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Michigan State
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This is to certify that the
thesis entitled
THE DEVELOPMENT OF A BIOLOGICAL PLATELET FACTOR 4

PURIFIED SUBSTRATE ASSAY

presented by

Suzanne C. Estry

has been accepted towards fulfillment
of the requirements for

[21.8. .degreein .Bathology.

 

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Major professor

Date/M

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THE DEVELOPMENT OF A BIOLOGICAL PLATELET FACTOR 4

PURIFIED SUBSTRATE ASSAY

By

Suzanne C. Estry

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1978

lAl ESSJKI
GAO“

ABSTRACT
THE DEVELOPMENT OF A BIOLOGICAL PLATELET FACTOR 4
PURIFIED SUBSTRATE ASSAY
By

Suzanne C. Estry

Released during platelet aggregation is a heparin neutral-
izing protein known as platelet factor 4. The physiologic func-
tion of this protein is not completely understood, but it does
exhibit anti—heparin activity in viva. Measurement of this ac-
tivity has been the subject of this study. With current techniques
the biological activity has been difficult to quantitate because of
the variability in blood cofactors necessary to assay platelet fac—
tor 4 activity. The new assay developed is a thrombin clotting time
utilizing a purified substrate which helps to eliminate this vari-
ability. The purified substrate is composed of predetermined stan—
dardized amounts of fibrinogen and antithrombin III. Used with this
purified substrate is a standardized concentration of thrombin. The
end point of the thrombin clotting time is the thrombin catalyzed
generation of fibrin from fibrinogen. The activity of thrombin is
decreased by the presence of antithrombin III-heparin complex and
this complex is in turn regulated by the competition of platelet
factor 4 for heparin. The final thrombin clotting time is a reflec—
tion of platelet factor 4 activity only if all other factors are
present in optimum controlled amounts. This new Biological Platelet
Factor 4 Purified Substrate Assay takes into account this need for

optimum levels of all other factors.

DEDICATION

To my husband, Doug, for his encouragement and advice and to

my family for their support.

ii

ACKNOWLEDGMENTS

I wish to express my appreciation to the following people:

Joan C. Mattson, M.D., my major professor, for all her technical
guidance, and personal encouragement given to me throughout my Master's
program.

Thomas G. Bell, D.V.M., Ph.D., for his technical assistance and
for serving as a member on my committee.

Earl W. Campbell, Jr., M.D., for his willingness to serve as a
member on my committee.

Martha T. Thomas, M.S., M.T.(ASCP), my academic advisor, for her
guidance in my graduate study.

Belinda J. Oxender for her excellent typing.

To all the people who generously donated their blood to this

worthy cause.

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . . . . . . 2
Platelet Factor 4 . . . . . . . . . . . . . . 2
Antithrombin III . . . . . . . . . . . . . . 11
MATERIALS AND METHODS . . . . . . . . . . . . . . l6
Substrate and Test Plasma . . . . . . . . . . . l6
Platelet Factor 4 Assays. . . . . . l7
Platelet Factor 4 Thrombin Clotting Time. . . . . 17
Modification of Plasma Heparin Neutralizing
Activity Assay . . . . . . . . l9
Biological PF4 Purified Substrate AsSay . . . . . . 20
Fibrinogen Curve . . . . . . . . . . . . 23
Assay for Antithrombin III . . . . . . . . . . . 23
RESULTS . . . . . . . . . . . . . . . . . . . 24
Effects of Anticoagulants on Thrombin Clotting
Time Assays for PF4 . . . . . . . . . . . 24
Effect of Other Plasma Constituents . . . . 27
Effect of Antithrombin III in the Test Plasma on
PF4 Values . . . . . 28
Sensitivity and Reproducibility of Available PF4
Assays. . . . . . . . . . . . 30
Development of Controlled Purified Substrate . . . . . 31
Biological PF4 Purified Substrate Assay. . . . . . 36
Effects of Other Plasma Constituents on the New
Assay . . . . . . . . . . . . . . . . . 40
DISCUSSION . . . . . . . . . . . . . . . . . . 42
CONCLUSION . . . . . . . . . . . . . . . . . . 47
APPENDIX. . . . . . . . . . . . . . . . . . . 48
LITERATURE CITED . . . . . . . . . . . . . . . . 49
VITA . . . . . . . . . . . . . . . . . . . . 55

iv

Table

10

ll

12.

LIST OF TABLES

Page

Amino acid composition of heparin—neutralizing
protein . . . . . . . . . . . . . . . . 6

Plasma PF4 levels with EDTA and sodium citrate as
anticoagulants and heating of specimen utilizing
modification of Penner's method . . . . . . . . 24

Plasma PF4 levels with EDTA and EGTA as anti-
coagulants utilizing modification of Penner's
method. . . . . . . . . . . . . . . . . 25

Plasma PF4 levels with varying concentrations of
EGTA utilizing modification of Penner's method . . . 26

Comparison of Handin's anti—release cocktail with
sodium citrate on plasma PF4 levels utilizing
modification of Penner's method . . . . . . . . 26

Plasma PF4 levels before and after plasma absorption
with calcium phosphate utilizing modification of
Penner's method. . . . . . . . . . . . . . 27

The effects of incubation of 60 C on antithrombin III
levels of activity measured by method of Penner . . . 28

Plasma PF4 levels from ten normal subjects utilizing
Dana et al. method. . . . . . . . . . . . . 30

Units of heparin neutralized per milliliter in the
substrate plasma curves obtained by method
Dana et al . . . . . . . . . . . . . . . 31

Plasma PF4 levels on three samples from a normal
subject utilizing Dana et al. method. . . . . . . 31

Effect of antithrombin III concentration on clotting
times (seconds) utilizing the purified substrate
assay . . . . . . . . . . . . . . . . . 35

Effect of thrombin concentration (8 u/ml) with various
dilutions of heparin on clotting times (seconds)
utilizing the purified substrate assay . . . . . . 35

Table

l3

14

15

l6

l7

18

Page
Effect of increased thrombin concentration (12.5 u/ml)
with various dilutions of heparin on clotting times
(seconds) utilizing the purified substrate assay. . . 36
Units of heparin neutralized per milliliter by
purified substrate alone. . . . . . . . . . . 36
Plasma PF4 levels from normal subjects utilizing the
Bio PF4 purified substrate assay . . . . . . . . 37
Repeated plasma PF4 levels in normal subjects obtained
on different days . . . . . . . . . . . . . 37
Comparing plasma PF4 levels of three samples from a
normal subject with the Dana et al. method and the
Bio PF4 purified substrate assay . . . . . . . . 38
Units of heparin neutralized per milliliter expressed
in micrograms per milliliter of PF4 . . . . . . . 40

vi

Figure

1

LIST OF FIGURES

Curves from the biological PF4 purified substrate
assay . . . . . . . . . . . . . . .

The effect of incubation times on the levels of
antithrombin III activity measured by method of

Penner. . . . . . .

Effects of increasing concentration of fibrinogen
on the thrombin clotting time

Effects of increasing concentration of fibrinogen
on the maximum velocity of fibrin formation . .

Units of heparin neutralized by serial dilution of
purified PF4. . . . . . . . . . .

vii

Page

22

29

33

34

39

INTRODUCTION

Platelets play an important role in hemostasis not only in the
formation of a platelet plug but also in the release of certain com-
ponents which aid in clot formation and eventually resolution.
Platelet factor 4 is a platelet specific heparin neutralizing protein
and is released when platelets aggregate. Preliminary studies by
other investigators have established that plasma levels of platelet
factor 4 are significantly increased in clinical situations involving
in vivo systemic platelet aggregation and suggest that platelet factor
4 may function as a sensitive parameter to study localized thrombotic
events, such as myocardial infarction.

The research project was designed to develop a biological assay
for platelet factor 4. In doing this, it became obvious that an im-
portant naturally occurring inhibitor in the blood system known as
antithrombin III must be taken into consideration. Antithrombin III
can inhibit certain coagulation factors and uses heparin as a cofactor.
Since these two proteins are closely interrelated, one cannot be

evaluated without consideration of the other.

 

LITERATURE REVIEW

Platelet Factor 4
History

The heparin neutralizing activity of platelets was first described
by Conley g£_§l, in 1948. They observed that thrombocytopenic patients
had a prolonged heparin effect as compared to those patients with
normal platelet counts (1). In 1951, Van Creveld and Paulssen described
a platelet factor that neutralized heparin. This heparin neutralizing
activity was initially thought to be associated with platelet procoagu-
lant activity (2, 3). It was soon shown to be distinct from platelet
factor 3 by JUrgens in 1954 (4). This antiheparin activity was termed
platelet factor 4 (PF4) by Deutsch §£_§l, in 1955 (5). This first
attempt at isolation and characterization of PF4 was made by Deutsch
and Franke, 1957; they concluded that PF4 had characteristics of a
protein (6).
Biochemistry

Research work done on PF4 during the sixties centered around per—
fection of techniques for isolation and characterization of the protein.
Deutsch g£_§l, in 1961, using DEAR-chromatography, increased the con-
centration of PF4 activity lOO-fold in comparison with previous work
(7). In 1964, Poplawski and Niewiarowski succeeded in isolating PF4
from pig platelets in higher concentration, but platelet factor 2 (PF2)

was present as a contaminant in their end product (8). They ultimately

 

achieved further purification and complete separation from all other
platelet factors by fractionation on DEAE-cellulose (9).

Niewiarowski g£_a1., in 1965, demonstrated that their purified
PF4 could neutralize antithrombin VI (fibrinogen breakdown products)
(10). They concluded that the two activities, 1) antiheparin activity
and, 2) the ability to neutralize antithrombin VI may reside in the
same protein. Their purified PF4 did not influence the fibrinogen-
fibrin conversion by thrombin indicating that PF2 and PF4 were not
the same substance.

Farbiszewski §£_§1, reported in 1969, that PF4 induces a para—
coagulation reaction, a non-enzymatic clotting of soluble fibrin monomer
complexes (11). However, this was later disproven in a study done by
Ka'ser—Glanzmann §_§_a_1_l_. in 1973 (12) .

The question of where PF4 resides in the platelet remains a con—
troversial point with no definite answer as yet. Using sucrose gradient
fractions of platelet homogenates and testing each fraction for heparin
neutralizing activity (HNA), Day §£_§1, in 1973 concluded that PF4 might
be localized in the platelet dense bodies (13). In 1974, walsh and
Gagnatelli assayed platelet release products for antiheparin activity
to help determine how PF4 is stored and released (14). They found that
the release of HNA from normal platelets by thrombin and collagen was
slower than that of serotonin. Lysosomal enzymes were not released by
collagen whereas under the same conditions HNA was released. They con-
cluded that PF4 either could be stored in and released from dense
granules by mechanisms different from those for other dense body con-
stituents, or that PF4 could be stored and released from a morphologically

similar but functionally distinct group of dense granules. Their evidence

 

seems to exclude the possibility that PF4 resides in alpha granules
which contain lysosomal enzymes. Isolating PF4 from pig platelets

and studying sucrose gradient fractions, Joist et_§1, indicated that
PF4 was distributed among all of the subcellular fractions except for
the dense granules (15). Two more studies in 1965, one by Broekman
gnggr (16) and another by DaPrada §£_§1, (17) suggest that PF4 resides
in the alpha granules. Another concept which needs experimental veri-
fication is that PF4 is attached in some way to the membrane of the
platelet. This idea has been suggested by O'Brien §£_gl. (18), Moore
§£_§1, (19), and Mui (20) based on the finding that some antiheparin
activity was found in fractions containing mostly membranes and in
those containing mitochondria and granules of lesser density.

Methods for isolation and purification of PF4 along with its
characterization improved during the seventies. One such experiment
by Barber §£_§1,, has shown that PF4 is released in the form of a
high molecular weight proteoglycan-PF4 complex (21). This complex
dissociates at high ionic strength into an active component (MW 29,000)
and a proteoglycan carrier. Upon further purification, the carrier has
been shown to consist of chondroitin 4-su1fate with a molecular weight
of 59,000. The molecule contains four chondroitin 4-su1fate chains
(MW 12,000) in covalent linkage to a single polypeptide. The complete
complex has a molecular weight of 350,000 which suggests that four
moles of PF4 are bound per mole of proteoglycan and that the carrier
occurs in the form of a dimer consisting of eight moles of PF4 and two
moles of proteoglycan. The isolated chondroitin 4-sulfate moieties
combine with PF4 at a binding ratio of one mole of PF4 per carbohydrate

chain. Heparin completely displaces PF4 from both the saturated

 

proteoglycan and chondroitin 4-su1fate complexes. The relative bind—
ing capacities of glycosaminoglycans for PF4 as shown by Barber §£_§1,
are: heparin [100], heparitin sulfate [75], chondroitin 4-su1fate
[50], dermatan sulfate [50], and chondroitin 6-sulfate [50]. Hy—
aluronic acid does not combine with PF4 (21). Handin and Cohen (22)
also studied the ability of the sulfated glycosaminoglycans to bind
to the protein by employing the use of [3H] heparin. Their results
differed slightly from those of Barber g£_§1. (21). They concluded
that dermatan sulfate has a higher binding capacity than chondroitin
6-su1fate and chondroitin 6—sulfate has a greater binding capacity
than chondroitin 4-su1fate (22).

Evidence from other works indicate that the active component of
the complex (PF4) exists, at physiologic ionic strength and pH, as a
tetramer (19) with a molecular weight of the subunit ranging from
6,000 to 11,600 (19, 22, 23). Handin and Cohen have presented evi—
dence that it is the lysine residues on the protein that interact
with the heparin (22).

The amino acid analysis of PF4 has been reported in several
different publications with similar results (19, 22, 23, 24).
Table 1 lists the amino acid composition given by Handin and
Cohen (22).
Clinical Implications

Although PF4 has been demonstrated to neutralize heparin added
to plasma in vitro and its level in plasma has been shown to be in-
creased in certain pathological states, the role of this protein in
normal or abnormal hemostasis is unknown. The presence of PF4 with

its heparin neutralizing ability in the circulation may have potential

 

Table 1. Amino acid composition of heparin—neutralizing protein.

 

 

amino acid residue/mole amino acid residue/mole
Alanine 5 Lysine 7
Arginine 3 Methionine 0
Aspartic acid 6 Phenylalanine l
Cysteine 2 Proline 4
Glutamic acid 9 Serine 4
Glycine 4 Threonine 4
Histidine 2 Tryptophan 0
Isoleucine 4 Tyrosine 1
Leucine 8 Valine 3

 

 

significance in modulation of coagulation reactions, particularly the
interaction of heparin with antithrombin III. In addition, the fact
that PF4 can bind sulfated aminoglycans present on cell surfaces and
in plasma suggests that it may also play a role in other physiologic
processes.

There have been several attempts to deveIOp assays to measure the
plasma level of PF4 in order to define the role of PF4 in hemostasis
and thrombosis. The different assays currently in use can be divided
into two main approaches, 1) immunologic determination of PF4 protein,
and 2) biological assays of heparin neutralizing capacity.

Immunologic Assays

 

In 1974, Gjesdal developed an electroimmunoassay based on the
Laurell rocketry technique (25). One problem encountered with this
procedure is that it cannot detect PF4 in platelet poor plasma with-
out first concentrating the plasma, though serum levels can be measured.

A radial immunodiffusion method has been developed by Niewiarowski
g£_al., 1976, using the Mancini technique (26). They tested a normal

individual's platelet free plasma and platelet rich plasma which had

 

 

‘5

 

been treated to cause release of PF4. The results from this study
showed that PF4 was a sensitive marker of platelet release in vitro.

Another experimental method is the radioimmunoassay (RIA) (27,
28, 29). Bolton and associates found an increased level of PF4 in
patients who had received prosthetic heart valves (28). Handin
g£_al, used the RIA to compare the levels of PF4 in normal individ—
uals to those of patients with coronary artery disease, chest pain
with no myocardial infarction, and chest pain with myocardial in—
farction. They found a significant increase of PF4 level in all
groups of patients studied, with those of myocardial infarction
being the highest, 5—fold (29).

From these results and others obtained by using the immunologic
technique to measure PF4, it seems that the quantitation of such a
protein might be a useful diagnostic tool. These assays, however, do
not assess the biological activity of the protein molecule measured.
The biological activity may be an equally important diagnostic tool
for determining how PF4 functions physiologically. The currently
available system for measuring biological activity of PF4 involves the
addition of known quantities of heparin to plasma and the measurement
of the capacity of the plasma to neutralize heparin's anticoagulant
effect. The heparin effect may be quantitated by utilizing a thrombin
time assay or by measuring the rate of inactivation of Factor Xa.

The assay for Factor Xa inactivation is based on the ability of
residual heparin, not neutralized by PF4, to potentiate the inactiva-
tion of Factor Xa by a plasma inhibitor (30, 31). There is evidence
that, antifactor Xa, is antithrombin III (32). If this is true, the

method of Factor Xa inactivation adds another step to that of the

method of a thrombin clotting time. Even though the sensitivity of
Factor Xa-inhibitor-heparin reaction seems to be much higher (30),
than that of the thrombin-antithrombin III—heparin reaction, the end
point of both assays is a clot formation which must include thrombin
and fibrinogen.

Many variations to the thrombin time assays for PF4 have been
developed. The sequence of events in this kind of assay is as
follows: 1) heparin is added to the test system, 2) PF4 binds up
(neutralizes) the heparin, 3) any residual heparin will complex with
the antithrombin III, and 4) the antithrombin III-heparin complex
can inactivate the thrombin. The amount of thrombin inactivation is
measured by the amount of time it takes to convert fibrinogen to a
fibrin clot. One of the simplest assays of this type was deve10ped
by O'Brien and associates in 1974 (33). In the test system, heparin
is added to the test plasma and a thrombin clotting time is done.

The results are reported in seconds and a shortening of the clotting
time means a higher level of heparin neutralization. O'Brien and
associates used this Heparin Thrombin Clotting Time (HTCT) to study
different groups of patients. They found a shortening of HTCT in myo-
cardial infarctions, atherosclerosis, deep vein thrombosis, and sec—
ondary diffuse intravascular coagulation (33, 34). There was a pro—
longed HTCT in patients with idiopathic thrombocytopenic purpura (33).
Concerning the patients with myocardial infarctions, the HTCT returned
to a normal level within one to three months after infarction (34).

A thrombin clotting time method developed by Harada and Zucker
utilizes a series of heparin dilutions added to the test plasma (35).

With each dilution of heparin, a thrombin clotting time is done. The

 

heparin concentration giving a clotting time of over 20 seconds is
used as the end point. After this point the clotting time becomes
unreliable because the PF4 capacity to bind heparin has been exceeded. .
The excess heparin complexed with antithrombin III completely inacti-
vates the thrombin. The results of the assay are reported out as
units of heparin neutralized.
In 1976, Dana and associates modified the assay of Harada and
Zucker (35) by the addition of normal pooled plasma ”substrate plasma"
in order to provide a surplus of fibrinogen and antithrombin III (36).

The substrate plasma and test plasma are added together along with

 

serial dilutions of heparin. This mixture is incubated, after which
thrombin is added and a clotting time determined. The results are
reported in units of heparin neutralized per milliliter. Utilizing
this method, Dana g£_al. found a decrease of heparin neutralizing
activity (HNA) in patients with bone marrow depression, idiopathic
thrombocytopenic purpura, and myeloproliferative disorders with
thrombocytosis (36). The conditions in which an increased HNA was
found included diffuse intravascular coagulation, secondary thrombo-
cytosis, acute and chronic coronary insufficiency, and chronic coro—
nary artery disease (36, 37).

Another technique for assaying PF4, described by Fuster g£_al.,
uses a heparin thrombin clotting time in which a ratio of clotting
times of the test plasma to that of distilled water is utilized (38).
The ratio is plotted on the ordinate with percent of PF4 on the ab-
scissa. Results obtained with serum are used as 100% PF4, results
obtained with platelet poor plasma from a thrombocytopenic patient

(less than 5,000 mm3 platelet count) are used as 0% PF4. Also in

10

this procedure the test plasma has been heated at 60 C for 10 minutes
to remove the fibrinogen and antithrombin III which is not needed.
The pooled normal substrate plasma utilized in this test contributes
optimal amounts of antithrombin III and fibrinogen (38).

Saleem and Fretz in 1977 created another modification to the
basic heparin thrombin clotting time in which they used a lyophilized
commercial plasma for the substrate plasma (39). The lyophilized
plasma is a normal pooled plasma utilized as a normal control in rou-
tine coagulation procedures. The authors claimed improved sensitiv-
ity and precision of the assay due to the fact the lyophilized plasma
had a low sensitivity to heparin, permitting use of larger concentra-
tions of heparin that still had reproducible clotting times.

Protamine sulfate has the ability to neutralize heparin. This
substance was used by Poplawski and Niewiarowski (1965) to standard—
ize their assay of measuring antiheparin activity (40). The method
involved a thrombin clotting time in which a serial dilution of
protamine sulfate is substituted for the test specimen in calculat—
ing a standard curve. The clotting time of the test specimen is
compared to the curve of the protamine sulfate with results being
expressed in mean equivalents of protamine sulfate standard solution.
Bovine citrated platelet poor plasma is used as a substrate plasma.

In 1977, Levine g£_§l, measured PF4 activity by the method of
Poplawski and Niewiarowski (40) using hexadimethrine bromide (Poly-
brene <:)) as a standard instead of protamine sulfate (41). The
authors stated that Polybrene (:) proved completely stable when di-
luted whereas protamine lost activity progressively after dilution

and was stable for less than one hour. This assay was used to measure

 

ll

PF4 activity in platelet poor plasma prepared from platelet rich
plasma which had been subjected either to controlled wall impact
surface injury or to shear stress force in a jet stream injection
device. The results indicated that this assay was a sensitive index
of mechanical platelet damage.

The assays mentioned thus far have dealt with measuring either
the antiheparin activity of PF4 or its antigenic levels. Another
unique assay has been developed to measure the effect of PF4 on
collagenase activity (42). Collagenase degrades the protein colla—
gen at neutral pH and physiological temperature. Regulation of col-
lagen metabolism by collagenase is important in both normal and dis—
ease states (42). Collagenase activity can be measured by the re—
lease Of 14C glycine peptides from guinea pig skin collagen (43).

It has been shown that PF4 inhibits human skin collagenases which
suggests a possible physiological role for this protein in connec—
tive tissue metabolism (42).

Antithrombin III

 

History

Before antithrombin 111 can be discussed, a little background on
antithrombins in general must be given. 'In 1905, Morawitz first ap—
plied the term "antithrombin" to a substance in the circulating blood
which inhibited thrombin by progressive inactivation (44). In 1939,
Brinkhouse and associates, also reported a substance which in con-
junction with heparin prevented thrombin formation (45). Throughout
the years different antithrombins have been described and at one time
there were thought to be six different antithrombins.

ANTITHROMBIN I: Seegers demonstrated in 1947 that fibrin could
adsorb thrombin (46).

 

12

ANTITHROMBIN II: Astrup and Darling, 1943, described a heparin
cofactor with antithrombin activity (47).

 

ANTITHROMBIN III: Astrup and Darling, 1943, also reported an anti-
thrombin which inhibited thrombin progressively
without heparin (47).

ANTITHROMBIN IV: Seegers and associates, 1952, described a pro-
gressive antithrombin seen only in ether—treated
defibrinated plasma having no other antithrombin
activities (48). Others have not been able to
confirm this finding.

ANTITHROMBIN V: Loeleger and Hers, 1957, talked about this anti-
thrombin as the cause of a hemorrhagic diathesis
in a patient with rheumatoid arthritis (49).

ANTITHROMBIN VI: Niewiarowski and Kowalski, 1958, characterized
this as a degradation product of fibrinolysis
(50).

 

In 1967, Abildgaard from Norway showed that antithrombin II and anti-
thrombin III are the same protein (51). This inhibitor is now called
only antithrombin III or antithrombin-heparin cofactor. The term,
antithrombin II, is no longer in usage. Since antithrombin III is a
physiologically more significant natural inhibitor than the other
known antithrombins and since it has a progressive and irreversible
action on thrombin with its interaction with heparin, it has been
studied extensively.

Biochemistry

 

Antithrombin III (AT 111) is an alpha-2-globulin with a molecu-
lar weight of approximately 64,000 (52, 53). The mode of action of
AT III has been extensively studied by Rosenberg (32). He has demon—
strated that AT III can inactivate not only thrombin but also other
serine proteases of the coagulation-fibrinolytic system including
coagulation factors XIIa, XIa, Xa, IXa, and plasmin of the fibrino-

lytic system. When AT III and thrombin or other serine proteases

 

l3

interact, a stable complex is formed. Complex formation has been
documented by sodium dodecyl sulfate—polyacrylamide gel electro—
phoresis (SDS-PAGE). Both AT III and thrombin migrate in SDS-PAGE .
as single bands with molecular weights of 62,000 and 33,800, respec-
tively. When these two proteins are incubated together these two
single bands disappear and a complex with a molecular weight of
88,700 is formed.
If thrombin is treated with di—isopropyf1uorophosphate which
alkylphosphorylates the unique serine residue, no interaction be-

tween AT 111 and thrombin takes place (32). Rosenberg has shown

 

that it is the arginine residue on the antithrombin that is involved
in the interaction of serine proteases. Inhibition of the serine
proteases can be accomplished with or without heparin as a cofactor.
In the case of thrombin, if heparin is not present, AT 111 can in-
activate a larger quantity of thrombin but at a much slower rate than
if heparin were used as a cofactor. In the presence of heparin, in-
activation of thrombin is almost instantaneous. It has been shown
by Rosenberg that heparin binds to lysyl residues on the AT 111.
The dramatic increase in reaction rate is probably due to a heparin
dependent conformational alteration of the AT 111 which renders the
reactive arginine site more accessible to the active serine center
of thrombin (32).
Clinical Implications

With the recognition that AT III complexed with heparin is re—
sponsible for neutralization of thrombin and other serine proteases,
it has become evident that it is a very important feedback system in

the coagulation-fibrinolytic process. A decrease in plasma AT III

14

levels is of major clinical importance since such a decrease is
thought to promote thrombogenesis. There are two categories of
decreased AT III, one of which is inherited and another of which
is acquired and found in certain pathological conditions.
Inherited

Studies done on several families reporting congenital defi-
ciencies of AT III have supported the thesis that low levels of
AT 111 can lead to thrombosis. In 1965, Egeberg reported a com—
prehensive study on the coagulation system in members of a
Norwegian family with a high incidence of thrombotic disease in
which episodes most often occurred as deep venous thrombosis in
the leg (54). Since the procoagulant factor activities were with-
in the normal range, attention was centered around AT 111. Plasma
AT III activity was abnormally low in members with a history of
thrombosis and in some of their children. The average level for
all members was 72% of normal and 50% of normal for those with a
history of thrombosis. These episodes occurred most often with
trauma, surgery, pregnancy, and inflammation. It was seen in this
family that AT III deficiency can manifest itself with thrombosis
in children as well as adults. As the deficiency was found in both
males and females and seemed to be inherited from either parent,
Egeberg concluded that the inheritance pattern was one of autosomal
dominance. Other family studies have reported similar data to that
of the Norwegian family (55, 56).
Acguired

With the acquired states of AT III deficiency, the data is less

conclusive. Not all patients exhibit the same finding with the same

 

15

disease states. There appears to be a tendency toward a decrease in

AT III activity in the circulating blood in coronary—artery disease

(57), hepatic disease (58), venous thrombosis (59, 60), extensive .
pancreatic carcinoma (61, 62) and disseminated intravascular coagu-

lation (63). There are also reports of reduced AT III levels and

an increased risk of thrombotic manifestations in women taking oral

contraceptives (63, 64). However, AT III levels are not uniformly

depressed in these groups of patients. The question of why AT 111

activity is decreased in some but not all patients having these con-

ditions has not been resolved.

 

MATERIALS AND METHODS

SUBSTRATE AND TEST PLASMAS

 

Preparation

 

The substrate platelet poor plasma was prepared by collecting
blood from normal subjects in plastic syringes. Blood samples were
anticoagulated with a 1:10 ratio by volume of 3.8% trisodium citrate.
Blood was then centrifuged at 2500 rpm for 10 minutes. The platelet
poor plasma was removed with a plastic pipette and placed in plastic
test tubes.

The test plasma was prepared in a similar manner. After separa—
tion, 1 m1 aliquots of test plasma were heated at 60 C for 10 minutes
and recentrifuged to obtain the defibrinated supernatant. If the
sample was not to be assayed immediately, the plasma was aliquoted
and stored at -30 C before heating at 60 C.

Anticoagulants

 

In tests on the prevention of platelet release, several anti—
coagulants were evaluated. In addition to citrate, ethylenediamine—
tetraacetic acid (EDTA) and ethyleneglycol-bis (B-aminoethyly ether)
N, N'-tetraacetic acid (EGTA) were studied. Both were obtained from
Sigma Chemical Company, St. Louis, Missouri, and used at concentra-
tions ranging from 0.2 mg/ml to 3 mg/ml. An anticoagulant cocktail
developed by Robert I. Handin was also used which included the follow-

ing:

16

l7

Acid citrate dextrose (ACD) solution, NIH formula A

EDTA 1 mM
Prostaglandin E1 1 uM
Adenosine 1 mM

One milliliter of the Handin ACD cocktail was added to 5 ml of whole
blood.
Absorption of Plasma

Platelet factor 2 accelerates the conversion of fibrinogen to
fibrin (66). Calcium phosphate can absorb PF2 but not PF4. In some
studies calcium phosphate was added to the heat inactivated plasma
at a ratio of 1 mg to 1 m1 of test plasma. The mixture was heated
at 37 C for 10 minutes with frequent mixing and centrifuged to ob-
tain supernatant.

Alpha-Z-macroglobulin

 

In tests of the effect of other antithrombins, performed only
with the newly developed assay, a 2-macroglobulin was evaluated.
Purified a 2-macroglobulin, obtained from the National Red Cross,
Bethesda, Maryland was used at a concentration of 3 mg/ml. It was
added either to the imidazole buffered saline to be tested with the

purified substrate alone or added to the defibrinated test plasma.

PLATELET FACTOR 4 ASSAYS
Platelet Factor 4 Thrombin Clotting Time (PF4 TCT)

The PF4 TCT was a modification of a thrombin clotting time de-
scribed by Penner (65). The thrombin used was Fibrindex from Ortho
Diagnostics, Inc., Raritan, New Jersey. It was diluted in 0.1 M
calcium chloride until a TCT of 8-9 seconds was achieved. Citrated

saline was made by adding one part of 3.8% trisodium citrate to five

18

parts of 0.9% sodium chloride. Upjohn's sodium heparin (1000 units/m1)
was used; 1 ml of 1000 u/ml was diluted in 100 ml of citrated saline
giving a stock of 10 u/ml. This heparin stock was diluted further in
citrated saline to obtain dilutions from 1 u/ml to 6 u/ml in l u/ml
increments. The pooled substrate and test plasma were prepared as
previously described.

The test procedure requires the construction of a PF4 TCT stan—
dard curve. The heparin concentrations previously prepared were
diluted 1:10 in substrate plasma giving final concentrations of 0.1
u/ml to 0.6 u/ml of heparin in 0.1 u/ml increments. A TCT was done
on each heparin dilution. After a reasonable curve was obtained
using an individual substrate plasma, a pooled substrate plasma was
obtained from 10 normal male donors to produce a final constant
curve. The curve was plotted on semi—log paper with the clotting
time (seconds) on the ordinate and the units of heparin concentra-
tions on the abscissa.

In the performance of the assay of the test plasma, the substrate
plasma with a final concentration of 0.3 u/ml of heparin was used.
The reaction mixture consisted of 0.2 m1 of the substrate plasma—
heparin mixture, and 0.1 ml of test plasma. The test mixture was in-
cubated at 37 C for 3 minutes. One-tenth milliliter of thrombin was
then added and a clotting time determined.

The results were reported as units of heparin neutralized per ml
by the PF4 in the test plasma. The results were calculated by first
determining the heparin concentration of the test plasma. This was
obtained from the standard curve utilizing the clotting time of the

test plasma. The heparin concentration of the test plasma was then

l9

subtracted from the known amount of heparin (0.3 u/ml) added to the
substrate plasma.

Modification of Plasma Heparin Neutralizing Activity Assay, Dana et al.
(36)

One vial of thrombin (Fibrindex) was diluted with 3 ml of veronal
buffer (pH 7.35) to give a final concentration of 16.7 u/ml. Upjohn's
sodium heparin (1000 u/ml) was diluted in 0.9% sodium chloride to give
concentration from 0.1 u/ml up to 1.0 u/ml in increments of 0.1 u/ml.

Substrate plasma and test plasma were prepared as previously de—
scribed. The assay was performed using the Fibrometer, BBL Bioquest,
Cockeysville, Maryland. A standard curve using pooled substrate plasma
was prepared using substrate plasma with increasing heparin concentra—
tions. The reaction mixture consisted of 0.1 m1 veronal buffer, 0.1
ml pooled substrate plasma and 0.05 ml of saline (BLK) or heparin
dilution. This reaction mixture was incubated for one minute at
37 C. Fifty microliters of diluted thrombin was then added and the
time required for clot formation recorded. This procedure was re-
preated with increasing concentrations of heparin until a clotting
time of over 20 seconds was obtained. The test plasma was assayed in
the exact same way except that 0.1 m1 ofdefibrinated test plasma was
used in place of the 0.1 m1 veronal buffer.

The standard (substrate) and test curves were plotted on linear
graph paper with the clotting times on the ordinate and the units of
heparin on the abscissa. The units of heparin, as read on the stan-
dard curve at 20 seconds, were subtracted from the units of heparin as
read on the test plasma curve at 20 seconds. This value was reported

as units of heparin neutralized.

2O

BIOLOGICAL PF4 PURIFIED SUBSTRATE ASSAY

The subject of this thesis is the development of a biological
assay for PF4. The assay as finally developed is described here.

This assay uses only purified plasma components in place of a pooled
substrate plasma. First, a concentration of 12.5 u/ml of thrombin was
obtained by diluting thrombin (Fibrindex) in 4 ml of imidazole buffered
saline. Fibrinogen from General Diagnostics, Morris Plains, New Jersey,
was reconstituted with distilled water to a concentration of 6 mg/ml.
Dilution of the heparin in increments of 0.05 u/ml were prepared as
described for previous assays. Antithrombin III obtained from the
American National Red Cross in Bethesda, Maryland or purified by a
modification of the technique of Thaler g£_§l, (67) was used at a
concentration of 1.35 mg/ml.

Assayed with this procedure were either test platelet poor plasma,
prepared as previously discussed, or purified PF4 obtained from the
American Red Cross, Lansing, Michigan. Purified PF4 in a concentra-
tion of 13.5 ug/ml was diluted at 1:10, 1:20, and 1:40 for use in
this assay.

Both a standard curve (obtained with purified substrate compo—
nents which lacked PF4) and a test curve (obtained with test plasma
containing PF4) were done. For the standard (0 PF4) curve, 0.05 ml
of fibrinogen, 0.05 ml of AT 111, 0.1 m1 imidazole buffered saline,
and 0.05 ml saline (BLK) or heparin dilution were pipetted into a pre—
warmed fibro cup. This mixture was incubated for one minute at 37 C
and the fibrometer timer started with the addition of 0.05 ml of
thrombin. This procedure was repeated, using increasing heparin con—

centrations until the clotting time was over 45 seconds. The same

21

steps were followed as above for the test plasma except that 0.1 m1
of defibrinated test plasma was used in place of 0.1 ml imidazole
buffered saline.

In the calculation of results, all curves were brought to zero by
subtracting the clotting time of the zero heparin from the clotting
times of each one of the increasing heparin dilutions. This was done
for both the standard (0 PF4) curve and test plasma curves. Curves
were then plotted on linear graph paper with the zeroed clotting

times on the ordinate and heparin concentrations on the abscissa.

 

 

Example:
u/ml Standard Curve (0 PF4) Test Plasma #1 Test Plasma #2
nggrin Clot Times zeroed Clot Times Zeroed Clot Times Zeroed
0 12.2 sec. 0 16.8 0 14.7 0
0.10 13.7 1.5 17.2 0.4 15.8 1.1
0.20 18.7 6.5 19.2 2.4 16.3 1.6
0.25 20.3 8.1 22.2 5.4
0.30 34.8 22.6 23.7 6.9 18.2 3.5
0.35 49.2 37.0 27.3 10 5
0.40 >120 34.3 17.5 19.8 5.1
0.45 38.8 22.0
0.50 49.7 32.9 24.8 10.1
0.55 >120
0.60 28.3 13.6
0.70 37.3 22.7

An example of the curves are shown in Figure 1.

After the curves were plotted, the results were determined by reading
the units of heparin directly off the curves at 15 seconds. The units
of heparin obtained from the standard (0 PF4) curve were then sub-

tracted from the units of heparin obtained from the test plasma curve.

The results were reported as units of heparin neutralized per m1.

CLOTTING TIME (SEC)

 

22

 

40 F
35 __ std.
\ test I
30 *-
25 t’
test 2
20 - \
m—— ————————— ——- —————————————
IO r-
55..
l l L_1 l l l
0! 0'2 03 0'4 05 0'6 07
UNITS of HEPARlN/ml
Figure 1. Curves from the Biological PF4 Purified Substrate Assay.

23

FIBRINOGEN CURVE

 

The reagents needed for construction of a fibrinogen curve are
thrombin and fibrinogen. One vial of thrombin (Fibrindex was diluted
with 5 ml of imidazole buffer (pH 7.2) to a concentration of 10 u/ml.
The fibrinogen (Ab Kaba from Sweden) used in this study contained
1.2 grams citrate and 0.4 grams sodium chloride per gram of fibrino-
gen. It was reconstituted with distilled water to a concentration
of 6 mg/ml and further diluted with 0.9% sodium chloride to the fol-
lowing concentrations: 5 mg/ml, 4 mg/ml, 3 mg/ml, 2.5 mg/ml, 2 mg/ml,
1.5 mg/ml, 1 mg/ml, 0.5 mg/ml, and 0.25 mg/ml.

A thrombin clotting time was performed using each concentration
of fibrinogen on the Coagulation Profiler CP—7, Bio-Data Corporation,
Willow Grove, Pennsylvania. The reaction mixture consisted of 0.1 m1
of fibrinogen to which 0.1 m1 of thrombin was added after 5 minutes
incubation at 37 C. The time and rate of clot formation was recorded

by the instrument on linear graph paper.

ASSAY FOR ANTITHROMBIN III (AT 111)

The biological activity of AT 111 was measured by two different
assays; the Thrombo-Screen AT III Kit by Pacific Hemostasis Labora-
tories, Inc., Los Angeles, California, and the Antithrombin III Assay

described by Penner (65).

RESULTS

Effect of Anticoagulants on Thrombin Clotting Time Assays for PF4

The following results were obtained using the modified assay of
Penner's thrombin clotting time. Table 2 shows that there is no dif-
ference in sensitivity between the two different anticoagulants used
but there is an improvement by heating the plasma to 60 C for ten
minutes. The improvement was not consistently found with every sample,
however, this could be a reflection of the inherent variability of the
method. In each instance serum levels of PF4 were determined to ob-
tain a theoretical 100% value of PF4 following platelet activation.
Table 2. Plasma PF4 levels* with EDTA+ and sodium citrate as anti-

coagulants and heating of specimen utilizing modification
of Penner's method.

 

EDTA Citrate

 

Sample EDTA Heating Citrate Heated Serum
1 0.17 0.14 0.145 0.145 0.47
2 0.16 0.155 0.175 0.15 0.50
3 0.16 0.115 0.13 0.13 0.43
4 0.09 0.05 0.105 0.095 0.41
5 0.065 0.085 0.085 0.12 0.37
6 0.195 0.115 0.195 0.15 0.45
7 0.145 0.13 0.135 0.16 0.42
8 0.10 0.75 0.14 0.105 -—-—

 

*Results in units of heparin neutralized/m1.
+Concentration added, 1 mg/ml of whole blood.

24

25

Other comparisons of anticoagulants involved the use of EDTA and
EGTA. There were no consistent results that indicated an improve-
ment in measuring baseline plasma levels of heparin neutralizing

activity (Table 3) by use of any one anticoagulant.

Table 3. Plasma PF4 levels* using unheated and heated plasmas with
EDTA+ and EGTA+ as anticoagulants utilizing modification
of Penner's method.

 

EDTA EGTXT

 

Sample EDTA Heated EGTA Heated
1 0.195 0.115 0.055 0
2 0.145 0.13 0.060 0
3 0.10 0.075 0.170 0.105
4 0.125 0.10 0.135 0.125

 

*Results in units of heparin neutralized/ml.
+Concentration added, 1 mg/ml of whole blood.

There seemed to be an inhibitory effect or a decrease in values with
EGTA, so an experiment utilizing various concentrations of EGTA was
done. This was to see if there was an optimum level of EGTA and to
rule out the possibility that either a too high or too low concentra-
tion caused the inhibitory effect (Table 4).

The anticoagulant cocktail developed by Robert Handin was evalu-
ated to determine if it was superior to other anticoagulants in its
ability to protect platelets from activation during preparation of
the sample for assaying. Blood was drawn and placed into either the
Handin cocktail or standard 3.8% sodium citrate. Samples were then
treated identically. The heparin neutralizing activity of the sample

in the anti-release cocktail was higher than that in the standard

26

citrate anticoagulant. This indicated that the cocktail was not
beneficial in reducing release due to handling (Table 5). Again
the serum levels were determined to obtain a theoretical value for

100% PF4 following platelet activation.

Table 4. Plasma PF4 levels* with varying concentrations of
EGTA utilizing modification of Penner's method.

 

 

Concentration Specimen Specimen

of EGTA mg/ml Unheated Heated
0.2 specimen clotted -----
0.6 0.165 0.015
1.0 0.140 0.05
2.0 0.12 0.05
3.0 0.135 0.015

 

*Results in units of heparin neutralized/m1.

Table 5. Comparison of Handin's anti-release cocktail with sodium
citrate on plasma PF4 levels* utilizing modification of
Penner's method.

 

 

Citrate Cocktail
Sample 'Unheated 'Heated CaHPO4 Unheated Heated CaHPO4
1 0.11 0.095 0.11 0.175 0.140 0.135
2 0.135 0.115 0.185 0.155 0.095 0.140

 

*Results in units of heparin neutralized/ml.

27

Effect of Other Plasma Constituents

Platelet factor 2 (PF2) accelerates the conversion of fibrinogen
to fibrin (66). Since calcium phosphtae can absorb PF2 but not PF4,
calcium phosphate was added to plasma to determine the extent of PF2
influence on the PF4 assay. Samples were treated the same except
that, after heating, one aliquot was treated with calcium phsophate
with the results shown in Table 6. The absorption of PF2 from the

plasma did not influence the measurement of PF4 in this assay.

Table 6. Plasma PF4 levels* before and after plasma absorption with
calcium phosphate+ utilizing modification of Penner's method.

 

 

Sample Citrate gigggze EDTA c52§04 EGTA 03:;34
1 0.095 0.05 0.05 0.08 ——————————
2 0.12 0.11 0.085 0.10 ----------
3 0.15 0.15 0.115 0.125 0 0.055
4 0.16 0.13 0.13 0 125 0 0.075
5 0.095 0.110 ____________________
6 0.11 0.11 ____________________
7 0.095 0.10 0.02 0.04 ——————————
8 0.10 O 08 ____________________
9 0.085 0.085 ____________________

10 0.10 0.095 ____________________
11 0.105 0.115 0.075 0.085 0.105 0.105
12 0.12 0.15 0.10 0.09 0.105 0.105
13 0.095 0.11 ____________________
14 0.13 0.085 ____________________

 

*Units of heparin neutralized/m1.
+Concentration added, 1 mg/ml of defibrinated plasma.

28

Effect of Antithrombin III in the Test Plasma on PF4 Values
Although, apparently at the threshold of reproducibility for
this method, the results from measuring PF4 levels on unheated
plasma versus heated plasma seemed to indicate an improved sensi—
tivity due to the loss of antithrombin 111. To further support
this, an experiment was performed measuring the biological activ—
ity of antithrombin III. Plasmas were heated at 56 C to defibrin—
ate the plasma (method procedure) and 60 C to remove antithrombin
III activity. The antithrombin III values given in Table 7 show
that there is a definite loss of antithrombin III activity after

heating the plasma to 60 C for 10 minutes.

Table 7. The effects of incubation at 60 C on antithrombin 111
levels* of activity measured by method of Penner.

 

 

Sa le Heated at 56 C+ Heated at 60 C
mp for 5 minutes for 10 minutes
1 100 40
2 145 35
3 150 40
4 137 35
5 85 50

 

*Values are in percent of normal.
+To defibrinate plasma.

Since the antithrombin III biological activity dropped on the
majority of samples to about one third of the original level, samples
were heated for longer periods of time to determine if the loss of

activity was dependent on time of incubation (Figure 2). Optimum

29

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

“F
IOO-
l.
>_ 80
t:
Z
t.
0 60-
<[
C _.
4 4O
o\°
20r-
TEMP(C) 56 60 - 60 60 60 '
TIME(M!N) 5 IO 20 30 40
Figure 2. The effect of incubation times on the levels of anti-

thrombin III activity measured by method of Penner.

30

inhibition of antithrombin III was achieved after 30 minutes.
Longer incubation did not cause the antithrombin III to fall
further.

In a separate experiment, plasmas were heated to a higher
temperature (65 C) to determine if further reduction in anti-
thrombin III could be achieved. Proteins denatured at 65 C making
it impossible to assay the samples. Therefore, 60 C appears to be
the optimum temperature for antithrombin III inactivation.

Sensitivity and Reproducibility of Available PF4 Assays

 

The sensitivity of the modified method of Dana et al. was
tested. A group of ten normal subjects were assayed with the

results given in Table 8.

Table 8. Plasma PF4 levels* from ten normal subjects utilizing
Dana et a1. method.

 

Range 0.095 to 0320

Mean 0.287
S.D. 0.129

 

*Units of heparin neutralized/m1.

The same variation in values found with normal controls were

also noted in the substrate plasma curves (Table 9).

31

Table 9. Units of heparin neutralized per milliliter in the sub-
strate plasma curves obtained by method of Dana et al.

 

Number of Curves 13
Range 0.16 to 0.46
Mean 0.335
S.D. 0.093

 

To further test the reproducibility and variability of the modi—
fied Dana g£_a1, assay, three normal subjects were used. Three veni—
punctures (15 minutes apart) were done on each individual with each
sample being number coded. After all nine samples were drawn, they
were treated identically and tested for plasma heparin neutralizing
activity. The results given in Table 10 show that there is a great

deal of variation from sample to sample from the same individual.

Table 10. Plasma PF4 levels* on three samples from a normal subject
utilizing Dana et a1. method.

 

 

 

Sample
Subject 1 2 3
l 0.55 0.45 0.39
2 0.39 0.48 0.40
3 0.47 0.43 0.51

 

*Units of heparin neutralized/m1.

The Development of a Controlled Purified Substrate
In order to develop a more sensitive and reproducible biologi-

cal assay, it was felt that a carefully controlled substrate was

32

essential. It was decided to utilize purified plasma components
which provide optimum amounts of the needed plasma reactants. In
the development of the purified substrate, the optimum concentra—
tion of the components (fibrinogen and antithrombin 111) had to be
determined. Initially, a fibrinogen curve was constructed, using
the Bio—Data Coagulation Profiler CP-7. This instrument not only
gave results of the thrombin clotting time in seconds (Figure 3)

but also provided the maximum rate of velocity at which the fibrino—
gen was polymerizing (Figure 4). From the clotting times it seemed
that a concentration of at least 3 mg/ml was necessary for the assay.
However, utilizing the maximum velocity of the fibrinogen curve, the
highest concentration (6 mg/ml) tested appeared best.

The antithrombin III obtained from the National Red Cross had a
protein concentration of 13.5 mg/ml and an activity of 43 u/ml. In
order to find the best concentration for the assay, the antithrombin
III was tested at various dilutions. From the initial results
(Tables 11 and 12) it appeared that the amounts of thrombin and
antithrombin III had to be adjusted to find the optimum amounts for
this system. Adjustment of the antithrombin III (Table 12) gave
only a small range of measurable clotting times before total throm—

bin inactivation resulted in no clot formation.

CLOTTING TIME (SEC)

33

 

 

..
25 -—
201—
15 —
10 ~—
5 .—
L L L 1 1 1L :1!
I 2 3 ‘4 5 6
mg/ml of FIBRINOGEN
Figure 3. Effects of increasing concentration of fibrinogen on

the thrombin clotting time.

FIBRINOGEN POLYMERIZATION (Vmox)

 

34

 

 

"F
55..
41..
3 r-
2 r-
I
I...
O I I l I I
2 3 4 5 6
mg/ml of FIBRINOGEN
Figure 4. Effects of increasing concentration of fibrinogen on

the maximum velocity of fibrin formation.

 

35

Table 11. Effect of antithrombin III concentration on clotting times
(seconds) utilizing the purified substrate assay.+

 

Fibrinogen AT 111*

 

mg/ ml undiluted 1‘ 2 1‘ 4 1: 8 1:10
3 no clot no clot no clot 14.2 14.3
6 no clot 10 --— -__ ___

 

*13.7 mg/ml protein concentration.
+8 u/ml thrombin concentration.

Table 12. Effect of thrombin concentration (8 u/ml) with various
dilutions of heparin on clotting times (seconds) uti—
lizing the purified substrate assay.

 

Antithrombin III diluted 1:10 (1.35 mg/ml)

 

Fibrinogen u/ml of heparin
mg/ml 0 0.1 0.2 0.3
3 14.1 no clot —-— ---
6 9.1 10.9 11.3 no clot

 

Antithrombin III diluted 1:20 (0.685 mg/ml)

 

Fibrinogen u/ml of heparin
mg/ml 0 0.1 0.15 0.17 0.19 0.20 0.25
6 10.0 9.6 11.4 12.9 12.1 18.1 no clot

 

Because it would be desirable to have a greater range of heparin dilu—
tions with measurable thrombin clotting times, the thrombin concentra-

tion was altered (Table 13).

36

Table 13. Effect of increased thrombin concentration (12.5 u/ml)
with various dilutions of heparin on clotting times
(seconds) utilizing the purified substrate assay.

 

Antithrombin III diluted 1:10 (1.35 mg/ml)

 

 

Fibrinogen u/ml of heparin
mg/ml 0 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.50
6 11.2 12.2 14.5 16.0 19.5 23.2 33.7 40.8 no Clot

 

Biological PF4 Purified Substrate Assay

 

Based on the results of experiments described thus far, an im—
proved assay has been developed which utilizes a purified substrate
with 6 mg/ml of fibrinogen, 1.35 mg/ml of antithrombin III and 12.5
u/ml of thrombin. The following results are from this newly developed
assay. In this assay, heparin clotting times are performed on the
purified substrate alone with these results forming the baseline for
the test. The results for the purified substrate (0 PF4) are given
in Table 14. This set of results includes the use of two different
preparations of antithrombin 111, one from the National Red Cross,
Bethesda, Maryland and another prepared at the American Red Cross,

Lansing, Michigan. There is very little variation in the results.

Table 14. Units of heparin neutralized per milliliter by purified
substrate alone.

 

 

Number of runs 12
Range 0.25 to 0.325
Mean 0.293

S.D. 0.021

 

37

A group of 10 normal subjects was assayed with this method
(Table 15). The results indicate that the assay is very sensitive

for there is a small range of the results.

Table 15. Plasma PF4 levels* from ten normal subjects utilizing
the Bio PF4 purified substrate assay.

 

 

Range 0.035 to 0.17
Mean 0.105
S.D. 0.045

 

*Units of heparin neutralized/m1.

Four of these normal subjects were assayed on different occa-
sions, to test the daily variance in an individual and to determine

the reproducibility of the assay (Table 16).

Table 16. Repeated plasma PF4 levels* in normal subjects obtained
on different days.

 

 

 

 

 

 

Sample
Subject 1 2 3
1 0 065+ 0.045+ 0.115
2 0.17 0.16 0.14
3 0.14 0.12 _____
4 0.09 0.105 -----

 

*Units of heparin neutralized/m1.
+Specimen was lipemic.

To further test the reproducibility of this procedure, three

samples drawn a few minutes apart from a normal subject were number

 

38

coded and tested by the new Bio PF4 assay and the modified Dana
g£_§1, method. All of the blood samples were handled in exactly
the same manner. After platelet poor plasma was obtained from
centrifugation, platelet counts were done and the plasma aliquoted
and frozen to be assayed by both methods at a later time. Platelet
counts on the plasmas were 7,000/mm3 or less. The results are

given in Table 17.

Table 17. Comparison of plasma PF4 levels* of three samples from a
normal subject using the Dana et al. method and the Bio
PF4 purified substrate assay.

 

 

 

 

 

Bio PF4 purified substrate assay Dana §£_§l, method
Sample Sample
Subject 1 2 3 1 2 3
1 0.13 0.14 0.14 0.17 0.225 0.25
2 0.09 0.09 0.105 0.125 0.23 0.195
3 0.10 0.10 0.12 0.135 0.33 0.25

 

*Units of heparin neutralized/m1.

Also tested with the new assay was purified PF4 with a protein
concentration of 13.5 ug/ml. Platelet factor 4 is not soluble in low
salt concentrations, so the purified protein had to be assayed in
higher salt solutions. Unfortunately, high salt concentrations in-
terfere with the test system so dilutions had to be used which main-
tained solubility but decreased the higher salt content. The results
of testing a 1:10, 1:20, and 1:40 dilution of the purified PF4 are

shown in Figure 5.

UNITS of HEPARIN/ml

39

   

 

 

5 _.

4 ..

3 __

2 .—

I .a

I I . 1 L L . I
0.2 0-4 06 O-8 l-O I2 I'4
Ug/ml 0f PF4

Figure 5. Units of heparin neutralized by serial dilutions of

purified PF4.

H II"

40

From the graph (Figure 5), it is possible to interpolate re—
sults from the new assay expressed in units of heparin neutralized
per milliliter into micrograms per milliliter of PF4 present in
plasma. The results from Table 15 have been converted to micro—

grams per milliliter of PF4 and shown in Table 18.

Table 18. Units of heparin neutralized per milliliter expressed
in micrograms per milliliter of PF4.

 

 

 

5131332; “’ 3.3228152?“ ug/m of a.
Lower range 0.035 0.155
Upper range 0.17 0.195
Mean 0.105 0.175

 

Effects of Other Plasma Constituents

 

Even though antithrombin III is considered the main plasma inhibi-
tor of thrombin, there are other antithrombins present in the plasma.
In order to determine if plasma levels of a 2—macroglobulin would in—
terfere with the new assay, it was added to the test system. The units
of heparin neutralized per ml by the purified substrate alone was 0.33
u/ml, with 3 mg/ml of a 2—macroglobulin added to the purified substrate
was 0.34 u/ml. This indicates no interference with a 2—macroglobulin
with the use of the purified substrate.

Another experiment included the addition of a 2-macroglobulin to
the test plasma before assaying. The results are as follows: 1) test
plasma with no a 2-macroglobulin neutralized 0.045 u/ml of heparin,
and 2) test plasma with 3 mg/ml of a 2—macroglobulin added neutralized

0.05 u/ml of heparin. Again, these results indicate no interference

41

with the new assay. The amount of a 2—macroglobulin added to the

test plasma represents an elevated plasma level of a 2-macroglobulin.

 

DISCUSSION

The main problem inherent in all of the available assays for PF4
which use a pooled normal substrate plasma is the uncontrolled reac—
tion of the plasma components. This reaction includes not only the
blood coagulation factors but also the inhibitors of the coagulation
system. When assaying for the heparin neutralizing activity of PF4,
there can be interactions within the test system which can lead to
unreliable results. The reactions involved in the measurement of PF4
heparin neutralizing activity are summarized as follows: 1) PF4 and
the added heparin form a complex, 2) any residual heparin forms a com—
plex with antithrombin III which causes rapid inactivation of thrombin,
and 3) the remaining unneutralized thrombin will cause the conversion
of fibrinogen to fibrin which is the end point. The important compo-
nents (antithrombin III and fibrinogen) of the pooled substrate plasma,
such as used in the Dana g£_§1, procedure (36), would be in competi-
tion with the antithrombin III and fibrinogen in the test plasma for
participation in the reactions of the test system. Even though, in
the procedure of Fuster §£_§1. (38), the test plasma is heated to re—
move the antithrombin III and fibrinogen, it can only be assumed that
the pooled substrate plasma has optimum levels of antithrombin III and
fibrinogen. In the procedure of Saleem g£_§1. (39) utilizing a lyophi-
lized commercial plasma as a substrate, there is still an assumption
that there is an optimum level of antithrombin III and fibrinogen.

In the newly developed assay utilizing the purified substrate, the

42

 

43

concentration of fibrinogen and antithrombin 111 have been tested to
ensure optimum levels. Based on the results of these experiments,
it is felt that the proper concentration of substrate constituents
have been achieved for the reaction taking place.

Another consideration in the use of the pooled plasma for a sub—
strate is the presence of other potential antithrombins and coagulation
factors. It has been shown by Rosenberg (32) that antithrombin 111
can inactivate other serine proteases besides thrombin. So in the
case of the pooled plasma, interactions between antithrombin III and

other coagulation factors could cause interference in the enzymatic

 

 

reaction of the test system. In the Dana et a1. method, heparin

neutralizing activity of the control group was reported to be quite
low (0.1 i 0.1 u/ml of heparin neutralized) (36). However, it was
also reported that all test plasmas were screened for any unrecognized
antithrombin activity; if any were found the samples were discarded
and not tested. Heating the test plasma to remove antithrombin III
and optimizing the antithrombin III concentration in the purified sub-
strate assay, make it unnecessary to take into consideration variations
in the level of antithrombin III in the test plasma. The reaction in
the purified substrate assay is more defined than in any of the pre—
viously available biological systems. Unexpectedly higher PF4 levels
might be present in substrate plasmas. This possibility is also
avoided by the use of a controlled substrate which contains only
antithrombin III and fibrinogen.

The results of testing the new assay indicate enhanced sensi—
tivity and an improved reproducibility over existing methods. By

assaying the plasma of a normal subject drawn at three different

44

intervals using both the Dana gt_§l, method and the new method, it
was possible to show that there is greater variability with the

Dana g£_§l, method than with the new assay. Even though the heparin
neutralizing activity of PF4 can be detected by other biological pro-
cedures, it is clear that the use of plasma as a substrate introduces
variables that contribute to a loss of sensitivity.

Statistical analysis of the data obtained from assaying test
samples with the new assay and the Dana §£_§1, method (Table 17) is
used to compare the two methods. Two parameters used in analyzing
the data are: 1) the student t test which compares the accuracy, and
2) the F test which compares the precision of the two methods. The
results from the t test (p < 0.005) show a significant difference be—
tween the two assays, and indicate that the Dana g£_§l, method is not
as accurate. Having establishing that there is a significant differ-
ence between the methods and that the accuracy of the new assay is
greater, a comparison of the precision of the two procedures was made
using the F test. Results from the F test show again a significant
difference at the 95% confidence level between the precision of the
two methods. The values found for these two tests are: df = 8,

t = 4.727, and F = 10.24.

Another indication of the improved precision of the new assay is
the ability of this assay to use smaller increments of heparin. A
difference of 0.05 u/ml of heparin neutralized can be detected with
the use of the purified substrate. In the Dana g£_§1, method even a
change of 0.1 u/ml of heparin will sometimes not be reflected by a
change in the clotting time. Small changes in heparin concentration,
indicating the binding of PF4 to heparin, can be detected in the new

assay.

 

45

Expression of results in all of the biological methods used
is based on the amount of neutralized heparin added to the test
system. Assaying purified PF4 with the controlled substrate in-
troduces another possible way of expressing plasma PF4 activity.
The results of the normal subjects expressed in units of heparin
neutralized per milliliter (Table 15) have been converted to micro—
grams of PF4 by use of the curve obtained from assaying purified PF4
(Figure 5). These values are given in Table 18 with the mean of the
normal subjects having a level of 175 ng/ml of PF4. Levine and

Krentz, using a RIA method for assaying PF4, reported various levels

 

depending on the speed of centrifugation of the specimen and kind of
anticoagulant used. The range of values they reported, utilizing a
citrate anticoagulant and centrifugation at 2400 g, was 48—136 ng/ml
of PF4 (27). They also reported that the higher the speed of cen—
trifugation, the lower the levels of PF4. Comparison of 175 ng/ml
(new assay with specimen centrifuged at 625 g and citrate anticoagu—
lant) to that of 88 ng/ml (mean for the RIA method with specimen cen—
trifuged at 2400 g and citrate anticoagulant) suggests that the new
biological assay might be as sensitive as the RIA methods. Several
investigators (27, 28, 29) have measured the levels of PF4 in platelet
poor plasma utilizing the RIA method. They used anti—release reagents
in acid citrate dextrose as the anticoagulant. Values of PF4 in these
cases ranged from 5 to 16 ng/ml depending on the speed on centrifuga-
tion of the specimen.

The experiment in which a 2-macroglobu1in was added to the assay
system to evaluate its antithrombin activity showed no inhibition.

It has been reported that a 2-macroglobu1in contributes significantly

46

to the inactivation of thrombin. However, the reaction time for
this inhibition is much longer than with antithrombin III and

a 2-macroglobulin does not use heparin as a cofactor (51). Since
the assay utilizes short incubation times, any interference from
a 2-macroglobulin should be avoided. Data supports this as no
changes in PF4 values, expressed as heparin neutralization, could
be detected when exogenous a 2—macroglobulin was added to the sys-
tem.

There seems to be no improvement in the detection of PF4 with
different attempts to reduce platelet release due to specimen han-
dling. Even though the assay used to evaluate the different anti—
coagulants (citrate, EDTA, EGTA, Handin's cocktail) did not have a
great sensitivity, no significant differences were found and 3.8%
trisodium citrate was used throughout.

The absorption of the test plasma with calcium phosphate to
remove PF2 did not cause a change in the heparin neutralizing ac-
tivity of the test sample. Even though the role of PF2 is the ac-
celeration of the conversion of fibrinogen to fibrin, the experiment

suggests no interference by PF2 in the test system.

 

IIJ'

CONCLUSION

The development of the Biological Platelet Factor 4 Purified
Substrate Assay has proven to be a sensitive and reproducible assay

for measurement of PF4's heparin neutralizing activity. The pre-

 

cision of the assay has been optimized to the extent that results
on individual test subjects show superior reproducibility with the
controlled substrate than with any biological assay in general use.
Even though the physiological function of PF4 is not completely un-
derstood, research indicates that it may be involved in coronary
artery disease, thromboembolic disorders and in pathological states
which exhibit thrombocytopenia and thrombocytosis. Utilization of
the newly developed assay may help to elucidate the role of PF4 in

these disease states.

47

 

Mr

 

 

APPENDIX

APPENDIX A

Preparation of General Reagents

0.9% Sodium Chloride

Dissolve 0.9 grams of sodium chloride in 100 m1 of distilled water.

3.8% Trisodium Citrate

 

Dissolve 3.8 grams of trisodium citrate in 100 m1 of distilled water.

0.1 M Calcium Chloride

Dissolve 14.7 grams of calcium chloride in 1000 ml of distilled water.

Acid Citrate Dextrose (ACD), NIH Formula A

22.0 grams of trisodium citrate,

8.0 grams of citric acid,

24.5 grams of dextrose, are dissolved in distilled water and diluted
to 1 liter.

Veronal Buffer

 

11.75 grams of sodium diethyl barbital,

14.67 grams of sodium chloride, are dissolved in 1570 ml of distilled
water. Mix, then add 430 ml 0.1 N HCl and pH to 7.35.

Imidazole Buffer (pH 7.2-7.4)

Dissolve 1.72 grams of imidazole in 90 m1 of 0.1 N HCl and then dilute
to 100 ml with distilled water.

Imidazole Buffered Saline

A 30% solution is prepared by combining 30 m1 of imidazole buffer
(pH 7.2-7.4) and 70 m1 of 0.9% sodium chloride.

48

 

 

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10.

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VITA

VITA

The author was born in Chicago, Illinois on July 10, 1947. The
author graduated from Eastern Illinois University with a Bachelor of
Science in Medical Technology in September of 1969. She completed a
medical technology internship at Decatur Memorial Hospital, Decatur,
Illinois. The author worked four years at Boyd Memorial Hospital,
Carrollton, Illinois, and two years at Mercy Hospital, Urbana,
Illinois as a staff technologist. In September 1975, she was admitted
to the graduate program in Clinical Laboratory Science at Michigan
State University.

The author is married to Douglas William Estry.

55

"IIIIIIIIIIJIIIIIIIII