QUANTWATWE ESHMATES 0F
PROTHROMBEN CGMPONENTS

 

Thesis for the Degree of M S.
MICMCMN STATE UMVERSKY
CHARLES E. HEAR
1989

  

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ABSTRACT
QUANTITATIVE ESTIMATES OF PROTHROMBIN COMPONENTS

by Charles E. Hiar

The formation of thrombin from prothrombin involves the inter-
action of Factors V, VII, X, and prothrombin. Physicochemical studies
on these clotting factors have been published by various authors.

Many of their results have not been in agreement. The work summarized
in this report concerns the heat stability of the "prothrombin complex".
Normal and artificially reconstituted plasmas were incubated at 45 C.
for varying lengths of time. The effect of this incubation is evalu-
ated. In addition, a method is presented by which the concentration
of the heat labile Factor V can be determined by this incubation.

The presence or absence of the four factors are detected by the
one-stage prothrombin time. Specially prepared plasmas and serums are
used to vary the levels of the factors. These reagents, when used in
conjunction with the automated prothrombin time procedure, provide a
sensitive method of detecting differences in levels of the clotting
factors. The study indicates that incubation at 45 C. can be used to
measure the concentration of Factor V, while the other 3 factors are

not adversely affected.

QUANTITATIVE ESTIMATES OF

PROTHROMBIN COMPONENTS

By

Charles ES Hiar

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1969

/
Zea

/@

To Ruth Ann

for her constant encouragement
over the past two years

11

ACKNOWLEDGEMENTS

I would first like to express my appreciation and gratitude to
my major professor, Dr. A. E. Lewis, of the Department of Pathology.
He has given me much assistance and encouragement throughout my
graduate work. Without his insight into various research problems,
and his knowledge of mathematical and statistical computations, this
work could not have been completed.

I am grateful to Dr. Charles Sander of the Department of Pathology,
and Dr. Anthony Bowdler of the Department of Human Medicine, for their
assistance and advice in preparing the manuscript.

I would like to express my thanks to Dr. C. C. Morrill, Chairman
of the Department of Pathology, for providing the funds and facilities
necessary to do my graduate work. I would also like to thank Dr.
Morrill and those involved in administrating the U. S. Public Health
Service Allied Health Traineeship, for providing the monetary support
for my family and myself during my graduate study.

To Mr. Gary Hammerberg I am grateful for the transportation he
provided in obtaining supplies and blood samples, and for his willing-
ness to assist me whenever needed.

I would like to thank the numerous students in the Department of
Pathology and School of Medical Technology for providing me with blood

samples for testing.

111

I am especially grateful to Mrs. Margaret Shick and her fine staff
of the laboratory in the Olin Memorial Health Service. Their willing-
ness to lend me the use of their automated equipment and laboratory
space, in addition to obtaining blood samples, is greatly appreciated.

I am grateful to Mr. James Osburn and Dr. Robert Foy of the labora-
tory staff at Sparrow Hospital, and to Mr. Robert Brooks of the Depart-
ment of Pathology at Michigan State University. They willingly lent
me supplies and allowed me to use their facilities when time was a
factor in completing my project.

To Mrs. Gretchen King, Miss Sue Darling, and Mrs. Vel Chesser I
am grateful for the fast and efficient way they handled the stenographic
work that I needed to have done. I would also like to thank Mrs. King
for her knowledge and assistance with the various administrative require-
ments in obtaining my degree.

I would like to thank Mr. Robert Modica, Technical Director of the
Laboratory at St. Mary's Hospital in Grand Rapids, Michigan, for his
encouragement, advice, and sincere interest in the advancement of
medical technology.

Finally, I am deeply indebted to my wife Ruth Ann. My returning
to the life of a student meant many hardships for her. Her constant
encouragement was a great inspiration for me, and her role of wife,
mother, and often father to our son in my absence is deeply appreciated.

It is to her that this work is dedicated.

iv

INTRODUCTION .

REVIEW OF LITERATURE

MATERIALS AND METHODS.

TABLE

Preliminary Procedures.

OF CONTENTS

Experiments Concerning the Heat

Prothrombin Complex.

Stability

Experiment Concerning DevelOpment

RESULTS.

DISCUSSION .

SUMMARY AND CONCLUSIONS.

REFERENCES CITED .

APPENDIX

VITA

Page

12

12

13
15
16
29
33
34
37

4O

Table

LIST OF TABLES

Comparison of prothrombin times of normal human plasma
before and after incubation for 30 minutes at 56 C.

Effect of incubation on Factors VII and X.

Effect of incubation on Factors VII and X.

Effect of incubation on prothrombin. . . . .

Concentration of Factor V restored to normal plasmas.

Percentages represent final concentrations . . . .

Statistical evaluation of estimating the Factor V con-

centration in an unknown plasma.

vi

Page

20
22
23

24

27

28

Figure

LIST OF FIGURES

Page

Destruction of the labile factors in the plasma of
3 normal subjects . . . . . . . . . . . . . . . . . . . 21

Demonstration of intersecting point of dilution

curves. Lines were calculated by the method of least
squares. Lines I-VI denote Factor V concentrations

of 0, 1.25, 2.5, 5.0, 10.0 and 20.0%, respectively. . . 25

The linear relationship of Factor V concentration

to a. Factor V conc. = 67.0-3.5 G. . . . . . . . 26

vii

LIST OF APPENDICES
Appendix Page

1 Preparation of reagents rich in the clotting fac-
tors of the prothrombin complex . . . . . . . . . . . . 37

viii

INTRODUCTION

As candidates for the Master's Degree in Clinical Laboratory
Science, we are mainly interested in the clinical aspects of pathology.
One of our major goals is to devise easy and practical methods of
solving problems arising in clinical laboratory procedures. As the
field of clinical pathology advances, new methods are continuously
being sought to augment existing ones. Procedures must also be found
to measure substances which could previously not be assayed. Such is
the problem encountered in oral anticoagulant therapy with respect to
the four factors of the prothrombin complex.

The prothrombin time is universally used in the clinical laboratory
to study hemorrhagic disorders in Stage II of the clotting mechanism.
The test is simple in concept, easy to perform, and can be adapted to
a variety of uses. It was originally thought that only prothrombin de-
ficiencies could be detected by the test. It is now known that the pro-
thrombin time may reflect deficiencies in a complex of interacting
factors. These are the Factors V, VII, X, and prothrombin itself.

Factor V is probably the least understood of the four factors
involved. No one is certain as to where it originates or exactly what
role it plays in coagulation. The factor can be assayed by several
methods. Each of these has its disadvantages, however. The relation-
ship or ratio of Factor V to Factors VII, X, and prothrombin has been

the prime concern of this work. A change in ratio of the Factor V

concentration to the others which are affected by coumarin therapy might
indicate a more complete picture of the patient's progress.

Various investigators have tried to isolate the clotting factors
and analyze them to determine their function, chemical, and physical
properties. Varying results have been achieved. These have been, in
large part, due to difficulties in reagent standardization, and the
inherent instability of the clotting factors. Technical errors have
also affected reproductibility and accuracy and have played a large
part in leading to conflicting results.

The author was concerned with the unreliability of this physico-
chemical data presented on the factors of the prothrombin complex.

With this in mind, the thermostability of these factors was
reinvestigated.

The study was carried out using the one-stage prothrombin time
adapted to an automated procedure. Standardized commercial reagents
were used whenever possible. This eliminated, to a large degree, the
technical difficulties encountered in testing, and allowed for greater
sensitivity and reproductibility of results. The study was carried out
with the purpose of measuring the thermal decay of the factors involved
in the prothrombin complex. With this in mind we hoped to shed addi—
tional light on the physicochemical properties of these clotting
factors. Knowledge of the extent of decay should give an estimation
of concentration of the labile factors present. Our second purpose,
then, was to devise a simple procedure for the detection of concentra-

tion of the labile Factor V using thermal incubation.

REVIEW OF LITERATURE

Since several prominent investigators have postulated mechanisms
for the coagulation of blood, it would be impossible to review the
prothrombin mechanism without first relating it to the overall coagu-
lation scheme. The simple explanation by Morowitz was an accumulation
of all the known facts about coagulation up to the end of the last
century. His classical theory stated that:

Thrombokinase + Ca2+ + Prothrombin-—9 Thrombin
This simple theory lay dormant until the develOpment of the one-stage
prothrombin time by Quick (1937) whereby the classical theory of
Morowitz was put to practical use. Since then the Morowitz theory
has become extremely inadequate, but the usefulness of his basic prin-
ciples still prevails.

Seegers (1962) postulated a theory that prothrombin plays the
central dominant role in the blood clotting scheme, and that all other
factors except Platelet Co—factor I (Factor VIII) and Ac-globulin
(Factor V), are derivatives of it.

A third scheme by Macfarlane (1964), called the "cascade" theory,
and a similar one by Davie and Ratnoff (1964), the "waterfall" sequence,
differs from that of Seegers. They postulated that clotting is initia-
ted by the activation of Factor XII. The active Factor XII acts as an
enzyme activating Factor XI. This in turn activates other factors in
a sequence resulting in the formation of thrombin from prothrombin, and
finally the formation of fibrin from fibrinogen.

3

Prothrombin

 

Seegers, Macfarlane, Davie and Ratnoff all agree on one fact in
their prOposed clotting mechanisms. They have found that prothrombin
exists in only the free or readily activated state. Anderson and
Barnhart (1964) found prothrombin in isolated microsomal and soluble
fractions in bovine and canine liver parenchymal cells. The workers
found subcellular prothrombin in these fractions using specific
fluorescent antiprothrombin homologous with the species. Induced hypo-
prothrombinemia reduced or eliminated microsomal prothrombin.

This led them to conclude that the site of synthesis of prothrombin
is the microsomes of the liver cells.

Various techniques have been used to isolate and study prothrombin
in a relatively pure state. Tishkoff and Williams (1967) isolated
prothrombin as a barium complex containing protein 21.5%, barium 32%,
and citrate 8.9%. They discovered prothrombin to be an a-2—g1yc0protein
found in a high degree of purity as a true prothrombin-metal complex.
The molecule was stable in this form. This work agrees with other
investigators to the extent that prothrombin is a glchprotein.

Seegers (1940) observed the protein-carbohydrate complex to con-
tain about 4% carbohydrate, while Tishkoff at al. (1960) found 10.4-
14.72 in bovine prothrombin. Whether or not there is a species dif-
ference has not been conclusively determined.

Tishkoff and his co-workers (1960) found that sialic acid comprised
the largest constituent in the carbohydrate fraction of their bovine
preparations. Their electrophoretic studies on starch gel showed pro-

thrombin to be the least mobile, when compared to Factors VII and X.

These other factors contained little protein but substantial quantities
of sialic acid.

The conversion of prothrombin to thrombin is an undisputed fact.
Whether or not the prothrombin itself converts autocatalytically to
thrombin, or whether it is comprised of different subunits which inter-
act to form thrombin, has not been decided. Seegers (1968) presented
a paper which postulated this conversion. His version of the mechanism
is as follows: (1) prothrombin dissociates to form mainly prethrombin
and Aut0prothrombin III; (2) AutOprothrombin III converts to AutOpro-
thrombin C; (3) AutOprothrombin C converts prethrombin to thrombin.

The AutOprothrombin C and III were isolated by Seegers and Marciniak
(1965). This view is not shared by those who believe prothrombin itself
is converted to thrombin in the classical theories of coagulation.

Physicochemical data published in Todd and Sanford (1963) state
that prothrombin is precipitated from oxalated plasma by 50% saturated
(NHQ)ZSOH. It is adsorbed by the usual adsorbing agents Mg(OH)2,

BaSO Al(OH)3, and Ca3(POu)2. Seitz filtration will also remove it

4’
from plasma. The substance is relatively labile between 40 C. and 60 C.
and inactivated at 85 C. It is a sulfur containing glchprotein. Pro-
thrombin is consumed almost completely in normal clotting. It is present
in quantities of about 0.2 mg./ml. and represents 0.14% of the plasma
proteins. It converts gradually upon standing with sodium citrate anti-
coagulant to thrombin. Seegers and Alkjaersig (1953) agree with the
conversion and Marciniak and Seegers (1962) state that AutOprothrombin

C is stable for 30 minutes at 56 C. As stated previously, this Auto-

prothrombin C may or may not be a component of prothrombin.

Prothrombin is affected by the common oral anticoagulant drugs of
coumarin origin. These compounds are thought to compete with vitamin K
in reactions leading to prothrombin synthesis. The relationship was
first described by Weddell and Guerry (1939). The first congenital
deficiency was reported by Rhoads and Fitz-Hugh (1941) and deficiency
in the newborn by Brinkhous et a2. (1937). Congenital defects, however,
are rare, while those associated with liver disease or inability of the

liver to produce the factor are common.

Factor V
Factor V, or Labile Factor, is also the Ac-globulin referred to
by Seegers. This factor is believed to be involved in acceleration of
the conversion of prothrombin to thrombin. Physicochemical data sumr
marized in Todd and Sanford (1963) show that this factor is precipitated
by 33% (NHs)230a and by a pH less than 4.0 and greater than 10.0. It

is not adsorbed from plasma by the usual adsorbing agents BaSO BaCO3

4’
or Al(0H)3. The factor is readily inactivated by heat and also by
storage even at 0 C. It decays much faster when oxalate is used as an
anticoagulant rather than citrate. An antibody to Factor V has been
found by Ferguson at al. (1958). The antibody acted as an inhibitor
to Factor V, was non—dialyzable, and remarkably stable to temperature,
pH, and fat solvents.

It has been postulated that Factor V interacts in some way with
metal ions to enhance the coagulability of blood. Two studies have
been carried out to this effect. Leikin and Bessman (1956) studied the

effect of EDTA in lengthening the prothrombin time of normal plasmas.

The workers found that the prolonged prothrombin time could be partially

or completely corrected by the addition of prothrombin-free human plasma.
They concluded that the EDTA complexed some metal and that Ca2+ was not
principally involved. In this study the Mn2+, Ba2+, or Zn2+ ions had no
effect on the prothrombin time. Fe2+, Fe3+, or Ca2+ lengthened the time
while N12+ seemed to protect the Factor V activity.

Other alkaline earth cations (Ba2+, Sr2+, Mg2+) can also be sub-
stituted experimentally for calcium to achieve clotting. Ferguson at
al. (1967) found that these ions in a concentration of 16-20 mM, at a
pH of 6.2-7.3, may substitute for calcium. Factor V still converted
prothrombin to thrombin in their presence. Their study also showed
that Factor V showed no activation prior to its participation with
thrombokinase (thromboplastin) in the final prothrombin conversion.
Using an enzyme free prothrombin plus Ca2+, and cephalin, they could
achieve no conversion with Factor V until some thrombOplastic enzyme
was added. They concluded that the role of the cation is to complex
with thrombokinase and thus determine its true prothrombin activator
function.

In a study by Hougie (1957) indications were found of a sedimen-
table complex formed in the reaction of AHF (Factor VIII), PTC (Factor
IX), Stuart factor (Factor X), and platelets. The Factor V was found
to interact with this complex. If it was left out, the complex had very
little activity. They found Factor V to enter the scheme subsequent to
I the coagulant formed by the other factors. This complex may be the
thromboplastic enzyme mentioned by Ferguson and his co-workers (1967),

and would support their findings.

I \ll||. |1J i]

The question has been asked whether or not an increase in amount
of one factor of the prothrombin complex would effect a decrease in
another factor. Since the discovery that the prothrombin time will
detect deficiencies in any one of four factors involved, this poses an
interesting question. Ferguson and Patch (1956) presented work in which
they artificially reconstituted plasmas with various factors of the
prothrombin complex. Their studies found that moderately low ranges of
prothrombin and Factor VII can be compensated by high levels of Factor
V. This level of Factor V was found to be in the range of ZOO-300%
of normal. Factor VII in increased amounts at fixed concentrations of
prothrombin and V exerted relatively minor effects. Increasing levels
of prothrombin exerted little effects also, and only a large excess of
Factor V offset any deficiencies of the other factors. No mention was
made of the fourth factor (X), however.

Factor V, unlike prothrombin, VII, and X, is not affected by the
coumarin anticoagulants. Shanberge (1956) found no change in Factor V
levels during the entire course of Dicumarol therapy on his patients.
Additional remarks published in Todd and Sanford (1963) say that Factor
V levels ih plasma are not affected by dietary lack of vitamin K, cou-

marin type medication, or biliary obstruction.

Factor VII

 

It is interesting to note that although Factor VII has been found
deficient in certain hereditary hemorrhagic disorders, it is not included
in the scheme of Macfarlane or by Davie and Ratnoff. The "cascade" or
"waterfall" schemes make no allowance for it. Seegers, however, does

claim that Factor VII (Autoprothrombin I) and Factor IX (Autoprothrombin

II) are both derivatives of prothrombin that reinforce enzyme activity
for the production of thrombic activity.

A patient with a severe hereditary deficiency in Factor VII has a
normal prothrombin time. Conversely, a patient with a severe heredi-
tary hypoprothrombinemia often has normal levels of Factor VII. This
was noted by Quick and Hussey (1962).

In vitamin K deficiency or after a vitamin K antagonist such as
Dicumarol has been administered, both Factor VII and prothrombin de-
crease. This, according to Quick (1966), does not mean that one is
a precursor to the other, but their site of synthesis is probably the
same. In fact, several authors state that Factor VII is more sensitive
to Dicumarol therapy than either prothrombin or Factor X. Alexander
(1959) found Factor VII to be the first to decrease under therapy and
that it responds more quickly to vitamin K than does prothrombin. This
agrees with Georgatsos et a1. (1958), who found Factor VII to be de—
pressed to a greater extent than prothrombin after Dicumarol therapy.

The importance of Factor VII is not in agreement. Ackroyd (1956)
says that, although prothrombin conversion is slower in Factor VII
deficiencies, there is no incontrovertible evidence at present to show
that it is needed in the coagulation of blood. This disagrees with
Alexander (1959), who found only a slow or insignificant thrombin forma-
tion during in vitro studies of the factor, and agrees with Keller et
a1. (1951), who say that Factor VII increases the rate but not the
amount of thrombin formed. These findings fail to explain the hemor-
rhagic symptoms found in congenitally deficient patients or why, in the

presence of tissue thrombOplastin, Johnston et a2. (1959) and Johnston

10

and Hjort (1961) found increases in the level of 2-1/2 to 3 times that
found before clotting. Factor VII must therefore have a physiological
function however obscure it may be.

Todd and Sanford (1963) summarize physicochemical data showing
Factor VII to be relatively stable. It is resistant to heat at 37 C.
in serum for at least four hours and to 45 C. for at least six minutes.
These findings agree in part with the results of this study (see dis-
cussion). Factor VII is also readily adsorbed by the usual adsorbing
agents. It can be precipitated at a pH less than 5.0 and greater than
9.0. Hogenaur and Deutsch (1968) found that Factor VII probably con-
sists of two polypeptide chains much like Factor X. Their adsorption
techniques and fractionations on DEAE cellulose gave RF values of 0.54
and 0.70 for Factors VII and X, respectively. These values were ob-
tained with polyacrylamide electrOphoresis. The N and C terminal ends
of both polypeptide chains were composed of glycine and serine. Finger-
prints of the amino acid composition of the two factors gave similar
results. These findings suggest that the two factors have similar
chemical compositions and may account for the difficulty in separation

of one from the other.

Factor X
The physicochemical properties of Factor X are very similar to
those of Factor VII. According to data published in Todd and Sanford
(1963), both are present in serum and in plasma and are adsorbed by the
same agents. The stability of Factor X to heat is somewhat diaputed.
Deficiency of Factor X has been described in both hereditary and acquired

conditions. Some investigators feel that it is involved in most

ll

hemorrhagic complications of oral anticoagulant therapy. Factor X is
believed to be a converter of prothrombin to thrombin. Studies by
Lanchantin et a1. (1969) show that inactive Factor X is converted to
the active form. At a pH of 7.4, 0.01 M Ca2+, and a temperature of
23 C., the activated Factor X converts prothrombin to thrombin, the
reaction following first—order kinetics.

Hougie et al. (1956) and Telfer et al. (1956) found that Factor X
is necessary in both the intrinsic and extrinsic systems, and is con-
cerned with the formation of a prothrombin activator. Other work has
been published in which Factor X has been consumed in blood activated
by tissue thromboplastin, but not by blood activated merely by surface
contact. Niemetz (1967) found that blood allowed to clot in siliconized
glassware showed no activation of Factor X. This verifies the claim
by the previous workers in that this factor is involved in the extrinsic
system. Niemetz found it to be involved in the intrinsic system but
only if a phospholipid or AHG (Factor VIII) was added. He stated that
Factor X is consumed during in vitro coagulation only when surface acti- ’
vation, and either bovine AHG or phospholipid, were added. Neither sur-
face activation, AHG, or phospholipid alone will result in a significant
consumption of Factor X.

Factor X deficiencies can be separated from those of Factor VII by
the action of Russell's Viper Venom. The observation that the venom
restores blood clotting in Factor VII defiCiencies, but not in Factor X
deficiencies, has been observed by various workers. This thrombOplastic
activity in the presence of added cephalin makes a useful diagnostic test

to differentiate the two clotting abnormalities.

MATERIALS AND METHODS

Preliminary Procedures

Studies were made on normal human subjects. Blood was drawn in
Vacutainer* tubes containing sodium oxalate 0.1 M as an anticoagulant,
and sorbic acid 0.2 mg./ml. as an antimycotic agent. The specimens
were centrifuged immediately for 10 minutes at 2000 rpm. The plasma
was separated from the cells and placed in clean test tubes.

Plasma specimens requiring incubation at 37 C. for more than 3
hours (aged plasma) were collected in an aseptic manner according to
regulations described by the American Association of Blood Banks (AABB).
The blood was drawn into autoclaved Vacutainer tubes. The plasma was
then placed into sterile screw-tOpped tubes utilizing previously auto—
claved disposable pipettes. The plasma specimens were then incubated
at 37 C. until a one-stage prothrombin time greater than 32 seconds
was obtained. This prothrombin time was checked at various intervals
using aseptic technique to withdraw the plasma portions from the stock
mixture. Total incubation times varied from 48 to 56 hours. Upon
completion of the incubation, the plasma mixture was recentrifuged for
10 minutes at 2000 rpm. Gram stains and cultures on blood agar plates
were made of the sediment to check for bacterial contamination.

Other plasmas and serums were specially prepared to be rich in

clotting Factors V, VII, and X according to the methods of Eichelberger

 

*Becton, Dickinson, and Co., Columbus, Nebraska and Rutherford,
N.J.

12

13

(1965) with minor modification. These methods are described in detail
in Appendix I.

Prothrombin times were measured by the one-stage method of Quick
(1957) as adapted for use on the Clot—timer.* All determinations were
performed in duplicate or until the results agreed within i_0.3 seconds.
Exceptions were allowed when the time exceeded 35 seconds where devia-
tions of i;0.5 second were accepted. Simplastin** was used as the
thromboplastin—calcium reagent and was diluted according to the manu—
facturer's instructions.

With each new batch of reagents a commercial control was included
in the day's run to check the reliability of the reagents. Constant
temperature heating blocks and water baths were used for incubation and
their temperatures controlled to 1:0.5 C. All plasma specimens were

stored at 4 C. until ready for testing.

Experiments Concerning the Heat Stability of the Prothrombin Complex

 

Experiment I. This experiment was designed to find a temperature high

 

enough to destroy rapidly any labile factors of the prothrombin complex,
but low enough to prevent denaturation of any essential proteins. Seven
normal plasmas were incubated for 30 minutes at 56 C. Prothrombin times
were determined before and after incubation. The results are shown in

Table 1.

 

*Heller Laboratories, San Mateo, Calif.

**Warner—Chilcott Laboratories, Morris Plains, N.J.

l4

Experiment II. To determine if any factors were labile to heat, 3 nor-

 

mal plasma specimens were used. They were incubated for 90 minutes at
'45 C. At various time intervals from 0 to 90 minutes portions were
withdrawn and their prothrombin times measured. The curve illustrating

the effects of destruction are shown graphically in Figure 1.

Experiment III. To determine the effect of heat on Factors VII and X,

 

2 pooled plasma samples were utilized. They were heated at 45 C. for

30 minutes, during which prothrombin times were measured at various
intervals. At each time interval, 2 aliquots of 0.4 ml. of plasma were
removed. They were designated as Control and Test samples. On the
first run 0.1 ml. aged serum (Appendix I) was added to the Test samples
and nothing was added to the Control samples. On the second run, 0.1
ml. of physiologic saline was added to the Control samples and the aged
serum added to the Test samples. This was done to equalize the dilution
of the factors already present in the plasma. The results are presented

in Table 2.

Experiment IV. To determine further the effects of heat on Factors VII

 

and X, the following experiment was completed. Plasma samples from 4
normal individuals were pooled. They were collected aseptically and
incubated at 37 C. until the prothrombin time was increased to 32
seconds (see preliminary procedures). After this incubation the pooled
plasma was incubated at 45 C. for 60 minutes. At various time inter-
vals, 0.4 ml. portions were removed and placed in each of 2 test tubes.
To the Control tube was added 0.1 ml. saline and to the Test sample was
added 0.1 ml. BaSO“ adsorbed plasma. The results are summarized in

Table 3.

15

Experiment V. This eXperiment was designed to determine whether or not

 

prothrombin was labile to heating to 45 C. The same aged plasma as in
Experiment IV was used. To 0.7 ml. of the aged plasma, previously incu-
bated for 60 minutes at 45 C., was added 0.1 ml. aged serum and 0.2 ml.
BaSO” adsorbed plasma. These reagents were added at various time inter-
vals to portions removed from the stock supply. Their prothrombin times

were determined and the results are given in Table 4.
Experiment Concerning Development of the Factor V Assay

Experiment VI. Results of the previous experiments indicated that

 

Factor V was the only significantly labile factor in the prothrombin
complex. This experiment was designed to devise a test whereby the
concentration of Factor V could be estimated using thermal incubation.
Dilutions of BaSO“ adsorbed plasma were prepared ranging from an undi-
luted plasma (100%) to 6.25%. When added in the amount of 0.1 ml. to
0.4 ml. of plasma in the testing procedure, they represented Factor V
concentrations of 20, 10, 5, 2.5, and 1.25%. The results are shown

graphically in Figures 2 and 3 and in Tables 5 and 6.

RESULTS

Figure 1 represents graphically the destruction of the coagulation
factors over a period of time at 45 C. At this point it was not known
which factors were destroyed. It was assumed that only Factor V was
labile. The correction studies (Tables 2 through 4) showed this assump-
tion to be true.

Experiment VI represents the major emphasis of this study. The
explanation of Table 5 and Figures 2 and 3 may be found in the section
devoted to the derivation of the formulas used in this work. Table 6
summarizes the statistical evaluation of the estimation of the concen-
tration of Factor V as described by the formula: [V] B 67.0 - 3.5 O.
The derivation of this formula and a proposed method of obtaining the
relative concentration of the thermostable components are as follows.

1. A series of dilution curves were made of the incubated plasma
(45 C.). To these were added varying concentrations of Factor V in the
form of BaSO1+ adsorbed plasma. Table 5 lists the relative concentra-
tions of the thermolabile and thermostable components. Figure 2 repre—
sents the true value of these lines as calculated by the method of least
squares. Initial investigations relating concentration of Factor V to
the change in prothrombin time after heating did not yield linear results.

2. The data were treated in the following manner:

16

17

VI V I

¥II II
I
.1.
dil

i’////
’I////
(106/

[rig

96-91

 

 

prothrombin time

Lines I-VI represent additions of BaSO“ adsorbed plasma to obtain con-
centrations of 0—20% (see Table 5).

3. The lines were extrapolated to yield 0 values. These are the
most stable points on the line and eliminate effects due to hemodilution
or changes in concentration of the thermostable component.

4. By taking only one line of the series we can see the following

relationship:
._1_ 2+
dil
1-

 

 

 

prothrombin time

(a) fl=h=fl=% (b) 2(t1-e)=1(t2-e)

(c) 2t1 - 2 e = t2 - e (d) 2t1 - t2 = O

18

5. The values for 0 were plotted against Factor V concentration
(see Figure 3). This results in completion of the formula having the
form of y = a - bx.

The following is a proposed method of determining the thermostable
component of the prothrombin complex. We can see from Figure 2 that a
theoretical point of intersection can be calculated for all the dilution
curves of Experiment 6 in which varying amounts of Factor V has been
added. The proposed method is as follows:

1. A new set of axes is constructed, giving the intersection of

the lines a value of 80% (see the concentration values in Table 5).

VI V

_1__
dil

 

 

/ I
[gal/)g'
204 III
l/I/’ /
40- / ’//,’ prothrombin time

 

 

2. The lines above represent at least 80% activity of the thermo-
stable component. Taking one line and diluting the stable component in

half we have the following case:

 

 

l9

undiluted

’ diluted

 

 

(%) prothrombin time

This method can then be applied to all cases once the concentration of

Factor V is known.

20

Table 1. Comparison of prothrombin times of normal human plasma be-
fore and after incubation for 30 minutes at 56 C.

 

 

Prothrombin times (sect)

 

 

Patient Before incubation After incubation

1 14.0 >70*

2 16.0 >70*

3 15.6 >70*

4 14.5 > 70*

5 13.6 >70*

6 14.0 > 70*

7 14.6 *>70*
Control 13.0**

 

*Dense flocculent precipitate

**Normal value for control = 11-15 seconds

Pro-

thrombin

time
(sec.)

21

31‘l

29.

274

25‘

23'-

 

21.4

19 d

 
    

17‘-

/

154

 

 

v I I l 1 I ' l ‘
10 20 30 4O 50 60 70 80 90

Time of incubation (min.)

Figure l. Destruction of the labile factors in
the plasma of 3 normal subjects.

22

Table 2. Effect of incubation on Factors VII and X

 

 

 

 

Time (min.) of Prothrombin times (sec.)

incubation Control Test

(a) O 15.0 13.5
10 16.5 17.0
20 17.5 18.0
30 18.0 20.0

Control = 12.5*

(b) O 16.6 13.5
10 17.0 16.6
20 19.8 19.0
30 23.5 22.3

 

*Normal range for control = 11-15 seconds

23

Table 3. Effect of incubation on Factors VII and X

 

 

 

 

Time (min.) of Prothrombin timesg(sec.)

incubation Control Test
0 32.3 15.5
10 32.6 16.0
20 33.6 16.0
30 36.3 16.5
60 39.3 17.0

Control = 11.5*

 

*Normal value for control = 11-15 seconds

24

Table 4. Effect of incubation on prothrombin

 

 

Time (min.) of

 

incubation Prothrombin time (sec.)
0 15.8
10 16.5
20 16.5
30 15.6
60 16.6

Control = 11.5*

 

*Normal value for control - 11-15 seconds

25

6-1
I
5-
VI
11
4'J V III
V

3.
_.1_.
dil 2‘!

 

 

O ' /. 1 I
10 ’ 20 30 40
-1.
prothrombin time (sec.)
221’

 

. Figure 2. Demonstration of intersecting point of
dilution curves. Lines were calculated by the method of
least squares. Lines I-VI denote Factor V concentrations
of 0, 1.25, 2.5, 5.0, 10.0 and 20.0%, respectively.

26

 

 

80 4
60 '
40 -
Factor V
(%)
20 1
u I l
0 5 10 15 20
0 (sec.)

Figure 3. The linear relationship of Factor
V concentration to 0. Factor V conc. . 67.0-3.5 0.

Table 5.

27

Concentration of Factor V restored to normal plasmas.
Percentages represent final concentrations

 

 

 

 

Mixtures :_ Incub. plasma Prothrombin time
Normal plasma % BaSO“ plasma % % (sec.)
100 --- --- 13.4
--- --- 100 25.8
--- 20 80 15.7
--- 20 40 19.0
--- 20 20 23.2
--- 10 80 19.0
--- 10 40 21.3
_-_ 10 20 27.0
--- 5 80 20.4
--- 5 40 24.4
--- 5 20 29.3
--- 2.5 80 22.5
___ 2.5 40 24.9
___ 2.5 20 34.3
--- 1.25 80 23.0
--- 1.25 40 28.0
--- 1.25 20 36.0
--- 0.0 80 25.5
--- 0.0 40 31.0
--- 0.0 20 43.3

 

28

Table 6. Statistical evaluation of estimating the Factor V concentra-
tion in an unknown plasma

 

 

0 Cone. of V Calculated conc.*

 

 

 

19.3 0.00 -0.55

19.0 1.25 0.50

18.1 2.50 3.65

17.8 5.00 4.70

16.2 10.00 10.30

13.5 20.00 19.75
S.D. - 0.70

*Calculated from: [V] = 67 - 3.5 O

DISCUSSION

The various data published on heat denaturation of proteins agree
with the results of Experiment 1. Each particular protein has its own
denaturation temperature. In addition, human serum inactivated at this
time and temperature for various serologic tests (VDRL, and heterOphile)
shows no precipitate. We must conclude that the precipitate found
after incubation was indeed fibrinogen.

Experiment 11 agrees with other published work in that at least one
factor of the prothrombin complex is labile to heat. We noted an increase
in prothrombin times in the normal plasmas after incubation. The decay
curve of Figure 1 reveals that at least one factor is being inactivated.

Table 2 reveals that factors other than V are stable during incuba-
tion at 45 C. The 2 runs differed only by the addition of 0.1 ml.
saline to the control tubes in Part b, while none was added in Part a.
In neither case did the addition of aged serum, rich in Factors VII and
X, bring a large correction in the prothrombin time when compared to the
controls. It appears from Experiment III that Factors VII and X are
stable for at least 30 minutes at this temperature. Part of the experi-
ment agrees with Hougie et al. (1957), who found Factor X to be stable
up to 56 C. and at a pH range of 6-9 in a serum medium.

Experiment IV was also designed to test the stability of Factors
VII and X. The addition of BaSO” adsorbed plasma corrected the pro-
thrombin time to normal or near normal levels in the test samples. The
purpose of aging the oxalated plasma was to destroy the labile Factor V.

29

 

30

Eichelberger (1965) presented the method used in this study, but due to
bacterial contamination certain modifications had to be used. With these
modifications it was found that Factor V was destroyed much more slowly
than stated by Eichelberger. 'The experiment showed that if plasma is
kept in a sterile, tightly stoppered container, Factor V is destroyed
much more slowly than previously thought. The slight increase in the
prothrombin times noted in Table 3 could be due to some destruction of
the other factors because of the lengthy heating time involved, but the
change is small when compared to differences in the Test and Control
samples obtained by correcting the aged heated plasma with BaSO” adsorbed

plasma.

Experiment V concerned the stability of prothrombin at 45 C. The
results shown in Table 4, after reconstituting plasmas in all factors
except prothrombin, show very small changes in prothrombin levels over
the 60 minute incubation period. In addition, the oxalated aged plasma
kept prothrombin at a stable level for a period of 24 to 56 hours at
37 C.

Experiment VI presents a method by which Factor V concentration
can be estimated. As demonstrated, incubation at 45 C. is not necessary
in the new method, but to develop this method the incubation and dilu-
tions of the plasmas described in materials and methods were necessary.
In addition, this method presents a way of estimating concentrations of
Factors VII, X, and prothrombin at the same time. Various methods have
been presented for estimating Factor V concentration. Eichelberger
(1965) used the aged oxalated plasma described earlier as a substrate

for the reaction and found the concentration from a Factor V dilution

 

 

31

curve. This is time consuming when one must incubate plasma at 37 C.
for several days and is subject to bacterial and other types of contami-
nation. Some methods estimate the severity of a defect in Factor V by
the difference in the uncorrected and corrected prothrombin times after
the addition of BaSO” adsorbed plasma. The method presented after the
work in Experiment VI combines some features of both methods, resulting

in a more useful and accurate determination.

By further consideration of the data of Experiment VI, several con-
clusions can be made. It has been shown that there is a linear relation-
ship between the reciprocal of "prothrombin" concentration and clotting
time after optimal amounts of Ca2+ and tissue thromboplastin have been
added. The first analysis of the data was designed to test the hypothe-
sis that the abscissal intercepts were a function of the labile factor
concentration.

The slapes of the lines relating the reciprocal of concentration
to "prothrombin time" are assumed to be mathematically related to the
relative concentrations of labile and stable factors. If these hypothe-
ses are correct, all the lines should meet at a single point when
extrapolated.

The procedure for estimating the relative concentrations of the
thermolabile and thermostable factors (i.e., Factor V and Factors VII,

X and prothrombin) may be summarized as follows. Essentially the
procedure consists of measuring two prothrombin times; one in the usual
way and one on plasma diluted with an equal volume of saline. The

time required for each of these specimens to coagulate after adding

calcium and thrombOplastin is recorded as t1 and t2, respectively.

 

32

First, it is necessary to complete the calculation of the concen-

tration of Factor V as follows:

v - 67 - 3.5(2t1 — t2),
where v represents the concentration of Factor V expressed as a percent
of normal levels.

The value of 2t1 — t2 has been shown to be identical to the value
0 (see results).

It is emphasized that a theoretical method of obtaining the thermo-
stable component is presented. Further work is necessary to obtain
additional data and more accurate dilutions of this component are
needed. It is believed, however, that this pr0posed mechanism will

hold true in all cases.

SUMMARY AND CONCLUSIONS

The work presented here brings out several basic facts. The fac-
tor called prothrombin is relatively stable to incubation at 45 C., as
are Factors VII and X. Factor V is indeed thermolabile as most workers
believe, although it is reasonable to assume that under prOper condi-
tions its decomposition may be retarded. This will only hold true at
37 C. At 45 C. it is rapidly destroyed.

The last finding of this study is the principle by which the con-
centration of Factor V in an unknown plasma can be estimated. A
theoretical method is also presented whereby the relative concentration

of the thermostable factors can be found.

33

REFERENCES CITED

REFERENCES CITED

Ackroyd, J. F.: The formation of Factor VII. Brit. J. Hematol.,
2:397-411, 1956.

Alexander, B.: Clotting Factor VII (Proconvertin): Synonymy, prOper-
ties, clinical, and clinicolaboratory aspects. New Eng. J. Med.,
260:1218-21, 1959.

Anderson, G. and Barnhart, M.: Intracellular location of prothrombin.
Proc. Soc. Exp. Biol. Med., 116:1-6, 1964.

Brinkhous, K. M., Smith, H. P., and Warner, E. D.: Plasma prothrombin

level in normal infancy and in hemorrhagic diseases of the newborn.
Am. J. Med. Sci., 193:475-80, 1937.

Davie, E. W. and Ratnoff, O. D.: Waterfall sequence for intrinsic blood
clotting. Science, 145:1310-11, 1964.

Eichelberger, J. W., Jr.: Laboratory Methods in Blood coagulation.
Hoeber Medical Division, Harper and Rowe Publishers, New York,
1965.

Ferguson, J. H., Ennis, E. G., Iatridis, P. G. and White, N. 3.: Basic
a3pects of blood clotting: Thrombokinase and co-factors in the
conversion of prothrombin to thrombin. Thromb. Diath. Haemorr.,
18:647-63, 1967.

Ferguson, J. H., Johnston, C. L., Jr., and Howell, D. A.: A circulating
inhibitor (anti-AcG) specific for the labile Factor V of the
blood clotting mechanism. Blood, 13:382-97, 1958.
I
Ferguson, J. H., and Patch, M. J.: Determinants of the so-called "pro-
thrombin time". Proc. Soc. Exp. Biol. Med., 93:193—96, 1956.

Georgatsos, J. G., Hussey, C. V., and Quick, A. J.: Synergistic action
of Russell's viper venom and tissue thrombOplastin. Proc. Soc.
Exp. Biol. Med., 97:674-76, Jan.-Apr., 1958.

Hogenaur, K. and Deutsch, E.: Isolation and characterization of blood
clotting Factors VII and X. Thromb. Diath. Haemorr., 19:304-06,
1968.

Hougie, C.: The role of Factor V in the formation of blood thrombo-
plastin. J. Lab. Clin. Med., 50:61-68, 1957.

34

 

 

 

35

Hougie, C., Barrow, E., and Graham, J. B.: The Stuart Factor: A
hitherto unrecognized blood coagulation factor. Proc. 6th Cong.
Int. Soc. Blood Transfusion. 1956, Boston.

Hougie, C., Barrow, E., and Graham, J. B.: Stuart clotting defect I.
Segregation of a hereditary hemorrhagic state from the hetero-
geneous group heretofor called "Stable Factor" (SPCA, Procon-
vertin, Factor VII) deficiency. J. Clin. Invest., 36:485-96,
1957.

Johnston, C. L., Jr., Ferguson, J. H., O'Hanlon, F. A., and Black,
W. L.: The fate of Factor VII and Stuart Factor during the
clotting of normal blood. Thromb. Diath. Haemorr., 3:367-74,
1959.

Johnston, C. L., Jr., and Hjort, P. F.: Development of increased Fac-
tor VII activity during the spontaneous coagulation of blood.
J. Clin. Invest., 40, part 1:743-51, Jan.-June, 1961.

Koller, F., Loeliger, A., and Duckert, F.: Experiments on a new clot-
ting factor (Factor VII). Acta Haematol., 6:1-18, 1951.

Lanchantin, G. F., Friedmann, J. A., Hart, D..W., and Aronson, D. L.:
Studies on the mechanism of human prothrombin activation by
Factor X. Abstracts of Fed. Proc., Amer. Soc. Exp. 3101.,
53rd Ann. Meeting, Apr. 13-18, 1969. p. 639.

Liekin, S., and Bessman, S. P.: The effect of various EDTA complexes
on coagulation. Blood, 11:916-23, 1956.

Macfarlane, R. G.: An enzyme cascade in the blood clotting mechanism
and its function as a biochemical amplifier. Nature (London),

Marciniak, E. and Seegers, W. H.: AutOprothrombin C, a second enzyme
from prothrombin. Canad. J. Biochem. & Physiol., 40:597-605,
1962 O

Niemetz, J.: Factors influencing the consumption of Factor X (Stuart—
Prower Factor) during intrinsic coagulation. Thromb. Diath.

Quick, A. J.: The coagulation defect in sweet clover disease and in
the hemorrhagic chick disease in dietary origin. A consideration
of the source of prothrombin. Am. J. Physiol., 118:260-71, 1937.

Quick, A. J.: Hemorrhagic Diseases. Lea and Febiger, Philadelphia,
1957.

Quick, A. J.: Current blood clotting schemes. Thromb. Diath. Haemorr.,
16:318-30, 1966.

36

Quick, A. J., and Hussey, C. V.: Hereditary hypoprothrombinemias.

Rhoads, J. E., and Fitz-Hugh, T., Jr.: IdiOpathic hypOprothrombinemias,
an apparently unrecorded condition. Am. J. Med. Sci., 202:662-
70, 1941.

Seegers, W. H.: Purification of prothrombin and thrombin, chemical
preperties of purified preparations. J. Biol. Chem., 136:
103-11, 1940.

Seegers, W. H.: Prothrombin. Harvard Univ. Press, Cambridge, Mass.,
1962.

Seegers, W. H.: History of prothrombin activation in sodium citrate
solution. 16th Annual Symposium on Blood (Abstract). Wayne
State Univ. School of Medicine, Detroit, Mich. Jan. 19-20,
1968.

Seegers, W. H., and Alkjaersig, N.: Comparative prOperties of puri-
fied human and bovine prothrombin. Am. J. Physiol., 172:
731-36, 1953.

Seegers, W. H., and Marciniak, E.: Some activation characteristics
of the prethrombin unit of prothrombin. Life Sciences, 4:
1721-26, 1965.

Shanberge, J. N.: The effect of Dicumarol on certain factors of blood
coagulation. J. Lab. Clin. Med., 48:218-22, 1956.

Telfer, T. P., Denson, K. W., and Wright, D. R.: A "new" coagulation
defect. Brit. J. Hematol., 2:308—16, 1956.

Tishkoff, G. H., Pechet, L., and Alexander, B.: Some biochemical and
electrophoretic studies on purified prothrombin, Factor VII
(proconvertin) and Factor X (Stuart). Blood, 15:778-90, 1960.

Tishkoff, G. H., and Williams, L. M.: Studies on the structure and
activity Of bovine prothrombin. 15th Annual Symposium on Blood
(Abstract). Wayne State Univ. School of Medicine, Detroit,
Mich. Jan. 20-21, 1967.

Todd and Sanford: Clinical Diagnosis by Laboratory Methods, 13th ed.,
ed. by Davidsohn, I., and wells, B. B. W. B. Saunders Co.,
Philadelphia and London, 1963.

'Waddell, W. W., and Guerry, DuP.: Effect of vitamin K on the clotting
time of the prothrombin and the blood. With special reference
to unnatural bleeding of the newly-born. J.A.MLA., 112:2259-63,
1939.

 

APPENDIX I

Preparation of reagents rich in the clotting
factors of the prothrombin complex

37

38

BaSO“ Adsorbed Plasma

 

Collect blood in oxalate or citrate anticoagulant.
Immediately centrifuge for 10 minutes at 2000 rpm.
To each m1. of plasma add approximately 100 mg. of chemically pure

BaSO“.

Place in 37 C. water bath for 10 minutes. Invert tube several
times during this incubation.

Centrifuge for 10 minutes at 2000 rpm.

Remove upper 2/3 of plasma. It should have a one-stage prothrombin
time of over 1 minute.

Readsorption may be carried out until prothrombin time of over 1

minute is obtained.

Aged Plasma

Strict aseptic technique must be used throughout this procedure.
Separate the plasma from the cells by centrifugation at 2500 rpm

for 10 minutes.

Using a sterile disposable pipette, transfer the plasma to a sterile
screw-tOpped test tube.

Incubate at 37 C. for 24-56 hours or until the one-stage prothrombin
time exceeds 32 seconds. If citrate anticoagulant is used, the
incubation time may be longer.

Check prothrombin times at various intervals using aseptic tech-
nique when removing plasma samples from the stock mixture.

At the end of the 37 C. incubation, recentrifuge for 10 minutes at

2000 rpm. Remove plasma from any sediment formed.

 

39

Make a smear for Gram's stain of the sediment and inoculate a por-

tion of it on a blood agar plate to check for bacterial contamination.

Aged Serum

Obtain serum samples after clotting of normal human blood.
Pool 3 or 4 of these samples.

Allow to stand at 4 C. for at least 48 hours before using.

VITA

The author was born in Petoskey, Michigan, on June 21, 1942. He
graduated from Pellston Public High School in June, 1960, and enrolled
at North Central Michigan College in September of the same year. Upon
completion of 2 years of undergraduate work, he transferred to Central
Michigan University and received a 3.8. in biology and chemistry in
June, 1964. He completed one year of internship at St. Mary's Hospital
School of Medical Technology in June, 1965. In July 1965 he passed the
registry for medical technologists given by the American Society of
Clinical Pathologists.

Before entering graduate school the author worked at St. Mary's
Hospital and Grand Rapids OsteOpathic Hospital for 3 years. He entered
graduate school in the program of Clinical Laboratory Science offered
by the Department of Pathology at Michigan State University in January,
1968.

The author was married to Ruth Ann Nyp of Grand Rapids, Michigan,
on October 8, 1966, and has a son 8 months old. He is a member of the
Lansing, Michigan, and American Societies of Medical Technologists and
is active in society functions at the state and local levels.

In February, 1969, the author accepted a position with the Michigan
Department of Public Healthand is affiliated with the Bureau of Labora-

tory Improvement.

40

'1'
III
'5
I'll
1|
1
Ill
5