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A CLEMCAL EVALUAHON OF
THE THROMBIN ENHEBETEON TEST

 

Thesfis for the. Degree of M. S.
MECHIGAN STATE UNWERSETY
DARLENE ELABNE CLINGENPEEL
1970

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ABSTRACT
A CLINICAL EVALUATION OF THE THROMBIN INHIBITION TEST

by Darlene Elaine Clingenpeel

A major disadvantage in the use of heparin therapy is the lack of
a convenient and accurate system of controlling dosage.

The thrombin inhibition test, a method for monitoring heparin therapy,
was modified and clinically tested.

A linear relationship was found to exist between the clotting time
of a fibrinogen solution and the reciprocal of the thrombin concentration.
There is also a linear relationship existing between the number of micro-
grams of heparin present and the number of inactivated thrombin units.
From these results the following formula was derived to calculate the

concentration of heparin:

Heparin concentration 2.11 - 19.7

t
(ug./ml.) (clotting time
in seconds)

This method has a variance of 0.0159, thus; 95% of the time the cal-
culated heparin concentration will be :_0.256 ug./ml. from the actual
concentration in.the plasma.

In the clinical evaluation of this assay method, the calculated
heparin concentration was compared to the Lee-White clotting time. A
linear relationship exists between these 2 methods. The precision and
accuracy of the thrombin inhibition test is much greater than that of the

Lee-White clotting time.

A CLINICAL EVALUATION OF THE THROMBIN INHIBITION TEST

By

Darlene Elaine Clingenpeel

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1970

C997 8/0
5 - 81'}~ 70

For

My Parents and Family

ii

ACKNOWLEDGEMENTS

I would like to express my appreciation and gratitude to Dr. A. E.
Lewis, Department of Pathology, for his continued support and guidance
throughout my graduate work.

My sincere appreciation goes to the members of my committee: Dr.

A. J. Bowdler of the Department of Medicine and Dr. C. H. Sander of the
Department of-Pathology, for their assistance and advice in the prepara-
tion of this manuscript.

To the-Chairman of the Department of Pathology, Dr. C. C. Morrill,

I would like to express my thanks, for providing the funds and facilities
necessary to do my graduate work. I would like to thank those involved
in administrating the Allied Health Professions Advanced Traineeship
Grant AHT-69—049 that provided support during my graduate study.

I am grateful to Mr. John Spuller and the Warner Chilcott Company
for providing reagents and technical materials used in this experiment.

I would like to thank Mr. Joe Cavanaugh and the Becton Dickinson
Company for lending me the Fibrometer System and the 0.5 m1. probe.. This
machine and the special probe enabled me to automate the thrombin inhi-
bition test.

I extend thanks to Mr. Tom Nicholson and the Rupp and Bowman Company
for providing the Fibrometer tips and tubes needed for this project.

I-am especially grateful to Dr. Leo Walker, Miss Bernice Rochon, and
their fine staff at St. Lawrence Hospital. Their willingness to obtain

blood samples from patients receiving heparin therapy is greatly appreciated.

iii

To Dr. Eleanor Berden, Dr. Joel Newton, and Mr. A1 Barrett and their
laboratory staff at Ingham Medical Hospital, I give recognition and thanks
for cOOperating and helping me obtain the blood specimens needed for this
research project.

I would like to thank Dr. John Young, Dr. John Finger, Pathologists, and
Mrs. Shirley Cresswell, Chief Medical Technologist at St. Luke's Hospital
in Saginaw, Michigan, for providing me with the necessary blood specimens,
and_for the use of their laboratory facilities. Their advice, encourage-
ment and sincere interest in the advancement of medical technology is
greatly appreciated.

I am grateful to Mrs. Margaret Shick and the laboratory staff at
the MSU Health Service. They willingly allowed me to use their labora-
tory equipment and space during this project.

To the numerous graduate students and the secretarial staff of the
Department of Pathology, I express my appreciation for providing me with
blood samples needed for my experiment.

I am especially grateful to Miss Leona Gielda for her technical
advice, encouragement, and her willingness to assist me whenever needed.

Finally, I would like to express my appreciation and gratitude to

my family and friends, for their encouragement, inspiration, and support.

iv

TABLE OF CONTENTS
' Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . .~. . . 2
Blood Clotting Mechanism. . . . . . . . . .4. . . . . . . . 2
Thrombin. . . . . . . . . . . . . . . . ... . . . . . . . . 3
Fibrinogen. . . . . . . . . . . . . .'. . . .‘. . . .‘. . . 5
Heparin . . . . . . . . . . . . . . . . . . . . .-. . . . . 6
Heparin Assay . . . . . . . . . . . . . . . . . . . . . . . 8
MATERIALS AND METHODS. . . . . ,. . '. . . . . . . . . . . . . . . . 11
Source of Plasma. . . . . . . . . . . . . . . . . . . . . . ll
Reagents Used in This Experiment. . . . . . . . . . . . . . ll.
Thrombin Activity . . . . .-. .g. . . . . .». . . .'. . . .' 12
Heparin Assays. . . . . . . . . . . . . . . . .'. . . . . . 12.
Plasma Stability. . . . . . . . . . . . . . . . . . . . . . 13
Clinical Experiment . . . . . . . . . . . . . . . . . . . . 13
RESULTS. . . . . . . . . . . .~. . . . . ... . . . . . . . . . . . 15
_Thrombin Activity . . . . . . . . . . . . . . . . . . . . . 15
Heparin Assays. . . . . . . . . . . . . . . . . . . . . . . 15
Plasma Stability. . . . . ... . . . . . . . . . . . . . . . 16
Clinical Experiment . . . . . . . . . . . . . . . . . . . . 17
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . .'. . . . 23
SUMMARY AND CONCLUSION . . . . . . . . . . . . . . . . . . . . . . 26
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

VITA _ I O O O O O O O O O O O O O O O O O O O O O O O O O O I O O O 31

LIST OF FIGURES
Figure Page
1 Thrombin dilution curve (summary of data) . . . . . . . . . l8
2 Thrombin activity curve . . . . . . . . . . . . . . . . . . l9

3 The number of active thrombin units that remained after
the addition of various concentrations of heparin . . . . . 20

4 Comparison of calculated heparin concentration to the
Lee-White clotting time . . . . . . . . . . . . . . . . . . 21

5 Comparison of Lee-White clotting time to calculated
heparin concentration . . . . . . . . . . . . . . . . . . . 22

vi

INTRODUCTION

Heparin is used when a rapid anticoagulant effect is desired. One
great disadvantage in using heparin therapy is the lack of a convenient
and satisfactory control system. A relatively quick and simple test is
needed to determine reliably the heparin concentration in the patient's
plasma.

The common test for monitoring heparin therapy is the Lee-White
clotting time, which measures the time it takes for a known quantity of
whole blood to clot. This time-consuming and inconvenient method has
many variables which, if not closely controlled, can lead to erroneous
results.

Schwab (1969) developed a thrombin inhibition test to determine the
concentration of heparin in blood. The purpose of this project was to
continue the development of this method, in order to provide a useful

guide for the management of heparin therapy.

REVIEW OF LITERATURE

Blood ClottinggMechanism

The phenomenon that blood coagulates was beyond human comprehension
and was.not the subject of investigation before the late 1600's. In 1666,
Malpighi discovered that pale fibers remained, when blood clots were
washed in water. This substance which formed these fibers was called
"fibrin". He was the first of many researchers trying to discover the
physical and chemical changes that cause a fluid substance, blood, to
turn to gel. The experimental data, collected by these researchers,
gave rise to numerous theories on the mechanism.of blood coagulation.
The exact mechanism, however, is still the subject of controversy.

At present, there are 2 schools of thought about the coagulation
of blood. Macfarlane and his associates in Britain (1964) and Davie and
Ratnoff in the U.S.A. (1964) believe that all the reactions in blood A
coagulation are enzymatic. Coagulation is first initiated by contact
with foreign material. Then a cascade of proenzyme-enzyme transforma—
tions take place until the final substrate, fibrinogen, is reached.

This hypothesis is widely accepted today.

However, Seegers (1964) strongly Opposes this theory. According to
him, some of the clotting factor precursors thought to be converted to
enzymes may not exist. gHe believes that prothrombin contains 2 enzymes
in precursor form, thrombin and autOprothrombin C (Factor X). The pro-

teolytic degradation of prothrombin activates this molecule. Seegers

3
concludes that the activation of prothrombin occurs by an autocatalytic
process rather than an enzymatic cascade.

If each step in coagulation of blood is taken individually, it pre-
sents a set of intricate problems. The-last step in coagulation. alone,
involves thrombin, fibrinogen, fibrin, and certain other factors in the
plasma. When any one of these factors is inhibited, normal clotting does

not occur.

Thrombin

Many theories have been prOposed to explain or summarize the studies
on coagulation; most of these were listed in an extensive review of the-
literature up to 1935 by Howell. It has generally been accepted that
thrombin is formed from some chemical union of prothrombin, calcium, and
tissue factors. Thrombin, in turn, initiates the final conversion of
fibrinogen to fibrin by the hydrolytic effect.

Because of the numerous questions involving the chemistry of coagu-
lation, it was important to isolate and purify the various proteins
involved without altering their preperties. Seegers, Brinkhous, Smith,
and Warner.(l938) purified prothrombin from oxalated beef plasma, then
by the addition of thromb0plastin, thrombin was obtained. They defined
a unit of thrombin as the amount that will.clot 1 cc. of a standardized
fibrinogen solution in 15 seconds. If dried or stored at -30 C., thrombin
will retain its activity indefinitely. Thrombin was further purified
by Seegers and McGinty in 1942.

Certain impurities have been found in bovine thrombin commercially
prepared by Parke, Davis & Company. The eXperiments of Yin and Wessler
(1968) show that this commercial thrombin is contaminated with a large

amount of Factor X. These researchers reported that the results obtained

4
in the studies of coagulation or enzyme kinetics may be misleading when
this product is used.

Many substances that have the properties of thrombin can be formed
from prothrombin by several methods. When these substances were tested,
they exhibited chemical and physical differences which were important to
understanding the activation and inactivation of prothrombin and thrombin
(Lamy and Waugh, 1954).

Lamy and Waugh (1954), using ultracentrifugation and electrOphoresis,
tried to explain the transformation of prothrombin into thrombin. They
concluded that this transformation was so complicated that no definite
scheme could be proposed.

It was found by Therriault, Gray and Jensen (1957) that the rate
of conversion of prothrombin was accelerated in the presence of trace
amounts of thrombin. Apparently thrombin speeds up its own formation by
activating a plasma accelerator—globulin (Factor V). However, the spe-
cific mechanism of this-action is not completely resolved.

The action of thrombin as an enzyme in the conversion of fibrinogen-
to fibrin is widely accepted (Laki, 1953; Sherry, Troll and Gluek, 1954).
Thrombin hydrolyzes 4 peptide bonds from the fibrinogen molecule (Laki,
1953). Thisproteolytic enzyme was shown by Laki and Gladner (1964) to
have a specificity for certain peptide bonds formed between arginine
and glycine residues. The molecular weight of thrombin was estimated
to be 8000 by their experiments.

Besides acting as an enzyme, thrombin has another important func-
tion. It induces platelets to aggregate, contract, and to release sero-

tonin, nucleotides, potassium, and other substances (Ganguly, 1969).

5
Fibrinogen

Fibrinogen is a protein present in plasma in the concentration of
1 to 8 mg./ml. with a molecular weight of 330,000 (Laki and Gladner,
1964). The isolation of this protein is simplified considerably, because
of its unusual solubility characteristics. Since fibrinogen is easily
denatured and is extremely labile, purification is difficult (Jacques,
1943; Ware and Lanchantin, 1954).

Lorand in 1954 considered the fibrinogen-thrombin reaction to be
the most striking phenomenon in protein chemistry. The addition of trace
amounts of thrombin to a solution of fibrinogen results in a gel mesh-
work. It has been reported that only minute amounts of thrombin are
necessary to clot a very large quantity of fibrinogen (Eagle, 1935; Laki,
1953). Because of the results of several researchers, fibrinogen is
considered to be the primary substrate for thrombin.

The chemical difference between fibrinogen and fibrin indicates that
peptide bonds are split from the fibrinogen molecule by thrombin, result-
ing in the formation of fibrin (Laki, 1953). By 1954, it was reported
that the proteolytic removal of fibrino-peptide from the fibrinogen mole-
cule was necessary to activate it. The activated fibrinogen then spon-
taneously but reversibly polymerizes and fibrin is formed (Ferry, 1954;
Lorand, 1954). The previous results of Lorand and Middlebrook (1952)
were in agreement with this view.

Laki and Gladner (1964) examined fibrinogen from 6 species. They
suggested that the 4 peptide bonds of fibrinogen hydrolyzed by thrombin
are between.arginine and glycine residues. This is in accordance with
the earlier experiments of Sherry, Troll and Gluech (1954). The fibrino-
peptides are by-products once they are removed and are not needed for the

polymerization that follows (Laki and Gladner, 1964).

6

The currently accepted concept of the conversion of fibrinogen to
fibrin suggests that this conversion occurs in 3 steps. The first step,
as described above, is the enzymatic action of thrombin on fibrinogen,
resulting in the formation of.a fibrin monomer plus fibrino-peptides.

In the next.step, fibrin monomers combine to form intermediate fibrin
polymers. Finally, there is a secondary aggregation of the intermediate.
polymers to form the clot network (Ellias and Iyer, 1967). The formation
of the fibrin clot occurs by 3-dimensional polymerization. Electrostatic
forces and hydrogen bonding are believed to be responsible for this type
of polymerization (Ferry, 1954; Laki and Gladner, 1964).

The liver has long been regarded as the source of fibrinogen. The
precise cellular localization of synthesis remained uncertain. It was
shown by an immunologic technique that fibrinogen was contained in the
liver. Barnhart and Anderson (1962) reported that fibrinogen is synthe-
sized by the microsomes and stored in the soluble part of the parenchymal
cell. This was consistent with the experiments of Forman and Barnhart
(1964). The rate of formation of this protein is adjusted-to its plasma

level.

Heparin~
In the early 1900's, McLean discovered the first physiologic anti-

coagulant while trying to extract cephalin from livers. The-term, heparin,
and the description of the material were reported later by Howell and

Holt (Vigran, 1965). Heparin, a polysulfated mucOpolysaccharide, is a
naturally occurring anticoagulant found in most tissues of the body. The
highest concentrations were found in the liver and lung, with only minute
amounts in the blood (Eiber and Danishefshy, 1957). The large metachro-
matic granules of mast cells are loaded with heparin. The term heparino-

cyte is applied to these cells (Jorpes, 1962).

7

Heparin is one of the most potent inhibitors of blood coagulation
and, because of this prOperty, it has been used extensively in the clini-
cal treatment of thrombosis (Eiber and Danishefsky, 1957). This anti-
coagulant, together with a cofactor in the plasma, acts as an antithrombin
and will inhibit the action of thrombin on fibrinogen. It prevents plate-
let stickiness and agglutination (Vigran, 1965). Heparin also interferes
with thrombOplastin generation and the conversion of prothrombin to
thrombin (Lenahan, Frye, and Phillips, 1966).

The clotting time is rapidly increased after an intravenous injec-
tion of heparin (Jaques and Ricker, 1948). The experiments of Eiber and
Danishefsky (1957) demonstrated that the clotting time would return to
normal within 5 hours. According to their work, the quick return of the
normal clotting time was due to rapid elimination of heparin rather than
deactivation.

Heparin also has a lipolytic effect. Triglycerides are cleared from
the serum of animals and men when heparin is administered by any inject-
able route. This clearing activity is the result of the release of an
enzyme, Zipoprotein lipase (Vigran, 1965).

Since the discovery of heparin, there has been active controversy
about the mode of action of this anticoagulant. Howell, in 1935, believed
heparin to be an antiprothrombin, because heparin did not inhibit puri-
fied thrombin's action on purified fibrinogen. Quick (1936) reported
that heparin is not an antiprothrombin. His experiment demonstrated that
heparin, itself, was not an antithrombin, but will react with an unknown
substance in the plasma to form a strong antithrombin. Hence, he called
heparin an antithrombogen. By 1957, Monhouse and Clarke noted that
heparin did not significantly increase the thrombin clotting time, unless

there was a heparin cofactor present in the plasma.

8

Astrup and Darling (1942) used antithrombin, prepared frmm ox plasma,
in their experiments. When they plotted the amount of antithrombin added
against the percent of thrombin inactivated, a straight line was obtained.
Since they were able to obtain a quantitative measurement of-naturally
occurring antithrombin, they applied the same principle to antithrombin
formed from.heparin (1943). The results were not too satisfactory, because
the curves obtained were not always straight lines. From their data
they concluded that the thrombin inhibitor which is released by the addi-
tion of heparin is not identical with naturally occurring antithrombin.

Seegers (1954) reported 4 different categories of antithrombin:
Antithrombin I is concerned with thrombin adsorption on fibrin; Anti-
thrombin II is associated with heparin and heparin cofactors; Antithrom-
bin III directly inhibits thrombin activity; Antithrombin IV inhibits
prothrombinfs activity.

It was demonstrated by Henstell and Kligerman (1967) that antithromr
bin in the plasma is bound to an inhibitor, leaving little or no free
antithrombic activity. Since heparin increases antithrombic activity,
it was prOposed that heparin splits the bond between antithrombin and
its inhibitor. A new bond is formed between heparin and antithrombin,
which may react with any thrombin present.

Abildgaard (1968) suggested that the increase in clotting time is
caused by the formation of thrombin-heparin-fibrinogen complexes. At
present, his results confirm that heparin inhibits the enzymatic action

of thrombin on purified human fibrinogen by the formation of a complex.

Heparin‘Assay

 

Heparin was first used as an in viva anticoagulant by Best in 1935.

Because of its rapid action and its relatively nontoxic properties, it

9
has been used widely in the control of thromboembolic disorders (Vigran,
1965). The recommended intravenous dosage of heparin ranges from 200-300
mg. every 12 hours (Miale, 1967) to 30,000 units per day (Penner, 1967).
It is necessary to determine periodically whether the patient has an ade-
quate level of anticoagulant in his blood.

The method used universally in controlling heparin therapy is the
Lee-White clotting time. This method has many variables such as the diame-
ter of the tube used, the frequency of tilting the tube, environmenta1~
temperature, and the venipuncture technique. These variables cause the
end results of the Lee-White clotting time to be neither reproducible
nor reliable (Vigran, 1965).~ Sufficient amounts of heparin will have a
prolonged effect on several of the coagulation systems. Many researchers
have suggested that the Lee-White clotting time could be replaced by one
of the following tests: whole blood recalcification time, activated
whole blood, the partial thromboplastin time, or the thrombin clotting
time (Spector and Corn, 1967).

The relationship of the clotting time to heparin dosage was studied
in 1948 by Jaques and Ricker. When they plotted the heparin dosage
against the logarithm of the clotting time, a straight line was obtained.

Blomback, Blomback, Corneliusson and Jorpes (1953) investigated the
reliability of the following methods used in heparin assays: a whole
.blood method, a thrombin method on plasma, the method of U.S.P. XIV, 1950,
and the sulphated whole blood method. They found that the thrombin clot-
ting time method gave the best results, varying only 2-52 from the standard
heparin concentration. The other methods gave concentrations 5-102 lower
than the thrombin method.

The data obtained by Rappaport and Ames.(1957) indicates that a,

properly standardized plasma thrombin time can be used effectively to

10
follow heparin therapy. They suggested the use of platelet-poor plasma
to rule out residual heparinemia and platelet-rich plasma to check the
therapeutic heparin level. Since platelet-rich plasma is less sensitive
to heparin, Schatz and Hathaway (1963) recommended it as an indicator of
therapeutic prolongation of coagulation.

Penner (1967) measured heparin by its antithrombic activity in his-
1aboratory. The assay required the addition of a measured amount of
thrombin to the patient's blood. He found the normal thrombin clotting
time to range from 18 to 24 seconds. In the presence of heparin, this
clotting time is lengthened. His results were correlated with values
obtained using the Lee-White clotting time.

Lenahan et al. (1966) recently proposed that the activated partial
thromboplastin time could monitor heparin therapy. Because sufficiently
standardized and sensitive reagents are not available, this test cannot
be adOpted for routine clinical use (Harmeling and Cordray, 1967).

Schwab~(l969) prOposed a one—step thrombin clotting time method for
assaying accurately heparin concentration in platelet-rich plasma. A
relationship was found between the time it took the fibrinogen in the
plasma to clot and the amount of thrombin added. This procedure could
easily be adapted for a clinical laboratory to provide a useful guide for
the management of heparin therapy. However, Rezansoff and Jaques (1967)
point out that in vitro tests may fail to reflect the effect of heparin

on coagulation in vivo.

MATERIALS AND METHODS

Source of Plasma
A 1.5 inch, 21-gauge needle was used to draw blood from human sub-
jects. Four and one-half milliliters of blood were added to a tube con-
taining 0.5 m1. of 0.1 M sodium.oxalate. Platelet-rich plasma was.

obtained by centrifuging the whole blood for 20 minutes at 60 G.
Reagents Used in This Experiment

Thrombin. Topical Thrombin (bovine origin)* was used in all tests. Each
vial contained 1000 N.I.H. units** of dried thrombin. A stock solution
of thrombin was prepared using the method of Rappaport and Ames (1957):
5 ml. of oxalated saline (1 part 0.1 M sodium oxalate and 5 parts 0.85%
sodium chloride) was added to the dried contents of one vial. The solu-
tion was then absorbed with 100 mg. of barium sulfate for 20 minutes at
37 C. After centrifuging at 750 G. for 10 minutes, the supernatant was.
transferred to a.lO ml. siliconized volumetric flask. To this solution,
2.5 m1. of glycerol was added. Physiologic saline was then used to dilute
the mixture to 10 ml.

The stock thrombin solution (100 units/ml.) was stored at 4 C. for

1 day before it was used. This solution was stable for 1 week when stored

 

*TOpical Thrombin (Bovine Origin) - Parke, Davis & Company,
Detroit, Michigan.

**National Institute of Health Units - the amount required to clot
1 m1. of standardized fibrinogen solution in 15 seconds.

11

 

1 1 . , , I’ll..-
II b u .I i I W4 a H. , . I v. . . o . | —
:AEIP . as... d \- b. 5. .

 

12

at 4 C. and for 3 months if frozen in polyethylene tubes (12 x 75 mm.).

Heparin. A stock heparin solution containing 100 units (1 mg./m1.) was
prepared from a heparin ammonium salt* solution of 1000 units by diluting
with deionized water. This stock solution was stable for 1 week if stored

at 4 C.

Thrombin Activity
Using a stock thrombin solution (100 units/ml.) a series of dilutions
were prepared with cold physiologic saline (4 C.) to give the following
concentrations: 20, 15, 10, 8, 6, 5, 4, and 2 units/ml.
Approximately 2 m1. of pooled platelet-rich plasma was incubated in
polyethylene tubes (12 x 75 mm.) for 3 minutes in a 37 C. water bath.
Next, 0.4 ml. of incubated plasma was placed in each of 4 prewarmed plas-

* To each reaction cup, 0.1 ml. of the diluted thrombin

tic reaction cups.*
was added and the clotting time*** in seconds was recorded. Each dilu-
tion was repeated 16 times and the average clotting time was calculated.

A thrombin activity curve was constructed by the method of least squares.

Heparin Assays
The stock heparin solution (1 mg./ml.) was diluted with deionized
water to make solutions of.0.01, 0.02, 0.03, 0.04, 0.05 mg./ml. To iced
polyethylene tubes (12 x 75 mm.) 1.9 ml. of pooled platelet-rich plasma
and 0.1 ml. of-diluted heparin were added. The final concentration of
heparin was then 0.5, 1.0, 1.5, 2.0, and 2.5 ug./ml. of plasma. Platelet-

rich plasma (1.9 ml.) was placed in an iced polyethylene tube and used

 

*Heparin (Ammonium Salt) - Biological Research, Inc., St.
Louis, Mo.

**Fibrotube; BD, Division of BioQuest, Cockeysville, Md.

***Fibrometer System; BD, Division of BioQuest, Cockeysville, Md.

13
as a control. Each tube was kept in crushed ice until thrombin times
were performed. Next, each tube was incubated 3 minutes in a 37 C. water
bath. To 0.4 ml. of incubated control plasma or heparin-containing
plasma, 0.1 m1. of thrombin (8 units) was added and the thrombin times
recorded in seconds. This was repeated 16 times for each heparin dilu-

tion and the control.

Plasma Stability

 

Pooled platelet-rich plasma was stored at 4 C. in 12 air-tight poly-
ethylene tubes (12 x 75 mm.). One tube was removed from storage every.
hour and 4 thrombin times were performed. This procedure was repeated
12 times and the average clotting time was calculated for each hour the
plasma was stored at 4 C. The thrombin concentration was 8 units for

this experiment.

Clinical Experiment

 

Blood of patients* receiving heparin therapy was used for this experi-
ment. An oxalated sample of blood was obtained when a Lee-White clotting
time was performed on these patients. Four thrombin times were determined
for each recorded Lee-White clotting time. The average thrombin time was
determined for each sample. The heparin concentration was calculated by
the following formula:

Heparin concentration - 2.11 - 1242_
t

(ug./ml.) (clotting time
in seconds)

 

*Patients were from St. Luke's Hospital, Saginaw, Mich., St.
Lawrence Hospital, Lansing, Mich., and Ingham.Medical Hospital, Lansing,
Mich.

14
This formula was determined from the results of the heparin assay.

experiment (page 12).

RESULTS

Thrombin Activity

The reciprocal of the thrombin units added was plotted against the
time in seconds it took 0.4 ml. of platelet-rich plasma to clot, and a
straight line was obtained. The equation of the line was determined by
the method of least squares. The results of the first series of determi-
nations are summarized in Figure 1.

Figure 2 is a thrombin activity curve constructed from the above
results. The equation for this curve is y -'Kx in which y is the recip-
rocal of the thrombin units that was added and x is the observed clotting
time. Using the data from the 16 experiments (8 dilutions of thrombin]
experiment) K.was calculated to be 0.0092. K represents the factor the
clotting time can be multiplied by to obtain the reciprocal of the con-
centration of.thrombin present. This was the procedure used for determin-

ing the residual activity of thrombin alone or in the presence of heparin.

Heparin Assays
For.the next set of determinations 0.1 m1. of varying concentrations
of heparin were added to 1.9 ml. of iced platelet-rich plasma and the
thrombin times determined as before. A control was performed for each
series of heparin dilutions and the concentration of thrombin (8 units)
added, remained constant for these'experiments.‘ From the first series
of determinations, one concludes that the clotting time multiplied by the

thrombin concentration equals a constant (t x T - k) where t is the clotting

15

16
time in seconds, T is the number of active thrombin units, and k is a
constant). The k was calculated from the control experiments, and the
number of active units of thrombin that remained after the addition of
known amounts of heparin was determined (T = k).

The number of active units of thrombin was.plotted against the con-
centration of the heparin added. Using the data from 16 separate experi-
ments (3 concentrations of heparin/experiment) a straight line was ob-
tained by the method of least squares. A.summary of the results is rep-
resented by Figure 3.

If the number of active units of thrombin equals 8 (number of units
of thrombin added) minus Kh (some constant times the heparin concentration
added) then a formula for determining heparin concentration can be writ-
ten as 8;— k_- h. This formula can be rewritten as y - B.- Ax (where y
is the fiepafin concentration and x is the reciprocal of the clotting time).
The values for A and B were calculated by the method of least squares
(A -.19.7, B - 2.11).

When-0.1 m1. of thrombin (8 units) is added to 0.4 ml. oxalated

platelet-rich plasma, the heparin concentration can be calculated by the

following equation: Heparin concentration = 2.11 - 19.7
t
(ug./ml.) (clotting time

iniseconds)
This method has a variance of 0.0159 with 95% confidence limits of

i 0.256 ug./ml.

Plasma Stability
The effect of storage of platelet-rich plasma was studied. Thrombin
times were not prolonged when stored 12 hours‘at 4 C. in air-tight poly-

ethylene tubes. The slight variation that occurred was the same as

l7
exhibited by fresh platelet-rich plasma. For 48 determinations per-
formed, the average thrombin clotting time was 10.5 seconds with a stan-

dard deviation of 0.77.

Clinical_Experiment

The purpose of the following studies was to evaluate the clinicall
application of this assay method. The calculated heparin concentration
in 18 patients was compared to their Lee-White clotting times. Their
heparin concentrations were plotted against their Lee-White clotting
times. A straight line was obtained by the method of least squares.
The linear relationship that exists between the calculated concentration
and the Lee-White clotting times is summarized in Figure 4.

If the Lee-White clotting times are compared to the calculated
heparin concentration, there is also a linear relationship. This is

shown in Figure 5.

18

 

 

60”
500
40”

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a

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o

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0 301

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w

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w-l

u

H

O

v-I

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10W

 

' 0.1 03 0.53 ' 0.1. ' 03
Reciprocal of Thrombin Units

Figure 1. Thrombin dilution curve (summary of data).

 

*Each point represents the average of 16 different determinations.
Ninety-five percent confidence limits 1:5.5 seconds.

19

0.6

Reciprocal of Thrombin Units

 

 

10 2'0 3'0 40 5‘0

Clotting Time (seconds)

Figure 2. Thrombin activity curve.

 

*Each point represents the average of 16 different determina-
tions. Ninety-five percent confidence limits :_0.054 reciprocal units
of thrombin.

Number of Active Thrombin Units

 

 

0.3 ‘1.Lo ' 135 ‘ 2T0
Concentration of Heparin (ug./ml.)

Figure 3. The number of active thrombin units that remained after
the addition of various concentrations of heparin.

 

*Each point represents the average of 16 different determinations.
Ninety-five percent confidence limits 1:1.4 thrombin units.

21

70 ‘_

60L

50.

40

30 y

Lee-White Clotting Time (minutes)

 

20
O St. Lawrence Hospital

 

 

A Ingham Medical Hospital

I St. Luke's Hospital '

(DJ.—

L ‘ L
2 4 6
Calculated Heparin Concentration (118./ml.)

Figure 4. Comparison of calculated heparin concentration to the
Lee-White clotting time.

 

Ninety-five percent confidence limits 1 8.4 minutes.

Rank correlation coefficient 0.703.

Calculated Heparin Concentration (ug./ml.)

12

10,

22

 

O St. Lawrence Hospital
A Ingham Medical Hospital

I St. Luke's Hospital

 

J n - 1 ‘ L ——‘

30 ' 40 50 60
Lee-White Clotting Time (minutes)

Figure 5. Comparison of Lee-White clotting time to calculated

heparin concentration.

 

"Ninety-five percent confidencenlimits : 1.59 ug./m1.

DISCUSSION

The replacement of the Lee-White clotting time by some other method
has been reported by many researchers. Due to the disadvantages and
inadequacies of the whole blood clotting time, a substitute procedure
that rapidly and accurately assays heparin in plasma was sought.

Coagulation is a complicated process that depends on the presence
of many substrates, enzymes, and cofactors. The visible or last step
requires fibrinogen, thrombin, and plasma factors to result in the forma-
tion of a fibrin clot. The in viva formation of thrombin requires many
steps. If thrombin is added to plasma, all these steps can be bypassed.

Heparin acts at several stages of the coagulation system, but one
of the more effective sites is‘its inhibition of the action of thrombin
on fibrinogen. By relating the time required for clot formation to the
reciprocal of thrombin concentration, it became possible to measure
thrombin activity. Then by adding a known amount of thrombin to an
unknown amount of heparin, it should be possible to estimate the concen-
tration of heparin. Fortunately, simple linear relationships exist
between: (1) the clotting time of fibrinogen and the reciprocal of the
thrombin concentration, and (2) the number of micrograms of heparin pres—
ent and the number of units of thrombin inactivated. These relationships
are summarized in Figures 1, 2, and 3. In concentrations of heparin
above 1.5 ug./ml. the relationship no longer holds true. When the con-
centration is over this value, the original sample should be diluted with
normal plasma, and a thrombin time repeated.-

23

24

After determining the activity of thrombin, the effect of heparin
on this enzyme.can be summarized by the equation y - B - Ax. By using
the value of the 2 constants and the thrombin clotting time, the heparin
concentration can be calculated. This method has a variance of 0.0159;
therefore, 95% of the time the calcuIated heparin concentration will be
:_0.256 ug./m1. from the actual concentration in the plasma.

Schwab in 1969 reported that platelet-rich plasma was needed for
the thrombin inhibition test. This assured that a sufficient amount of
fibrinogen.and heparin cofactor were present. It was stated early that
heparin is not a strong antithrombin unless a cofactor is present.
According to the literature, the fibrinogen«leve1 rarely decreases in
concentrations low enough to interfere with coagulation.. In several
trial tests, a platelet factor* was added to platelet-poor plasma in an
attempt to reproduce the results obtained by platelet-rich plasma. The
platelet substance failed to reproduce the same effect as platelet-rich
plasma in assaying thrombin.activity.

The data in Figure 4 suggeSts a linear relationship_between the
calculated heparin concentration and the Lee-White clotting time. The
precision and accuracy of the heparin assay, using a thrombin inhibition
test, is greater. This test is relatively simple and requires only a
few minutes to perform.

A similar linear relationship exists between the Lee-White clotting
time and the calculated heparin concentration. Figure 5 summarizes this
relationship. This would provide one with a method of estimating heparin
concentration from published Lee-White clotting times. If the Lee-White
clotting method is used to determine heparin concentration, there is a

variance of 0.222.

 

*Platelin; Warner—Chilcott Company, Morris Plains, N.J.

25

The time-saving element of this method makes it an improvement over
the time-consuming Lee-White test.v The potential sources of error lie
in the preparation of the plasma samples and the dilution of the thrombin.
A centrifuge with predetermined settings for platelet-rich plasma will
eliminate plasma inaccuracy. Stability of the dilute thrombin can be
insured by making the dilution in polyethylene tubes and using the result-
ing mixture within 20 minutes.

With a method available that assays the concentration of heparin
in blood, a dosage schedule may be determined. This could be predicted
on the bases of kinetic studies of the rate of heparin clearance from
the circulatory system for each individual patient. Research is needed

in this area to prove the validity of this hypothesis.

SUMMARY AND CONCLUSION

A major disadvantage in the use of heparin therapy is the lack of
a convenient and accurate system of controlling dosage.~

The purpose of this project was to continue the development of a
method for the assay of heparin in blood that would provide a useful
guide for the management of heparin therapy.

A linear relationship was found to exist between the clotting time
of a fibrinogen.solution and the reciprocal of thrombin concentration.
There is also a linear relationship that exists between-the number of
micrograms of heparin present and the number of inactivated thrombin
units. From these results, the following formula was derived to calcu-

late the concentration of heparin in plasma:

Heparin concentration ~= 2.11 - 19.7
t
(ug./ml.) (clotting time

in seconds)

To evaluate the clinical application of this assay method, the
measured heparin concentration was compared to the Lee-White clotting
time. A linear relationship exists between these 2 methods.

There is a variance of 0.0159 if the heparin concentration is de-,
termined by using the thrombin inhibition test. However, if the Lee-
White clotting time is used to predict the heparin concentration there

is a variance of 0.222.

26

27
The thrombin inhibition test is a one-step procedure requiring a
relatively small amount of time to perform. The precision and accuracy
appears to be higher than the Lee-White clotting test for monitoring

heparin therapy.

REFERENCES

REFERENCES

Abildgaard, U.: Inhibition of-the thrombin-fibrinogen reaction by heparin
in the absence of cofactor. Scand. J. Haemat., 5: 432-439, 1968.

Astrup, T., and Darling, 8.: Measurement and prOperties of antithrombin.
Acta Physiol. Scand., 4: 293-308, 1942..

Astrup, T., and Darling, S.: Antithrombin and heparin. Acta Physiol.
Scand., 5: 13-29, 1943.

Barnhart, M., and Anderson, 8.: Intracellular localization of fibrino-
gen. Proc. Soc. Exp. Biol. & Med., 110: 734-737, 1962.

Blomback, B., Blombfick, M., Corneliusson, E., and Jorpes, J. E.: On
the reliability of the methods used in the.aasay of heparin. J.
Pharm. Pharmacol., 5: 1031*1040, 1953.

Davie, E. W., and Ratnoff, 0. D.: waterfall sequence for intrinsic.
blood clotting. Science, 145: 1310-1312, 1964.

Eagle, H.: The formation of fibrin from thrombin and fibrinogen. J.
Gen. Physiol., 18: 547-554, 1935.

Eiber, H., and Danishefsky, 1.: Clearance of injected heparin from
the blood. Nature, 180: 1359-1360, 1957.

Ellias, T. P., and Iyer, G. N.: Some.inhibitors of the fibrinogen-
fibrin conversion. Thromb. Diath. Haemorrh., 18: 499-509, 1967.

Forman, W., and Barnhart, M.: Cellular site for fibrinogen synthesis.
JOAOMOAO' 187: 128-132, 19640

Ferry, J. D.: Polymerization of fibrinogen. Physiol. Rev., 34: 754-
760, 1954.

Ganguly, P.: Studies of human platelet proteins. Blood, 33: 590-597,
1969.

Harmelling, J. 0., and Cordray, D. R.: The activated partial.thrombo-
plastin time as a control in heparin therapy. 8. Med. J., 60:
594-598, 1967.

Henstell, H., and Kligerman, M.: The nature of heparin antithrombin
' action. Thromb. Diath. Haemorrh., 18: 167-178, 1967.

28

29

Howell, W. H.: Theories of blood coagulation. Physiol. Rev., 15: 435-
470, 1935.

Jaques, L. B.: The reducing prOperties of fibrinogen. Biochem. J., 37:

Jaques, L. B., and Ricker, A. G.: The relationship between heparin dosage
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Jorpes, J. E.: Heparin, its chemistry, pharmacology and clinical use.
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Laki, R.: The clotting of fibrinogen. Blood, 8: 845-856, 1953.

Laki, K., and Gladner, J. A.: Chemistry and physiology of the fibrinogen-
fibrin transition. Physiol. Rev., 44: 127-160, 1964.

Lamy, F., and Waugh, D.: Transformation of prothrombin into thrombin.
Physiol. Rev., 34: 722-729, 1954.

Lenahan, J. C., Erye, S., and Phillips, G. E.: Use of the activated
partial thrombOplastin time in the control of heparin administra-

Lorand, L.: Interaction of thrombin and fibrinogen. Physiol. Rev.,
34: 742-752, 1954. '

Lorand, L., and Middlebrook, W. R.: Studies on fibrino—peptide. Biochim.
BiOphys. Acta, 9:.581-582, 1952.

Macfarlane, R. G.: An enzyme cascade in the blood clotting mechanism
and its function as a biochemical amplifier. Nature, 202: 498-
499, 1964.

Miale, J. B.: Laboratory Medicine-Hematology, 3rd edition, C. V. Mosby
COO. Ste LOUiB, 19670

Monkhouse, F. C., and Clarke, D. W.: Further studies on the purifications
of antithrombin and heparin cofactor. Canad. J. Biochem. &
Physiol., 35: 373-382, 1957.

Penner, J.: Management of heparin therapy. Ann Arbor, Mich., personal
communication, 1967.

Quick, A. J.: Is heparin an antiprothrombin. Proc. Soc. Exper. Biol.
& Med., 35: 391-392, 1936.

Rappaport, 8. 1., and Ames, S. B.: Clotting factor assays on plasma.
from patients receiving intramuscular or subcutaneous heparin.
Amer. J. Med. Sci., 234: 678-686, 1957.

Rezansoff, W. E., and Jaques, L. B.: An investigation of the assay of
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18: 150-160, 1967.

3O

Schatz, I. J., and Hathaway, J. C.: Heparin therapy and thrombin times.
J.A,M.A., 186: 740, 1963.

Schwab, M. L.: Heparin assay using thrombin inhibition. M.S. Thesis,
Michigan State University, East Lansing, 1969.

Seegers, W. H.: Prothrombin and fibrinogen related to the blood clotting
mechanisms. Physiol. Rev., 34: 711-713, 1954.

Seegers, W. H.: Enzyme theory of blood clotting. Fed. Proc., 23: 749,
1964.

Seegers, W. H., Brinkhous, K. M., Smith, H. P., and Warner, E. D.: The
purification of thrombin. J. Biol. Chem., 126: 91-95, 1938.

Seegers, W. H., and McGinty, D. A.: Further.purification of thrombin:
Probable purity of products. J. Biol. Chem., 146: 511-518, 1942.

Sherry, S., Troll, W., and Glueck, H.: Thrombin as a proteolytic enzyme.
Physiol. Rev., 34: 736-741, 1954.

Spector, 1., and Corn, M.: Control of heparin therapy with activated
partial thromboplastin times. J.AJM.A., 201: 157-159, 1967.

Therriault, D. 0., Gray, J. L., and Jensen, H.: Influence of thrombin
on-rate of prothrombin conversion. Proc. Soc. Exp. Biol. and
Med., 95: 207-211, 1957.

Vigran, M.: Clinical Anticoagulant Therapy. Lea & Febiger, Philadelphia,
1965.

Were, A. C., and Lanchantin, G. F.: Purification of fibrinogen, pro-
thrombin and thrombin. Physiol. Rev., 34: 714—721, 1954.

Yin, E. T., and Wessler, 8.: Bovine thrombin and activated Factor X.

VITA

The author was born in Ithaca, Michigan, on March 1, 1944. She
graduated from Ithaca High School in June, 1961, and enrolled at Alma
College in September of the.same year. In June of 1965, she received
a Bachelor of Science Degree in biology. The next year, she completed
a one-year internship at St. Luke's Hospital School of Medical Tech-
nology, in Saginaw, Michigan. She passed the registry for medical
technologists given by the American Society of Clinical Pathologists
in August of 1966.

Before entering graduate school, the author worked at St. Luke's
Hospital for two years. She entered graduate school in the program
of Clinical Laboratory Science offered by the Department-of Pathology
at Michigan State University in September, 1968.

The author is a member of the Lansing, Michigan, and American
Societies of Medical Technologists.' She is also a member of the Michigan

and American Associations of Blood Banks.

31

  
  

     

      

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