solution. They consisted of phosphorylase A,
albumin, gamma globulin, and cytochrome c. The mobility of
the known proteins was plotted against the log of their
molecular weights. Unknowns were compared to the standard

curve to obtain an apparent molecular weight.

25

Factor VIII Inhibitor Bypass Activity (FEIBA) Assay

An assay was developed based on the correction of the
abnormal APTT of Factor VIII inhibitor plasma after a
l-minute and 40-minute incubation time. The inhibitor assay
consisted of adding 0.1 m1 of Factor VIII Inhibitor plasma
to 0.1 m1 of highly purified Factor X, Factor II, Factor IIa
or' a combination of Factor" X’ and. IIa and. allowing* the
proteins to incubate at 37C for 1 minute and 40 minutes. At
the appropriate times, cu1.:m1 of activated cephalaplastin
(ACTIN) was added for an additional 2 minutes incubation.
After allowing the lipid to interact, 0.1 ml of 0.02M
calcium chloride was added, a stop watch started and the
clotting time recorded. Dilutions of proteins in the assay
were :made ‘with sodiunl barbital. buffered saline, pH’ 7.4.

(VBIS).

Chromogenic Substrate Assay

 

Direct absorbance assays were utilized and the reaction
was as follows:

0.1 m1 purified Factor X (0.4 clotting units of

activity in the assay)

0.1 ml Tris Imidizole buffer (pH 7.4) or

0.1 m1 purified thrombin (0.015 clotting units in

assay)

0.1 ml activated Cephaloplastin (SIGMA)

0.1 ml 0.02M calcium chloride

26

27

0.1 ml purified human Factor V (SIGMA)

The above reactants were pipetted into a pre-warmed
cuvette (37C) and allowed to incubate for various times. At
the appropriate time, 0.1 m1 of purified human antithrombin
III (22 ug in assay) and 0.1 m1 sodium heparin (UPJOHN) were
added for an additional 15 minute incubation. At this
point, 0.1 ml 82238 (Ortho) diluted to 0.1 mM in the assay
was added and the recorder turned on. An endogenous rate
was established during a 2-minute period, which was followed
by the addition of 0.1 ml highly purified human prothrombin
(250 units of activity in the assay). The rate of
para-nitroaniline production was followed at 405 nm.

Calculations for converting the spectrophotometric data
into linear plots were based on Kosow's kinetic analysis
(106) in which the following formula was used:

X — VO t + 1/2 at2
where VO equals the velocity at zero time and is the
endogenous rate of hydrolysis, t equals time, a equals
acceleration, and X equals absorbancy. When Vot is
subtracted from the absorbancy (X) this value will be X
corrected. The X corrected value is then plotted against
(time)2 to) obtairl a straight line. Acceleration, a, is

proportional to the velocity of the reaction.

Preparation of Blue Dextran Resin

 

Preparation of this affinity column was performed

according to the procedure of Vician and Tishkoff (107).

Preparation of Sephadex G-200

 

Sephadex G-200 was prepared according to the

manufacturers' recommendations.

Preparation of DEAE-52

 

The anion exchange resin was prepared according to

Methods in Enzymology (108).

Preparation of Heparin Sepharose

 

Preparation of this chromatographic material began with
30 ml of washed Bio Gel A-15M mixed with 30 m1 of 2M sodium
carbonate. With rapid stirring, 3 ml (0.05 volumes) of
cyanogen bromide - acetonitrile solution was added and
stirred vigorously for 1 to 2 minutes. The cyanogen bromide
- acetonitrile solution was prepared by adding 50 m1 of
redistilled acetonitrile to 1009 of cyanogen bromide. The
slurry was then washed with 5-10 volumes of 0.2M sodium
bicarbonate buffer, pH 9.5 and distilled water. The washing
was done in less than 90 seconds. The slurry, plus one
volume of a 2M sodium bicarbonate, was placed in a plastic

bottle with 8-10 mg/ml of heparin.

28

29

The activated agarose — heparin mixture was stirred
constantly for 18-20 hours at 4C. Unreacted groups were
treated by adding’ 1 molar glycine and stirring for an
additional 3 hours at room temperature. The coupled agarose
was subsequently washed using sequentially 20 volumes each
of 0.01M sodium bicarbonate/0.5M sodium chloride (pH 10.0),
0.1M sodium acetate/0.5M sodium chloride (pH 4.0), and 2M
Urea/0.5M sodium chloride. The equilibrating buffer
contained SmM potassium chloride, 3mM calcium chloride, 10mM

sodium acetate and 10mM Triethanolamine - HCl (pH 6.4).

Preparation of Hydroxyapatite Column

Hydroxyapatite (BIO RAD) was hydrated with 0.2M
phosphate buffer, pH 6.8. The resin was defined and
degassed and then poured all at once into a jacketed column,
2.5cm x 45cm. The column was equilibrated with 0.02M

phosphate buffer (pH 6.8).

Preparation of Thrombin Sepharose
Thrombin Sepharose was prepared according to Rosenberg,
gt a_l (109) with modifications. Fourteen m1 of packed

Sepharose 4B was washed with distilled H O and then added to

2

a beaker with 14.0 ml distilled H20, stir bar, pH electrode
and thermometer. Finely ground cyanogen bromide (CnBr) was
added all at once and the coupling reaction began. The

temperature was kept between 18C-20C and the pH at 11.0.

30

Sodium hydroxide, 4M, was used to adjust the pH. The
reaction proceeded for approximately 12 minutes at which
time excess ice was added to the beaker and the gel washed
with 500 ml of cold 0.1M NaHCO (pH 8.5) and 14.5 ml of

3
thrombin in 0.1M NaHCO (pH 8.5). The washing step was

3
completed in less than 120 seconds. The slurry was gently
stirred overnight. One molar ethanolamine - HCl, pH 8.0,
was added and stirred with the gel for 2 hours, after which
the thrombin sepharose was washed with distilled H20. The
gel was then stirred for 30 minutes in 0.1M carbonate
buffer/1.0M! NaCl, pH 8.90. The slurry' was filtered and

washed four more times with the carbonate buffer and stored

at 4C.

Preparation of Thrombin

 

Prothrombin was activated to thrombin according to the
method Lanchantin, gt it (110) with minor modifications.
Australian Taipan venom (oxyranus scutellatus scutellatus)
was partially purified (M) .a DE52 column to remove
nonspecific proteolytic enzymes. Approximately 3 mg/ml of
Australian Taipan venom was placed on a 1 x 10 cm column and
run at 20 ml/hour, 6 minutes/tube. The protein was eluted
with a buffer gradient of 0.02M TRIS pH 7.2, at 25C and
0.02M TRIS/1.0M sodium chloride. The venom was then eluted
from the column at a conductivity reading of approximately

4.5 mmOHMs. The purified venom was reacted with purified

31

prothrombin in a ratio of 0.7 mg venom to 15 mg of Factor
II. The reaction took place at 28C for 30 minutes with
constant agitation.

The reaction mixture was then placed on a Bio Rex 70
(100-200 mesh) column (1.6 x 70cm) which had been
equilibrated with two bed volumes of 0.1M acetate buffer (pH
7.0). The Bio Rex was swollen directly in the buffer
without preliminary washing procedures. It was poured in
three sections to allow for better uniformity. The column
was run at 8 minutes/tube, 12 ml/hour. Protein was eluted
with a buffer gradient consisting of 120 ml of 0.1M acetate
buffer pH 7.0 and 200 m1 of 0.1M acetate buffer/1.0M sodium
chloride. Conductivity measurements and thrombin clotting

assays were performed on the protein peaks.

Preparation of AT-III Sepharose Column

 

The Antithrombin III (AT-III) obtained from the
American Red Cross, Bethesda, Maryland, was prepared from
fresh frozen plasma by the method of Wickerhouse and
Williams (114). A Sephadex G-100 column was packed and 20
m1 of the reconstituted AT-III was placed on the column.
Imidizole buffered saline, pH 7.35, was used to elute the
protein. Protein was monitored at an absorbance of 280 nm
and AT-III assayed in terms of its capacity to inactivate
thrombin during an incubation time. The AT—III was then

dialyzed against sodium carbonate buffer, pH 8.5, for 7

32

hours. It was then attached to cyanogen bromide activated

Sepharose 4B.

Purification of Factor X (Vician-Tishkoff's method)

One liter of cryo-precipitate poor plasma plus
inhibitors (benzamidine - HCl, polybrene, and soybean
trypsin inhibitor) was thawed at 37C in a water bath. The
thawed plasma was poured through gauze into a plastic
container on ice. After thawing, the plasma was stirred at
4C for 60 ndnutes with the addition of 100 nfl.<xf cold 1M
barium chloride. The plasma was then centrifuged at 3500
rpm for 30 minutes at 4C. The supernatant was discarded and
the pellet resuspended in 500 nfl.<xf cold distilled water.
Dowex (30g of 50X 8, 20—30 Na mesh) was added to the
resuspended protein and stirred for 30 minutes, after which
it was filtered through a coarse sintered glass funnel. It
was washed 3 times with 15 ml each of cold 3.1% sodium
citrate. The protein solution was once again stirred for 30
minutes in an ice bath with the addition of 56 ml of cold
barium chloride. It was then centrifuged in three plastic
centrifuge tubes for 30 minutes at 3500 rpm. The
supernatant was discarded and each pellet resuspended with
80 ml of 0.85% sodium chloride, and mixed for two minutes
only. The centrifuge tubes were spun once more at 3500 rpm
for an additional 30 minutes. The supernatant was discarded

and the precipitate resuspended with 120 ml of cold

33

distilled water. Dowex (309) was once again added to the
protein solution and stirred for 30 minutes followed by
filtering and washing with a total of 45 ml of cold 3.1%
sodium citrate. The protein solution. was then dialyzed
overnight (approximately 16 hours) at 4C against 7 liters of
phosphate/NaCl buffer, pH 5.8. After dialysis, the solution
was passed through a chelex column which had been pretreated
by washing with H

O, 0.5M acetate buffer, (pH 5.4), H O, and

2 2
phosphate NaCl buffer (pH 5.8) and then concentrated in a
Bio Rad hollow fiber beaker at 4C. The concentrated protein
was centrifuged at 15,000 rpm in corex tubes and filtered
through glass wool. After approximately 30 liters of plasma
were processed this way, the last step involving
concentration of the protein by the hollow fiber technique
was changed. An ammonium sulfate precipitation step was

substituted. After the chelex column, solid (NH4)ZSO was

4
added to the protein solution to yield a 0-40%
precipitation. The (NH4)ZSO4 was added slowly, at 4C, for
15 minutes with constant stirring. The pH was adjusted with
2M NaOH to approximately 6.2. The protein solution was then
centrifuged in small plastic tubes at 16,000 rpm for 30
minutes at 4C. The supernatant was saved and the appropriate
amount of (NH4)ZSO4 was added to yield a 40-75%

precipitation. The (NH SO4 was once again slowly added at

4’2
4C, with constant stirring. Once all the solid was in the

solution it was stirred for 30 minutes. The pH was again

34

adjusted to approximately 6.2. The protein solution was
centrifuged at 16,000 rpm for 30 minutes. The supernatant
was discarded and the pellets resuspended in about 5 m1 of
Sephadex phosphate/NaCl buffer plus inhibitors. The
solution was dialyzed and then placed on the gel filtration
column, Sephadex G-200.

The isolated prothrombin complex was first
chromatographed on a Sephadex G-200 column. The fraction
collector was run at a flow rate of 20ml per hour and
fractions collected at intervals of 15 minutes per tube.
Protein was eluted with phosphate/NaCl buffer (pH 5.8) plus
inhibitors and monitored at an absorbance of 280 nm.
Clotting assays were performed on the eluted protein. The
pooled protein was concentrated and dialyzed against 0.1M
acetate buffer (pH 6.0) overnight at 4C.

The second column utilized in the purification of
Factor X was a Blue Dextran agarose affinity column prepared
as previously described. The column was run at 20 ml/hour
and 12 minutes/tube. Protein was eluted with a stepwise
gradient of acetate buffer (pH 6.0) consisting of: 0.01M
sodium acetate/acetic acid, and 0.4M sodium acetate acetic
acid plus 0.2M sodium chloride. The column was regenerated
with 300 ml of 0.1M sodium carbonate/1M NaCl (pH 8.8) and
then reequilibrated with 0.01M sodium acetate buffer (pH
6.0).

35

The final column used in this purification process was
an anion exchange column, DE52. The protein was eluted with
an exponential buffer gradient using sodium phosphate, pH
6.1. The flow rate was 20 m1/hour at 4C.

Conductivity measurements and clotting assays were
performed on the eluted protein. Factor X eluted from the
final column was pooled, dialyzed against sodium barbital

buffered saline, pH 7.4 and stored at -80C.

Purification of Factor X (Kosow's method)

 

Factor X was also purified according to Kosow's method
(111) which utilized three types of chromatographic
material: DEAE-A50, heparin. agarose, and. hydroxyapatite.
The procedure began with thawing 2 liters of cryo-poor
plasma plus inhibitors, collected as pmeviously described.
The plasma was stirred at 4C for one hour with 3.79 of
preswollen DEAE-A50 which had been swollen and defined in
the following buffer: 10mM triethanolamine - chloride, 0.75
mM potassium chloride, and 2mM EDTA. The protein was then
poured into a Bel Art funnel and washed with the following
buffer: 10mM triethanolamine - chloride, 0.2M potassium
chloride, and 2mM EDTA. The protein solution and resin were
then poured into a 2.5cm x 45 cm column and the gel allowed
to settle. The column was run at 20 minutes/tube, 10
ml/tube and 30 ml/hour. Protein was eluted with a buffer

gradient of 500 m1 of starting buffer which consisted of:

36

10mM triethanolamine - Cl, 0.2M KCl, 2mM EDTA; and 500 ml of
limit buffer consisting of: 10 mM triethanolamine - Cl,
0.5M KCl, 2mM EDTA. Factor X clotting assays were performed
and tubes with the highest specific activity were pooled.
Two (NH4)ZSO4 precipitations were done on the pooled

protein. The first involved adding dry (NH 804 to yield a

4’2
0-40% precipitation. The dry solid was added slowly at 4C
and stirred for one hour. The pH was adjusted to 7.2. The
protein was then centrifuged at 16,000 rpm for 30 minutes.
The supernatant was saved and (NH4)ZSO4 added to yield a
40-75% precipitation. The solution was once again stirred,
pH adjusted and centrifuged for 30 minutes at 16,000 rpm.
The pellet was resuspended in starting buffer for the
heparin agarose column which consisted of: 10mM

triethanolamine - acetate, 5mM KCl, 3mM CaCl (pH 6.4). The

2
resuspended protein was dialyzed against the heparin agarose
starting buffer overnight.

The protein was then applied to the heparin agarose
affinity column *with a flow rate of' 30 :ml/hour and 12
minutes/tube. The buffer gradient was applied after the
first protein peak was eluted. The buffer gradient was 300
ml of starting buffer (listed above) and 500 ml of limit
buffer which was like the starting buffer with the addition
of 500 mM KCl. Conductivity measurements and Factor X, Xa

clotting assays were performed on the eluted protein. Tubes

with the highest specific activity were pooled and dialyzed

37

against starting buffer for the last column, the
hydroxyapatite column.

The protein was applied with a flow rate of 30 ml/hour,
12 minutes/tube. The protein was eluted with 500 m1 of 0.2M
KHPO and 500 m1 of 0.4M KHPO

4 4
degassed, deionized water. Conductivity measurements and

(pH 6.8) both prepared with

clotting assays ‘were performed. Tubes ‘with the highest
specific activity were pooled and concentrated with an
Amicon system, membrane PMlO. The concentrated protein was
then dialyzed against sodium barbital buffered saline and

stored at -80C.

Isoelectric Focusing

 

Isoelectric focusing’ was performed according' to LKB
Application Note 250: Analytical Electrofocusing in Thin
Layers of Polyacrylamide Gel.

The standards run with the unknown proteins were
catalase, ovalbumin, ribonuclease and cytochrome c mixed

together for a final protein concentration of 0.03 mg/ml.

Reduction and Alkylation of Factor X

The Factor X was reduced and alkylated according to
Summaria (112) with minor modifications. The procedure was
first done with gamma globulin to ensure proper reduction
reactions. The reducing reaction mixture consisted of 5.2

ml of globulin (approximately 0.5 mg/ml), plus 1.0 m1 of DTE

38

(0.0779 DTE in 1.0 ml of barbital buffered saline, pH 6.0).
The final pH of this reaction mixture was 6.95 at 23C. The
reaction took place for 20 minutes at 23C. The alkylating
step involved adding 1.24 ml of iodoacetate (0.1M in
combination with gamma globulin and DTE) and adjusting the
pH to 7.0 with 1M NaOH. The reaction was complete when
there was a decrease in pH change. The reaction mixture was
then. dialyzed. overnight. against SOdiUfll barbital. buffered
saline (VBIS), pH 7.4, at 4C.

Factor X was reduced and alkylated in a similar manner.
Factor X, 2.8 mg/ml, was reacted with 0.48 ml DTE (77 mg/ml)
at 23C for 20 minutes. The alkylating step involved adding
0.66 ml of 0.5M iodoacetate and adjusting the pH to 6.0 with
0.2M NaOH. The reaction mixture was likewise dialyzed

overnight at 4C against VBIS.

Activation of Factor X

 

Factor X was activated (113) in a reaction with
purified Russell's viper venom and 0.2M CaClz. The weight
ratio of X to protease was 14.3:1. The reaction time was
approximately 5 minutes. The activated Factor X was then
placed on a Sephadex G-100 column and the protein was eluted

with sodium phosphate/sodium chloride buffer, pH 6.1.

Antisera Production

Factor X used for injection was purified by Kosow's
method. Protein concentration was 0.32 mg/ml. On ml was
diluted with 2 ml of phosphate buffered saline, pH 6.0.
Therefore, final Factor X concentration was 107 mg/ml. The
protein was then diluted 1:1 with Freunds Complete Adjuvant
(Grand Island Biological Company). Two white male rabbits
(2kg) were each injected subcutaneously along the back with
10.5 ml of this mixture. The second injection was given six
weeks later. The rabbits were bled. on days 12 and 34
following the second injection. They received boosters one

month after the second bleeding.

39

Results

Purification of Human Factor X - Vician and Tishkoff's
Method

Factor X was successfully purified 2,000 fold by Vician
and Tishkoff's method (116).

The prothrombin complex extracted from cryo-precipitate
poor plasma or from a commercial concentrate was first
chromatographed on a gel filtration column, Sephadex G-200.
The prothrombin complex was conveniently eluted in the
second peak. Prothrombin complex chromatographic patterns
off the G-200 column are illustrated in Figure 5 and Figure
6. Samples containing Factor X activity, and some Factor II
and VII activity, were then pooled, concentrated and
dialyzed against starting buffer for the second column, Blue
Dextran agarose (BDA).

The BDA column exhibited a unique affinity for
separating out the Factor X from the remainder of the
prothrombin complex. The typical procoagulant profile in
which Factor X is conveniently eluted in the first or second
peak, while Factors II, VII and IX are eluted at higher
ionic strengths is illustrated in Figure 7. Approximately
95 liters of cryo-precipitate poor plasma were processed by

this method. Some of the fractionated patterns did show

40

ABSORBANCE (280m)

41

 

 

 

 

 

 

I T I 3
6-200 g
PROTHROMBIN COMPLEX 3
x
1.0— a ,-
0 _.
PROTEIN "‘ 5 \E
‘ " 9 1.;
0.81— I .- g
0 2 3
0.6— I, 3— 6.0 3
z ’ 8
x I 1000-- a <
I .. g “'
0.4.__
I {A-‘“”§.,‘ sou—.9
0.2— d, / . . O._2.o_ 0.2
' " AHA ‘ *
#1 I / 7 .0 \
0 / 20 30 4o
FRACTION NUMBER
Figure 5. Procoagulant profile of prothrombin complex

eluted from Sephadex G-200

42

(IN/51W") IIIA HOLOVd

 

 

 

«a e. N
O O O
I T l
(IN/51W") X 80.10%!
c. a. c. e.
co no u: N
l I I I
(IN/51W") NIBWOHHLOUd
E g
I T
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I
VIII

   

 

 

 

 
 

 

 

L— m
LU
m
5
2
E Z
— z
o
t: .9
l +—
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__ <
a:
IL
5
m D
— e N
o I
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IL I
O
_— o I
J 8|- '
\x I:
~ o<
I I I I I
=2 «2 w. e. “I °
F O O O C

(“WOBZI BONVBUOSBV

Figure 6. Procoagulant profile of Auto Factor IX
(HYLAND) chromatographed on Sephadex G-200

43

 

I T 7 1 T

A B

(74

T I r fl
I
I

 

 

 

 

I
1 759—.0 o1M—.+._a 14M__+_o fur—+0000 zumqul 1750
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E
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. «500 1 - 50 o
. : s z:
I . I 4
I :L I;
, «250 ~ 25
' I
I
___.L_L__I
90

 

FRACTION NUMBER

Figure 7. Pooled prothrombin complex chromatographed
on Blue Dextran resin

44

patterns where the Factor X was eluted with only the first
or second buffer. Several times, Factor VII was separated
from Factor II. In these cases, Factor VII was eluted prior
to the prothrombin complex. This occurred more frequently
when the prothrombin complex was absorbed from
cryo-precipitate poor plasma and subsequently precipitated
with ammonium sulfate. Clotting assays were performed and
samples with the highest amount of Factor X activity were
pooled, dialyzed against VBIS, pH 7.4, and stored at -80C.
Several preparations of Factor X were then thawed, pooled,
and dialyzed against the starting sodium phosphate buffer
for the DE52 column.

Approximately 5.0 to 10.0 mg of protein was placed on
the IMEXZ ion exchange column. Conductivity readings and
clotting assays were done. A typical elution pattern is
illustrated in Figure 8. Factor X fractions containing the
highest specific activity were immediately dialyzed against
VBIS (pH 7.4) and stored at -80C.

Data in Table 2 summarizes results from several
preparations and is a representative sampling of the Factor
X purification.

The purification process listed in Table 2 involved the
hollow fiber concentrating step and not the ammonium sulfate
precipitation. Factor X in the final product, is devoid of

Factor II, VII and IX clotting activity. Factor Xa activity

45

 

 

 

 

 

'3: I ' 1 I I I
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3 ACTIVITY
E ‘ “
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Ill
ELUTION -125

1.25— PATTERN _
A _ ' x _ ‘20
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o u o
3 . < z

.0 u. o

< 0.25:" ‘_'2.5 _5 U

 

    

 

 

 

/ 4o 50 so 70 so
FRACTION NUMBER

Figure 8. Chromatography pattern of Factor X protein off
ion exchange resin (DE52)

46

 

o.HmmH I

om.m I

o~.o

Amman.

omoHsHHoU

ludma

 

o.~om~ o.hv

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MH

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:muuxoo

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cowumoflmwusm .uoo .owdm

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x uouowm

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Aw.

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HE\: >uo>ooou cemuoua

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:wmuoum

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mESHo>

mcofluwumawum.amuo>mm mo >umEE:m d
can cmflofi>v COHumonwusm x nouomm ewes:

waumumz

A mam<9

47

is less than 0.01 u/ml. Specific activity is approximately
30-50 units/mg protein.

Factor X appeared homogeneous on polyacrylamide gels
performed according to the method of Fairbanks (117) with
intact disulfide bridges; a single band is seen for both
molecular species of X (the two peaks off the BDA column).
Following disulfide reduction, Peak 1. (off BDA), Factor )(
evidenced a 2-chain species, while the second peak is
predominantly a single chain species of Factor X with a

molecular weight of 85,000 daltons (Figure 9).

Purification of Human Factor X - Kosow's Method

 

Factor X purified by Kosow's method (118) yielded a
product with a specific activity of approximately 100
units/mg of protein.

The chromatographic pattern of Factor X off the
DEAE-A50 is illustrated in Figure 10. Fractions containing
the highest specific activity of Factor X were pooled and
two ammonium sulfate precipitations were performed.

The protein elution pattern of Factor X off the heparin
agarose column is shown in Figure 11. Peak Factor X
activity fractions were pooled, dialyzed against 0.2M
potassium phosphate buffer for 2 hours, and concentrated
with an Amicon system, membrane PMlO.

The concentrated protein was applied to the

hydroxyapatite (HT) column and protein eluted with a linear

_- I‘mfi—rT—A w

48

Figure 9. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of purified Factor X
(Vician-Tishkoff's method)

49

 

(onus/ml)

° FACTOR x

l_
‘79 I I I
DEAE—A50
A GRADIENT
E
é {vilg .—»' "&-1
2 .75— PROTEIN gHINY
w I” 1 T 8
U I
Z .-- o
3 .so— .. ' 6
8
8
m 4
< .25 —
2

   

 

 

 

 

I
07// 10 20 30 40 so
FRACTION NUMBER

Figure 10. Elution pattern of prothrombin complex
from DEAE-A50

p
O

CON DUCTANCE ("In")

 

50

 

 

 

 

 

I I I T A
5 g,
"' HEPARIN ‘5' 74
4 AGAROSE ;; E
“I
8
3__ ’PROTEIN 9 ‘2’
U E
v If ‘5
D
fi/ x ""16 z
\ .- O
A1-4— . \- —14 U
s -26
3 1 2.. GRADIENT I L 12
Q . I '—
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::LOT— ‘! i) ._10
z -18
508— F J /-:8
/
O . -14
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o‘ ‘6
0.2 E. O -.../... f. \ 2
1 o ~“o—o—!o -. \ _2
o / 30 40 so so

FRACTION NUMBER

Figure 11. Protein profile of Factor X from heparin
agarose column

51

gradient of 0.2M-0.4M potassium phosphate, pH 6.80. A
typical profile of Factor X eluted from the HT column is
presented in Figure 12. The final product was devoid Of
Factor II, VII and IX activity. Factor Xa activity was less
than 0.01 units/ml. The specific activity of the purified
Factor X was approximately 100 units/mg of protein. Sodium
dodecyl sulfate gels showed Factor X to have intact
disulfide bridges and a molecular weight of approximately
70,000 daltons. There appeared to be a high molecular
weight contaminant present on most preparations. These
results are consistent. with. Kosow's observations (Figure

13).

Activation of Prothrombin to Thrombin

 

The elution pattern of Australian Taipan venom off an
ion exchange column is illustrated in Figure 14. This
procedure was performed several times, with similar elution
patterns being obtained. The venom was eluted when
conductivity readings were equal to the standard curve
readings for 0.02M TRIS/0.1M NaCl. As shown in Figure 14
the second peak was the partially purified venom.

The activation took place as described in materials and
methods, the ratio of venom to prothrombin by weight was
0.7 mg:15 mg, respectively. Data presented in Figure 15
illustrate the Bio Rex elution pattern. Thrombin assays,

conductivity readings and absorbance measurements were done

52

 

9
a»

go
or tn

9
0!

.°
g

.9

ABSORBANCE (280 nm)
N

.9
N

P
p

 

 

I

HYDROXYAPAUTE

PROTEIN\

 

N

l
0‘! 0’
FACTOR X (units/ml)

h

w

M

 

 

Figure 12.

 

10 20 30

FRACTION NUMBER

Factor X pattern off of Hydroxyapatite
column

53

Figure 13. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of purified Factor X
(Kosow's method)

54

 

 

 

 

 

 
   

 

 

 

14 .02M TRIs I .on TRIs- .O2M TRIS/1.0M NSCI
1.2 -— ,L 12
PROTEIN /
1.0 — / .~° _10
GRADIENT /
O
a .‘o :
I
i / d
“I 0.6 — .0. _ 6 3
2 / -
< . U
a E
3 ACTIvE ..
I" 04 - U
can VENOM -4 8
“ 2
O
U
0.2 — — 2
0 1o 20 3o 40
FRACTION NUMBER
Figure 14. Australian Taipan Venom chromatographed

on DE52

55

 

 

 

 

 

 

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2 2 1 .
r825 3252528 . . _ . _
w m m m m :Enzizazouzp m .
0
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7
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4. 3. 3 3. 3 2. u 2. z 1. 1. L o. o o

 

 

 

 

 

 

 

Ascoonvmuz<nco

L.
mm<

FRACTION NUMBER

Thrombin chromatographed on Bio-Rex 70

Figure 15.

56

on individual fractions. Specific activity' of time peak
fractions was calculated to be 3,000 to 4,000 units of
activity/mg of protein. The thrombin tubes were dialyzed

against VBIS, pH 7.4, and stored at -80C.

Clotting,Studies

 

With the availability of purified coagulants and
procoagulants, in yitgg testing was begun. An attempt was
made to elucidate a coagulant mechanism explaining the
hemostatic effect of Factor IX concentrates.

As described in materials and methods, the Factor VIII
inhibitor assay is based on a modified APTT.

The first clotting studies were done with Factors X and
II (Figure 16). When Factor X, single chain or double chain
species, was combined with highly purified Factor II,
significant clotting activity was generated. This activity
was referred to as Factor VIII inhibitor bypass activity
(FEIBA). This activity was the same whether Factor X was
purified from E1 commercial concentrate (n: purified from
cryo-precipitate poor plasma as previously described.
Optimal conditions were established by maintaining Factor X
at a constant level and decreasing the Factor II activity
(Figure 17), or maintaining Factor II as a constant and
decreasing Factor X activity (Figure 18). It appeared that

maximum generation of FEIBA required approximately a 300%

57

cowumumcwm

«mHmm How mummmmow:
:HnEounuoum mo mOCOOEm
Edaflumo mcflumasoamu

.25; 52; ZO_h<mDUZ_
ow om

.sH musmflm

 

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(335) 3W”. DNILLO‘IO

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58

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:55 22: 29226:.
AC Am 2&—
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X I .I I0
OT 5528 V meow.

 

.ma musmflm

59

increase in Factor X activity and 100% increase in Factor II
activity compared over the levels of these activities in the
original Factor VIII inhibitor substrate plasma.

FEIBA was shown to be Factor V dependent by modifying
the inhibitor assay in which the inhibitor substrate was
depleted of Factor V activity. The inhibitor plasma was
incubated at 37C for 24 hours to deplete it of Factor V
activity. The depleted substrate (Figure 19) was employed
with the addition of Factors X and II, no correction of the
abnormal PTT was demonstrated. The addition of BaSO4
treated plasma to the reaction mixture as a source of Factor
V completely restored FEIBA coagulant activity.

The question first addressed was whether trace
quantities of activated clotting factors, such as Factors Xa
or IIa, could be causing the clotting activity. Prothrombin
was passed through a small sulphopropyl sephadex column to
remove all traces of IIa. Factor X combined with treated
Factor II exhibited impaired FEIBA generation when compared
to Factor X combined with untreated Factor II (Figure 20).
Presumably, the impaired generation was a result of the
removal of traces of thrombin. A more direct approach to
this problem was achieved by studies using highly purified
Factor Xa and IIa. Factor Xa alone or in combination with
Factor II failed to generate FEIBA (Figure 21). However,

Factor X in combination with IIa (0.05 u/ml) evidenced

60

mHH + x suwz cowumumcmm

«mHmm uanHmflcmHm .Hm musmfim
cowumumcwm «mHmm OOHAMQEH .ow musmflm

 

 

 

 

 

 

ASE. mic. 20.55302. 3E. m2.» 20.55302.
ov on .o
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:mb—xm mx 8. :36 x
:mb—xm ax 3am...“ a IqIIHIHI\IIIhO
:86 2. I I I I I l l\.... I Ioow
.6528 $8

 

61

significant FEIBA generation. Thrombin alone was completely
inactive.

Factor X, whether prepared by Vician and Tishkoff's
method or by Kosow's method, corrected the inhibitor plasma
with the addition of trace amounts of thrombin.

A study was undertaken to establish whether the same
bypass mechanism would apply to pdasma congenitally
deficient in a clotting factor. Plasma from patients
deficient in Factors VIII, IX, or X were used; the remaining
reactants ‘were the same (ACTIN, 0.02Nl CaClZ). The only
addition was BaSO4-treated plasma, as a source of Factor V,
to the Factor X deficient assay. A preliminary FEIBA assay
was done using Factor X (purified on BDA and then HT, and
also Factor X purified on Heparin Agarose, then HT) and IIa.
These clotting times pertain only to results after a
2-minute pre-incubation. The results are shown in Table 3
and demonstrate that the by-pass mechanism involves a
pathway which enters the coagulation cascade (Figure 1)
after Factor VIII. The results are inconclusive with regard
to Factor X deficient plasma since reactants contain Factor

X.

62

TABLE 3: The interaction of Factors X and IIa in different
congenitally deficient plasma in the FEIBA Assay.

 

 

A. Reference assay employing Factor VIII Inhibitor plasma:
1 minute clot time 40 minute clot time
(sec) (sec)
VBIS 125 140
X (.4u*) 77 81
IIa (0.0025 u*) 118 120
X + IIa 35 40

 

B. Congenitally Deficient Plasma: 1 minute FEIBA

 

 

Factor VIII Factor IX Factor X
deficient deficient deficient
plasma plasma plasma
Clot time Clot time Clot time
(sec) (sec) (sec)

X** 50 44 53

11a 77 94 90

X + IIa 47 35 55

VBIS 102 91 95

X*** 64 73 52

IIa 87 120 90

X + IIa 61 70 40

Normal Plasma 52 75 41

1110

 

*Units represent clotting activity in the assay system.

**X purified on BDA, followed by HT column.

***X purified on Heparin Agarose followed by HT column.
Another question addressed was whether X + IIa combined

evidence de novo FEIBA activity or whether FEIBA is

generated by a time-dependent reaction between Factor X and

63

IIa. Factor ){-+ IIa were incubated with and without the
inhibitor substrate for various time intervals. The
substrate employed was the inhibitor plasma from hemophiliac
patients. The assays and results are in Table 4 and clearly
demonstrate that the generation of FEIBA is not time
dependent.

Table 4A: Preincubation of X + IIa with the inhibitor

substrate for various time intervals, and
proceeding with a standard "1 minute" FEIBA

 

 

assay.
Incubation Time Clotting Time
(min) (sec)
1 39
3 40
5 50
10 50
15 51
20 60

4O --

 

Table 4B: Preincubation of X + IIa without the inhibitor
substrate for various time intervals, and
proceeding with a standard "1 minute" FEIBA

 

 

assay.
Incubation Times Clotting Time
(min) (sec)
1 39
3 42
5 42
10 62
15 55
2 62

40 62

 

64

In a continuation of the time study X + IIa were

pre-incubated without the inhibitor substrate for various

times, and a "40-minute" FEIBA was done. The results
confirm the fact that the X - IIa interaction is not time
dependent.

TABLE 5: Preincubation of X + IIa followed by a
"40-minute" FEIBA.

 

 

Incubation times 40-minute FEIBA
of X + IIa clot time
(min) (sec)
2 60
40 60
60 61
90 61
120 76

 

The involvement of Factor IX was examined by utilizing
purified Factor IX in the FEIBA assay in place of Factor X.
Factor IX was donated by Dr. S. K. Chung, Chapel Hill, North
Carolina, and contained 50/ l of Factor IX in 0.1M
TRIS-HCl/0.15M NaCl and 0.02% sodium azide, 0.1mM
Benzamidine - HCl (pH 7.0). Factor IX was dialyzed against
VBIS, pH 6.0. FEIBA assays were performed. The clotting
times are recorded in Table 6 and indicate that Factor IX is
not responsible, in this assay system, for the generation of

FEIBA activity.

65

Table 6: The effect of Factor IX in the FEIBA assay.

 

 

"1 minute" "40 minute"
FEIBA FEIBA
VBIS 1455 1505
X 785 97s
IIa 908 1118
X + IIa 56s 66s
IX 103s 107s
IX + IIa 1045 1205

 

Another in vitro clotting study was done to investigate
whether or not human serum, a source of Factors VII, IX, X,
XI, and XII, has any combined role in generation of FEIBA.

Results of studies employing serum are shown in Table 7.

Table 7: The effects of human serum in generation of

 

 

FEIBA*.
"1 minute" "40 minute"
FEIBA FEIBA
VBIS 1305 1605
X 835 1225
IIa 1265 1523
X + IIa 405 805
X + IIa + 0.50 ml
1:10 human serum 405 805

 

*The standard FEIBA assay was used employing Factor VIII
inhibitor plasma as substrate.

66

MOLECULAR STUDIES

 

As evidenced from the above in yitrg clotting studies,
X + IIa generated FEIBA, but apparently not in a time
dependent reaction (see Table 3,4 or 5). The question
arises whether X and IIa form a molecular complex with
unique coagulant properties or Fctor X species had
molecularly changed by the action of IIa. The first
molecular study was designed so that a X - IIa complex would
be readily detected by its exclusion properties from. a
Sephadex 100 column. The "altered Factor X" possibilities
were experimentally examined by testing Factor X with
immobilized IIa so that the IIa species would be reserved as
reactant after its interaction with Factor X.

In the first experiment a G-100 column, K15/30 was
packed and equilibrated with VBIS, pH 7.4. Purified Factor
X (Vician-Tishkoff'S' method) and thrombin (Bio Rex
preparation) were incubated for 40 minutes at 37C in a ratio
of 1300:1. The mixture was then placed on a G-lOO column,
and fractions collected at 2.2 ml/tube. Clotting assays for
Factors X and IIa were done, as well as 40 minute FEIBA
times on the protein fractions eluted from the column. Tube
number 19, which was the peak protein tube as well as the
peak tube for Factor X activity (4.6 u/ml) exhibited the
shortest FEIBA time of 88 seconds after a 40 minute
incubation. The void volume for the column was calculated

to be 15.2 ml.

67

Follow-up FEIBA clotting studies were performed to
compare the Factor X eluted from G-100 (tube #19) with
Factor X not preincubated with IIa or applied to the G-100
column. The results are recorded in Table 8.

Table 8: FEIBA results on X + IIa fractionated off Sephadex
G-100.

 

 

4W1 minute" W40 minute“
FEIBA FEIBA
VBIS 136$ 1358
X DE52 (starting
material for G-100
experiment) 130s 1505
IIa Bio Rex 1375 1385
X DE52 + IIa BIO REX 708 905
X G-100 #19 63s 875
X G-100 + IIa BIO REX 403 555

 

SDS gel studies utilizing the treated Factor X plus IIa
failed to reveal any Factor X intermediate.

Molecular studies continued with reacting Factor X with
immobilized thrombin. The Factor X was obtained from peak 2
BDA and further purified on DE52. Thrombin Sepharose was
prepared as previously described. The following reaction
mixtures were placed in plastic tubes in an automatic

shaking water bath for 40 minutes at 37C:

68

Reaction A:

0.19 ml ){-+ 0.1 ml IIa—Sepharose + 0.71 rml VBIS
(0.4 units)

Reaction B:

0.19 ml X + 0.5 ml IIa-Sepharose + 0.31 ml VBIS
(0.4 units)

After the 40 minute incubation, the reaction mixture was
separated from the resin using a small disposable column.
The supernatant was collected in plastic tubes and assayed
as shown below.

FACTOR X ASSAY Xa ASSAY IIa
ASSAY

 

Reaction A 3.2 u/ml 0.01 u/ml no clot
formation
at 10'

Reaction B 3.2 u/ml 0.01 u/ml no clot
formation
at 12'
FEIBA assays on the supernatants were performed to yield
the data listed in Table 9:

Table 9: FEIBA results from the interaction of X with
immobilized thrombin.

 

1 minute 40 minutes
VBIS 1355 1255
X DE52 495 955
X DE52 + IIa 305 325
Reaction A 1:1 405 405

Reaction B 1:1 545 975

 

69

From the estimation of IIa attached to the Sepharose (181
units of activity/ml of slurry), Reaction A had
approximately 18 units of IIa and Reaction B had
approximately 90 units of IIa. This is assuming that all
the IIa attached remained active.

SDS gels were run on the two reaction supernatants, and
on the Factor X and IIa used in the experiment. The
apparent molecular weights for the various bands were
calculated and are listed in Table 10.

Table 10: SDS gel results on the interaction of X with
immobilized thrombin.

 

 

Reduced Non Reduced
xznnsz 85,000 daltons 73,000 daltons
54,000
20,000
IIa 28,000 daltons 32,000 daltons
Reaction A
supernatant 73,000 daltons 73,000 daltons
53,000
19,000
Reaction B 80,000 daltons 76,000 daltons
Supernatant 53,000 daltons
20,000

 

Attempts to separate out the FEIBA components continued
with the preparation of an AT-III Sepharose column. Human
AT—III was attached to cyanogen bromide activated Sepharose

4B. Preliminary studies included a reaction with AT-III

70

Sepharose (2.5 ml of slurry) plus 170 units of IIa. The
mixture was incubated at 37C for 30 minutes with constant
agitation. The supernatant was then forced through a
quick-separation column and IIa assays were performed.
Particles appeared an: 14 minutes/3 seconds, strands at 15
minutes/45 seconds. The AT-III Sepharose was therefore
capable of inhibiting IIa clotting activity.

Purified Factor X and IIa were then incubated for 1
minute at 37C in a ratio of 4:2 respectively. Half of the
protein mixture (0.5 ml) was placed on the AT-III Sepharose
column, and 0.5 ml was run through a control column of
activated Sepharose plus ethanolamine. One minute and 40
minute FEIBAs were done with the following results:

Table 11: FEIBA results of X + IIa incubated with AT-III
Sepharose.

 

 

1 minute 40 minutes
Control column
supernatant 1025 1325
AT-III Sepharose
supernatant 1055 1275
X + IIa before
AT-III Sepharose 355 66s

 

Reduction and Alkylation of Factor X

 

Efforts to characterize the mechanism of the inhibitor
bypass activity led to an experiment to determine whether
the clot promoting activity of FEIBA could be attributed to

the double or single chain of the Factor X molecule. The

71

Factor X was reduced and alkylated as previously described
and FEIBA assays were performed. Sodium dodecyl sulfate
gels were run to verify proper reduction. The FEIBA clot

times are found in Table 12.

Table 12: FEIBA results of reduced and alkylated Factor X.

 

1 minute 40 minutes

X (0.4 u) 1255 1205
IIa Bio-Rex (0.0163 u) 1425 1625
X + IIa 625 705
X (reduced, alkylated,

dialyzed overnight

BBS pH 6.0) 1425 1395
X (reduced, alkylated) + IIa 1275 1625

 

SDS Gel Electrophoresis

 

Numerous SDS gels were run in an attempt to identify an
intermediate which could be responsible for the clot
promoting activity in the Factor IX concentrates.

Factor X (peak 2 BDA) further purified on DE52, was
incubated with IIa from a BIO—REX column for various times
at 37C. Reduced and non-reduced gels were run. Photographs
in Figure 22 do show evidence of a Factor X intermediate
formed with a X + IIa interaction on two separate occasions.

Attempts to further identify the intermediate or to
make certain it was not Factor Xa were unsuccessful due to

the presence of Factor IIa in the reaction mixture.

72

Figure 22. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of Factor X intermediate

73

In the next experiment, after preincubation of Factors
X + IIa, the IIa was neutralized with AT-III. The reaction
mixture was then run on SDS gels, the intermediate band cut
out, and a FEIBA assay performed. For this experiment,
FacUmr X was purified from Kosow's method, and IIa from a
BIO REX column. Preliminary work involved running SDS TRIS
gels in order to study the formation of the ATIII—IIa
complex (Rosenberg (119)). The lower gel consisted of 7.5%
acrylamide. The incubating solutions and reaction mixtures
were made according to Rosenberg (119). Approximately 0.8
m1 IIa (0.131 mg) and 0.8 m1 ATIII (0.128 mg) were combined
and incubated at various times, after which 12 nfl.<mf the
reaction mixture was taken out and applied to the gel. The
amount of protein on the gel was calculated to be 5.339
ATIII and 5.439 IIa on each gel. After a 60 second and 2
minute incubation, the bands seen were Complex I (97,000
daltons), Complex II (85,000), ATIII (66,000), and IIa (very
light band 32,000). After the 10 minute incubation of IIa +
ATIII, the IIa had disappeared from the gel and only the
complexes and ATIII bands remained. Therefore, it was
possible to run the gels without IIa interference.

Subsequently Factor X and IIa were preincubated for 20
minutes. Then 0.8 nfl.<mf the coagulant protein mixture was
incubated with AT-III for 20 minutes and SDS gels were

prepared. Results of the gels are listed in Table 13.

74

Table 13: Apparent molecular weights of bands appearing on
SDS gels.

 

Molecular weights
(daltons)

 

1) X + IIa Non-reduced 67,000
55,000
34,000
19,000
(contaminant)

2) X + IIa ATIII 87,000
68,000
56,000
33,200
19,000
(contaminant)

3) X

+

IIa ATIII Reduced 95,000
84,000
62,000
49,800
31,500
19,000
(contaminant)
13,500

4) X Non-reduced 69,000
19,800
(contaminant)

5) X Reduced 49,000
19,500
(contaminant)
13,400

6) IIa Non-reduced 38,000

7) IIa Reduced 33,000

8) ATIII Non-reduced 58,000

9) ATIII Reduced 65,000

Chromogenic Substrate Assays

 

After' many' i2_‘yit£g studies involving gels, column
chromatography and clotting studies, chromogenic peptide
assays were undertaken to investigate the kinetics of the
coagulant activity. This assay system, in which all
components were highly purified, was used in an attempt to
elucidate the FEIBA mechanism as well as identify whether
FEIBA has amidolytic properties.

$2238, a sensitive and soluble synthetic peptide, was
utilized. Numerous chromogenic assays were performed
according to the described reaction (Materials and Methods,
page 26) or with slight modifications. The source for
Factor V was varied and included purified Factor V (SIGMA)
or BaSo4 treated plasma.

The addition of trace amounts of thrombin to Factor X,
in the assay system, did not consistently accelerate the
kinetics of the reaction.

Additional Experiments on Factor X

 

Ouchterlony Diffusion

 

The double immunodiffusion technique was employed to
test the reactivity of Factor X purified from
cryo-precipitate poor plasma with antisera manufactured in
rabbits. The precipitate line formed was similar whether
the Factor X was purfied from Vician-Tishkoff's or Kosow's

method.

75

76

One preparation of Factor X was unusual in that it did
not exhibit FEIBA activity. However, this particular Factor
X was run on the Ouchterlony plates and exhibited the same
precipitate arcs against anti-X as the active Factor X
preparations.

Isoelectric Focusing

 

The electrophoretic technique of isoelectric focusing
was utilized in an attempt to demonstrate the difference
between the two Factor X preparations. Both preparations

exhibited an isoelectric point of 4.5.

DISCUSSION

Human Factor X was purified 2000 fold by
Vician-Tishkoff's (120) method and approximately 5000 fold
by Kosow's (121) method. Both purification schemes yielded
a Factor X which was free of other vitamin K dependent
clotting factors and relatively homogeneous on
polyacrylamide gels. The final products showed 30-50 units
of specific activity/mg (M? protein (Vician-Tishkoff's
method) and approximately 100 units of activity/mg of
protein (Kosow's method). The molecular weight of human
Factor X was consistent with data reported in the literature
(122, 123, 124).

Successful activation (xf purified prothrombin to
thrombin correlated well with that reported by Lanchantin,
et al (125), and yielded a highly purified thrombin with a
specific activity of 3,000 to 4,000 units/mg of protein.

Utilizing the purified procoagulants in the FEIBA
assay, it was concluded that the bypassing activity was due
to an interaction between Factor X with trace amounts of

thrombin. Factor V, calcium, and lipid were essential for

77

qr ”5*??- .~—

78

this reaction to take place. Elsinger (126) also concluded
that Factor V was essential for the bypassing activity.
Both Factor X preparations were equally effective in
combining with thrombin to significantly reduce the NAPTT of
the inhibitor plasma.

In vitro testing was utilized to examine the
interaction of activated clotting factors. The data
presented suggests that Factor Xa, by itself or in
combination with prothrombin, was not responsible for the
clotting mechanism. The data, however, does show a
"'Xa-like' activity". This is in contrast to an early
report by Kurcyznski (127), stating that Xa could be
directly responsible for the bypassing activity. DeWitt and
Feinstein (128) also reported that the hemostatic effect was
most probably due to activated clotting factors. The
clotting assay used in this study however, demonstrated the
ability' of’ the Ibypassing' activity' to remain after' a 40
minute incubation. This is sufficient time for protease
inhibitors (Alpha-Z-macroglobulin, alpha-l-antitrypsin,
AT-III) in the substrate plasma to neutralize Xa. In
support of our data, Elsinger (129, 130) presented evidence
to) exclude Xa as the single factor* necessary for' FEIBA
generation. He treated Xa and Fraction FEIBA (Fraction R)
with insolubilized soybean trypsin inhibitor. After 60
minutes, the FEIB-Activity of Xa disappeared while Fraction

FEIBA still showed a definitely shortened APTT.

79

Thrombin was shown to interact with Factor X to produce
an impressive reduction in the NAPTT of the inhibitor plasma
in this investigation. Thrombin alone was completely
ineffective. Elsinger (131, 132) also excluded the
involvement of thrombin as the sole factor responsible for
FEIBA generation because: (1) the thrombin content of the
Prothrombin Complex preparation is too low to shorten the
PTT of the inhibitor plasma; and (2) in a controlled
experiment, FEIB—Activity of thrombin disappeared completely
when treated with insolubilized SBTI, as compared to the
FEIB-Activity of treated Fraction R. The activated factor
activity of the prothrombin complex prepared in this study
was also very low.

The fact that the presumed action of the bypass
activity requires or includes more than one factor is also
supported by Elsinger (133) and Tishkoff (134). According
to Elsinger, gel filtration studies show the molecular
species of the coagulant activity to be larger than Factors
II, IX and X. He states it is probably greater than Factor
IX, but less than 160,000. Tishkoff, in similar gel
filtration experiments, located a coagulant that exhibited
Factor VIII-like activity in a standard one-stage Factor
VIII assay that also had an apparently larger molecular
weight.

The effects of purified Factor X and Factor X + IIa to

"correct" a plasma congenitally deficient in VIII, IX or X

80

was seen in data presented in Table 3. It is interesting to
note that Factor X alone was quite effective in decreasing
the clotting time in comparison to that seen with the buffer
alone. Although the addition of IIa to the reaction mixture
reduces the time slightly more, it seems that thrombin in
these studies may be merely an accelerator of the reaction
and not a primary component of the bypass mechanism.

From results of studies of the involvement of the
inhibitor substrate in the FEIBA assay, the conclusion is
drawn that the substrate is not contributing directly to the
active principle of the mechanism. Data are presented in
Table 4A. which show that incubation with the inhibitor
substrate will produce slightly faster clotting times than
preincubation without. We assume these subtle differences
are merely due to the factors contributed by the substrate
to the clotting mechanism: Factor V, fibrinogen, and
prothrombin. These will certainly enhance the formation of
a clot, but are not the active FEIBA components. These
results clearly demonstrate that the X - IIa interaction is
not time dependent.

We do not suspect a direct involvement of Factor IX.
Contamination with Factor IX would not be significant enough
to activate the bypass mechanism (see Table 6).

It can be summarized from the data in Tables 3 through

7 that:

1)

2)

3)

4)

5)

81

The possible contribution of contaminated Factors
IX or VIII to the unknown mechanism can be
eliminated.

The possibility of the inhibitor substrate as
contributing the key components (other than V,
fibrinogen, and prothrombin) to the mechanism can
be eliminated.

Purified Factor )( shows equalLy good correction
with a plasma congenitally deficient in Factors
VIII, IX or X.

There is a question as to whether thrombin in the
congenitally deficient plasma assay system is
indeed involved as the major active principle with
Factor X, or is merely an accelerator of the
reaction. However, data in Table 6 does support
the theory of the X + IIa interaction to produce
the most dramatic clotting results.

The X - IIa interaction for the generation of

FEIBA is not a time dependent reaction.

Assuming the activity of FEIBA is attributable to a

coagulant complex formed by the interaction of Factor X and

thrombin,

molecular studies were designed to isolate the

complex by separating out the active complex by column

chromatography or by reacting Factor X with immobilized

thrombin.

" pure I!

Thus, gel filtration would ultimately produce a

active component. Technical problems were

m—f--—.w-: .

82

encountered in which FEIBA could not be recovered from the
sepharose material (due to dilution effect of the column) or
in which thrombin leached off the sepharose and interfered
in the FEIBA assay. Thus, these effects were responsible
for inconclusive evidence in these sets of experiments.

The theory that the double chain of Factor X
contributes to the generation of the bypass activity was
supported by the loss of clot promoting activity when the
Factor X was reduced, alkylated and then utilized in the
FEIBA assay.

Further attempts at characterizing the FEIBA mechanism
led to a series of SDS gel experiments. If the molecular
model responsible for the bypass mechanism were not a binary
complex but rather involved a molecular change such that
Factor X would be split by thrombin, it was hOped to show
evidence of this new species on SDS gels. Indeed, an
intermediate band was seen on several gels but could not be
exclusively identified as a X intermediate (X or X ).
The possibility remained that it might be Factor Xa. The
interference of thrombin in the gel system was successfully
eliminated by electrophoresing an AT-III—thrombin complex.
However, results did not firmly indicate a new molecular
species.

Although purified in yitrg clotting systems revealed a
coagulant complex formed by the interaction of Factors X, V

and thrombin to be responsible for the bypass mechanism, it

83

was necessary to pursue a biochemical approach. Relating
FEIBA to kinetic parameters involved utilizing purified
procoagulant factors in a synthetic chromogenic substrate
assay to evaluate the clot promoting activity.

Elsinger (135) briefly compared the amidolytic
activities of FEIBA IMMUNO with those of thrombin and Factor
Xa. His conclusion was that neither Factor Xa nor thrombin
could be solely responsible for FEIB-Activity. The reaction
employed in this study, however, was more involved and was
aimed at proving that Factor X and IIa, in a purified
system, were capable of the generation of prothrombinkinase,
independent of the intrinsic or extrinsic cycle. Evidence
of increased kinetics with the addition of trace amounts of
thrombin to the chromogenic assay system was sought.
However, data collected was not sufficiently reproducible to
substantiate the data collected from the in. yitrg and
clotting assays.

Attempts at characterizing the mechanism of Factor IX
concentrates involved examination with plasma factors only.
Evaluation of the enhancement of Factor IX concentrates with
platelets, as reported by Vermylen (136), was not

investigated in this study.

CONCLUSION

A modified activated partial thromboplastin time was
devised to assay for Factor VIII inhibitor by-passing
activity (FEIBA) of Factor IX Concentrates. The activity
was based on the correction of the abnormal activated
partial thromboplastin time of Factor VIII Inhibitor plasma
after a one minute and forty minute incubation time. The
procoagulant and coagulant proteins employed in the bypass
assay were relatively homogeneous and were obtained from the
prothrombin complex by biochemical fractionation. FEIBA, in
our in m clotting system was found to be dependent on
the interaction of Factors X, IIa and V. It was not
established whether these proteins are reacting as a binary
complex or if thrombin is merely an accelerator of the
reaction.

While not enabling us to characterize an exact

mechanism, utilizing the chromogenic substrate assay

84

———"p ‘fl'ul'L- Ragnar

85

provided a technique for analysis of the kinetics of the
reaction.

In attempting to characterize the mechanism of Factor
IX concentrates, testing was developed in purified systems.
Although purification was presumed to create optimal
conditions, DeWitt and Feinstein (137) reported to the
contrary. They reported that the more the concentrates were
"cleaned up" the less effective and less potent they became.

Current state of knowledge does not allow designation
of the exact mechanism of the by-pass activity. Although
data shows strong evidence for the direct involvement of
Factors )( and. IIa, further investigation into the

biochemical mechanism is necessary.

_ ""7,

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Strauss, H.S. Ibid.

Piller, G., Hocker, P., Ludwig, E., Niessner, H.
(1976) Plasmapheresis: its role in the management
of inhibitor patients. Workshop on Inhibitors of
Factors VIII and IX, Vienna, Austria. Jan.,
1976.

Green, D. (1971) Suppression of an antibody to
Factor VIII by a combination of Factor VIII and
cyclophosphamide. Blood. 37, 381.

Ruggeri, Z.M., Mannucci, P.M., Allain, J.P., and
Frommel, D. (1975) Preliminary trial of
cyclOphosphamide fix: the management of
Hemophiliacs with Factor VIII inhibitors. Annals
of New York Academy of Sciences. 240, 412.

 

Rubin, R., Nyemetz, J., Estern, S. (1975) Use of
animal AHG concentrates (Factor VIII) in the
treatment of life threatening hemorrhage in
patients with Factor VIII antibodies. Annals of
New York Academy of Sciences. 240, 412.

 

 

Fekete, L.F., Holst, S.L., Peetoom, F., et a1
(1972) Auto Factor IX Concentrate: A new
therapeutic approach to treatment of Hemophilia A
patients with inhibitors. Fourteenth
International Congress of Hematology, Sao Paula,
Brazil. July, 1972.

Kurczynski, E.M., and Penner, J.A. (1974)
Activated Prothrombin complex for patients with
Factor VIII inhibitors. New England Journal of

 

Medicine. 291, 164.

Sultan, Y., Brouet, J.C. and Debre, P. (1974)
Treatment of inhibitors to Factor VIII with
activated prothrombin concentrate. New England
Journal of Medicine. 291, 1087.

 

 

Abilgaard, L.F., Britton, M. and Roberts, R.
(1974) Prothrombin complex (Konyne) for patients
with Factor VIII inhibitors. Seventeenth Annual
Meeting, American Society of Hematology. Abst.
87, 1974.

Sonoda, T., Solomon, A., Krauss, S. et al (1976)
Use~ of jprothrombin complex concentrates in
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73.

74.

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92

Goodnight, S.H., Common, H.H. and Lovreen, E.W.
(1976) Factor VIII inhibitors following surgery
for epidural hemorrhage in hemophilia: successful
therapy with a concentrate containing II, VII, IX
and X. Journal of Pediatrics. 88, 356.

 

Mannucci, P.M., Ruggeri, Z.M., Capitanio, A. and
Pareti, F. (1976) Clinical experience with a
prothrombin complex concentrate in the management
of Factor VIII inhibitors. Workshop on
Inhibitors of Factors VIII and IX, Vienna,
Austria. Jan., 1976.

Elsinger, F. (1976) Preparations with Factor VIII
by-passing activity. Workshop on Inhibitors of
Factor VIII and IX. Vienna, Austria. Jan.,
1976.

Preston, F.E., Dinsdale, R.C.W., Sutcliffe, D.J.,
et a1 (1977) Factor VIII inhibitor by-passing
activity (FEIBA) in the management of patients
with Factor VIII inhibitors. Thrombosis
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Stenbjerg, S. and Jorgensen, J. (1978) Activated
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treatment of Haemophiliac patients with antibody
to Factor VIII. Acta Medica Scandinavica. 203,
471.

 

Kingdon, H.S. (1970) Hepatitis after Konyne.
Annals of Internal Medicine. 73, 656.

 

Hellerstein, L.J. and Deykin, D. (1971) Hepatitis
after Konyne administration. New England Journal
of Medicine. 284, 1039.

 

 

Kasper, C.K. and Kipnis, S.A. (1972) Hepatitis
and clotting factor concentrates. Journal of
American Medical Association. 22, 510.

 

 

Menache, D. (1975) Ibid.

Preston, F.E., 3E_§l (1977) Ibid.

Stenbjerg, S. and Jorgensen, J. (1978) Ibid.
Mannucci, P.M., gE_§l (1976) Ibid.

Menache, D. gt_§l (1959) Ibid.

Blatt, P.M., Lundblad, R.L., Kingdon, M.D.,
McLean, G. and Roberts, H. (1974) Thrombogenic

materials in prothrombin complex concentrates.
Annals of Internal Medicine. 81, 766.

 

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Kingdon, H.S., Lundblad, R.L., Veltkamp, J.J. and
Aronson, D.L. (1975) Potentially thrombogenic
materials in Factor IX concentrates. Thrombosis
et Diathesis Haemorrhagica. 33, 627.

 

 

Blatt, P.M. et a1 (1974) Ibid.

Aronson, D.L. (1974) Inhibition of thrombotic
effects of Factor IX (letter). New quland
Journal of Medicine. 290, 861.

 

 

White, G.C., Roberts, H.R., Kingdon, H.S. and
Lundblad, R.L. (1977) Prothrombin complex
concentrates: Potentially thrombogenic materials
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Elodi, S. (1977) Thrombogenic jprOperties. of
prothrombin complex concentrates (letter).

Blood. 50, 961.

Blatt, P.M., et a1 (1974) Ibid.
Kingdon, et a1 (1975) Ibid.

Cash, J.D., Owens, R., Dalton, R.G., and
Prescott, R.J. (1978) Thrombogenicity of Factor
IX concentrates: _i_n vivo and _i_13 vitro rabbit
studies. Vox Sang. 35, 105.

Cash, J.D., et a1 (1978) Ibid.
Kurczynski, E.M., et al (1974) Ibid.
Elsinger, F. (1976) Ibid.

Vermylen, .J., Schet, .J., Semeraro, DL, and
Verstraete, DL (1976) Clinical and laboratory
experience with Fraction R: Attempts at
identifying the active principle. Workshop on
Inhibitors of Factors VIII and IX. Vienna,
Austria. Jan., 1976.

Tishkoff, G.H. (1976) Factor IX concentrates to
treat Factor VIII inhibitor: Biochemical studies
on its mode of action. Workshop on Inhibitors of
Factors VIII and IX. Vienna, Austria. Jan.,
1976.

Ware, A.G. and Seegers, N.H. (1949) Two Stage
Procedure for the Quantitative Determination of
Prothrombin Concentration. American Journal
Clinical Pathology. 19, 471-482.

 

l-..

101.

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Bachman, F., Duckert, F., and Koller, F. (1958)
The Stuart-Prower Factor Assay and its Clinical
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Haemorrhagica. 2, 24-38.

 

 

Quick, A.J. and Hussey, C.V. (1956) Hemophilia:
Quantitative studies of the coagulation defect.
A.M.A. Arch Int Med. 97, 524.

 

Proctor, R.R. and Rappaport, S.I. (1961) The
Partial Thromboplastin Time with Kaolin.

Jacoby, William, Colowick, Sidney (Editor in
chief) (1971) Methods in Enzymology: Enzyme
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Fairbanks, G., Steck, T.L., and Wallach, D.F.H.
(1971) Electrophoretic Analysis of the Major
Polypeptides of the Human Erythrocyte Membrane.
Biochemistgy. 10, 2606-2617.

 

Kosow, D.P., Furie, B. and Forastieri, H. (1974)
Activation of Factor X: Kinetic properties of the
reaction. Thrombosis Research. 4, 219-227.

 

Vician, L. and Tishkoff, G. (1976) Ibid.

Jacoby, William, Colowick, Sidney (Editor in
chief) (1971) Methods in Enzymology: Enzyme
Purification and Techniques, Vol. XXII, p 280.
Academic Press, Inc. New York, New York.

Rosenburg, R. and Damus, P. (1973) The
Purification and Mechanism of Action of Human
Antithrombin-Heparin Cofactor. J. Biol Chem.
248, 6490.

 

Lanchantin, G.F., Friedman, J.A., Hart, D. (1973)
Two Forms of Human Thrombin Journal of Biological
Chemistry. 248: 17, 5956.

 

 

Kosow, D. (1976) Ibid.

Summoria, L. and Robbins, K. (1976) Isolation of
a Human Plasmin derived, functionally active,
light (B) chain capable of forming with
Streptokinase an equimolar light (B) chain.
Journal of Biological Chemistry. 251, 18.

 

Fujikawa, L., et a1 (1974). The Mechanism of
Activation of Bov1ne Factor v Stuart Factor by

Intrinsic and Extrinsic Pathways. Biochemistry.
13, 26, 5290.

 

114.

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120.
121.

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125.

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128.

129.

130.

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Wickerhauser, M. and Williams, C. Large scale
method for the isolation of Antithrombin III. VI
International Congress of Thrombosis and
Hemostasis.

Clausen, J. Immunochemical Techniques. N.
Holland/American Elsevier.

 

Vician, L., and Tishkoff, G. (1976) Ibid.
Fairbanks, G. et a1 (1971) Ibid.

Kosow, D. (1976) Ibid.

Rosenberg, R. and Damus ( ) Purification
and Mechanism of Action of Human Antibodies -
Heparin Cofactor. Journal of Biological

 

Chemistry. 248, 18.

 

Vician, L. and Tishkoff, G. (1976) Ibid.

Kosow, D. (1976) Ibid.

DiScipio, R. gt_§l (1977) Ibid.

Kosow, D. (1976) Ibid.

Vician, L., Tishkoff, G. (1976) Ibid.

Lanchantin, G.F., §£_al (1973) Ibid.

Elsinger, F. (1977) Fraction FEIBA. Presentation
of XIIth Congress of World Federation of
Hemophilia. New York, June, 1977.

Kurcyznski, A. (1974) Ibid.

Dewitt, R. and Feinstein, D. (1977) Prothrombin
Complex Concentrates. Arch Int Med. 137, 1211.

 

Elsinger, F. (1975) Shortening of Clotting Times
of Hemophilia A Plasma with Inhibitors Induced by
Activated Factor IX Concentrates. Vth Congress
of International Society' of Thrombosis and
Hemostasis. Paris, France.

Elsinger, F. (1976) Preparations with Factor VIII
Inhibitor By-Passing' Activity. Workshop on
Inhibitors of Factors VIII and IX. Vienna,
Austria.

Elsinger, F. (1975) Ibid.

Elsinger, F. (1976) Ibid.

133.

134.

135.
136.

137.

96

Elsinger, F. (1977) Ibid.

Tishkoff, G.H. (1976) Factor IX Concentrates to
Treat Factor VIII Inhibitor: Biochemical Studies
on its mode of action. Workshop on inhibitors of
Factors VIII and IX. Vienna, Austria.

Elsinger, F. (1977) Ibid.

Vermylen, J. et a1 (1976) Ibid.

Dewitt, I“ and Feinstein, [L (1978) Editors
correspondence. Arch Int. Med. 138.

 

VITA

The author was born in Detroit, Michigan on May 4,
1951. She received a Bachelor of Science degree in Medical
Technology front Michigan State University in 1973. Her
internship was completed at Edward W. Sparrow Hospital in
Lansing, Michigan and became ASCP certified in 1974. The
author worked for five (5) years as Section Chief of the
Research Laboratory at the Great Lakes Regional Red Cross
Center in Lansing. In 1979 she was admitted to the graduate
program in Clinical Laboratory Science at Michigan State
University.

The author is married, has two (2) children, and lives

in East Lansing.

97