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An investigation of factors involved in the inception of blood coagulation.

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An investigation of factors involved in the inception of blood coagulation.

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Content A H IN V E S T IG A T IO N 0 3 ? F A C T O R S IN V O L V E D IN T H E IN C E P T IO N
O F B L O O D C O A G U L A T IO N
A Thesis
Presented to
the Faculty of the Graduate School
University of southern California
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
by
Gerard Franci s .Lanpha ntin
June 1954
UMI Number: DP21554
All rights reserved
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p h . D B^O ‘5 + L& .+?
This dissertation, written by
Gerard Francis Lanchantin
under the direction of. hXs.Guidan ce Committee, fyf#
and approved by all its members, has been pre
sented to and accepted by the Faculty of the
Graduate School, in partial fulfillment of re-
quirements for the degree of
D O C T O R O F P H I L O S O P H Y
Dean
D a te.. .y.lf
C om m ittee on Studies
ACKNOWLEDGEMENTS
This thesis could not have been written without
the aid of a great many people, most of w hom cannot be
properly credited on this page.
I should like to thank Professor Arnold G . Ware,
m y teacher and esteemed friend, whose contribution to
this work has been such as to make him almost a part of
To Drs. J. B. Field, R. S. Stragnell, and L.
Schwartz, I should like to express m y appreciation for
helping m e with a number of clinical cases. I am also
indebted to Dr. F. E. Davis and Miss A . A. Konugres of
the Transfusion Laboratory, Los Angeles County General
Hospital, for their cooperation in obtaining samples of
blood used in these experiments. For the suggestions
and comments from Dr. M . L. Lewis, Dr. R. I. McClaughry,
Prof. J. W . Mehl, and the students and staff of the
Department of Biochemistry, I am truly grateful.
The research reported in th is thesis was made
possible through the financial support of the United
States Public Health Service (R G 2684) and a grant from
the Medical Research and Development Board, Office of
the Surgeon General, Department of the Army, under
Contract No. BA.-49-007-M D-193.
TABLE OF CONTENTS
H IS T O R IC A L D EV ELO PM EN T.............................................................................
I. Introduction.......................................................................................
II. The Evolution of the Blood Clotting Theory . •
III. Present State of the Problem: The
Activation of Prothrom bin............................................
A. The Thromboplastic F a c to rs.......................................
B. Accelerators of Prothrombin Conversion . •
C. The Ant ithrombopla s t i n s ............................................
S T A T E M E N T O F T H E P R O B L E M A N D P L A N O F T H E E X P E R IM E N T
M A T E R IA L S A N D M E T H O D S .............................................................................
I. M a te ria ls ..............................................................................................
A. Fibrinogen . . . . . . ..................................................
B. Prothrombin . . .................................
G . Accelerator Globulin . . . . . . .......................
D . Thrombin.......................................................................................
E. Saline-Imidazole B u f f e r ............................................
F. Thromboplastin.......................................................................
G . Prothrombin-Free Beef Plasma (PFB) . ♦ . .
II. Methods
A. Physical Methods..................................................................
1. Dialyzing a p p a ra tu s ........................... . . .
2. Ultrasonic homogenization............................
iv
PAGE
3. Spectrographic analysis . . . . . . . 37
4. Zone electrophoresis............................................ 37
B. Biological and Chemical Methods . . . . . 38
1. W hole blood clotting time . . . . . . 38
2. Recalcified clotting t i m e ............................ 38
3. Determination of prothrombin
(prothrombin tim e )..................................' . . 39
a. One-stage methods . . . . . . . . 39
1). Quick determination....................... 39
2} • Owren m ethod................................. . 40
b. Two-stage method ............................ 40
4. Fibrinogen.................................. . ........................... 41
5. Accelerator globulin .................................... 41
6. Prothrombin consumption . . . . . . . 42
7. Thrombin ....................................... 43
8. Ant i thr esnbi n and heparin . . . . . . . 43
9. Protein . . . . . . . . . . . . . . . 43
44
11. Clotting system for demonstrating
thromboplastin a c t i v i t y ............................ 44
R E S U L T S A N D D ISC U SSIO N ............................................. 49
I. The Inhibition of Thromboplastin . . . . . . . 49
PAGE
A. Identification of a Thromboplastin
Inhibitor in Serum and in Plasma «... 49
1. Demonstration of thromboplastin
inhibitors in serum 49
2. Sedimentation of inhibited
thromboplastin and subsequent
reactivation by removal of
calcium • . . . • • • • • • • • • • • 53
3. Effect of oxalate upon the
demons tra ti on of thrombo-
plastin inhibitor in serum . .
4. The effect of barium sulfate
adsorption on antithrombo-
plastin a c t i v i t y .....................................
5. Identif ioat ion of anti thrombo
plastin in plasma......................................
6. Attempts a t isolation of an ti
thromboplastin • ................................,
7. Certain physical and biochemical
properties of the thromboplastin
inhibitor .......................................................
B. Clinical Studies on Antithromboplastin • • 74 j
*
58
59
63
65
70
vi
PAGE
1. Demonstration of antitbrambo-
plastin in v iv o ........................................... 74
2. Anti thromboplastin levels in
certain bleeding dyserasias.............. 77
G . An improvement in the Method for the
Identification of Antithromboplastin . • 78
D . A Report of a Case of Idiopathic
Antithromboplastinophilia , . . . . . . . . 85
1. H istory................................................. 86
2. Clinical laboratory findings and
coagulation studies ....................... 86
5* Therapy •••••••• ................................. • 90
4. Remarks.......................................................................... 95
II. The Activation of Thromboplastin by Calcium . . 95
A * The Activation of Thromboplastin
by Calcium as Measured in Crude
Clotting S y stem s.................................................... 95
B. The Activation of Thromboplastin by
Calcium as Measured in a purified
Clotting System ..................................................101
C. The Relationship Between Activated
Thromboplastin and Antithrombo plastin . • 104
vii
P A G E
III. An Investigation of the Anticoagulant
Properties of A nticephalin................................ 109
A. The Relationship Between the
Protein and Lipid Anticoagulants . . . . 118
17. Studies on Accelerator Globulin ............................. ISO
A. Mechanism of Action of Thrombin
in the Conversion of Plasma A cG
to scrum A cG ................................................ 120
B. Purification of Serum A cG ••*•...• 127
STE8MARY...................................................................................................... 1 3 2
BIBLIOGRAPHY................................................................................................ 136
LIST OF TABLES
T A B L E P A G E
I. Abbreviated Nomenclature . . . . . . . . . . . 25
II* Partial Chemical Analysis of a Semi-
Purified H um an Brain Thromboplastin
Preparation ............................................................. 52
III. Reagents in the Thromboplastin Assay
P ro cedure........................................................................................ 45
IF. Clotting Properties of Thromboplastin
After Incubation with Adsorbed,
Dialyzed Serum in the Presence and
Absence of Calcium •••*•.••••.•• 56
V . A Comparison of the Chemical and
Physical properties of Prothrombin,
Antithromboplastin, and Accelerator
Globulin................................ 71
VI. The Effect of Mixing Normal, Hemophilic,
and Antithrombopla sti nophili c Plasmas
on the Recalcified Clotting Time •••••• 88
VII. Coagulation Studies on a Case of
Idiopathic Anti thrombo pla sti nophi lia .... 89
ix
Various Dilutions of VIII. The Effect
H um an Brain Thromboplastin on the
Quick Prothrombin Time of Normal
91 Plasma
and
i of the 3X . Clotting System G
Purified Coagul
Isolated from H um an
on Pactors
and Tissues
LIST OF FIGURES
FIGURE PAGE
1. The Classical Theory for the
Coagulation of Blood................................................................. 4
2. Reactions Involved in the Formation
and Removal of the Blood C l o t ....................................... 7
i • • •
3. Factors Involved in the Activation
of Prothrombin................................................................................. 10
4. The Role of Accelerator Globulin in the
Conversion of Prothrombin to Thrombin .... 16
5. Flow sheet for the Preparation of
Prothrombin and Accelerator Globulin
from A C B Plasma . ........................... 23
6. The Ultraviolet Absorption Spectra
of a Semi-Purified H um an Brain
Thromboplastin . •................................................ 33
7. Dialysis Apparatus.............................................................................. 36
8. Clotting Times Produced by Various
Concentrations of Thromboplastin.............................. , 47
9. The Double Logarithmic Relationship
Between Thromboplastin concentration
and Clotting T im e ........................................... 43
xi
figure page
10. The Clotting Times Produced by
Untreated Serum, Thromboplastin,
and Calcium Chloride Incubated
Together at 37°C. and Added to a
Clotting system at In te rv a ls................................... . . 50
11. The Clotting Times Produced by Barium
Sulfate-Adsorbed Serum and Thrombo
plastin Incubated Together at 37°C.
with and without Calcium and Added
to a Clotting System a t Intervals . . . . . . 52
12. The Clotting Times Produced by Barium
Sulfate-Adsorbed Serum, Diluted
Thromboplastin, and Calcium Chloride
o
Incubated Together at 37 C. and Added
to a Clotting System at Intervals •••••• 54
13. The Effect of Oxalate on the Activity
of Thromboplastin Incubated with
Barium sulfate Adsorbed serum and
Calcium ................................................................. 60
14. The Relationships am ong Protein
Adsorption, Antithromboplastin
Activity, and Barium sulfate
Concentration in the Adsorption of Serum . . . 62
xii
F IG U R E P A G E
15. Tlie Inhibition of Thromboplastin
by Defibrinated Plasma and by
Serum from the Sam e Blood.................................................. 64
16. A Comparison of the Inactivation of
Thromboplastin by Barium Citrate-
Barium sulfate Adsorbed Serum and
Barium Sulfate Adsorbed serum . . . . . . . . SI
17. The Relationship Between Serum
Concentration and Thromboplastin
Inactivated........................................... 85
18. The Effect of Barium Citrate-Barium
Sulfate Adsorption of Plasma on its
Anti thrombo plastic Activity ....................... 84
19. The Effect of Incubation of Normal
and Antithromboplastinophilic Serums
with Calcium and Thromboplastin on
the Clotting Tim e and Thromboplastic
Activity ........................................................... 98
20. The Activation of Thromboplastin by
Calcium and its Dependence upon
Volume................................................................................................... 97
xiii
FIG U R E P A G E
21. The Double Logarithmic Relationship
of Thromboplastin Concentration to
Clotting Time During O ne Minute and
Sixty Minutes of Incubation of Thrombo
plastin with Calcium and B uffer.................................. 98
22. The Effect of Incubating Thromboplastin
and Calcium on the Clotting Time and
Thromboplastic Activity of a Purified
Clotting System Composed of Purified
Factors Isolated from H um an Blood and
Tissues ................................... . . . . . . . . . . . . 103
23. The Effect of Added Thromboplastin on
Inhibited Thromboplastin and Anti-
thromboplastin on Activated Thrombo
plastin .......................................................................................... * • 106
24. The Effect of the Activation of Thrombo
plastin by Calcium on the Inhibited
and Uninhibited Clotting System.................................. 110
25. The Effeet of Various Concentrations of
Lipid Anti coagulant on the Clotting
Times of T w o Thromboplastin Assay Tests . . . 112
xiv
F IG U R E P A G E
26. The Effect of Incubation of a
0.02 Per Gent Suspension of Lipid
with. Thromboplastin'and Calcium
on the Clotting Times of T w o Thrombo
plastin Test Procedures ........................... 114
27. The Effect of Prolonged Exposure of a
O ne Per Gent Lipid Suspension to O ne
Megacycle Ultrasonic W aves on some
of Its Physical and Biochemical
Properties ...................................... 116
28. A Diagrammatic Representation for the
Effect of the Lipid and Protein
Moieties, of Antithromboplastin on
the Inhibition of Tissue Thrombo
plastin . . .... . ... • ... . • . • • 121
29. A Working Hypothesis for Future
investigations on the Role of
Antithromboplastin in the Activation
of Prothrom bin.................................................................................. 122
30.. The Activation of Various Plasma
Accelerator Globulin Concentrations
by Thrombin ...................................................... 125
X V
F IG -D E E P A G E
31. The Relationship Between Proteolytic
Activity of Thrombin and its
Activation of plasma Accelerator
Globulin ................................................................... 126
32. Purification of serum Accelerator
Globulin by Chromatography on an
Amberlite IRA-400 Column .................................................. 130
H IS T O R IC A L D E V E L O H C E N T
I. Introduotion
Any historical development of the theories for the
coagulation of blood is necessarily limited in a paper of
this nature. The literatu re dealing with the blood clot
ting field is so extensive that only the major contribu
tions and developments concerning the more salient features
of this research problem can be discussed. In any review
of the literature in the coagulation field, one finds i t
requisite to be quite c ritical and selective in the choice
of references and the evaluation of certain data. This is
necessitated by the fact that the early literature is
crowded with reports of experiments conducted at a time
prior to the discovery of certain essential clotting factors.
M any publications deal with experiments with blood clotting
components isolated from several species which are not com
parable to the same components found in the human. S till
other reports l is t data, the nomenclature of which is con
fusing and often times misleading to the reader. I t is for
these reasons that the historical introduction to the pro
blem presented herein will be brief and primarily concerned
with the mechanism of blood clotting in the human speoies
as has been unequivocally substantiated up to the present
a
time.
Practically a ll ofx i the literature prior to 1940 has
been quite ably reviewed by Wohliseh (1). Owren has also
discussed many of the early experiments and amplified them
greatly (2). References to publications up to 1951 have
been presented by Elynn and Coon (5), while a thorough
treatment of the entire field can be found in a monograph
by Biggs and Maofarlane (4). Specific fields of endeavor
in enzyme chemistry (6,7,3), clinical procedures (9), and
therapeutic applications (10,11,12) have been published
recently. A complete development of the theoretical
approaches to the mechanism of coagulation since its early
beginnings has been presented by Milstone (15).
I I . The Evolution of the Blood Clotting Theory
Since the monumental discovery of the circulation
of the blood by William Harvey in 1616, physiologists have
speculated about the mechanisms controlling hemorrhage.
During the 18th century, numerous scientific approaches to
this problem resulted in the general belief that blood
ceased to flow after a vessel was traumatized due to a
constriction of the vessel its e lf and the formation of a
coagulum. Even as early as this w e find controversies as
3
to which of the two mechanisms was of primary importance.
By the advent of the 19th century, Jones* concept of the
events leading to hemostasis (14) took their roots and i t
was soon realized that the factors involved in the control
of the fluidity of the blood were quite complex. I t was
at this point, also, that surgeon and physiologist went
their separate ways, one attempting to control hemorrhage
by an investigation of the mechanical control of bleeding,
and the other attempting to form a chemical explanation for
the formation of the blood coagulum.
Earlier, Malpighi had discovered the fibrin thread
(15) but i t was not until 1877 that Hammarsten had found
that fibrinogen formed fibrin through the aetion of
thrombin (16). B y 1895, sehmidt discovered that thrombin
was formed from a precursor, prothrombin (17), and this
pieee of evidence coupled with the recognition of the
essentiality of calcium for the clotting of blood (18)
resulted in the formulation of the classical theory of
blood coagulation proposed by Morawitz in 1905 (19). This
scheme is presented in Figure 1. Although Morawitz »s
theory corroborated the ideas of Fuld and Spiro (20), i t
was challenged by the work of Nolf (21), Howell (22), and
Bordet (23). However, even though the classical theory
4
CLASSICAL THEORY
(MORAWITZ, 1905)
(l) Prothrombin ♦ Calcium +
Thromboplastin — >Thrombin
(£) Fibrinogen ♦ Thrombin
> Fibrin
FIG U R E 1
T H E C L A S S IC A L T H E O R Y F O R T H E C O A G U L A T IO N O F B L O O D .
5
]bas been amended by many workers, the basic reactions have
remained unabridged up to the present time. During the
early part of the 20th century, the chief controversies
centered around the role of thromboplastin and its relation
to platelets in the coagulation scheme. The discovery of
heparin (24) led Howell to postulate a sensitive heparin-
prothrombin complex which was responsible for the fluidity
of the blood in the circulation (25). Upon injury and
trauma with subsequent hemorrhage, the blood platelets
would break down, thereupon releasing a thromboplastio
substance which would in turn release prothrombin from
its anticoagulant complex allowing i t to be converted to
thrombin.
B y 1940, the role of vitamin K in the synthesis of
prothrombin was discovered and heparin and dieumarol were
finding use as therapeutic anticoagulants. After World
W ar II, a new factor, accelerator globulin, was discov
ered (2,26,27) and added to the essential components of
the classical theory. During this period of time, clot
ting antagonists such as anti thrombin and antithrombo
plastin received added emphasis as did the reactions
involved in the retraction and dissolution of the fibrin
clot its e lf. Further work on factors involved in the
formation of thrombin are, at present, s t i l l in a state
6
of flux as are the numerous roles that have been attributed
to the blood platelets. The elucidation of a ll of the
above factors involved in the production and removal of
the fibrin clot, when added to the classical Morawitz's
scheme, give a picture similar to that presented in
Figure 2.
Figure 2 depicts only the major reactions involved
in the blood clotting theory and is, at most, an over
simplification. In spite of a ll the work that has been
done on the mechanism of clot formation, there is s t i l l
comparatively little known. The removal of the fibrin
clot is , in fact, more of a mystery than is its formation.
The problem of clot retraction and removal has been amply
reviewed elsewhere (29,50,51) and need not be discussed
further. The factors involved in the conversion of
fibrinogen to fibrin are fairly well documented (5,52,53),
and hence only brief mention will be made of this reaction.
The real problem in the further elucidation of the coagula
tion mechanism concerns the factors involved in the incep
tion of clotting, i .e ., the activation of prothrombin.
7
F IG U R E 2
R E A C T IO N S IN V O L V E D IN T H E F O R M A T IO N A N D R E M O V A L
O F T H E B L O O D C L O T .
After Seegers (28)
CLOT FORMATION
Half of blood coagulation is concerned
with clo t formotion. Inhibitors pa rtici
pate in this mechanism and tend to
counterbalance.
PROTHROMBIN
Calcium
Thromboplastin
Ac-globulin
Platelet derivatives
Other factors
Inhibitors of
activation
Y
FIBRIN- th r o m b in fib r in
OGEN THROMBIN CLOT
+
Antithrombin
Y
Inactive thrombin
346
CLOT REMOVAL
The mechonism provided for the removal
of a clot is not prim arily an inhibitor of
clot formation. It can remove a clot and
probably serves other metabolic functions.
PROFIBRINOLYSIN
Fibrinokinase
Other factors
Inhibitors of
activation
FIBRIN
CLOT
FIBRINOLYSIN
FIBRIN
-► DERIV-
FIBRINOLYSIN AT|VES
Antifibrinolysin
Y
Inactive fibrinolysin
9
I I I . Present state of the Problem:
The Activation of Prothrombin
Perhaps the best method of introducing the problem
of prothrombin activation is to present the prevailing
scheme for the theory of blood d o ttin g as a protocol for
further discussion and investigation. At the present
time, Owren» s theory as to the factors involved in the
inception of blood coagulation (34,55) is generally
accepted by the majority of workers in the field. Owren»s
theory is depicted in Figure 5. In general, the nomencla
ture employed in Figure 3 w ill bo used in further refer
ences with the exception of the terms "proaecelerin" and
"aceelerin" in whose place "plasma accelerator globulin"
(plasma A cG ) and "serum accelerator globulin" (serum A cG )
w ill be substituted. Since the publication of ©wren’s
work the nomenclature in the field has been standardized
(36) while suitable synonyms for the terms used in th is
text m ay be found elsewhere (57,38).
In the Owren.outline of hemostasis as presented in
Figure 3, the sequence of events leading to prothrombin
conversion is in itiated by.contact, i.e . trauma. This m ay
occur internally or externally to the blood vessel through
injury, veni-puncture, sludging of blood, or the
10
8 . B L O O D CO AG ULA TIO N: T h e o r y o f P . A . O w ren (1951)
[Vasoconstrictor]
I Substance I
Contact
Platelet
Disintegration
antihemophilic ?
Globulin Tissues
Calcium
Contact
Proconvertin
Proaecelerin
Prothrombin
Thrombin
Fibrinogen Fibrin
F IG U R E 3
F A C T O R S IN V O L V E D IN T H E A C T IV A T IO N O F F R 0T H R 0 M B IN .
After Owren from Albritton (34).
' 1 1
t
! nondescript formation of thromboemboli. Upon such contact,
J
: a variety of problematical events take place of a physical 1
! I
I and biochemical nature. Tissue juice, containing thrombo-
j plastin enters into contact with the clotting proteins and
i catalyzes the early formation of thrombin from prothrombin.
!
1 Also, contact results in the breakdown of blood platelets
!
I with the release of a vasoconstrictor substance, serotonin :
I ;
j (5-hydroxytryptamine). The pharmacology of this substance
ihas been discussed elsewhere (39). Platelet disintegra-
: tion also results in the release of a clot-accelerating
t substance. I t was previously thought that this platelet
i
material was thromboplastin (40); however, isolation pro- ;
! cedures have failed to substantiate this point of ;
1 i
view (41,42). The most generally-accepted hypothesis ;
i . I
j reasons that this platelet material is a thromboplastin
co-factor which, when combined with a plasma globulin
(antihemophilic globulin), forms the plasma thrombo- 1
t
plastin (34). Therefore, at this point in the sequence
; of events, w e have the formation of thromboplastin from
i
the blood components and from the tissues. Thromboplastin i
in the presence of calcium ions then acts e n z y m a t ically in
. two ways. F irst, i t converts another plasma factor, pro
convertin, to a rather potent accelerator, convertin (not
1 12
! :
I
• diagrammed in Figure 3). Tills substance along with throm- ;
i i
i -
boplastin and calcium then converts a small portion of ,
' prothrombin to thrombin. Upon the formation of thrombin
i J
; in minute quantities another clotting factor is evolved
i
i
I from a precursor stage, plasma A cG (proaecelerin), to a
i .
j highly potent accelerator of prothrombin conversion, serum (
| A cG (accelerin). Serum A cG , thromboplastin, calcium, and j
' convertin are then instrumental in the rapid conversion of i
I !
! prothrombin to thrombin which subsequently polymerized
I
j fibrinogen to the insoluble protein, fibrin. These
i
i
i reactions leading to thrombin production are s t i l l some-
<
i i
; what controversial, particularly the mechanism of the
proconvertin to convertin reaction. ,
i I
| A. The Thromboplastic Factors j
i • t
The above scheme for the clotting theory has been j
substantially demonstrated by in vitro experimentation.
However, when one attempts to translate this sequence of
i i
: events into a reasonable explanation of in vivo mechanisms, :
1 a number of difficulties are encountered. If i t were not j
i
' for a number of bleeding disorders, which leave major gaps >
!
in our knowledge of the clotting mechanism, the whole pro- ,
; blem of blood coagulation would be simplified to the scheme!
i I
] of events presented in Figure 3. I
13
Brinkhouse et al. have recently summarized the
various types of hemophilia (43), The classical type of
the disease which is characterized by a prolonged clot
ting time and poor prothrombin consumption is probably
due to the lack of a clotting co-factor, antihemophiliac
globulin (A H C J). This substance appears to be a pg-globulin
isolated in Cohn fraction I (44,45). The disease is fur
ther complicated by the unusual stab ility of the hemophilic
platelets which do not lyse normally upon exposure to
foreign surfaces (46). This m ay be due to the deficiency
of a platelet lysing agent in hemophilic bloods. If such
is the case, i t can be readily seen from Figure 3 that the
hemophiliac suffers from the malfunction of certain re
actions necessary for the activation of prothrombin. H ow
ever, Tocantins believes that the anomalous clotting time
of hemophilic blood is due to an increase in a naturally-
circulating anticoagulant which he can isolate in a form
many times more active from hemophilic blood than
normal (47). If this be the case, then i t is difficult to
explain the experimental fact that less than one part of
normal blood or plasma w ill correct the prolonged clotting
time of nine parts of hemophilic blood.
The recent recognition of several other hemophilioid
14
states has complicated the further understanding of the
thromboplastic mechanisms. Deficiency diseases associated
with the lack of unrecognized clotting factors such as
plasma thromboplastin component (PTC ) and plasma thrombo
plastin antecedent (PT A ) have pointed out the necessity of
further research into the events occurring prior to the
conversion of prothrombin (48,49). In a ll probability,
A H G , PTC , and P T A as well as the factor in blood platelets
are essential components of the plasma thromboplastic
enzyme. The isolation of this enzyme (50,51) has dispelled,
many of the objections to its existence. Presumably, tissue
thromboplastin is a complete enzyme containing a ll the
necessary co-factors and activators.
From what has been said above, i t is quite obvious
that the thromboplastic mechanisms are quite complex. For
research purposes, tissue thromboplastin is the enzyme of
choice in the investigation of the blood clotting reactions.
This enzyme is quite ubiquitous in nature and i t is gener
ally believed that i t exists in a ll the cells of the
animal body. Since thromboplastin from tissues is capable
of extreme catalytic potency (52) in the conversion of
prothrombin to thrombin i t is perhaps wise to elucidate
the action of this enzyme in vitro before investigating
15
tlie circulating plasma thromboplastic factors.
B. Accelerators of Prothrombin Conversion
The proposed mechanism of action of accelerator
globulin (A cG ) is well known (55,54,55) and need not be
detailed here. That A cG serves to govern only the rate of
thrombin evolution can be seen from the reactions depicted
in Figure 4. A o g and thromboplastin are interchangeable
in regulating the rate at which prothrombin is converted,
i . e . , one can be substituted for the other in a modified
clotting system and the rate of clotting will remain the
sam e (5,56). However, a small quantity of thromboplastin
is absolutely necessary in order for activation to take
place while th is does not appear to be the case with AcG.
Thromboplastin and calcium alone w ill convert prothrombin
to thrombin slowly in the virtual absence of A cG , but A cG
and calcium alone w ill not activate prothrombin. Thus, i t
is seen that thromboplastin will activate the prothrombin
molecule and will also regulate the rate at which thrombin
is evolved, while A cG has only the la tte r property. Para
hemophilia is the term used to designate a lack of A cG in
approximately sixteen cases of this bleeding diathesis (45).
The role of convertin in the clotting scheme is
highly controversial. There is som e reason to believe
CALCIUM IONS
THROMBOPLASTIN
I. PROTHROMBIN----------------------- ^ THROMBIN
(slow reactio n )
The throm bin form ed by the interaction of these
three factors activates plasm a ACG as follow s:
THROMBIN
II. PLASMA ACG ----------------------- >-SERUM ACG
(inert) (active)
The serum ACG then accelerates reaction I as
follow s:
CALCIUM IONS
THROMBOPLASTIN
III. PROTHROMBIN-----------------------> ► THROMBIN
SERUM ACG
(rap id reactio n )
F IG U R E 4
T H E R O L E O F A C C E L E R A T O R G L O B U L IN
IN T H E C O N V E R S IO N O F P R O T H R O M B IN T O T H R O M B IN .
that this substance is nothing more than another form of
prothrombin (57) which has lost its ability to be con
verted to prothrombin but retains the ability to in itia te
the conversion of other prothrombin molecules to thrombin
in the presence of thromboplastin, calcium, and AcG. Most
of the physical and biochemical properties of convertin
parallel those of prothrombin and no convertin preparation
to date has been completely freed of minute traces of pro
thrombin. Since prothrombin its e lf m ay exist in several
states of reactivity (55,58) i t is difficult to clarify
this problem. And if convertin or its precursor, procon
vertin, are nothing more than derivatives of prothrombin,
how does one explain the bleeding dyscrasias associated
with a deficiency of this factor (59)?
C . The Antithrombopla stins
Thromboplastin inhibitors in the blood have received
J
l i t t l e attention until recently. Tocantins has presented
evidence interm ittently during the past decade which sup
ports the view that a thromboplastin inhibitor exists in
normal plasma (60,61). His work has culminated in the iso
lation of a lipid inhibitor from normal human plasma which
he named ”anti-cephalin" (47). This factor is a strong
inhibitor of clotting apparently acting as a thromboplastin
18
antagonist. I t is isolated as the methyl alcohol-soluble,
ether-soluble fraction of plasma and is obtained from the
hemophiliac in a form several times more active than normal.
This fundamental information is made even more interesting
by Tocantins* recent observations that hemophilic plasma
euglobulins can promote clotting in whole hemophilic plasma
if the inhibitors therein are made inactive by proper dilu
tion (63).
Overman and Wright described an inositol phosphatide
which was separated from plasma as well as from tissues and
from soy beans (65,64). This lipid is an inhibitor of clot
ting which was thought to act in a complex manner involving
thromboplastin. A method of preparation of the inhibitor
was not published but i t is assumed that the procedure was
similar to that described by Tocantins (47). yiala has
described a protein inhibitor of the firs t stage of clot
ting which is adsorbed from oxalated plasma by barium car
bonate, barium sulfate, and celite (65,66). The manner of
action of this inhibitor was not described.
M uch more impressive as an indication of a thrombo
plastin inhibitor in serum is the work of Schneider (67)
and of Thom as (68). These investigators demonstrated inde
pendently that human serum can effectively inactivate
19
tissue thromboplastin. Both used an in vivo assay teehnie
for thromboplastin which was based on the ab ility of this
substance to lei11 mice when injected intravenously. The
rapid death which resulted from injection of toxic doses
could be prevented by preliminary incubation of the throm
boplastin with serum. The inhibitor in serum was found to
be heat labile. Thom as was able to show that serum had no
effect on thromboplastin when calcium was removed by dialy
sis or by addition of oxalate* H e isolated thromboplastin
by high-speed centrifugation after i t had been incubated
with serum in the presence of calcium. The sedimented
material had no effect when injeeted into mice but its
clotting activity ms completely restored by addition of
oxalate. The inhibitor was separated from serum with
am m onium sulfate between the levels of 25 to 50 per cent
saturation and was not dialyzable through cellophane
membranes.
Thomas1 experiments provided proof that thrombo
plastin is inactivated by incubation with serum in the
✓
presence of calcium (68). Peculiarly enough, however,
neither Schneider nor Thom as was able to demonstrate this
effect successfully when in vitro thromboplastin assay
systems were used. The incubation of thromboplastin with
2 0
serum and calcium appeared to result in acceleration of
d o ttin g .
Making use of Thomas * observations, McClaughry suc
ceeded in identifying a thromboplastin inhibitor from
bovine lung extract (69). The thromboplastin in this pre
paration, which contained large amounts of blood, was firs t
sedimented b y * high-speed centrifugation in the presence of
calcium ions. The inhibitor was then dissociated from the
sediment by addition of oxalate followed by subsequent sep
aration from the thromboplastin. I t was then shown to have
the property of inactivating thromboplastin in the presence
of calcium ions and i t had no demonstrable effect on other
purified clotting components. The inhibitor was non-
dialyzable, and gave positive tests for protein.
In the work to be presented, an in vitro technic is
described for identification of a thromboplastin inhibitor
in human serum and in treated human plasma. The factor is
heat labile, non-dialyzable, and can be separated from
human serum by am m onium sulfate fractionation. I t was
found to be quite stable in serum at room temperature. I t
requires calcium ions in order to inactivate thromboplastin
and its effect is rapidly reversed by removal of the
calcium.
S T A T E M E N T O F T H E P R O B L E M A N D P L A N O F T H E E X P E R IM E N T
The purpose of the research was to study the factors
involved in the inception of the clotting of human blood.
L ittle is known concerning the events which occur in the
coagulation mechanism prior to the activation of prothrom
bin. The elucidation of these early changes in the eoagu-
lative properties of blood is of more than aeademie
interest since further knowledge of these ohanges will
eventually aid in the understanding of pathological bleed
ing, and in m any instances, provide a sound basis for
effective therapy.
The bleeding dyserasia in hemophilia is due to an
unknown factor or factors involved in the early stages of
blood clotting which are imperfectly understood. As m en
tioned previously, "there has been som e evidence presented
in the recent literature which indicates that a circulat
ing antithromboplastin m ay be responsible for the hem o->
philie syndrome.
I t was the intent of this research investigation to
identify and isolate antithromboplastin in human blood,
study som e of its biochemical properties, and investigate
its occurrence in certain hemorrhagic disorders. Studies
were also extended to the role of calcium in the clotting
22
mechanism as well as investigations of the bio chemical
characteristics of human accelerator globulin.
MATERIALS AND METHODS
The preparation of materials and the methods employed
throughout th is research project w ill not be given in
detail. Adequate references are presented in the text where
specific procedures were used. However, a short summary of
details and som e of the theory behind the use of certain
preparations and experimental assay procedures which were
employed routinely during the course of this investigation
are listed.
An amplification of the definition of the word
"purity” as used in the text dealing with the preparation
of materials is worthy of comment at this juncture. W hen
one speaks of purified enzymes or proteins, particularly
in reference to blood d o tting factors, either one of two
possible definitions is implied. Purity in this sense
refers to chemical and molecular homogeneity or may refer
to biological activity, several of the clotting factors
are pure according to the la tte r concept, i .e ., free of
other clotting factors, but they are certainly not free of
contaminating inert protein. Eor example, the best prepa
rations of fibrinogen thus far available (70) are devoid
of other clotting enzymes but many such preparations are
tainted with extraneous protein. O n the other hand,
24
prothrombin which has been brought to a point of high
activity (71) and by m any commonly-accepted standards
is chemically homogeneous (72,75,74), is s t i l l believed
by som e workers (75) to be contaminated with small amounts
of clot-promoting substances. This problem is certainly
d ifficu lt to circumvent since many of the clotting agents
exist in different stages of activity and certain "inert
contaminants" may, under specified conditions, give rise
to highly active coagulation factors.
Throughout the subsequent text, purity w ill be
used in reference to biological activity and distinguished
by i ts freedom from other known clotting factors by the
commonly accepted methods of assay.
For the sake of brevity and convenience, a number
of abbreviations w ill be used to designate clotting com
ponents and reagents. These abbreviations are presented
in Table I.
I. Materials
A. Fibrinogen
H um an fibrinogen was prepared from A C D plasma by
the freeze-thaw method of Ware, Guest, and Seegers (76).
H um an fibrinogen solutions were stored in a concentration
T A B L E I
A B B R E V I A T E D N O M E N C L A T U R E
Abbreviation Definition
A C D Plasm a Plasm a collected by tbs A m erican R ed Cross
containing U S P Acid-Citrate-Dextrose anti
coagulant
P ?B Prothrombin-free Beef Plasm a
A c G . Accelerator Globulin
A H O Antihemophilic Globulin
P T C Plasm a Throm boplastin C o m p o n en t
' P T A Plasm a Throm boplastin Antecedent
Thpln. Throm boplastin
Antithpln. Antithromboplastih
T C A Trichloro-aeetxc A cid
I
26
of 2 per cent olottable protein/ml. in 1.8 per cent sodium
chloride at -20°C. and diluted in half with d istilled water
before use.
Bovine fibrinogen was prepared from Cohn Fraction I
(Armour Laboratories, Chicago, 111.) by the method of
Laki (70). Bovine fibrinogen was stored as 1 per cent
olottable protein solutions in aquous 0.3 M K G 1.
Crude bovine fibrinogen preparations were made by
dilution of Cohn Fraction I to give a 1 per cent olottable
protein solution in 0.9 per cent sodium chloride. Such
preparations are contaminated with small amounts of pro
thrombin.
B. Prothrombin
Purified hum an prothrombin was prepared from freshly
collected A G D plasma by the method of Lewis and W are (77).
This procedure involves the adsorption of prothrombin on
barium citrate precipitates followed by elution and further
purification by am m onium sulfate fractionation. The final
product m ay be further concentrated by precipitation at
pH 4.8. The final yield of prothrombin activity was found
to be in the neighborhood of 50 to 70 per cent of the
original starting plasma with a protein concentration of
3.5 to 5.0 mg./lOO ml. of starting plasma. The specific
27
activity of such produets was approximately 2,000 two-
stage units/mg. protein. Electrophoretically, human pro
thrombin preparations isolated by this procedure demon
strated two peaks in the Tiselius apparatus. The flow
sheet for the preparation of prothrombin from human plasma
is presented in Figure 5 along with the protocol for the
preparation of hum an accelerator globulin from the sam e
starting material.
Purified bovine prothrombin.(71) #530818 having
approximately 2,530 two-stage units/mg. was kindly supplied
through the courtesy of Dr. Walter H . Seegers of W ayne
Univ. School of Medicine, Detroit, Michigan.
C. Accelerator Globulin
Plasma A cG was prepared by the method of Lewis and
W are (77) the flow sheet of which is presented in Figure 5.
A description of the isolation and purification of serum
A cG w ill be discussed in a later section of this paper.
D . Thrombin
Bovine thrombin was obtained from a commercial
source (Park Davis and Co.) in ampoules containing 5,000
units of topical thrombin. This material was found to be
contaminated with detectable amounts of prothrombin and
PP T A C -H
DISCARD S U PE R -PR 3L
DISCARD
p p t p r n r
DISCARD
s u p e r A c n r
DISCARD SU PER P R E
OiSCARD
P P T AC I E
DISCARD
P P T AC 3ZT
DISSO LV E AND DIALYZE
S U P E R AC m
DISCARD
SU PER PR HI
ADD S A S T O % SATN
CITR A TEO PLA SM A
ADD B * C L
SU PE R I
ADD N ^ S O ^ , CENTRIFUGE
S U P E R AC 3E
ADD NEUTRAL S A S
TO J j f e SA TN
P P T PR 1 2
DIALYZE, A D JU ST p H ,
CENTRIFUGE
P P T I
D ISSO LV E IN CITRATE
ADD B *C L
SU P E R A C - H
DILUTE 2 0 X : ACIDIFY
TO p H 5 . 5
ppt a c n r
DISSOLVE. A D O SODIUM
CITRATE TO 2 5 % CONC.
P P T AC 1 2
DISSOLVE, ADD NEUTRAL
S A S TO % SATN
p p t p r u
DISSOLVE AND
DIALYZE
ADO S A S TO H SATN
F IG U R E 5
F L O W S H E E T F O E T E E P R E P A R A T IO N O F P R O T H R O M B IN
A N D A C C E L E R A T O R G L O B U L IN F R O M A O D P L A S M A .
After Lewis and W are (?•?).
29
required further purification. Prothrombin was removed by
dissolving the contents of each ampoule in 2.5 ml. of
0.9 per cent sodium chloride containing 0.02 M potassium
oxalate, adsorbing with 20 m g. of barium sulfate/ml. for
30 minutes, centrifuging, recalcifying the supernatant with
exactly 0.1 ml. of 0.5 M calcium chloride and adding c.p.
glycerol to a final volume of 5 ml. This standard thrombin
solution was found to be stable at refrigerator tempera
tures for several months. I t had a protein concentration
of approximately 10.74 mg./ml. which was 5 to 5 mgs. of
protein/ml. less than the original unpurified thrombin
preparation when diluted to 5 ml. volumes.
E. Saline-Imidazole Buffer
Buffer of p H 7.4 was prepared by dissolving 3 grams
of imidazole, G.P., (Edoan Laboratories) in 975 ml. of
0.7 per cent sodium chloride solution and 25 ml. of 0.5 N
hydrochloric acid.
E. Thr ombopla st in
Most of the worh to be described has been done with
thromboplastin of human origin, although other brain throm
boplastins have been demonstrated to behave similarly.
H um an brains, removed from cadavers not more than 24 hours
post mortem, were used as a source of thromboplastin. The
brains were freed of blood vessels, minced in a Waring
Blendor, and dried with acetone by standard tecimics.
Five grams o f.dried brain were extracted with 100 ml. of
0.9 per cent sodium chloride containing 0.002 M potassium
oxalate. The inclusion of a small amount of oxalate in
the saline solution results in a more active product as
has been demonstrated by others (82). The extraction was
carried out at 45 to 50°C. for approximately 50 minutes
with occasional stirrin g . The suspension of brain was then
freed of gross particles by light centrifugation.
The work to be described demanded a semi-purified
thromboplastin preparation which could be sedimented by
high-speed centrifugation and recovered in high yields.
This was accomplished by centrifuging the crude product
described above for 2 hours a t 28,000 G - . in an angle head.
The white pellet of thromboplastin was then resuspended in
its original volume of oxalated saline with the aid of a
Potter-Elvehjem glass homogenizer. This process was then
repeated two more times. After the la st centrifugation,
the sediment was taken up in saline-imidazole buffer to
10 times its original volume. This preparation was almost
water clear and was found to retain its activity when
stored for several months at -20°0.
I A partial chemical analysis of one preparation of
: thromboplastin suspension described above is presented in
i
Table II. The discrepancy in protein values as determined
from the modified Biuret method and that calculated by
i
f nitrogen analysis is* in a ll probability* due to som e
nitrogen-containing phospholipids and the varied percentage
I of nitrogen in different proteins. The ultraviolet adsorp
tion curve for human brain thromboplastin suspensions is
depicted in Figure 6.
[
| G . Prothrombin-Free Beef Plasma (PFB)
I
| This reagent was used in a thromboplastin assay pro
cedure to provide a stable source of fibrinogen and aocel- '
erator globulin. I t had been used quite successfully for !
j
a number of years in a modified one-stage prothrombin test '
i • :
t (82). Freshly-collected beef blood was added to a solution:
of 0.1 M potassium oxalate (proportion of 9 to 1). The j
plasma was removed after centrifugation and mixed with ^
i
1 freshly-precipitated barium sulfate. Equimolar solutions
|
; of barium chloride and sodium sulfate were mixed and the
I precipitate was washed twice with distilled water. T w o
t
t grams of centrifuged packed barium sulfate (dry weight)
> were added to each 100 ml. of plasma, thoroughly dispersed,
i
: and allowed to stand a t room temperature for about SO
TABLE II
PARTIAL CHEMICAL ANALYSIS GF A SEMI-FURIFIED
H U M A N BRAIN THROMBOPLASTIN PREPARATION
Constituent
Protein
Nitrogen x 6.25
Total phosphorus
Total cholesterol
Trace elements:
C opper
L ead
C alcium
Silicon
j3gMg*/frLU
580
8 1 0
26.5
.,-1 8 .. I;-.
M ethod
M odified biuret (78)
Kjeldahl
Fisks and S u b b a R o w (7?)
Schoenheim er and Sperry (80)
Spectrographic analysis
Butt et al. (81)
33
0.5
0.4
p 0.3
o
X
u 0 2
y
u_
< _ >
Ld
& 0.1
0.0
3 2 0 280 3 00 2 6 0 240 220
WAVE LENGTH CnuO
FIG U R E 6
T H E U L T R A V IO L E T A B S O R P T IO N S P E G T R A
O F A SEM I-PU RIFIED H U M A N B R A IN T H R O M B O P L A S T IN .
34
Legend to figure 6
Specific extinction, K = log. Ip /l
cl
"W here c = Nitrogen in mg./ml., 1 » cell length in em .
and log I 0/ l s optical density read on a Beckman D U
quartz spectrophotometer. Thromboplastin preparation
in 0.153 M sodium chloride, p H 7.0.
35
minutes. The barium sulfate was then removed by centrifu
gation and each 100 ml. was run through a Seitz f ilte r of
40 to 50 per cent asbestos content. The plasma was then
. dialyzed against saline u ntil free of oxalate and diluted
with an equal volume of 0.9 per cent sodium chloride con
taining 0.6 per eent imidazole. The resultant solution
was then adjusted to p H 7.4 with 0.5 M hydrochloric acid
and frozen at -20°C. Prothrombin-free beef plasma treated
in the above manner had a protein concentration of 35 mg./
ml.
II. Methods
A. Physical Methods
1. pialyzing apparatus. The dialysis apparatus and
membranes employed throughout this study were essentially
those of Lewis and Ware (77). The dialysis contrivance is
presented in Pigure 7.
2. Ultrasonic homogenization. Throughout this inves
tigation an ultrasonic homogenizer was necessary to a c ti
vate or denature certain preparations. The machine em
ployed during the major part of this research was a Hyper
sonic Generator Model BU-204 manufactured by Brush Develop
ment Go., Cleveland, Ohio. I t had an output of 300 to
STIRRING MOTOR
HEAVY
RING STAND
SIDE VIEW
^ — COUNTER WEIGHT
STIRRING MOTOR
HOLLOW BRA SS TUBE BEARING
LIGHT WEIGHT BAR
L ITER
CYLINDERS
DIALYSIS BAG
-WEIGHTED CLAMP
FRONT VIEW
FIG U R E 7
D IA LY SIS A P P A R A T U S .
Af ter Lewis and W are (77).
37
1000 kc. with 250 watts of power and was operated a t 22°C.
at a cathode current of 225 milliamps (B.C.) with a 400 or
1000 kc. transducer. Materials to he treated were placed
in 15 ml. weighing bottles and suspended in water over the
geometrical center of the focused ceramic howl. In earlier
experiments, a similar instrument using an oil medium as
wave conductor and operating at 400 kc. was used through
the courtesy of the California Institute of Technology.
3. spectrographic analysis. All analyses for trace
metals were conducted hy the method of Butt et a l. (81) on
an Applied Research Laboratories (Glendale, California)
Spectrograph with a 1.5 meter grating. Samples, after
ignition and dilution in suitable buffer, were placed on
high purity electrodes and exposed for 13 to 17 seconds
to a 7.5 to 12.5 amp. (D.C.) arc. The intensity of a
given line spectra was then read on a densitometer and the
values interpolated from a standard ourve.
Zone electrophoresis. Electrophoresis on f ilte r
paper was carried out by a method similar to that of Kunkel
and Tiselius (83), The electrophoretic strips were devel
oped for protein by the method of these authors while a
lipid staining procedure was modified from the Nile Blue
Sulfate method previously used for histological
58
slides (84,85).
B * Biological and Chemical Methods
1. Whole blood clotting time. The performance of this
test is based on the original experiments of Lee and
White (86). Several modifications of this method were
utilized but a ll were based on the technic of Fahey (87).
The determination, in brief, is as follows. Blood was
taken by direct phlebotomy into a syringe and aspirated
into 5 glass tubes. The tubes, at 37°C., were tilte d every
30 seconds one at a time starting with the f ir s t until a
clot formed. This procedure was followed with the second
and then the third tube. The clotting time of the la st
tube was then taken as the whole blood clotting time. The
clotting time of normal blood, when determined by this pro
cedure, ranges from 8 to 15 minutes. In severe cases of
hemophilia, however, the whole blood clotting time m ay
exceed several hours.
2. Recalcified clotting time. In this procedure, 9
parts of blood are added to one part of 0.1 M potassium
oxalate or 3.2 per cent sodium citrate. The plasma is
obtained by centrifugation and diluted with saline to an
appropriate volume. Calcium chloride is then added to a
final concentration of 0.02 M and the clotting time noted.
59
Normal clotting values differ depending upon volume and
concentration of plasma in the final clotting system. In
the method used in this research, using volumes of 0.2 ml.
plasma, and 0.2 ml. of calcium solution, the normal value
was approximately 90 to 120 seconds.
3. Determination of prothrombin (prothrombin time).
a. One-stage methods.
i) • On!ck determination. This method was
originally designed by Quick In 1938 (88), and more details
have been given in a later publication (8). The method is
based on the principle that if excess thromboplastin is
added to recalcified plasma, the limiting factor that will
determine the velocity with which a clot w ill form w ill be
prothrombin. Therefore, ealcium and a high concentration
of thromboplastin in the form of a tissue extract are added
to plasma and the clotting time noted. In normal Indivi
duals the prothrombin time will be 12 seconds when rabbit
brain thromboplastin is used and approximately 15 seconds
using human brain preparations. Unfortunately, several
other clotting factors have been discovered since the test
was devised, such as A o g and hyper-active prothrombin,
which markedly effect the assay. I t is also quite obvious
that any change in fibrinogen concentration w ill also
40
vitiate the determination of prothrombin by this method.
The Quick Tim e is, at best, only an indication of the
coagulability of plasma rather than an accurate assay of
any one clotting factor.
2). Qwren method. This test was f irs t de
scribed by Owren (89) and later modified by W are and
StragneU (82). A stable, dried standard of prothrombin
is used as a control and exogenous sources of A cG and
fibrinogen are added to diluted plasma samples upon which
the determination is to be made. Units of prothrombin are
expressed in per cent of the activity of normal plasma
(i.e>., 100 per cent), as in the one-stage method. One-
hundred one-stage units are equal to the activity of a
1 to 10 dilution of normal plasma which will give a clot
ting time equal to the control prothrombin preparation.
I t must be emphasized that this method measures the rate
of prothrombin conversion and equates this velocity to
the concentration of prothrombin. This method was used
routinely throughout the course of this research.
Ji* Two-stage method. This method is based on the
publication of W are and Seegers (90) as modified by Lewis
and W are (77). I t is based on the to tal concentration of
prothrombin rather than the rate of conversion as is the
41
one-stage method. In this assay, diluted plasma or a pro
thrombin sample is incubated with thromboplastin extracts,
calcium and buffered bovine serum as a source of AcG. At
intervals of time, aliquots of this mixture are added to a
fibrinogen solution and the clotting time noted. Such a
procedure demonstrates the rate at which thrombin is
evolved in the incubation mixture. After a period of time
this rate of thrombin production is m axim um and the veloc
ity of fibrinogen clotting remains constant. A two-stage
unit is equal to that concentration that w ill clot a stand
ard fibrinogen solution in 15 seconds after the m axim um
rate of thrombin production has been achieved.
4. Fibrinogen. Fibrinogen was determined by the
method of Ware, Guest, and Seegers (76). A fibrinogen
solution was diluted and clotted with thrombin. The
fib rin clot was then dissolved in alkali and analysis for
protein, tyrosine, or nitrogen employed to determine con
centration. The concentration of a fibrinogen solution is
expressed in terms of per cent dottable protein, viz. ,
gms. clottable protein/100 ml.
5. Accelerator globulin. Plasma A oG and serum A cG
were determined by the method of Lewis and W are (91). This
method is based on the acceleration of an AoG-poor clotting
42
system when samples of A cG preparations are introduced.
O ne plasma or serum A eG unit is equivalent to 0.01 the
activity of normal human plasma.
§ .• Prothrombin consumption. The prothrombin consump
tion test (92) is based on the utilization of prothrombin
during the clotting of blood. W hen blood clots, not a ll
of the prothrombin is immediately converted' to thrombin.
Only 10 to 20 per cent of the prothrombin in normal blood
is in itia lly converted to thrombin, which is more than
enough to catalyze the fibrinogen to fibrin reaction. I t
is not until an hour after coagulation has taken place that
80 to 90 per cent of the prothrombin is in the form of
thrombin. In som e serums, several hours old, there is as
much as 10 per eent of the original prothrombin remaining.
In the running of this te st, blood was allowed to. clot in
glass, and a t intervals of time, aliquots of the serum were
taken and treated with citrate to arrest the clotting
mechanism. The fibrin clot was removed and the serum
freed of cells by centrifugation. The resulting fluid
was then assayed for prothrombin. In hemophilia, as in
other bleeding dyscrasias, prothrombin consumption is
poor and much of this precursor enzyme remains in the
serum m any hours after coagulation has taken place. In
many instances, the prothrombin activity of the serum will
increase as much as 300 to 300 per cent of its original
activity due to the production of a hyperactive type of
prothrombin as was previously discussed.
Z * Thrombin. Thrombin was measured simply by its
ability to clot a standard fibrinogen solution. A unit
of thrombin (Iowa unit) is that concentration of thrombin
which w ill clot a standard fibrinogen solution in 15
seconds (90). — ^
8. Anti thrombin and heparin. Anti thrombin was meas
ured by the modification of the method of G riffith (95).
I t consisted of incubating antithrombin solutions with
thrombin and measuring the thrombin activity in a given
time interval. The procedure followed was merely quali
tative. Heparin was measured by its ab ility to catalyze
the antithrombin reaction. This method was easily quanti
tated while heparin activity was measured qualitatively
by its loss of activity upon reversal with protamine.
9. Protein. Protein was measured by the modified
£ /
-/The definition and measurement of the thrombin
unit are given in detail in the M inim um Requirements of
Dried Thrombin, Second Revision, September, 1946, issued
by the Division of Biologies Control of the National
Institute of Health, Bethesda 14, M d.
44
Biuret method of Daughaday, Lowry, Rosebrough, and
Fields (78).
10. Calcium. All calcium solutions were standardized
by the assay of Lewis (94).
11. Clotting system for demonstrating thromboplastic
activity. Prothrombin-free beef plasma was used as a
stable source of fibrinogen and accelerator globulin. As
a source of prothrombin, freshly-reconstituted dried human
plasma was employed. The plasma had been originally dried
in 1945 and was found to contain a normal amount of pro
thrombin which remained stable for many hours after recon
stituting (82). For the purpose of this test i t was
diluted with d istilled water to 10 times the volume of the
original plasma. Thromboplastin and an optimal amount of
calcium were also added to m ake a complete system which
was quite sensitive to changes in thromboplastic activity.
The entire system was easily reproduced on successive
working days and gave remarkably constant clotting times
with thromboplastin of known activity. All tests were
carried out at 37°C. The reagents were preheated to 37°C.
and then added to 75 x 10 m m . te st tubes in the volumes
and order noted in Table III.
In the absence of thromboplastin, this system
TABLE III
R E A f iE N T S IN 'T H E T H R 0 M 3 Q P L A S T I N A S S A Y P R O C E D U R E
V o lu m e
Reagent
added
C o m p o n en t added to
; clotting system
0.1 ml. 1:10 dilution of
A C D Plasm a
Prothrom bin
0.1 nil. Pr othrom bin-JT re e
beef plasm a .
Accelerator globulin
and fibrinogen
0.1 m l. ~
4 V •
Imidazole-saline ;
buffer, p H 7 .1 * 0
0.1 ml. Thromboplastin Throm boplastin
0.1 ml. 0.015 H C aC l2 C alcium
clotted in approximately 320 to 480 seconds, depending upon
the prothrombin concentration of the reconstituted A cD
plasma. With a thromboplastin which gives 12 to 13 seconds
•with normal plasma in the Qjuich prothrombin time, this
system.will clot in approximately 33 seconds. Using the
purified thromboplastin described above, clotting times of
about 40 seconds are obtained. Dilutions of this thrombo
plastin result in progressively longer clotting times which
are indicated in Figure 8. Comparable to the findings of
Glendening and Bage (95) and Mills (96), the logarithm of
the thromboplastin concentration in this system was found
to be a straight line function of the logarithm of the
clotting time over a wide range, as seen in Figure 9. The
curve in Figure 9 was constructed from the average of 63
clotting times of different thromboplastin concentrations.
460 ~ j
300-
280-
260-
240-
W
220 -
P 200-
o 1 8 0 "
g 160-
P 140-
1 2 0 -
l O O -
80-
60-
40-
2 0
1 0 0 80 60 70 90 40 50 2 0 30 1 0
. 200
PERCENT O F THROMBOPLASTIN
FIG U R E 8
C L O T T IN G T IM E S P R O D U C E D B T
V A R IO U S C O N C E N T R A T IO N S O F T H R O M B O P L A S T IN .
Clotting system described in text.
CLOTTING T IM E (seconds)
48
4 0 0
3 0 0 -
2 0 0
4 0
20-
10
10 too Q 0 1 lO O O 0 1 1 lOOOO
PER CENT OF THROMBOPLASTIN
IT IC rU E R 9
T H E D O U B L E L O G A R IT H M IC R E L A T IO N S H IP B E T W E E N
T H R O M B O P L A S T IN C O N C E N T R A T IO N A N D C L O T T IN G T IM E
RESULTS AND DISCUSSION
I. The Inhibition of Thromboplastin
A . Identifioation of a Thromboplastin Inhibitor in Serum
and in Plasma
1. Demonstration of thromboplastin inhibitors in
serum* W hen normal human serum was substituted in the
clotting system above in place of the buffer, a small
amount of acceleration of clotting was usually noted.
W hen the thromboplastin, calcium chloride, and serum
were allowed to incubate a t 3?°C. before being added to
the other two components of the clotting system, variable
responses were obtained with different sera. As a general
rule, an acceleration phase developed during the f ir s t few
minutes which was then followed by inhibition. Fresh
serum usually exhibited more acceleration than aged serum.
A typical experiment is presented in Figure 10.
I t was suspected that the acceleration phase was
dependent upon the amount of prothrombin remaining in the
serum as well as upon the evolution of an accelerator which
was not present in our test system. Such a factor has been
described recently in the literature (97,98,99,100). since
this faetor, as well as prothrombin, is known to be
adsorbed on barium sulfate i t was of interest to te st the
CLOTTING TIME (seconds)
50
iao - 1
n o -
1 0 0-
90-
80
70-
60-
50-
40-
30
ao
60 50 40 30 2 0
INCUBATION TIME (minutes)
1 0
FIG U R E 10
T H E C L O T T IN G T IM E S P R O D U C E D B Y U N T R E A T E D S E R U M ,
T H R O M B O P L A S T IN , A N D C A L C IU M C H L O R ID E IN C U B A T E D T O G E T H E R
A T 37 °C. A N D A D D E D T O A C L O T T IN G S Y S T E M A T IN T E R V A L S .
51
inhibition with serum which had been adsorbed with barium
sulfate.
Previous experience had shown that the accelerator
factors can be removed more efficiently with barium sulfate
when oxalate is present. Therefore, oxalate was added to
the serum to a strength of 0.02 M and freshly-precipitated
barium sulfate was added at a concentration of 2 grams
(dry weight) per 100 ml. of oxalated serum. The suspen
sion was dispersed thoroughly, allowed to stand a t 37°0.
for 45 minutes, and the barium sulfate was removed by
centrifugation. The serum was then dialyzed against
saline u n til free of oxalate and adjusted to pH 7.4 with
0.5 per cent acetic acid.
’ W hen the barium sulfate adsorbed serum was incu
bated with thromboplastin and calcium chloride, a marked
inhibition of thromboplastin activity was apparent almost
immediately. W hen calcium was eliminated during the incu
bation period, the inhibition of thromboplastin was m uch
less. The results of a typical experiment are presented
in Figure 11. W hen expressed in terms of thromboplastin
activity (ordinates to the right of Figure 11) i t is
apparent that the serum was capable of inactivating more
than 95 per cent of the thromboplastin.
t* ofed>
w
p , p , p,
ca G O 09
o o o o
0»1>i4
O 'O 'O 'O '
O C D ( D < 0
*c( P iP ip i
H
P C Q C O to
M ® ® ®
cfH
O
P
%
P P
o y P e *
p P .& P *
H 4
O O e f o
H.JB jjj-g
Q O
P
%
%
< t >
H
I S'S1
• o w
Htt
P H *
C O P
c + '*
H *
J3 P
P
p,
O
P
H
O
H *
W
a
>■
0 3
F IG U R E 11
CLOTTING TIME (seconds)
O '
O '
G
w
O -
PERCENT OF CONTROL THROMBOPLASTIN ACTIVITY
C
53
i
1 W hen a smaller amount of thromboplastin was inou-
i
j bated with adsorbed serum in the presence of calcium, the
: inhibition was increased to a further extent (Figure 12).
I The thromboplastin used for this experiment (Figure 12) >
. was one-tenth the concentration employed previously. The
j clotting activity was reduced to a level of 213 seconds j
i
after a 100-minute incubation period with serum. W hen the
■ * j
| more concentrated thromboplastin was used (Figure 11), the
i ;
; inhibition reached a m axim um at a clotting activity of 107 ;
seconds. The difference in actual thromboplastin Goncen- ,
: tration between 107 and 213 seconds is approximately five-
1 fold (see Figure 10). Thus, the activity of the thrombo-
i
| plastin remaining after incubation appears to be propor- i
I I
i tional to the quantity of thromboplastin used. I
I
[ 2. Sedimentation of inhibited thromboplastin and sub- ;
* t
sequent reactivation by removal of calcium. The phenom
enon demonstrated in Figures 11 and 12 is actual inhibition^
i 1
I of the thromboplastin as indicated by the following experi-j
i |
, ments. O ne volume of barium sulfate-adsorbed serum was I
i incubated with one volume of thromboplastin and one volume !
of 0.015 M calcium chloride for a period of 2 hours at
| 37°C. The thromboplastin was then sedimented at 28,000 x
; g for 2 hours at 0°0. At the end of this period the
i i
a>
< &
©
P
< *
w
M
g
p !
d -
0 )
p .
« 4 > 3 i
I s a
* O S S
O'*
INCUBATION TIME (minutes)
CLOTTING TIM E (seconds)
3 8 8 8 5 8 8 8 8 8 3 8 8 8 5 ‘8 8£S8
o ’
O "
O "
O ’
o*
O "
88 s n s o
PERCENT OF CONTROL THROMBOPLASTIN ACTIVITY
o »
55
supernatant •w a s carefully removed by decantation and the
thromboplastin m s resuspended in 5 volumes of buffer con
taining 0.005 M calcium chloride. The suspension was dis
persed with the aid of a glass homogenizer before its
clotting activity was tested. A duplicate experiment was
performed in which the thromboplastin was treated in
exaetly the same manner except that i t was resuspended
and homogenized in buffer alone, the -calcium chloride
being added immediately before testing its clotting
ability. T w o controls were also run; one in which throm
boplastin was incubated with calcium and buffer; and, the
other in which thromboplastin was incubated with serum and
buffer. Both were then sedimented, resuspended, and
homogenized in buffer containing 0.005 M calcium chloride.
The clotting times of the four thromboplastins
described above were then determined as before by adding
0.5 ml. of each resuspended homogenized sediment to 0.2 ml.
of an equal mixture of prothrombin-free beef plasma and
reconstituted hum an plasma. The clotting times are indi
cated in Table IV along with the apparent amount of throm
boplastin activity interpolated from Figure 10. Except in
experiment 2 of Table IV, the results are the average of
two experiments.
TABLE IV
CLOTTING PROPERTIES OF THROMBOPLASTIN AFTER INCUBATION
WITH ADSORBED, DIALYZED SERUM IN THE PRESENCE A N D ABSENCE OF CALCIUM
E x p erim en t
No*
M ix tu re
in c u b a te d
f o r 2 h o u rs
a t 37°C . :
T h ro m b o p la stin
se d im e n te d an d
r e c o n s t i tu t e d i n
C lo ttin g
tim e i n
se co n d s
P e r c e n t
T h ro m b o p la stin
a c t i v i t y
re c o v e re d
1 Serum
C alciu m :
TPLN *■
0,00$ U
C alcium
106
5
2 Serum
C aleiu m
TPLN
B u ffe r 1*2 85
3
B u ffe r
C alcium
TPLN
0 .0 0 5
C alciu m i n
B u ffe r
1 *0 100
k
Serum
B u ffe r
TPLN
O.OQ5 M
C a lc iu m i n
B u ffe r
1*0 100
$ '
S u p e rn a ta n t fro m Exp* 1
-which c o n ta in e d 0*00$ M
C alciu m an d b u f f e r
1 * 6 5 L e ss th a n 1
TPLN* - T h ro m b o p la stin
57
I t is apparent from the f ir s t experiment in
Table IV that serum actually inhibits thromboplastin in
the presence of calcium. Only 5 per cent of the activity
remained whereas, in experiment 2, thromboplastin not
incubated with serum was dem onstrated to be completely
recoverable. I t is of interest that there was no appar
ent difference in the quantity of the thromboplastin
sediments in experiments 1 and 5. This indicates that
the inhibitor does not change appreciably the physical
properties of the thromboplastin. The action of the inhi
bitor was completely prevented by eliminating calcium
during the incubation period (experiment 4) and the throm
boplastin was actually recovered in 100 per cent yield.
Experiment 2 provides confirmation of Thom as* observations
that the effect of the inhibitor can be reversed by re
moval of calcium (68). In this experiment* the serum and
thromboplastin were incubated in the presence of calcium
but the thromboplastin sediment was resuspended in a
calcium-free medium. This resulted in the regeneration
of most of the thromboplastin activity.
The supernatant serum from experiment 2 was exam
ined for clotting properties after the thromboplastin had
been sedimented. The clotting time of 465 seconds was
58
almost the same as the clotting time of the system without
serum or thromboplastin (480 seconds). This'indicates
that very l i ttle active thromboplastin remained in the
supernatant and also'eliminates the possibility of the
production of an inhibitor when thromboplastin, serum,
and caioium were incubated together.
® • Effect of oxalate upon the demonstration of
thromboplastin inhibitor in serum. An assay system was
originally used which contained oxalate in the prothrombin-
free beef plasma. This was compensated for by adding
enough calcium chloride in the final step, to react with
the oxalate and to provide an optimal amount to achieve
the shortest clotting time. I t is of interest that this
system did not demonstrate as m uch inhibition of thrombo
plastin as the oxalate-free system described earlier. This
was interpreted to mean that the oxalate caused partial
reversal of the inhibition during the short period when
the thromboplastin and the prothrombin-free beef plasma
were in contact before clotting took place.
In order to demonstrate the effect of oxalate, pro-
thrombin-free beef plasma was prepared containing two dif
ferent concentrations of potassium oxalate, 0.015 M and
0.05 M . The clotting system was examined using untreated
59
thromboplastin to find the optimum level of calcium
chloride* This was found to be 0*025 M for both oxalate
concentrations and the clotting times were found to be
slightly shorter when oxalate was present. The thrombo-
* •*
plastin was incubated with equal volumes of barium sulfate
adsorbed serum and 0*025 M calcium chloride. At intervals,
aliquots were removed and tested for clotting activity as
before except the oxalate containing prothrombin-free beef
plasma was used. The results are indicated in Figure 15,
and compared to the oxalate-free system. I t is apparent
that thromboplastic activity is influenced by the presence
of soluble oxalates. M uch better success has been
achieved in demonstrating inhibition of thromboplastin
when oxalate-free systems are used.
4. The effect of barium sulfate adsorption on anti-
thromboplastin activity. The experiments cited above have
\
shown that when aged serum is adsorbed for 30 minutes with
20 m g. of barium sulfate/ml. in the presence of 0.02 M
potassium oxalate with subsequent dialysis to remove the
oxalate, the solution obtained had the property of inac
tivating thromboplastin in the presence of calcium. Fur
ther experiments with fresh serum demonstrated the fact
that inhibition was sometimes preceded by an acceleratory
60
1 2 0 - i
U O -
100-
■3 ao-
w
2 80-
70-
50
6
40
30
1 0 30 40 50 60 70 80 90 lOO HO ISO
INCUBATION TIME (minutes)
F IG U R E 13
T H E E F F E C T O F O Z A IA T E O N T H E A C T IV IT Y O F T H R O M B O E L A S T IN
IN C U B A T E D W IT H B A R IU M S U L F A T E A D S O R B E D S E R U M A N D C A L C IU M
A. N o oxalate present
B. 0,015 M oxalate in the prothrombin-free beef plasma
C. 0.03 1 oxalate in the prothrombin-free beef plasma
Optimum calcium was provided during the measurement of
the clotting time.
6 1
phase of clotting. The reason for this effect was ascribed
to the presenoe of residual prothrombin in the fresh serum
which was not removed by the barium sulfate. I t was there
fore of interest to test the effect of barium sulfate in
the demonstration of inhibitor activity by serum. Figure
14 (A .) presents a typical experiment in which varying
quantities of barium sulfate were employed in the adsorp
tion of fresh serum. Within the range of 20 mg./ml. to
200 mg./ml. the barium sulfate appears to adsorb protein
on a log.-log. basis, as one might expect from other known
adsorption isotherms. The data in. Figure. 14 (A ) have been
confirmed by Alexander (75). Also, (Figure 14 (B)) the
thromboplastin inactivated by the serum in the presence of
i f
calcium at the various times of incubation shows a similar
relationship. However, after equilibrium has been reached
in 50 minutes incubation time, this proportionality no
longer holds and the thromboplastin inactivated appears
to be independent of the barium sulfate employed to adsorb
serum.
r
I t would appear, therefore, that som e substance,
which is adsorbed on barium sulfate, is antagonizing the
rate at which the serum is inhibiting thromboplastin.
This substance does not affect the final equilibrium,
62
2.4
£ 2.0
0 0
IB
cT
CO
m 1.6
c o
l O
1. 0 1. 5
M S . PROTEIN ADSORBED /
ML. SERUM
0.5
i 5 30
M IN U T E S INCUBATED
TH PLN * S E R U M . C A LCIU M
1.5 1.0 0.5
LOG. % THPLN.
INACTIVATED
F IG U R E 14
T H E R E L A T IO N S H IP S A M O N G PR O T E IN A D S O R P T IO N ,
A N T IT H R O M B O P L A S T IN A C TIV ITY , A N D B A R IU M S U L F A T E
C O N C E N T R A T IO N IN T H E A D S O R P T IO N O F S E R U M .
65
indicating that i t is consumed during th is process. From
other evidence, to he discussed la te r, this material
appears to be prothrombin. Such a finding suggests two
modifications in the method used to identify antithrombo
plastin in serum! (1) Clotted blood or clotted plasma
should be allowed to stand at least one hour to allow as
complete prothrombin consumption as possible, and (2) the
serum thus obtained should be adsorbed with at least
100 m g. barium sulfate/ml. to insure the smallest possible
prothrombin contamination in the system.
O ne other observation in connection with Figure 14
is worthy of note. Above a level of 100 m g. barium sulfate/
ml., serum appears to have a reduced antithromboplastin
tite r. This m ay be due to the fact that such high levels
of barium sulfate produce rather non-specific adsorption
affin ities for the serum proteins so that antithrombo
plastin its e lf m ay be adsorbed at this level. This possi
b ility appears to be supported by some of the work of
Fiala (66) w ho finds a clotting inhibitor adsorbed on
barium carbonate.under a slightly different set of condi
tions.
5. Identification of antithromboplastin in plasma.
Figure 15 demonstrates that the antithromboplastin activity
CLOTTING TIME (secon d s)
O '
o
*
o
o -
HH 5 0 .
z 0
o
S
>
d 0 .
o o
5S
O "
3
3
S -
B >
tA
I ,
PERCENT OF CONTROL THROMBOPLASTIN
0>
65
* ■
exhibited in plasma is of the same order of magnitude as
that in serum from the sam e source- In this experiment,
the plasma was defibrinated with 20 units of thrombin/ml.
and allowed to stand 2 hours before barium sulfate adsorp
tion. serum, obtained from the same blood, was treated in
a similar manner.
< 5 . Attempts at isolation of antithromboplastin. In
previous work by other investigators (68,69) and the
author (102), i t was found that the inhibited thrombo
plastin moiety could be isolated from an incubation mix
ture of adsorbed serum, thromboplastin, and calcium by
high-speed sedimentation. This complex of inhibitor and
enzyme could then be disrupted by placing- i t in a calcium
free medium or in a solution containing a calcium binding
salt. Sedimentation of this mixture and reconstitution
then produced a thromboplastin with an activity comparable
to that of the uninhibited enzyme. O ne would suppose,
then, that this finding would be an excellent method for
isolating antithromboplastin. Therefore, a series of
experiments was carried out to study this possibility.
Thromboplastin, calcium, and adsorbed serum were incubated
for a period of time until the thromboplastin was more
than 90 per cent inhibited. The mixture was then
sedimented for 2 to 4 hours at 31,000 x g. The sediment j
was reconstituted in a calcium medium and tested for throm- j
I - ;
j boplastin activity. The enzyme was found to he 95 per cent;
i
I inactive. If, on the other hand, the thromboplastin was
\ i
! reconstituted in a calcium-free medium or in oxalate, the
i enzyme regained its in itia l 100 per cent activity. H ow -
i '
ever, the supernatant had no inhibitor activity. These i
experiments were repeated several times with the same '
I
; resu lt.
I
i
| I t appears from these results that som e substance
attaches itse lf to the thromboplastin molecule and that
the resulting complex is broken dow n or easily dissociated ,
. . j
| in the absence of calcium. Inhibited thromboplastin shows
ja significant increase in nitrogen and phosphorus above
I
i that for the active enzyme. Also, the ultraviolet adsorp- ;
!
tion spectrum shows an increase in the peak a t 280 mu
Indicative that som e substance is being adsorbed on throm- j
! boplastin which contains one of the aromatic amino acids. I
! - I
i i
; However, if the inhibited enzyme is repeatedly washed with [
1 calcium solution and subsequently sedimented, the ultra- j
i I
> . *
S violet adsorption peak at 280 mu disappears but the enzym e j
i
remains inhibited.
, Since the above methods were unsuccessful in the i
67
isolation of antithromboplastin, attention was directed to
more conventional isolation procedures. I t had been pre
viously found by Thomas (68) that anti thromboplastin could
be precipitated from serum by am m onium sulfate between the
levels of 25 to 5 G per cent saturation. In this work, the
majority of inhibitor activity of adsorbed serum was found
in the fraction precipitated between 40 to 50 per cent
saturation. However, the fractions thus obtained were
extremely unstable and demonstrated an inductive or latent
period of inhibitor activity upon subsequent incubation
with enzyme and ealcium. This m ay be suggestive that two
components are involved in the antithromboplastin moiety,
or that the antagonist to antithromboplastin mentioned
above m ay have been quite active in these preparations.
There has been some evidence reported that Reineke
salt is an effective clotting accelerator and th is finding.
has been interpreted to mean that i t precipitates a clot
ting inhibitor in the blood (102). i t was of interest to
investigate the possibility that this material might be
effective in isolating antithromboplastin from serum. I t
was subsequently found that Reineke salt was an effective
antagonist to inhibitor activity. Serum containing 10 m g.
of Reineke salt/ml. demonstrates considerably less
6 8
inhibitor activity than serum alone.
Upon fractionation of serum at room temperature
with Reineke salt, i t was found that inhibitor activity
resided in the precipitate formed at 5 per cent salt con
centration. • The precipitate thus obtained had to be
exhaustively dialyzed against water before antithrombo
plastin activity could be demonstrated. This fraction
dissolved in buffer solution demonstrated a latency period
of activity- similar to the am m onium sulfate fractions.
Salting-out with sodium chloxide, adsorption on
activated alumina and acid precipitation of the am m onium
sulfate fractions of antithromboplastin,were found to be
ineffective in the isolation and purification of this
material from human serum. Protein fractions, isolated
by the low temperature-alcohol method of Cohn (103) and
supplied through the courtesy of Cutter Laboratories
indicated that the inhibitor resided in fractions IV-1
and IV-3,4 of human plasma. These fractions also contain
the lipid anticoagulant described by Tocantins (47) and
were, therefore, investigated further. Subsequent experi
ments demonstrated the fact that the antithromboplastin
activity of these plasma fractions was increased upon
ultrasonic homogenization. Such a finding has led
69
Lanchantin and W are to speculate that the protein anti
coagulant reported herein is in a ll probability a co-factor
to the lipid anticoagulant of "anticephalin” described by
Tocantins (104). However, Field, Lanchantin, and Ware were
not able to isolate any of the lipid inhibitor from the
am m onium sulfate fractions of adsorbed serum (105). This
m ay explain w hy such salt fractions demonstrate a latency
period before they are able to inactivate thromboplastin.
In experiments on the lipid anticephalin, to be described
in a later section of this paper, i t was discovered that
this anticoagulant loses its inhibitory properties after
incubation with calcium and thromboplastin. This fact,
coupled with.the evidence described.above, strongly suggests
that antithromboplastin is a moiety composed of both lipid
and protein entitles, each of which is an inhibitor of
thromboplastin.
The protein fractions containing antithromboplastin
activity which were isolated from serum with Reineke salt
or am m onium sulfate are water soluble and appear to be
pseudoglobulins. The inhibitor isolated in the above
manner is inactivated when frozen in solution and stored
at -20°G., but the majority of the activity is retained
if i t is lyophilized immediately from the frozen state.
70
The sam e instability of antithromboplastin is also demon
strated. in adsorbed, dialyzed serum when frozen.
£. Pertain physieal and biochemical properties of the
thromboplastin inhibitor. Table V indicates that anti
thromboplastin shares m any similar physical and chemical
properties ascribed to prothrombin and A cG . I t is.fo r this
reason, perhaps, that considerable difficulty is encount-.
ered when one attempts to identify or isolate this material
in serum or plasma.
N o antithromboplastin activity can be detected in
serum heated to 56°C. for 30 minutes. The inhibitor is
nondialyzable for 5 days at 0°C. and has an optimum activ
ity between p H 7.0 to 8.0. I t has no demonstrable effect
on the rate of conversion of fibrinogen to fibrin and its
antithromboplastic activity cannot be reversed by protamine
sulfate. The amount of thromboplastin inactivated by the
inhibitor appears to be independent of AcG'and contingent
only on the prothrombin concentration in the incubation
mixtures of thromboplastin, calcium and serum or plasma.
In the final clotting system, however, the amount of throm
boplastin inactivated is independent of the prothrombin
c one entra ti on.
There appears to be no relationship between
TABLE V
1 C O M P A R I S O N O F T H E C H E M I C A L A N D P H Y S I C A L P R O P E R T IE S O F
P R O T H R O M B I N , A N T I T H R O M B O P L A S T I N , A N D A C C E L E R A T O R G L O B U L I N
Property Prothrombin
Antithrombo-
plastin
Accelerator
globulin
Water solubility Soluble Soluble Soluble
Salt solubility Soluble in dilute Soluble in dilute Soluble in dilute
Ppt. by asm. sulfate
\ % saturation)
50-66 1 iQ -5o
3045
p H of acid ppt. M
<3.5 5 4
Adsorption on B a S O ^
BaCOj, CajtFO^g etc.
Adsorbed Unadsorbed Unadsorbed (precursor)
adsorbed (active form)
Heat stab ility (56°C.
fo r 30 minutes) -
P artially stable Labile Labile
Cohn Fraction H I-1 IV-1, -3 4 III
Adsorption on asbestos
or Seitz filte rs
P artially
adsorbed
Adsorbed Unadsorbed
Necessity of calcium
ion for effect
Necessary Probably necessary N o effect
Utilized in clotting ■ ■ „ ; .+ -
..
S tability in plasma Stable Stable Labile
72
antithromboplastin and antithxombin or the A cG inhibitor.
Ether extracted serum which is devoid of antithrombic
activity retains its propensity to inhibit thromboplastin
as m uch as the untreated serum. The A cG inhibitor,
described by Lewis and W are (55), has no demonstrable
effect on thromboplastin.
The antithromboplastin described herein is active
against a wide variety of thromboplastins (106) which in
clude extracts of horse, rabbit, and human brain as well
as rabbit lung preparations and Russell viper venom. The
inhibition of the la tte r thromboplastic preparation by
human serum or plasma does not appear to require calcium.
The same was found to be true for the inhibition of the
thromboplastie activity of trypsin. However> this anomaly
m ay be due to serum antitrypsin rather than antithrombo
plastin.
Antithromboplastin was also identified in lymph
obtained from a thoracic eannulated rat on a saline drink
ing ration. Rat lymph, with a protein concentration of
56.2 mg./ml. was treated with barium sulfate in a manner
similar to that described previously for serum and plasma.
It appeared to have approximately 50 per cent of the anti
thromboplastin activity of human serum.
75
.The thromboplastin inhibitor described herein is
apparently different from the inhibitor recently described
by Fiala (66). This investigator reported that his factor
■ w a s adsorbed on barium carbonate, barium sulfate, and
celite. The la tte r, -w hich was reported to be the most
efficient adsorbing agent, had no demonstrable effect on
the thromboplastin inhibitor of serum when treated as
Fiala described (66). I t does not appear, therefore, that
F iala's inhibitor can be the sam e as the thromboplastin
inhibitor. . . . .
The fact that antithromboplastin m ay consist of two
complimentary' en tities, is a distinct possibility. Throm
boplastin, which has been reactivated by citrate or oxalate
from the inhibited state, demonstrates two properties dif
ferent from the original uninhibited enzyme: (1) i t is
more easily inactivated by the addition of more inhibitor,
and (2) the reactivated enzym e can be partially inhibited
by the citrate eluate which was washed off the inhibited
thromboplastin when i t was reactivated again. This would
seem to indicate that reactivation of inhibited thrombo
plastin by citrate or other calcium binding salts sp lits
off a fragment from the inhibited enzyme leaving another
fragment or co-factor remaining on the reactivated
74
thromboplastin molecule.
B. Clinical Studies on Antithromboplastin
1. Demonstration of antithromboplastin in vivo. The
part played by this inhibitor in the maintenance of blood
fluidity m ay be of importance. Tocantins has demonstrated
that dilution of blood in vitro (62) or in vivo as the
result of severe hemorrhage (107) will accelerate d o ttin g .
This effect is very likely due to dilution of the thrombo-
!
plastin inhibitor. I t is also possible that thromboplastin
precursors in plasma exist as a thromboplastin-inhibitor
complex which liberates thromboplastin when calcium is
removed. Such a mechanism could readily explain the hyper
coagulability of freshly-collected oxalated or citrated
plasma. Recently, Honorato has found that normal and hem o
philic bloods demonstrate significantly faster clotting
times when the calcium concentration is decreased (108).
In fact, i t has been suggested by a number of investigators
that the intravenous injection of oxalate causes a coagu
lant effect (109,110). This has been reported to take \
place in the normal person as well as in the hemo
philiac (111,112,113).
In the experiments noted earlier, i t was found that
in vitro oxalate salts were capable of reducing the_ _ _ _ _ _ _ _ _ _ _
75
inhibitory property of antithromboplastin on the thrombo-
plastic enzyme. The same effect was also elicited by
citrate and fluoride ion. T o test this effect in vivo,
a ealcium binding agent, disodium-ethylene-diamine was
given intravenously to six hemophilics. The results of
such an experiment have been reported elsewhere (105) and
m ay be summarized as follows. Serum calcium levels in the
hemophilics were reduced from 15 to 50 per cent. A short
ening of the whole blood clotting time of approximately
50 per cent was noted in three of the individuals. The
reduction of clotting was accompanied by a slight but
significant increase in prothrombin consumption. This
response was only a fraction of that obtained from the
administration of whole fresh blood. The occurrence of
such an effect in only 50 per cent of the volunteer hem o
philics studied is unexplained. In approaching the prob
lem from the opposite direction, i.e.# increasing the
calcium concentration in the plasma of normal individuals,
the results proved negative. The intravenous administra
tion of as m uch as 15 gm . of calcium gluconate was with
out effect on the Lee-White clotting time. Such would be
expected if the circulating antithromboplastin were exert
ing its effect maximally and maintaining the plasma
\
! 76 '
t
t
j clotting mechanism refractory to tissue thromboplastin,
I
I The experiments reported above suggest that a throm-
I ' !
j boplastin-inhibitor complex exists in vivo and that the
| disruption of this complex by reduction of calcium concen- !
!
I tration m ay be of importance under certain conditions,
! ‘
j The problem remains as to how antithromboplastin is !
| involved in the overall clotting mechanism. I t must be j
i
j kept in mind that the clotting system described here em- ;
J ploys concentrations of the clotting components far removed[
i ;
! from those found in blood under normal conditions. This is
I i
j especially true for the concentration of thromboplastin.
! The best estimates which the author can make, upon repeti- ;
tion of som e of Chargaff * s work on the isolation of throm-
| boplastin from plasma (114), is that hum an blood contains
about one-tenthousandth the concentration of thromboplastin j
used in the assay described above. Such a small concen- j
tration of enzyme in the circulating blood would, in a ll i
!
I
probability, be bound to the inhibitor. Support for this !
j
j faet is apparent from the experiments cited earlier in
!
I which calcium binding agents seemed to reverse this com- 1
; bination in vivo. Antithromboplastin also probably acts
to keep thromboplastin in an unreaetive form when the
enzym e seeps into the circulation from the tissues during
77
trauma. A defect in this mechanism m ay possibly he
responsible for the occurrence of thrombo-emboli and
intravascular clotting.
E. Antithromboplastin levels in certain bleeding
dyscrasias. Since Schneider had reported that antithrom-
boplastiri, when measured by the in vivo mouse assay proce
dure, was elevated in the third trimester of pregnancy
(115), i t was then of interest to study this occurrence ■
by the in vitro method previously described. The follow
ing results were obtained: In preliminary studies i t was
found that serums from w om en in the third trimester of
pregnancy show a higher propensity to inactivate thrombo
plastin than the serums of normal females. However,
normal males show a greater tite r of antithromboplastin
over that of either gestating females or normal w om en.
These findings were somewhat vitiated by the fact that
the inhibitory effect produced on the enzyme by incubating
i t with serum and calcium was reversed after a period of
time. Sueh an occurrence makes the above results some
what suspect. This is, perhaps, due to the fact that
these serums under investigation had poor prothrombin
conversion which m ay have caused this substance to be
incompletely adsorbed from the serum and thus interfered
78
with the assay procedure for antithromboplastin.
In an investigation of a number of cases of hemo
philia that presented themselves at the Los Angeles County
General Hospital over a period of two years, there was no
indication that the serums from these patients had an
increased tite r of antithromboplastin over normal.
In a case of acute leukemia, complicated by thrombo
cytopenia and leucopenia (platelets 17,000, W B C 1,800) with
a prolonged do ttin g time, the serum antithromboplastin
tite r was found to be normal. Such was also found to be
the case in a patient with Waldenstrom purpura having a
prolonged clotting time and high serum gam m a globulin as
measured electrophoretieally.
C . An Improvement in the Method for the Identification of
Antithromboplastin
Since i t was found that residual prothrombin was
indirectly responsible for the anomalous results obtained
for the quantitative analysis of inhibitor in barium
sulfate adsorbed serum, a new method had to be devised
that would obviate this difficulty. Adsorption is a log
arithmic process and because of this fact i t is impossible
to render serum or plasma completely free of prothrombin.
However, better adsorption methods should make i t possible
79
to reduce the prothrombin content of these fluids to such
a negligible quantity that i t would be of no influence in
the assay procedure. The barium citrate-barium sulfate
adsorption method of Lewis and Ware (77) for the isolation
of prothrombin from plasma was found to be an elegant
method to solve this problem.
Clotted blood was allowed to stand at 37°C. for one
hour. I t was then centrifuged at 2,500 x g for 10 minutes
to separate the eells and plasma. Ten ml. of the super
natant serum were then treated with 0.2 ml. of 1.0 M sodium
citrate to give a final concentration of 0.02 M citrate.
The serum was placed a t 0°C. and 1.0 ml. of 1.0 M barium
chloride added slowly with stirring. The mixture was
allowed to stand 15 minutes at 0°C. and then centrifuged
for 10 minutes in the cold a t 31,000 x g. T o the super
natant liquid, 0.75 ml. of sodium sulfate was added slowly
to precipitate the excess barium. This mixture was also
allowed to stand in the cold for 15 minutes before centri
fugation at 31,000 x g for 10 minutes. The adsorbed
serum thus obtained was then tested for excess of barium
or sulfate ion and passed through a 1 x 8 cm . column of
Amberlite IR-100 (sodium cycle) prepared by the method of
Quick (8) or Amberlite IR-400 (chloride cycle) depending
80
on the excess of ion present. In later experiments i t was
found advantageous to dialyze the serum against saline to
remove the excess ions.
As can he observed in Figure 16, which shows a com
parison between the new method and that previously employed,
the antithromboplastin activity of the serum prepared by
the barium eitrate-sulfate method is m uch higher than pre
viously found. For comparison purposes the serum prepared
by the old method has been diluted to .80 per cent with
buffer to equal the serum concentration of the new method.
The serum thus treated is found to inactivate 100 per cent
of the thromboplastin in 10 minutes. The anticoagulaht
effect produced beyond this level is, in a ll probability,
due to the inhibition of the enzyme existing in the plasma
standard in the te st system.
In Figure 17 Is demonstrated the fact that the
thromboplastin inactivated in a short interval of time
(6 minutes) is directly proportional to the logarithm of
the serum concentration.
Figure 18, curve A, demonstrates the anticoagulant
properties of plasma adsorbed by the new method. The
in itia l inactivation of thromboplastin by plasma appears
to be similar to that in serum but in plasma som e reversal
CQ
8
3 3
O
C Q
W
>
03
Q
C Q
§
Q
C Q
a
B !
8
g
|
C Q
ts
C Q
§
§
o
H H
O
a
>3
H
O
H
Q
H
C Ji
Z
o
c
C P
>
H
o
z
H
2
m
z
|
z
c
H
m
C Q
CLOTTING TIME IN SE C O N D S
o
o
tvj
o
o
u >
O
O
o
o
o
o
O '
o
00
o
5
P E R CENT OF CONTROL
T H R O M B O P L A S T IN ACTIVITY
Legend to figure 16
A. Incubated equal volumes of barium citrate-sulfate
adsorbed serum, 0,015 M calcium chloride and 100 per
cent thromboplastin.
B. Incubated equal volumes of barium sulfate adsorbed
serum (100 mg./ml.), 0.015 M calcium chloride and
100 per cent thromboplastin.
C. Sam e as B but with 80 per cent serum.
Test system: 0.3 ml. incubation mixture tafcen
at intervals and added to 0.1 ml. FFB and 0.1 ml. plasma
standard.
% T H PL N . INHIBITED
8
84
400 i
C O
o
z
S 300 ■
U J
CO
z
^ 200 ■
i —
C D
z
I —
H -
100 9
o
_ l
o
60 40 50 20 30 10 0
INCUBATION TIME IN MINUTES
F IG U R E 18
T H E E F F E C T O F B A R IU M C IT R A T E -B A R IU M S U L F A T E
A D S O R P T IO N O F P L A S M A . O N ITS A N T IT H R Q M B 0 P L A S T IC A C TIV ITY .
A. Barium eitrate-barium sulfate adsorbed plasma.
B. O ne part of unadsorbed plasma added to 1,000 parts
of A .
C. O ne part of unadsorbed plasma added to 50 parts of A.
See text for explanation.
85
of this antagonism takes place. This is further accentu
ated if a portion of the original unadsorbed serum is added
to the adsorbed fluid. As l i t t l e as one part of unadsorbed
plasma in 1,080 partis of adsorbed liquid is sufficient to
reduce to a considerable extent the antithromboplastic
activity of the adsorbed plasma. At a level of 1 to 50,
the inhibitor tite r is obliterated in the early stages of
incubation but after the prothrombin in the plasma has
been utilized, the inhibition becomes evident. Attention
should be called to the similarity that curve 0 has to the
activity of unadsorbed serum (see Figure 10).
D , A Report of a Case of Idiopathic Antithromboplastin-
ophilia
Curing the course of this research project, a case
of a bleeding diathesis associated with a circulating anti
coagulant presented itse lf at the Los Angeles County
General Hospital. Further laboratory examinations and
coagulation studies on the patient’s blood resulted in a
diagnosis of an elevated antithromboplastin and anti-AHG
plasma tite r, since reports of such a finding never
appeared to have been documented in the medical literatu re,
i t is suggested that such a disease be tentatively termed
secondary or Idiopathic antithromboplastinophilia. The
86
results of coagulation studies on th is patient will be
presented in more detail elsewhere (116).
1. History. A sixteen-year old unmarried negress,
M .C. (PF #1385 494), entered the Los Angeles County General
Hospital with a preliminary diagnosis of museular hematoma
of the le ft thigh and pelvic region. In the performance
of routine clinical determinations, i t soon became obvious
that after phlebotomy the patient*s blood did not d o t in
a normal period of time. Subsequently, i t was found that
the Lee-White clotting time was markedly prolonged although
the bleeding time appeared to be normal. Previous history
of the patient indicated no unusual bleeding episodes in
earlier life , nor the occurrence of a bleeding diathesis
in any member of the family. The patient had delivered a
healthy normal child eleven months prior to the onset of
the hematoma and her menstrual periods after this time
were uneventful.
2. Clinical laboratory findings and coagulation
studies. Routine laboratory examinations indicated no
abnormal blood chemistry with the exception of a severe
anemia. Hemoglobin was 3 gms. per cent. Liver function
tests including prothrombin were normal. The preliminary
diagnosis of a circulating anticoagulant was made on the
87
basis of the experiment presented in Table 71, in which i t
is obvious that the patient’s plasma is capable of pro
longing the recalcified clotting time of both normal and
hemophilic plasmas. These experiments were repeated three
times with the plasmas of the patient and different normal
and hemophilic donors. The hemophilics whose plasmas were
employed in these experiments were of the classical type
since they could be differentiated from F T A and P T C hemo-
philioid states by the fact that normal barium sulfate
adsorbed plasma could correct their prolonged fecaleified
clotting time.
Coagulation studies on the patient’s blood are
summarized in Table VII. The results reported in the
Table are typical of that seen in a case of a circulating
anticoagulant in which a ll the clotting factors are appar
ently normal but the Lee-White whole blood clotting time
is prolonged and the prothrombin consumption practically
negligible. Protamine titra tio n , capillary frag ility
(Rumple-Leede), anti thrombin and the quality and number
of the platelets were normal. Normal clot retraction
further substantiated the apparent integrity of the plate
le ts. Electrophoresis of the patient’s serum in the
Tiselius apparatus showed a normal distribution of serum
TABIE 71
THE EFFECT OF MIXING NORMAL, HEMOPHILIC, AND ANTITHRQMBO-
PLASTINOPHILIC PLASMAS ON THE HEGALCIFIED CLOTTING TIME
Parts of plasm a from Recalcified
Patient M.C H em ophiliac Normal
Clotting time
(seconds)
1 ©
9
9
1
1
$
8
1 ©
9
9
mm
$
1
1 ©
1
1
' U -1
1*36
366
1 0 6
h2Q
1 1 2
33k
3 0 ©
185
T A B IE V II
C O A G U L A T I O N S T U D IE S O N A C A S E O P
ID IO P A T H IC A N T I T H R O M B O P L A S T I N O P H I L I A
Determination Patient If.C . N orm al
Prothrom bin (modified O irren) 1 2 0 5 6 lO O g
Prothrom bin time (Q uick) 16.7 sees. ; 16.9 secs.
Prothrom bin consum ption (l hour) 0 8 0 -9 0 5 6
Lee-W hite clotting time 90 mine.: 8 - 1 © mins.
Recalcified clotting time 1 « £ 0 secs. 8 7 secs.
T hrom bin time 2 $ secs. 32 secs.
j& c G 1 0 0 5 6 1 0 0 5 6
Fibrinogen O .U gm s.£ 0.3 gm s.56:
Bleeding time (Ivy) 1*5 m ine. 1-2 m ins.
90
proteins.
In attempting to further investigate the site of
action of the anticoagulant, i t is com m on practice in cases
of this kind to test the effect of various dilutions of
thromboplastin on the Quick prothrombin time of the
patient’s plasma. B y this method, i t is possible to deter-
mimine if the plasma in question is more refractory than
normal plasmas to dilute thromboplastin suspensions and
hence i t serves as a means of indicating whether the
patient’s circulating anticoagulant is antithromboplastie.
This did not appear to be the case with the patient under
study as evidenced from such a te st described in Table YIII
However, if the patient’s serum is treated in a manner as
described previously and assayed for antithromboplastin,
i t appears from such an experiment presented in Figure 19,
that the patient’s serum has a higher propensity to in
activate thromboplastin than normal serum. Figure 19 is a
typical example of three experiments using the patient’s
serum and different normal serums. A dilution of one part
of the patient’s serum in nine parts of normal serum in
creases the anti thromboplastin tite r of the normal serum.
3. Therapy. Seventy-five m g. of emulsified vitamin
and 100 m g. of A C T H A R gel intramuscularly daily were
TABLE VIII
THE EFFECT OF VARIOUS DILUTIONS OF HUMAN BRAIN
THROMBOPLASTIN ON THE. QUICK PROTHROMBIN TIME OF NORMAL
AND ANTITHROMBOFLASTINOPHILIC PLASMA
Per cent
Thromboplastin
Clotting time (seconds)
Normal Patient
1 0 0 .0 0 1 6 .9 1 6 .7
$ 0 .0 0 1 8 .6 l 8 .i i
2 $ .0 0 2 1.7 2 1 .2
1 0 .0 0 26.8 2 7 .0
5 .0 0 3 3 .0 3 1 .8
1 .0 0 5 5 .5 5U .0
0 .5 0 6 0 .2 5 8 .2
0 .1 0 1 1 7.6 1 1 0 .0
0 .0 5
132.8 125.it
0 .0 1 u a . o 1 6 9 .2
0 .005
n a i o 18 0 .5
0 .0 0 1 lhl.0 2 1 0 .0
0 .0 x u a . o 2 1 0 .0
C L O T T IN G T I M E A N D T H R O M B O F L A S T IG A C T IV IT Y
H
CLOTTING TIM E (seconds)
PERCENT THROMBOPLASTIN REMAINING
(0
to
93
! ineffective in reducing the prolonged recalcified clotting
time of the patient*s plasma. In one instance, a cross-
i
transfusion of 1.5 lite rs of whole, fresh blood shortened
i
! the reealcified clotting time of the patient’s plasma from
I 436 seconds to 223 seconds. However, two days after trans-
i fusion the clotting time increased to 250 seconds and was
! 360 seconds on the fifth day. Such a finding is indicative
' (
I of the ab ility of the anticoagulant to regenerate itse lf in |
! the patient’s circulatory system or to inactivate the i
i
jcoagulant effect of exogenously supplied blood.
! 4. Remarks. The inability of the patient’s plasma
|to correct the reealcified clotting time of hemophilic
j
; plasma has rather marked ramifications in elucidating the
i 1
i
i thromboplastic factors in the clotting theory. Such an
experiment indicates that the patient’s plasma does not
i '
contain A H G -, which is also absent in the hemophilic. Fur- !
i
thermore, in experiments reported elsewhere (116), i t was ,
i i
j subsequently discovered that the plasma from M . C. is cap-
! able of destroying the A H G in normal blood. This was
demonstrated by the ab ility of the patient’s plasma to
inactivate the corrective effect of normal barium sulfate
adsorbed plasma on the clotting time of the hemophilic.
!Such a finding excludes the possibility of the
anticoagulant being an antagonist to P T C or P T A ..
From the experiments presented above, i t appears
■ . . i
that the site of action of the anticoagulant in the blood j
i
of the patient under study is centered upon tissue thrombo-j
j
plastin and the A H G . Such a finding lends credence to the
purported theory that A S G and tissue thromboplastin are
synonymous. The mechanism of action of the anticoagulant
does not appear to be that of an antibody-antigen relation
ship since precipiten tests by the method of Craddock and
Lawrence (117) were negative.
The apparent discrepancy in the commonly-accepted
anti thromboplastin test as measured in the Quick system
and shown in Table VIII, and that reported in this paper
and diagrammed in Figure 19, is, no doubt, due to the
presence of oxalate in the former method. As mentioned
earlier, the thromboplastin inhibitor requires calcium for
its activity and any calcium binding environment w ill pre
vent the action of anti thromboplastin. I t is, therefore,
necessary that any in vitro te st for a thromboplastin
inhibitor be conducted in a system containing calcium.
I
Stieh a finding makes previously reported cases in which j
1
|
the Quick te st was employed somewhat suspect. j
i
The etiology of the disease, antithromboplastin- j
j
95
ophilia, is difficult to ascertain since this is the f ir s t
documented case of the type, since the patient had no
previous family history of any bleeding dyscrasia and since
coagulation studies on the patient’s offspring a ll proved
to be normal, the disease appears to be idiopathic or
spontaneous. Although the menstrual periods of M.C. were
normal, the possibility of placental retention exists
from the eleven months prior childbirth, such an instance
has been reported by Frick (118) and m ay suggest that the
elevated antithromboplastin and anti-AHG is due to the
continuous extrusion of placental thromboplastin into the
circulation for which the body builds up these natural
antisubstances or antibodies.
I I . The Activation of Thromboplastin by Calcium
The physiological role of calcium in the coagulation
of blood has remained a much disputed question for many
years (for a review, see Wohlisoh (1)). The experiments
to be reported below and elsewhere (119) appear to indicate
that calcium is an activator of the thromboplastic enzyme.
A. The Activation of Thromboplastin by Calcium as Meas
ured in Crude Clotting systems
I t has been a frequent observation in this
96
laboratory using a modified prothrombin method that incuba
tion of thromboplastin with calcium produces a more active
thromboplastin material (82). i t was then of interest to
investigate this phenomenon in more detail using the throm
boplastin assay procedure. If equal volumes of thrombo
plastin and 0.015 M caleium chloride are incubated together
and aliquots of this mixture introduced into the test
system, a marked increase in the velocity of coagulation
takes plaee. This effect appears to be dependent upon
volume as demonstrated in Figure 20. Incubation of equal
volumes of enzyme, buffer and calcium with addition to E F B
and plasma standard produces a more rapid coagulant effect
than ineubation of enzyme and calcium alone with addition
to buffer, PFB and standard plasma. Apparently, thrombo
plastin with an estimated particle weight of 167 million
(120), is such a large molecule that a solution of i t in
calcium must be of a certain ionic concentration in order
to allow spaee for a critical surface on the enzyme
molecule to become covered with calcium ions. This, per
haps, m ay explain the dependence of activation on volume.
The degree of activation also appears to be independent of
enzyme concentration below a c ritic a l value. This is
demonstrated in Figure 21. In this instance a
CLOTTING TIME I N SECON DS
97
6 0
THPLN +
BUFFER
5 5
50
THPLN +-
C ALCiUN/1
4 5
THPLN +
CALCIUM +
BUFFER
4 2
50 0 100 150 200
INCUBATION TIME IN MINUTES
F IG U R E S O
T H E A C T IV A T IO N O F T H R O M B O P L A S T IN
B Y C A L C IU M A N D ITS D E P E N D E N C E U P O N V O L U M E .
L
2 0 0
150
GO
^ 100 ;
80 ■
z
o
C D
UJ
«o
WITHOUT INCUBATION
U J
g 4 0
INCUBATED 6 0 MIN.
C D
Z
I —
I —
o
_ ! 20 ■
10
1 1 0 5 0 100 200300 5 1000
PER CENT THROMBOPLASTIN
F IG U R E 21
T H E D O U B L E L O G A R IT H M IC R E L A T IO N S H IP
O P T H R O M B O P L A S T IN C O N C E N T R A T IO N T O C L O T T IN G T IM E
D U R IN G O N E M IN U T E A N D SIX T Y M IN U T E S O F IN C U B A T IO N
O F T H R O M B O P L A S T IN W IT H C A L C IU M A N D B U F F E R .
99
thromboplastin assay curve was determined in the presence
of calcium in a manner similar to that shown in Figure 9.
However, as can be, seen from Figure 21* after one hour of
incubation the enzyme in the presence of calcium and
buffer has increased in activity to a marked degree. The
activated enzyme appears to follow the same log.-log.
relationship to the clotting time as the unactivated
thromboplastin. This experiment m ay thus answer the ques
tion as to the role of calcium in the coagulation mecha
nism. This property of calcium activation is not uncom m on
to biochemistry since this ion is also obligatory to the
activity of several other enzyme systems in the
body (121,122).
O ne of the necessary points that must be critically
examined before one m ay postulate an activating effect of
calcium on thromboplastin is that the thromboplastic pre
paration is not contaminated with som e other clotting com
ponent. This problem is somewhat complicated by the fact
that the enzyme has never been purified and l i ttle is
known concerning its chemical structure. However, such
evidence can be found indirectly. In the f ir s t place, the
preparation of the enzyme material its e lf seems to exclude
the possibility of a contaminating coagulant. Acetone
100
dehydration, heat extraction, and repeated centrifugation
would, to a ll intents and purposes, denature the other
sensitive clotting proteins. Secondly, no evidence for
prothrombin, thrombin, or A C G can be demonstrated.' And,
in the third place, an experiment employing differential
denaturation on the thromboplastic enzyme was designed to
rule out the presence of a clotting agent in the enzyme
preparation.
This experiment was carried out as follows: Throm
boplastin was irreversibly denatured by ultrasonic homoge
nization a t a frequency of 1,000 kc. The loss of thrombo
plastic activity was almost a straight line function of
the duration of exposure to ultrasonic waves. Conse
quently, by talcing aliquots of the treated material at
various time intervals, a series of partially denatured
enzyme solutions was obtained. These solutions were sub
sequently incubated with calcium and buffer and their
activity checked with untreated enzyme preparations diluted
to a comparable activity. I t was found that the degree of
activation of the treated and untreated material was the
same. That is to say, the denatured material was activated
to the sam e degree as the untreated enzyme at comparable
in itia l activity. I t is extremely unlikely that such
101
oould occur if the enzyme preparations were eontaminated
with som e other clot-promoting substance since no two
proteins are likely to be denatured at the same rate.
B. The Activation of Thromboplastin by Calcium as Meas
ured in a Purified Clotting system
To further confirm the results reported above for
the activation of thromboplastin in crude clotting systems,
i t was next of interest to investigate this phenomenon
using purified reagents. The calcium activating effect
on the thromboplastic enzym e isolated from hum an brain as
measured in a purified clotting system has been reported
elsewhere by Lanchantin and W are (119) and the results of
such experiments are summarized below.
The reconstructed clotting system was composed of
the reagents listed in Table IX which were added in 0.1 ml.
volumes to 75 x 10 m m . test tubes .at 37°C. The pseudo-
firs t order kinetic relationship of thromboplastin concen
tration to clotting time allowed standardization of the
thromboplastin activity. In this system, the arbitrary
value of 100 per cent thromboplastin activity was given
as that concentration of enzyme which would give a 13-
seoond clotting time in the reconstructed system. As can
be seen from Figure 22, prior incubation of thromboplastin
© a i v e r s i r y o t S o u t h e r n
TABLE IX
CLOTTING SYSTEM COMPOSED OF THE PURIFIED
COAGULATION FACTORS ISOLATED FROM HUMAN BLOOD AND TISSUES
Clotting
factor
Concentration Activity
Prothrom bin 0*09 m g * protein/ml. ll« 2 units ^
Plasm a A c G 0*18 m g * protein/ml* 91a ^
Throm boplastin 0*8l m g * protein/ml*^ 1 0 0 5 *
Fibrinogen 10 m g . elottable protein/ml. -
C alcium 0.008 M -
Activity com parable to norm al h u m a n plasma.
3 0
0 .5
f-
UNINCUBATBD
1. 0
S a 5 ‘
§
u
cn
8 .3 9 s
o— o
Q
5 3
1—4
S o
l d
s
H
U N INCUBATED
1 0
25
C J
( u
O
5 0
-100
-200
1 0
INCUBATION TIME IN MINUTES
FIG U R E 22
T H E E F F E C T O F IN C U B A T IN G T H R O M B O P L A S T IN A N D C A L C IU M
O N T H E C L O T T IN G T IM E A N D T H R O M B O P L A S T IC A C T IV IT Y O F
A PU RIFIED C L O T T IN G S Y S T E M C O M P O S E D O F PU R IFIED
F A C T O R S IS O L A T E D F R O M H U M A N B L O O D A N D TISSU ES.
104
with calcium produced a marked enhancement of clotting of
the purified system. The results of thromboplastin activa
tion by calcium in the purified clotting system, therefore,
appear comparable to those^findings reported above for
crude coagulation systems. ..
In the two-stage procedure for the determination of
prothrombin, thromboplastin which has been activated with
calcium appears to speed up the in itia l stages of the con
version of prothrombin to thrombin but i t does not effect
the final thrombin yield. Strontium ion, which coagulates
blood in a physiological manner though less efficiently
than calcium, also is active in the above clotting systems
by enhancing the activity of human brain thromboplastin.
J S * The Relationship Between Activated Thromboplastin and
Anti thr om bo pla stin
In view of the finding that thromboplastin was
activated in the presence of calcium, i t was then of
interest to investigate the relationship that this phenom
ena m ay have to the inhibition of thromboplastin by an ti
thromboplastin in serum as prepared by the new adsorption
technic. Consequently, equal volumes of adsorbed serum,
enzyme, and 0.015 M calcium chloride were mixed and incu-
bated at 37°C. At varying time intervals, aliquots of
105
this mixture were taken and tested in the clotting system
consisting of FFB and standard plasma. A typical thrombo
plastin inactivation curve was produced as shown in
Figure 23, curve A. In the span of 35 minutes, the enzyme
in the mixture was completely inactivated and a steady
state of inhibition realized. At point 1, shown in
Figure 23, a small quantity (0.1 ml.) of11,000 per cent
enzyme was added to 0.9 ml. of the incubation mixture to
obtain the same enzym e concentration that was originally
in the mixture before inhibition had taken place. Such a
seeding did not materially change the calcium and serum
concentrations in the incubation mixture. As is demon
strated in the Figure, the addition of enzyme to the incu
bation mixture produced a virtually complete reversal of
the inhibitory process immediately and the system returned
to its original control value 50 minutes later, such an
experiment is indicative of two things: (1) The inhibitor
is consumed during its inhibition of thromboplastin, and
(2) there is no other anticoagulant produced from serum by
the aetion of thromboplastin and calcium, i._e., anti
thrombin. The la tte r effect does not support the work of
Hartmann (123). such an experiment has been repeated m any
times. Serum which has inactivated thromboplastin loses
A M ’ X T H R G M B O P IA S T IN O N A C T IV A T E D T H R O M B O P L A S T IN
M
m h
H Q
8 *
s §
t)
H
to
w
C O
> ■ 3
H
3
C Q
► 3
H
3
CLOTTING TIME SECONDS
,5-m^.mIMNWNU
r o o i v i o i o y i N O i o o i ' j o
u i o c n o m o u i o u l o u i o
o i—O — fr
e e
o
O p o
— o
y — i -------- ----------------- ------------
K ► — * H u ,
o o o
8 °
PER CENT OF CONTROL THROMBOPLASTIN
ACTIVITY
107
Legend to Figure 23
A . Incubation mixture: equal volumes of adsorbed serum,
0.015 M calcium chloride, and 100 per cent thrombo
plastin.
B. Incubation mixture: equal volumes of 0.015 M calcium
chloride and 100 per cent thromboplastin.
1. 0.1 ml. of 1,000 per cent thromboplastin added to
0.9 ml. of incubation mixture A.
2. 0.5 volumes of adsorbed serum added to 1 volume of
incubation mixture B.
Test system: 0.3 ml. of incubation mixture, added
.to 0.1 ml. of FFB and 0.1 ml. of standard plasma.
108
its ability to inactivate more enzyme. This relationship
is not completely stoichiometric in this system. That is
to say, there is a sufficient quantity of inhibitor in
serum to inactivate more than 100 per cent thromboplastin
but not enough to inactivate 200 per cent of the enzyme
completely. I t remains to be determined what tite r of
antithromboplastin exists in serum by titratin g i t against
various quantities of enzyme to determine what quantity of
thromboplastin is sufficient to completely deplete serum
of inhibitor activity.
Curve B of Figure .25, shows the activation of enzym e
with time when thromboplastin and calcium are. incubated
together. In a span of 15 minutes, the enzyme has in
creased approximately 10 times in activity and remains,
from that time on, in a steady state. At point 2, shown
in Figure 23, adsorbed serum was added, upon the addition
of serum, thromboplastin was immediately inhibited at a
rate comparable to that for the system described in curve
A . However, in this case, the activated thromboplastin is
not completely inhibited. This experiment suggests two
facts: (1) The rate of inhibition of unactivated and
calcium activated enzyme is the same, and (2) the degree
of inhibition of thromboplastin holds som e proportionally
109
to its state of activity. There does not appear to be a
state of competition between calcium and inhibitor for any
of the active groups on the enzym e molecule. This is
demonstrated in Figure 24. I t appears from the Figure
that antithromboplastin does not antagonize the activation
of thromboplastin by calcium and the converse has been
shown in previous experiments which have demonstrated that
calcium is necessary for enzyme inhibition.
I I I . A n Investigation of the Anti coagulant
Properti es of Antioephalin
The lipid anticoagulant described in numerous publi
cations by Tocantins (47,60-63) and mentioned previously in
this work was investigated further in an attempt to deter
mine its relationship to the antithromboplastin described
in this paper. Several preparations of this material were
isolated from blood and brain by previously published
technics (47). Purified preparations of lipid were also
* '
supplied in dry form through the courtesy of Dr. Leandro
Tocantins of the Jefferson Medical' school of Philadelphia.
Most of the work to be described below has been conducted
with the purified product.
Preliminary experiments with this material confirmed
m i
w t s t s
m
m s m
S E P S I S
»uA - .~ .r 'M t‘
m m m ? » u S a s s »
^SRkdx&
w m m m
£ « 3 » e
& w .
2 7 0 m m m
i m »
si
awsai
mmmm
< r s > s
BHPMgg
^ M t V‘
m a ssa a t
m $ m
I M K
m s m
< m $
te e s
20 30 '40 50
INCUBATION TIME IN MINUTES
mvBtn
^fPISpapp
«!M i3!388^S*SSS& «
s Ps s e
0 i; 015 f?M ffoaic
Sj^ r * - r # -rti> Ji?.^-**--^ * * w ;
e * i e s f l 9 5 * j r a i 5
? j f e S 3 ®
SMMNH
mtattuiMi
I W W
I l l
the work of Tocantins in m any instances. The lipid
material was found to be a potent inhibitor of the modified
Quick prothrombin determination (47). I t is heat stabile,
its activity increased markedly upon ultrasonic homogeniza
tion, and its anticoagulant activity centered somewhere in
the in itia l activation of prothrombin. However, further
experiments in our laboratory have failed to eonfirm its
elevation in hemophilia in the six patients with this
disease that were studied. The fact that the lipid mate
ria l is adsorbed on glass could not be substantiated and
this finding has since been retracted by Tocantins (124).
A comparison of the anticoagulant properties of
various dilutions of lipid suspended in saline by u ltra
sonic homogenization for 15 minutes at 1000 kc. on the
Tocantins thromboplastin te st system and the thrombo
plastin assay procedure described previously is diagrammed
in Figure 25.
*
An attempt was made to determine i f the lipid mate
ria l was capable of inactivating thromboplastin by incuba
tion of the enzyme and inhibitor in the presence of
calcium at 37°C. for 30 minutes. The thromboplastin was
then sedimented by hi^h-speed centrifugation and resus
pended -by the method described previously. Unlike
i
112
o
LxJ
c n
z
U i
o
z
o
aoi o .i 1.0 1 0 . 0
MG. LIPID / ML. ADDED
FIG U R E 25
T H E E F F E C T O F V A R IO U S C O N C E N T R A T IO N S
O F LIPID A N T IC O A G U L A N T O N T H E C L O T T IN G T IM E S
O F T W O T H R O M B O P L A S T IN A S S A Y T E S T S
A * Thromboplastin assay procedure using 10$ thrombo
plastin.
B. Thromboplastin assay procedure using 100$ thrombo
plastin.
C. Tocantins* assay te st using 10$ thromboplastin.
G i Tocantins* assay te st using 100$ thromboplastin.
113
adsorbed serum, the lipid showed l i t t l e capability of
inactivating thromboplastin. The small amount of inhibi
tion, approximately 20 per cent, was due to the fact that
a portion of the lipid material was sedimented with the
thromboplastin.
The next avenue of approach was to investigate the
effect of incubating thromboplastin, lipid, and calcium on
both thromboplastin test procedures. The results can be
found in Figure 26, which shows a comparison between the
two test methods. I t is interesting to note that when
measured by the antithromboplastin assay procedure, the
lipid produces an in itia l inhibition of clotting, but this
antagonism is gradually reversed and the clotting time re
turns to the control value in approximately 140 minutes.
This does not appear to be the case with the Tocantins*
test system. In using this method, i t was quite difficult
to achieve duplicate values, the accuracy of each clotting
time being of the order of plus or minus 5 seconds. This
apparent discrepancy between the two tests is probably due
to the presence of oxalate in the Tocantins* system.
It was next of interest to determine the effect
of ultrasonic waves on the antiooagulant activity of the
purified lipid. Consequently, several 1 per cent
114
1 1 0
100 •
CO
o
g 9 0 -
o
U l
CO
z 8 0 -
u i
2
i —
o
z
I —
»—
o
o
20 4 0 6 0 80 100 120 140
INCUBATION TIME IN MINUTES
FIG U E E 26
T H E E F F E C T O F IN C U B A T IO N O F A 0.0B P E E C E N T S U S P E N S IO N
O F LIPID W IT H T H B 0 M B 0 P L A S T IN A N D C A L C IU M O N T H E
C L O T T IN G T IM E S O F T W O T H R O M B O P L A S T IN T E S T P R O C E D U R E S .
A . Thromboplastin assay procedure described in text.
Control • 45 secs.
B. Tocantins* thromboplastin assay te st.
Control =15 secs.
X15
suspensions of lipid in saline were prepared and exposed
to 1000 kc. at room temperature. A n example of three
typical experiments is presented in Figure 27. upon pro
longed exposure, the anticoagulant activity of the lipid
suspension increases, reaches a m axim um in about an hour,
and then seems to level off as measured in the Tocantins*
test and a modified Owren prothrombin time (H T B dialyzed
free of oxalate). The anticoagulant aotivity of th is
material thus treated is even more striking in that the
plasma prothrombin substrate used in both tests had a
Quick prothrombin time of 10 seconds with human brain
thromboplastin and a modified Owren determination of 180
per cent prothrombin.
The suspension of lipid in saline whieh had a pH of
7.0, in itia lly , showed a decrease in p H upon exposure to
1000 ke. This is suggestive of the freeing of acid groups
in the lipid material due to oxidation and cavitation of
the suspension.
I t was subsequently discovered that the material
thus treated formed a flocculent precipitate upon the
addition of calcium chloride to the medium. The material
isolated from the precipitate accounted quantitatively for
a ll the lipid in suspension. The lipid which was
1 1 6
^ 250
“ T»
S 200
Ul
•a
lu 150
• “ 1 0 0
S2 150 -a
t 100
a .
C B
80 100 GO 40 2 0
E X P O S U R E TIME IN MINUTES
FIG U R E 27
T H E E F F E C T 0F P R O L O N G E D E X P O S U R E O F A O N E P E R G E N T
LIPID S U S P E N S IO N T O O N E M E G A C Y C L E U L T R A S O N IC W A V E S
O N S O M E O F ITS P H Y S IC A L A N D B IO C H E M IC A L PR O PE R T IE S.
A. Increase in anticoagulant properties as measured
by the Tocantins* test.
B. Increase in anti coagulant properties as measured
by a modified Owren prothrombin te st.
C. Decrease in pH .
D . Mg./ml. of precipitate formed upon the addition
of calcium to the lipid suspension.
117
precipitated by calcium was evident after only 15 minutes
of exposure. There was no increase in snob, precipitates
after continued ultrasonic homogenization in spite of the
increase in anticoagulant activity of the suspension. The
solubility in certain organic reagents of the calcium and
sodium salts of such material isolated after 90 minutes
exposure to ultrasonic waves along with other data (1S4)
indicate that this substance has properties similar to
those of cephalin.
Sinee this homogenized material appeared to bind
y
calcium, this problem was investigated further. I t was
found that the thromboplastin assay procedure, which has
a calcium optimum of 0.003 M in the final do ttin g mixture,
demonstrated a lower requirement for calcium in the pres
ence of lipid. In this instance, the calcium optimum was
lowered to 0.001 M .
From the results described above, i t does not appear
that the lipid anticoagulant described by Tocantins is a .
truly physiological antithromboplastin by its e lf. Its
anticoagulant properties are, no doubt, related to its
sim ilarity to eephalin, and that i t thus m ay act competi
tively with a cephalin-protein liice thromboplastin for the
j #
active groups on prothrombin. However, this appears to be
118
merely a temporary adsorption phenomenon since this antag
onism is eventually nullified upon ineubation of lipid,
calcium, and thromboplastin in vitro. The same would be
expected to oecur in vivo. Here, again, is an instance in
which an in vitro clotting system employing oxalate salts
does not give a good translation into the events which m ay
be occurring in vivo where calcium is present continually.
A. The Relationship Between the Protein and Lipid Anti
coagulants
There appears to be som e relationship between the
lipid anticephalin described by Tocantins and the protein
anticoagulant described herein. I t is q,uite probable that
these two factors are in actuality separate moieties of a
lipoprotein anticoagulant. This seems to be the case from
the following known facts:
(1) A lipoprotein anticoagulant exists in blood.
The experiments of Tocantins (125,126) have shown that
during prolonged high-speed centrifugation of plasma,
certain light density lipoproteins float to the surface.’
This top plasma layer is hypocoagulable even after plate
lets are added and demonstrates a three- to seven-fold
increase in anticephalin activity. Higher yields of this
119
lipid material are also obtained from this top layer of
plasma. Plasmas thus treated have the ab ility to prolong
the reealcified clotting time of normally centrifuged
blood. These results have been essentially substantiated
in this laboratory and have been extended to include zone
electrophoresis of the various plasma layers. The proce
dure used was similar to that employed by Fasiola (84)
except that Nile Blue Sulfate was employed to develop the
electrophoretic strips by a technic formerly used for
histological staining (85). Several experiments employing
zone electrophoresis gave good indication that plasmas
subjected to prolonged high-speed centrifugation show an
increase in the lipid content of the p-protein fraction or
the uppermost layer of plasma.
(2) Plasma fractions IV-1 and ITr-3,4, isolated by
the method of Gohn, demonstrate both lipid and protein
anticoagulant properties as mentioned earlier and their
activity is increased upon ultrasonic homogenization.
(3) The protein fraction of serum which is pre
cipitated by am m onium sulfate has no detectable lipid
content and displays a latency period before i t is fully
active against the thromboplastin molecule. Tocantins*
lipid, on the other hand, which contains essentially no
1 2 0
protein, inhibits thromboplastin immediately but loses this
property upon incubation with the enzyme and oaleium. H o w
ever, when both the protein fraction and lipid are added to
the enzyme and ealcium simultaneously, the inhibition is
immediate and progresses further u n til equilibrium is
reached. Serum and plasma show essentially the same mani
festations. These effects on the previously-described c lo t
ting system for the identification of antithromboplastin
activity are diagrammed in Figure 28.
(4) From what has been said previously* the protein
moiety requires calcium for maximal inhibition, while the
lipid anticoagulant reduces the oaloium optimum for the
conversion of prothrombin.
From the foregoing statements and the experimental
observations recorded previously, a working hypothesis for
the*further investigation of the role of antithromboplastin
in the meohanism of prothrombin activation is diagrammed in
Figure 29.
IV. Studies on Accelerator Globulin
A. Mechanism of Action of Thrombin in the Conversion of
Plasma A Q G to Serum A o G
There has been considerable interest in the manner
-- - - - - - -1
131 |
3120 -
q
- s .o <
H
* i o
Tocantins lip id -
in h ib ito r
a d so rb ed , b o i
Cchn F r a c J tic .. __
4 .3 r a g s ./m l.
p r o t e in i s o l a t e d b y
40 -5 0 % s a t . o f s e r u m
w i t h a m r o . s u l f a t e .
1 0 m g s ./ m l .
INCUBATION TIM E (minutes}
F IG U R E 38
A D IA G R A M M A T IC R E P R E S E N T A T IO N F O R T H E E F F E C T O F T H E
LIPID A N D P R O T E IN M O IE T IE S O F A N T IT H R O M B O P L A S T IN O N
T H E INHIBITION O F TISSU E T H R O M B O P L A S T IN .
1 2 2
ANTI THROMBOPLASTIN
THROMBOPLASTIN PROTHROMBIN
PROTHROMBIN- Ca44-
THFLN complex
THROMBIN +
THPLN - Ca*4 compli
ANTTTHPLN- Ca44-THPLN
complex
PROTHROMBIN -—►PROTHROMBIN - Ca4
Glycoprotein
variou s states of
a ctiv ity
THPLN^compiex'
A N T IT H P L N
FIG U R E 29
A W O R K IN G H Y P O T H E S IS B O R F U T U R E IN V E S T IG A T IO N S
O N T H E R O L E O F A N T I T H R O M B O P L A S T IN
IN T H E A C T IV A T IO N O F P R O T H R O M B IN .
123
in which the clotting enzymes exert their action. F o r
example, what is the enzymatic activity of thromboplastin
in the conversion of prothrombin to thrombin? H o w does
thrombin polymerize fibrinogen and by what manner of action
does this enzyme activate A cG to a more potent accelerator?
Are the actions of these enzymes similar to other biochem
ical catalysts, i . < 3 . , hydrolytic, proteolytic, phosphor-
ly tic , etc.?
Answers to these questions have been suggested in a
few instances. Som e thromboplastins have a definite phos
phatase activity (127), although the thromboplastic activ
ity of trypsin appears to be proteolytic. Thrombin acts
to convert fibrinogen by a proteolytic splitting of fib rin
ogen end groups to form a peptide and a series of long
fibrin strands (128). Thrombin also has esterase activity
in splitting tosyl-arginine-methyl ester (129). There has
also been som e suggestion by Guest and W are (130) that
thrombin not only converts fibrinogen but slowly dissolves
the d o t as proteolytic e n z y m e s do.
Since fairly pure preparations of plasma A eG could
be prepared by the method of Lewis and W are (77), the
effect of thrombin in activating this material was studied
1 ' * ' »v
in more detail. The preparation of plasma A cG and the
124
i
purification of bovine thrombin was discussed previously.
An incubation system was devised in which one unit of
bovine thrombin was Incubated with serial in itia l activ i
ties of plasma A cG and at various periods of time aliquots
of the reaction mixture were taken and assayed for A cG
activity. Such an experiment is presented in Figure 30.
I t appears from the Figure, that the ratio of plasma
A cG to thrombin is quite critic al in order to e lic it a
m axim um activation. Thrombin not only activates plasma A cG
to a more potent substance, but also tends to destroy the
final product. This is , perhaps, w hy serum A cG is so
unstable when once elaborated from its precursor form by
thrombin.
A suggestion as to how thrombin might e lic it these
events is depicted in Figure 31. In this experiment,
350 units/ml. of plasma A cG was activated with 1 unit/ml.
of thrombin at p H 7.4. At varying time intervals, 2 ml.
aliquots of the reaction mixture were taken, precipitated
with 2 ml. of trichloroacetic acid (TCA), centrifuged,
the supernatant diluted with 3 ml. of 5 per cent T G A , and
the optical density read at 280 mu at p H 0.6 in a Beckman
Spectrophotometer.
O ne m ay surmize from the experiment presented in
SERUM ACG U N I T S / M L .
125
10%
0 5 10 15 20
INCUBATION TIME IN MINUTES
F IG U R E 30
T H E A C T IV A T IO N O F V A R IO U S P L A S M A A C C E L E R A T O R
G L O B U L IN C O N C E N T R A T IO N S B Y T H R O M B IN .
550, 87.5, and 17.5 Units of human plasma A cG each,
activated with one unit of bovine thrombin.
126
L d
<
> “
CO
.08 -
Q
C
u
10 15 20
INCUBATION TIME IN MINUTES
25 30
2
N
CO
Z
3
U >
U
<
•2
X
F IG U R E SI
T H E R E L A T IO N S H IP B E T W E E N P R O T E O L Y T IC A C T IV IT Y O F T H R O M B IN
A N D ITS A C T IV A T IO N O F P L A S M A A C C E L E R A T O R G L O B U L IN .
See text fdr explanation.
137
Figure 31, that thrombin splits off a fragment from plasma
Acs in converting i t to the serum form. This fragment has
a peak adsorption in the ultraviolet range in the neighbor
hood of 375 to 380 m ju and presents a curve throughout the
ultraviolet spectra similar to that of an ordinary protein
or peptide containing an aromatic amino acid. The la tte r
substance being more probable sinee i t is not precipitated
by T G A .
Thrombin did not display a similar proteolytic
activity on serum albumin at an equivalent protein concen
tration, and thus i t is probably graced with a certain
amount of substrate specificity.
A rather provocative question arises as to the
reason w hy serum A cG loses its activity upon dialysis.
This fact m ay suggest that serum A cG is actually the frag
ment of plasma A cG not precipitated by T G A or is a co-
\ - '
factor necessary for the protein which is precipitated by
acid.
B. Purif i cat ion of Serum A cG
A number of preliminary experiments by Lewis and
W are indicated the inadvisability of attempting to further
purify plasma A cG (K P T A C VI in Figure 5). This was,
indeed, unfortunate since purification of the precursor
128
form of A cG would be more advantageous for research, pur
poses than the serum form. This being the case, attention
was then directed to further purification of the serum
accelerator. Early experiments indicated that the acid
<
precipitate of barium citrate-barium sulfate adsorbed
human A O D plasma (FPT A G III) was an excellent starting
material since its A cG activity could be increased som e
10 to 50 times by the addition of 5 units of thrombin/ml.
of dissolved precipitate. I t was subsequently found that
the specific activity (A cG units/mg. protein) could be in
creased by chromatography with activated alumina and
elution with calcium citrate.
This technic resulted.in a fraction of eluate com
ing off the alumina column with a specific activity of
2000 units/mg. protein, approximately a twenty-two-fold
purification of serum accelerator activity. However,
better purification was found to result by chromatographic
adsorption on A^berlite ion-exchange' columns as suggested
by the preliminary experiments of Nakamura (131). This
elegant method was investigated further and a typical
experiment is presented below.
A preparation of Fraction PPT A G III was activated
with thrombin and diluted to 10 ml. with saline to give
129
2.4 m g. protein/ml. with a specific activity of 170 units
of AcG/mg. protein. This volume was then placed on a
24 x 1.2 cm . wet Amberlite IRA-400 (chloride cycle) column.
The column was then washed with successive 10 ml. volumes
of d istilled water 3 times followed by elution with 3 per
cent sodium chloride with a p H of 6.0 adjusted with 1 per
cent acetic acid o f* 0.001 M phosphate buffer. The results
of determinations for A cG and protein on the resulting
one ml. effluents collected from the column are presented
in Figure 32.
Several comments► in connection with the experiment
presented in Figur© 32 are worthy,,of.note. The firs t
fractions coming off.the column contain unadsorbed protein
associated with A cG activity which is also easily washed
off with water. The la tte r fractions show a peak of A cG
activity associated with little protein after subsequent
elution with 3 per cent sodium chloride. This presents
an apparent anomaly since theoretically one would not
expect two peaks of A cG activity if i t were a single sub
stance. This m ay be explained as follows: The f ir s t peak
is the result of plasma A © G which is not completely acti
vated to the serum form by thrombin before i t is placed on
the column. Plasma A cG is not adsorbed on Amberlite
_
150
~i 1 r
2000 r 1 0 M i_ s • a c g
PREP.2.4 MGS.
PR O TEIN / M L .
1 8 0 0 - OF 0 . 9 /- N a CL
S .A .= 1 7 0 u . / N / I G .
PRO TEIN
2 4 X 1.2 CM.
AMBERLITE IR 4 .0 0
COLUMN
L d
O I—
? O
a:
Cl
CO
o
8 0 2 0 4 0 6 0
M LS. OF E F F L U E N T -------
H20 -------+ ------------ 3 . 0 / NaCL
INFLUENT CHANGES
100
>
FIG U R E 32
PU R IFIC A T IO N O F S E R U M A C C E L E R A T O R G L O B U L IN
B Y C H R O M A T O G R A P H Y O N A N A M B E R L IT E IRA-400 C O L U M N .
131
i
i
1 columns. The second peak of activity is serum A cG of high
j specific activity. In this experiment the forty-fifth
| ml. of effluent had an activity of 5,335 units/mg. protein.
j Preliminary investigations indicate that the size and
l i
1 activity of the A cG fractions coming off the resin columns '
i
j depend upon the time the starting material is placed on
the column after activation with thrombin. In the experi- I
! ment presented above the starting material was placed on !
i ■ ;
! the column at 10 minutes after activation. Other runs
; indicate that better activity can be achieved in the serum
I
A cG peak if a considerably longer interval of time elapses
after thrombin activation to allow for a more complete
»
conversion of the precursor to the active form. Further j
investigations by this technic have led to the activity I
i
of A cG in 1 ml. of plasma being concentrated in 16 jugm s.
of protein. 1
Further experiments on human serum A cG of high
; specific activity have demonstrated the fact that i t does i
i
' not have antithrombic activity as postulated by Seegers
j working in crude clotting systems (132).
i
i
SUMMARY
W ith, the use of a reproducible assay procedure for
i the quantitative estimation of thromboplastie activity,
| evidence is provided which lends strong support to the
i existence of a factor in hum an blood which is capable of
inhibiting tissue thromboplastin. This physiological •
anticoagulant, antithromboplastin, can be identified in
j
human serum and plasma rendered free of prothrombin by j
i adsorption on barium citrate and barium sulfate preoipi- j
i tates. The enzyme antagonist requires calcium for maximal .
activity and is capable under certain conditions of com
pletely inhibiting tissue thromboplastin from several d if
ferent animal species and organs. Thromboplastin which
l
has been inhibited by th is factor in serum can be isolated j
i I
j by high-speed sedimentation and the enzyme-inhibitor com- ;
pi ex can be disrupted by the removal of calcium. The !
existence of a thromboplastin-anti thromboplastin complex \
in the human circulatory system which is easily reversed
I
' with oxalate, citrate, and fluoride salts provides an j
explanation for the coagulant properties of certain calcium;
binding salts in vivo.
j
Antithromboplastin can be partially isolated from
!
1 adsorbed serum between the levels of 40 to 50 per cent
saturation with am m onium sulfate at 0°C., by 5 per cent
saturation with Beinecke salt at room temperature, and is
found in plasma fractions IV-1 and IV-3,4 isolated by the
method of Cohn. The inhibitor is heat labile, non-
dialyzable, and appears to be a pseudoglobulin.
t
There is som e suggestion that the circulating an ti-
thromboplastin in vivo m ay be composed of two distinct
co-factors,,each having its ow n anticoagulant properties. j
O ne of these factors is a heat labile protein which appears!
to compete with prothrombin for the active groups on the
i
thromboplastin molecule in the presence of calcium. The
other factor is a heat stable lipid which shares many of j
the physical and chemical properties of cephalin and
|
apparently acts through some type of competitive mechanism j
i
with this thromboplastic substance during prothrombin
activation.
The reason why other investigators have been only
partially successful in demonstrating antithromboplastin
activity in vitro appears to be two-fold: (1) clot accel- '
erators which are produced by incubation of thromboplastin 1
i
with serum and calcium have masked part of the inhibition, j
I
and (2) the inclusion of calcium binding salts in the clot-'
ting systems causes a partial reversal of thromboplastin
I
134
inhibition.
j The antithromboplastin tite r in a number of patho-
; logical serums was investigated, serum antithromboplastin
I appears to have an increased rate of activity during the
third trimester of hum an pregnancy over that of serums
obtained from non-gestating w om en. There is no available
evidence which indicates that th is anticoagulant in ele-
t
vated in hemophilia.
A n unrecognized bleeding diathesis associated with
an increased serum antithromboplastin has been described
and tentatively named idiopathic or secondary antithrombo-
plastinophilia. A detailed investigation of this disease
has resulted in the hypothesis that the A H G - and thrombo
plastin m ay be synonymous terms for the same catalytic
factor in the activation of prothrombin.
Preliminary observations which indicated that the
thromboplastic enzyme may be activated by calcium were
extended. A clotting system composed of the purified
coagulation components isolated from human blood and t i s
sues was devised to further investigate this problem. The
data obtained by the use of this reconstructed system,
which simulated the activities of the clotting factors in
normal blood, strongly suggest that the role of calcium in
135 j
I
i
the clotting mechanism is to activate thromboplastin. j
The availability of purified preparations of plasma j
A cG prompted further studies on the role which thrombin !
plays in the activation of this clotting co-factor. The
results of such experiments indicate that thrombin has a I
proteolytic effect in activating plasma A c G - to serum A cG .
Methods for the purification of serum A cG by adsorp
tion on Amberlite IB-400 columns were extended further.
Preparations of serum A cG of high specific activity
obtained by th is method have no demonstrable antithrombin
activity as previously reported by others.
t
B IB L IO G R A P H Y
I
I
B IB L IO G R A P H Y
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U sttver-strr o t S o u tftem CaIlf808J$e
I

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Asset Metadata

Creator Lanchantin, Gerard Francis (author)

Core Title An investigation of factors involved in the inception of blood coagulation.

Degree Doctor of Philosophy

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Health Sciences, Medical Ethics,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Ware, Arnold G. (committee chair), [illegible] (committee member), Mehl, J.W. (committee member), Visser, Donald H. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-601950

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