Note: Descriptions are shown in the official language in which they were submitted.
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A THROMBIN BL000 FRAC?ION FOR USE IN A MEDICAL PROCEDURE.
1. FIELb OF T E INVENT ON
The subject invention relates to the use of
thrombin in a medical procedure in an animal. More
specifically, the subject invention relates to such
uae of thrombin wherein the thrombin is a thrombin
blood fraction, as defined hereinbelow.
2. B_ ACKG O~D OF THE INVENTION
one mechar. ~m for hemostasis, i. e. ,
prevention of blood loss, of an animal is the
formation of a blood clot. Clot formation, i.e. ,
15 blood coagulation, occurs by means of a complex
cascade of reactions with the final steps being the
conversion of fibrinogen by thrombin, calcium ions and
activated factor XIII to form the fibrin clot. For a
review of the mechanisms of blood coagulation and the
2o structure of fibrinogen, see C.M. ,Tackson, Ann. Rev.'
Biochem., 49:765-811 (1980) and B. Furie and B.C.
Furie, Cell, 53:505-518 (1988).
Thrombin, which is a prateolytic enzyme, is
derived from prothrombin. Prothrombin is converted to
25 thrombin by calcium and prothrombinase.
Prathrombinase is formed through a cascade of
reactions that begins with the proteins f actor XI and
factor XII.
A fibrin sealant is a biological adhesive
3o Whose effect im~.tates the final stages of coagulation,
thereby resulting in a fibrin clot. Conventional
f fibrin sealants generally consist of concentrated
human fibrinogen, bovine aprotinin and factor XIII, as
the first component and bovine thrombin and calcium
35 chloride as the second component. Application is
generally carried out faith a double-barrelled syringe,
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which permits simultaneous application of bpth
components to the site where one wants to form the
fibrin clot. Aprotinin is a fibrinolytic inhibitor
added to promote stability of _f fibrin sealants.
The ffibrinogen component of the fibrin
sealant is prepared from pooled human plasma: The
f fibrinogen can be concentrated from the human plasma
~by cryoprecipitation and precipitation using various
reagents, e.8., polyethylene glycol, ether, ethanol,
1o ammonium sulfate or _glycine. for an excellent review.
of fibrin sealants, see M. Brennan, Blcad Reviews,
5:240-244 (1991); J.W. Gibble and P.N. Ness,
Transfusion, 30:741-T47 (1990) ; H. Matras, J. Oral
Maxillofac Surg: , 43 : 605-611 ( 1985) and R. Lerner and
N. Binur, J. of Surgical Research, 48:165-181 (7.990) .
Recently, there has also been an interest in
the preparation of fibrin sealants that utilize
autologous fibrin. An ~autologous fibrin sealant is a
fibrin sealant wherein the fibrinogen component of the
fibrin sealant is extracted from the patients own
blood. The use of an autologous fibrin sealant is
preferred because it eliminates the risk of
transatission of blood-transmitted infections, e.8. ,
hepatitis B, non A, non 8 hepatitis and acquired
2S immune deficiency syndrome (AIDS ) , that could
otherwise be present in the fibrinogen component
., extracted from pooled human plasma. See L.E.
Silberstein et al., Transfusion, 28:319-321 (1988) ; K.
Laitakari and J. Luotonen, Laryngoscope, 99:874-97 s
(1989) and A. Dresdale et al., The Annals of Thoracic
Surgery, 40:385-387 (1985).
An infection can be transmitted by a f fibrin
sealant not only by means of the fibrinogen but also
by means of the bovine aprotinin and the bovine
thrombin component. Hovine thrombin has been known to
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carry the infectious agent bovine spongiform encephalitis
(sSfi) and other viruses pathogenic to mammals .
Furthermore, bovine thrombin is a potential antigen,
which can cause immunological reactions in humans. Thus,
the use of bovine thrombin could result in the recipient
of the bovine thrombin being adversely affected. See
D_M. Taylor, J_ of Hospital Infection, 18 (Supplement
A) :141-146 (199i) , S.B. Prusiner et al., Cornell Vet, 81
No. 2: 85-96 (1991) and D. Matthews, J_ Roy. Soc. Health, CtitGt)>
3-5 (February 1991).
Accordingly, there is the need for a sealant
that utilizes thrombin that can be delivered to a patient
without the risk of viral contamination or other adverse
affects _
3. SUN~IARY OFTF38 INVENTION
The subject invention relates to a composition
for use in a medical procedure in an animal, arhich
composition is a thrombin containing blood fraction which
comprises:
(a) a thrombin concentration of from about 1
NIH unit/ml to about 2,000 NIH units/ml,
and
(b) a specific activity of thrombin of from
~''~ 25 about 1 NIH unit/mg of blood protein to
about 200 NIFi units/mg of blood protein;
wherein said thrombin blood fraction is substantially
free of active antithrombin III.
The subject invention also relates to a method
for preparing a prothrombin blood fraction from whole
blood comprising:
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(a) diluting whole blood to an ionic
strength of less than about 100
millimolar;
(b) separating plasma from said whole
blood;
(c) lowering the pH of said plasma to
precipitate a prathrombin blood
fraction; and
(d) separating said prothrombin blood
l0 fraction from said plasma.
The prothrombin blood fraction is then
redissolved and converted to thrombin, i.e., the
thrombin blood fraction is formed, which can then be
utilized in a medical procedure in an animal, e.g., as
i5 a component of a fibrin sealant.
4. DETAILED DESCRIPTION OF TH~ INVENTION
The subject invention relates to the use of
a thrombin blood fraction, as defined hereinbelow, in
Zn a medical procedure, e.g., as a component of a fibrin
sealant, in an animal, preferably a mammal. Suitable
mammals include a human, a cow, a pig, a dog and a
rabbit, ar other mammals that have an adequate blood
volume to prepare the thrombin blood fraction. The
25 thrombin blood fraction can be prepared from whole
blood and is impure in that it contains blood proteins
.. other than thrombin. However, the thrombin blood
fraction can be prepared very simply arid rapidly,
a . g . , in less than about one or two hours , and is
3o believed to be as efficacious as a highly .pure
thrombin preparation.
The thrombin blood fraction carp be prepared
from whole blood. It is preferred that the whole
blood be obtained from a single =ndividual animal.
'35 Also, it is preferred that the thrombin blood fraction
r
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be administered to the same individual animal from
which the whole blood was taken.. thus, one aspect of
the invention is the use of an autologous thrombiul
blood fraction in a medical procedure: In this
embodiment, there is also no risk of transmission of
blood~transmitted infections because the thrombin
blood fraction is to be administered to the same
individual animal that donated the whole blood. Also,
for the same reason, the blood proteins other than
thrombin that are present in the thrombin blood
fraction would not be antigenic.
Furthermore, since the thrombin blood
fraction is preferred to be prepared from a single
individual animal and used in the same, generally
small volumes of whole blood are required, especially
since it is also preferred to prepare a thrombin blood
fraction far a single use. Also, it is preferred to
prepare the fraction within several hours of the time
of use. 1t is preferred that from about l0 ml to
2o about 50 ml, more preferably from about to ml to about
' 30 ml and most preferably from about 10 ml to about 20
ml of whole blood be utilized to prepare the throaabin
blood fraction of the subject invention.
The thrombin blood fraction ef the subject
invention has a thrombin concentration of from about l
NIH unit to about 2,000 NIH units, preferably from
about l00 NIH units to about 800 HIH units and most
preferably from about 1C0 NIIi units to about 500 N=H
units per ml of the thrombin blood fraction.
It is believed that at such concentrations
the thrombin blood fraction possesses a sufficient
thrombin concentration for the desired medical use.
of course, the preferred thrombin concentration
depends on the medical use of the thrombin blood
fraction.
Y
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6
The thrombin concentration of the thrombin
blood fraction can be determined by measuring the
coagulation time of a standard fibrinogen solution
after addition of the thrombin blood fraction in a
suitable diluted forma As a reference, standard
thrombin solutions, containing from 2 to 15 NIH
units/ml, can be utilised.
The thrombin blood fraction of the subj ect
invention has a specific activity of thrombin of from
to about 1 NIH unit to about 200 NIH units, preferably
from about 5 NIH units to about 100 NIH units and most
preferably from about 5 NIH units to about 50 NIH
units per mg. of-total blood protein. Such lower
specific activities of the thrombin blood fraction are
believed to be as effective in a medical procedure as
the more pure thrombin blood fractions. However, such
lower specific activity of the thrombin blood
fractions can be prepared more readily.
The specific' activity of the thrombin blood
fraction is, in essence, a measurement of the amount
of thrombin per amount of blood protein in the
thrombin blood fraction. Thus, the specific activity
of the thrombin blood fraction of the subj ect
invention is quite low relative to thrombin
preparations that have been prepared heretofore. For
example, United States Patent No. 5,143,838 discloses
., a thrombin preparation with a specific activity of at
least 800 NIH units per mg. of total protein. Also;
l3nited States Patent No. 5, 251, 355 discloses a
3o thrombin preparation with a specific activity of
greater than 1, 000 NIH units/mg. of total protein.
Although the specific activity of the thrombin blood
fraction of the subject is low, never the less it is
believed that the blood fraction is efficacious far
use in medical procedures.
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The specif is activity of the thrombin blood
fraction of the subject invention can be calculated by
measuring the thrombin concentration (NIH unitsJml)
and by dividing that number by the protein
concentration (mg/ml) measured by any standard protein
assay, e.g., W absarbance.
The thrombin blood fraction is also
substantially free of active antithrombin III. For
the purpose of the subj ect invention, "substantially
io free of active antithrambin III' means that the
thrombin blood fraction per unit volume contains an
amount of active antithrombin III that is less than
about 5o% by activity of antithrombin =II in nonaal
plasma per unit volume. It is preferred that such
i5 percentage be less than about 30%, more preferably
less than about 10% and most preferably less than
about 5%. It is essential that the antithrombin III
either be removed from or in--' ctivated in the thrombin
blood fraction. Otherwise, antithrombin III will
2o prevent the conversion of the prothrombin in the blood
fraction to thrombin and/or inactivate the thrombin
that is formed.
It is preferred that the thrombin blood
fraction also be substantially free of fibrinogen and
25 fibrin. For the purpose oP the subject invention;
substantially free of f fibrinogen and f fibrin means that
the thrombin blood fraction per unit voluiae contains
an amount of fibrinogen plus fibrin that is less than
about 1% by weight of fibrinogen in norZnal plasma per
3 o unit volume. It is pref erred that fibrinogen not be
present in the thrombin blood fraction because the
thrombin will convert --.he fibrinogen to fibrin, which
will polymerize to fo: a clot, thereby rendering the
thrombin blood fraction impracticable. The fibrinogen .
35 itself can be removed from the thrombin blood fraction
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.~ g _
or the fibrinogen can be removed as the fibrin clot,
thereby, of course, also removing the f fibrin.
HaWever , the f fibrinogen need net be removed if, f or
example, the thrombin is inactivated as described in
PCT publication No. W091/09641.
The thrombin blood fraction -can be utilized
immediately after it is prepared. If the fraction is
not utilized immediately after its preparation, the
fraction can be stored. Storage of the fraction
~o requires that the fraction be preserved by, for
example, freezing or lyophilizing the fraction or
holding the composition at 4 °C. The fraction in
frozen or lyophilized form will be stable for a period
of months. When the fraction is held at 4 °C, it is
i5 stable for at least a period of days.
=t the fraction is frozen, the fraction must
be tha~red at the time of use. If the fraction is
lyophilized, at time of use, it is preferred that the
fraction be reconstituted by addition of distilled
20 water.
The thrombin blood fraction can be in
virtually any form, for example, a solution,
suspension, emulsion or solid, with a solution being
preferred. Thus, for example, sucr fZaction can be a
25 liquid, gel, paste ar salve. Also, of course; the
traction can be in the form of a granule.
It the thrombin blood fraction is in a solid
f vrm, then the concentration of the thror.~hir, blood
f=action can be determined by dissolving it in a
30 solution and then measuring the thrombin
concentration. If the resulting thrombi~
concentration is from about 1 NIB unit/:al to about
2 , 000 lJIH units/ml, then the thrombin blood fraction
is within the scope of the subject invention.
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4.1. METHOD FOR PREPARATION OF T$E
T~O~BIN BLOOD FRACTION
The thrombin blood fraction of the subject
invention can be prepared by any method known or to be
developed. Also, the thrombin blood fraction can be
prepared in a device as described in the
PCT publication No. W091/I???8.
1O 4.Z.1. METHOD FOR PREPARATION OF TH~ T~iROMBIN
BLOOD FRACTION BY FORMATION OF THE
EUGLOB IN FRACTION FROM PLASMA
Whole blood can be withdrawn from an
individual animal, e.g. , a human, and preferably in
the presence of an anticoagulant. Any anticoagulant
l5 can be utilized so long as it does not act by directly
inactivating thrombin. Suitable anticoagulants are
heparin, EDTA, citrate or any other agent that can,,
directly or indirectly, prevent the formation ~of
thrombin, with citrate being preferred.
2o The plasma, zrhich contains the prothrombin,
is then separated from the Whole blood. Any
separation technigue can be utilised, Eor example,
sedimentation, centrifugation or filtration.
Centrifugation can be carded out at about 1, 500 to
25 about 3 , 000 g., for about 10 minutes. The supernatant,
which contains the plasma, can be removed by standard
techniques. If it is desired to obtain a thrombin
blood fraction that contains growth factors, then such
centrifugation should be at about ;25 g. far about 20
30 minutes or 1,000 g. for about.2 to 3 ainutes. The
thrombin blood fraction of the subject invention will
then contain growth factors, which are released from
the platelets during the conversion of prothrombin to
thrombin .
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The plnsma in then treated, for example, by
dilution with distilled water, followed by the
addition of acid, e.g., citric acid, to lower the
ionic strength to less than about 100 millimolar,
preferably less than about 50 millimolar and most
preferably from about 20 to about 40 iaillimolar and
lower the pH to about 4.5 to about 6 and preferably to
from about 5 to about 5.5. Lactic acid or acetic acid
are also suitable acids. Generally, a weight ratio of
1o the plasma: distilled water or acid of from about 1:5
to about 1:50, with 1:10 being preferred.
This treatment, i. e. , the icwerinq of the
ionic strength and pH of the plasma, results in the
formation of a precipitate that is generally referred
i5 to as the "euglvbulin fraction." The euglobulin
fraction contains the prothrombin, fibrinogen and many
other blood proteins, but is substantially free of
antithrombin III.
Rather than diluting the plasma to lower the
2o ionic strength, and before acidifying tha plasma, the
ionic strength can be lowered by dialyzing the plasma
by placing the plasma in a dialysis bag, which is then
placed is distilled water. The dialysis permits the
ions to diffuse out of the plasma, thereby lowering
25 the ionic strength. A suitable dialysi s bag is
composed of cellulose nitrate. Also, the ionic
strength can be lowered by diafiltration or ecalus ion
chromatography.
The euglobulin fraction can b~ prepared as
3o described in A. Quick, Production of Thrombin From
Precipitate Obtained by Acidification of Diluted
Plasma, Am. J. Physiol. , ,i,$1:114-118 ( 19 55 ) and R.
Biggs and R.G. Macfarlane, Human Blood Coagulation,
pages 3?5-3-76, Blackwells Scientific Pub lications,
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Oxford, 3rd Edition (I962).
The excess fluid can then be separated prom
the euglobulin fraction by, for example,
s centrifugation, tiltration or sedimentation.
Centrifugation can be carried at about 1, 50o g. for
about 2 to about 5 minutes.
The prcthrombin of the euglobulin fraction
is then redissolved and converted to thrombin, thereby
~o forming a thrombin blood fraction of the subject
invention. This can be carried out by solubilizing
the euglobulin fraction in a physiologically
acceptable solution, e. g. , saline, in an amount equal
to or preferably less than (about 10%) the original
15 amount of plasma. An alkaline buffer can be added in
an amount to raise the pH of the solution to about 6
to about 8 and preferably to about 6.5 to about 7.5.
Ncnlimiting examples of suitable alkaline buffers
include sodium hydroxide, potassium hydroxide, calcium
2o hydroxide, bicarbonate buffers such as sodium
bicarbonate and potassium bicarbonate, salts of acetic
acid and salts of sulfuric acid. Preferred alkaline
buffers include: Sodium carbonate/bicar'3~anaze pH ~ . o ,
Sodium bicarbonate/NaOH pH 7.0, 1.5M Glycine/NaOH pH
25 6.5-7.5, Bis hydroxyethylaminoethane sulphonic acid
(BES) pH 7.5, Hydroxyethylpiperazine propane sulphonic
acid (EPPS) pH 7.5, Tricine pH 7.5, Mcrphali.no propane
sulphanic acid (MOPS) pH 7.0, Trishydroxyinethyl
aminoethane sulphonic acid (TES) 'pH 7.0 and
30 Cyclohexylaminoethane sulphonic acid (GFiES) pH 7.0;
with sodiu.~ carbonate/bicarbonate pH 6:5 - 7.5 Bis.
hydroxethylaminoethane sulphonic acid (8~S) pH 7.5,
Hydrvxyethylpiperazine propane sulphonic acid (EppS)
pH 7.5 and Trishydroxymeth~~1 aminoethane sulphonic
35 acid (TES) pH 7.5 being most. preferred.
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Calcium is added to the neutral solution in
order to convert the prothrombin to thrombin. Of
course, the calcium can be part of the alkaline
buffer. Calcium can be added in the form of, for
example, calcium chloride. The amount of calcium
added should be sufficient to convert an amount of '
prothrombin to thrombin that is sufficient for theca
intended medical use. Furthermore, the reaction
should be permitted to occur for a period of time
sufficient to convert enough of the prothrombin to
thrombin that is sufficient for the intended medical
use. Generally, from about 5 millimolar to about 50
millimalar calcium chloride is sufficient.
Also, rather than adding a source of calcium
s5 ions, prothrombin activating enzymes from snake venoms
can be utilized. For example, snake venom from Eccis
carinatus or the Australian Tiger snake can be
utilized.
As the thrombin f orms ; it converts the
fibrinogen to fibrin, which forms a fibrin clot. It
is preferred to remove the fibrin clot, which can be
carried out by, for example, wrapping the fibrin clot
around a stirring rod or collecting the fibrin onto
glass beads.
4.1.2. METIiOD FAR PR~FARATION OF THROMBIN BLOOD
FR~:CTION BY DILUTING WHOL~ BF~OC~D~
In a preferred embodiment, the thrombir~
blood fraction can be prepared by initially preparing
3o a Prothrombin blood fraction from whole blood
comprising:
(a) diluting whole blood to an ionic
strength of lass than about 100
millimolar;
3s (b) separating plasma from said whole
blood;
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(c) lowering the pH of said plasma to
precipitate a prothrombin blood
fraction; and
(d) separating said prothrombin blood
~ fraction from said plasma.
The prothrombin blood fraction is then
redissolved and converted to thrombin, i.e., the
thrombin blood traction of the subject invention is
formed, which can then be utilized in a iaedical
to procedure in an animal , e. g . , as a component of a
f fibrin sealant. This method provides a thrombin blood
fraction of the subject invention, which can be
prepared in only about 45 minutes:
Specifically, whale blood is drawn from an
individual animal. The whole blood is then
immediately (within about five minutes) diluted in
order to lower the ionic strength to less than about
loo millimolar, preferably less than about 5o
millimolar and preferably to from about 20 to about 40
2o millimolar. It should be noted that since the whale
blood is diluted imtaediately, there is no need for the
use of an anticoagulant. Any physialogica lly
acceptable solution at physiological osuiotic pressure
can be utilized to lower the ionic strength, e.g., a
glucose aqueous solution such as a 5:5% isotonic
aqueous glucose solution.
The plasma is then separated from the whole
blood. Any separation technique can be utilized, for
example, sedimentation, centrifugation or filtration.
3o Centrifugation can be carried out at about .1, 500 to
about 3, 000 g: for about 5 to about 10 minutes. The
supernatant, Which contains the plasma, can be removed
by standard techniques.
The plasma fraction is then acidif ied,
3S thereby resulting in the formation. of a prothrombin
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blood fraction, which is generally referred to as the
euglobulin fraction, which is a precipitate. The
plasma fraction can be acidified with, for example,
citric acid, lactic acid or acetic acid. The pH
should be lowered to fro~a about 4.5 to about 6 and
preferab2y to from about 5 to about 5:5.
The excess fluid can then be separated from
the prothrombin blood fraction by, for example,
centrifugation, filtration or sedimentation.
Centrifugation can be carried out at about 1, 500 g: for
about 2 to about 5 minutes. This grothrombin blood
fraction contains prothrombin, fibrinogen and many
other blood proteins, but does not coma in
antithrombin FII. The prothrombin blood fraction is
then redissolved and the prothrombin is converted to
thrombin. Any physiologically acceptable solution,
e.g., saline, can be utilized to red3ss olve the
prothrombin fraction. Furthermore, only a small
volume of solution is required to redissolve the
2 o prathrombin fraction. It is believed that only from
about 0.4 ml to about 1 ml of solution is required if
about 17 ml of whole blood was initially drawn. An
alkaline buffer can be added in an amount to raise the
pH of the solution to about 6 to about 8 and
preferably to about 6.5 to about 7.5. Nonlimiting
exaiaples of suitable alkaline buffers ir~clude sodium
hydroxide, potassium hydroxide, calcium hydroxide,
bicarbonate buffers such as sodium bicarbonate and
gotassium bicarbonate,.tri-metal salts o f citric ac id,
salts of acetic acid and salts of sulfuric acid.
Preferred alkaline huffers include: , Sodium
carbonate/bicarbonate pH 6-8, Sodium biearbonate/NaOH
pH 6-8, Glycine/NaaH pH 6-8, 8is
hydroxyethylaminoethane sulphanic acid ( B~S ) pFl 6-8 ,
Hydroxyethyl.piperazine propane sulphonic acid (EPPS)
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pH 6-8, Tricine pH 6-8, Morpholino propane sulphonic
acid (MOPS) pH 6-B, Trishydroxymethyl aminoethane
sulphonic acid (TES) pH 6-8 and Cyclohexylaminoethane
sulphonic acid (CHES) pH fi-8; with Sodium
carbonate/bicarbonate pH 6-8 Bis
hydroxethylaminoethane sulphonic acid (HES) pH 6-8,
Hydroxyethylpiperazine propane sulphonic acid (EPPS)
pH 6-8 and Trishydroxymethyl aminoethane sulphonic ~~
acid (TES) pH 6-8 being most preferred.
i0 Calcium is added to the neutral solution in
order to convert the prothrombin to thrombin. Of
course the calcium can be part of the alkaline buf f er .
Calcium can be added in the form of form of, for
example, calcium chloride. The amount of calcium
i5 added should be sufficient to convert an amount of
prothrombin to thrombin that is sufficient for the
intended medical use. Furthermore, the reaction
should be permitted to occur fox a period of time
sufficient to convert enough of the prothrombin to
2o thrombin that is sufficient f~~r the intended medical
use. Generally, from about 5 millimolar to about 50
millimolar calcium chloride is sufficiezzt. It has
been observed that at about 25 minutes of reaction
sufficient amounts of thrombin are formed for mast
25 medical uses of the thrombin blood fraction of the
subject invention.
. Also, rather than adding a source of calcium
ions, prothrombin activating enzymes from snake venams
can be utilized. For example, snake venom from Eccis
3o carinatus or the Australian Tiger snake can be
utilized.
As the throzabin forms, it coral arts the
fibrinogen to fibrin, . which farms a fibrin clot. =t
is preferred to remove the fibrin clot, , which can be
35 carried out by, for example, wrapping the fibrin clot
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around a stirring rod or collection of the fibrin onto
glass beads.
4'.1.3. PREPARATION OF THROMBIhI BLOOD
6 FRACTION BY REMOVING OR INACTIVATING
ANTITHROMBIN III FROM PLASMA
In an alternative method, the thrombin blood
fraction of the subject invention can be prepared by
withdrawing whole blood from an individual animal,
e. g. , a human, and preferably in the presence of an
l0 ~ticoagulant. Any anticoagulant can be utilized.
Suitable anticoagulants are heparin, EDTA, citrate or
any other agent that can, directly or indirectly,
prevent the formation of thrombin, with nitrate being
preferred.
15 The plasma, which contains the prothrombin,
is then separated from the whole blood. Ax~y
separation technique can be utilized, for example,
sedimentation, centrifugation or filtration.
Centrifugation can be carried out at about 3,000 g.
for about 10 minutes: The supernatant, which contains
the plasma, can be removed by standard techniques.
Antithrombin III is then removed from the
plasma or is inactivated. For example, the pH of the
plasma can be lowered to at least to about 5. Such
25 lowering of pH inactivates antithroa~bin III; otherwise
antithrombin III would prevent the conversion of
prothrombin to thrombin. Antithrambin =II can be
inactivated by any technique. For example, by the
addition of 0.05 ml of 5 mol. /liter HCl per 1.0 ml .
plasma. After about 10 to about 20 minutes of
incubation, the plasma can be neutralized with 0.05 ml
of 5 mol./liter NaOH per ml of plasma.
Rather than inactivating the antithrombin
III, the antithrombin III can be removed from the
3S
plasma by passing the plasma through a column that
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binds antithrombin III, e.g. , a heparin column or a
column with antibodies to antithrombin II2:
The plasma fraction, which contains
prothrombin, is then treated to convert prothrombin to.
thrombin. This can be carried out by, far example,
the addition of a source of calcium ions , as described
above,. or by the addition of 0.1 ml CaCl: of 0.36
mol. /liter per ml of plasma. Also, rather than adding
a source of calcium ions, prothrombin activating
enzymes from snake venoms can be utiliz ed . For
example, snake venom from Eccis carinatus or the
Australian Tiger snake.
As the thrombin farms, it converts the
fibrinogen to fibrin, which farms a fibrin clot. It
is preferred to remove the fibrin clot; which can be
carried out by, for example, wrapping the fibrin clot
around a stirring rod or collection of the fibrin onto
glass beads.
The resulting plasma is a thrombin blood
fraction of the subject invention.
4.1.4. METHOD FOR THE PRE..pARATION OF A
THROMBIN BLOOD FRACTION HY DILUTING
WITt1 AN ACID SOLUTION DIRECTLY
8y the known process for preparing thrombin
from plasma, plasma is diluted in the ratio 1:10 with
water, whereatter a pFi-reducing acid is added, such as
acetic acid, with the result thatw the pH-value is
about 5.0 to 5.3. The mixture is then centrifuged for
20 min. at 2,000 g. The resulting precipitate
contains different coagulation factors, such as inter
olio prothrombin and fibrinogen. When the excess
fluid has been removed, the precipitate is dissolved
in a physiological solution, preferably a 0.9~ sodium
chloride solution, whereafter a pH-value,-.increasing
agent, such as sodium carbonate, is added until the
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pH-value is about 7 . 9 . When the precip hate has been
dissolved, calcium chloride is added and causes the
conversion of the prothrombin into thrombin by a
conventional, so-called coagulation cascade. The
s resulting thrombin causes a conversion of fibrinogen
into fibrin, whereafter the thrombin is separated by
centrifuging and then subjected to a succeeding
purification (column purification).
Another aspect of the subj act invention is
that the diluting step ~is performed directly With a
diluted acid solution, e.g. , a.4g acetic acid, by the
centrifuging step being performed in a container with
a relatively large flat precipitation surface, by the
physiological solution, with. the agent for increasing
pH, and the calcium chloride being added at the same
time to the precipitate as a mixture, whereby said
precipitate is dissolved and then coagulates while
forming fibrin, and by the thrombin then being
removed.
As a result, it is now possible to prepare
thrombin from plasma of autologous blood in a
relatively quick way, which is in particular due to
the fact that the precipitation is perf ormed in ~ a
container with a relatively large flat precipitation
surface. A quick and complete dissolution is thereby
ensured of the precipitate containing prothrombin and
fibrinogen. The complete dissolution is important
before the presence of calcium chloride causes the
conversion of prvthrombin to thrombin. A too early
3o thrombin formation leads to fibrin formation and the
fibrin will bind the added fluid, whereby the
dissolution of the precipitate stops and cannot
continue unti l said f luid has again been passed out of
the fibrin. The relatively small precipitate with a
3s relatively large surface area relative to the amount
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causes a complete dissolving of said precipitate by
addition og the combined mixture of physiological
solution, with the agent far increasing pH; and
calcium chloride before the thrombin has been formed
S through said coagulation cascade and starts the
formation of fibrin.
Moxeover according to the invention, the w
mixture may be admixed with a plasminogen catalyst ,
such as streptokinase, before being added to the
to precipitate with the effect that the following
separation of thrombin from fibrin is promoted.
According to the invention, the mixture
added to the precipitate can advantageous 1y be set , to
increase the pH-value to 6.5, whereby the resulting
15 active thrombin is found to obtain the best keeping
qualities as the thrombin has a tendency to: became
inactive on standing, which is usually the case in
connection with enzymes. In addition, the dissolved
precipitate can be transferred to a flexible material
. Z0 before the coagulation starts, said flexible material
presenting a relatively large surface upon which the
f i.brin resulting from the coagulation can be
deposited, whereafter the thrombin may be pressed out
of the fibrin by the flexible material being subjected
25 to a compaction. Exactly the obtained complete
dissolving of the precipitate prior to for~aation of
fibrin turned out to allow a quick transfer of the
solution to a flexible material prior to said
formation of fibrin. The flexible material ensures
30 that the fibrin is deposited across a particularly
large surface, the compaction of which f acilitates the
separation of thrombin from fibrin.
It is particularly preferred ~.rhen the
flexible material is a sponge with open pores.
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The flexible material can be placed fn a
syringe, into which the dissolved precipitate can be
absorbed and from which the thrombin can be squeezed
out by an activation of the piston of the syringe. In
this manner, a particularly easy separation of the
thrombin from the flexible material is ensured.
Moreover, the centrifuging may according .to
the invention suitably be performed at about 1,500 g
for about 5 min, which also aeceferates the preparing
of thrombin.
Thus, the sub j ect invention comprises a
process for preparing thrombin from human blood
plasma, whereby the blood plasma is diluted to about
10 to 17%.with water or another ion-intensity-reducing
~s fluid and an acid to reduce pH to about 5.0 to 5.3,
Thereafter the mixture is centrifuged, and whereby the
precipitate resulting from the centrifuc3ing is admixed
a physiological solution and calcium chloride, said
physiological solution increasing pH to about 6 to 8,
characterized by the diluting step being performed
directly with a dilated acid solution, by the
centrifuging step being per:ormed in a container with
a relatively large plane precipitation surface, by the
physiological solution, with the agent for increasing
pH, and the calcium chloride being added at the same
time to the precipitate as a mixture, whereby said
precipitate is dissolved and then 'coagulates while
f orming f fibrin, and by the thrombin then being removed
f rom the f fibrin .
35
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4.2. ACCELERATION OF THROMBIN FORMATION
BY CONTACTING BLOOD OR PLASMA WITH
A SURFACE THAT ACTIVATES BLOOD
COA~TLATION FACTORS XI AND X22
Another aspect of the subject invention is
the preparation of a thrombin blood fraction wherein
whole blood or plasma is contacted crith a surface that
activates blood coagulation factors XI and XII. It is
believed that a negatively charged surface such as
glass or kaolin, with glass being pref erred, can
provide such activation. Nonlimiting examples of
glass surfaces are glass beads, glass Drool, glass
filters and glass capillary tubes..
The whole blood or plasma should contact the
surface for a period of time sufficient to activate
such blood coagulation factors, e.g., for about 5 to
about l0 minutes. If the whole blood or plasma has
not been treated with an a: ~.:.icoagulant , then the whole
blood or plasma should be in contact With such a
surface for not more than about 60 seconds, preferably
less than about 30 seconds and more preferably about
seconds. Without the anticoagulant , and if the
contact of the whole blood or plasma with the surface
is too long, then fibrin clots will form prematurely.
It is preferred that plasma be exposed to a
25 surface of about 4 cmZ to about 60 cmi, preferably from
about 10 cm to about 30 cm? and most praferably about
. 20 cm~ of such surface for each milliliter of plasma.
Also, it is preferred that whole hlood be exposed to a
surface of from about 2 cm' to about 3o cm~, preferably
3Q from about 5 cm~ to about 15 cmi and most preferably
about 10 cm= of such surface for each milliliter of
whole blood.
It is believed that the contacting og the
plasma or whole blood with a surface that can activate
factors XI a.nd XII. accelerates the conversion time of
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prvthrombin to thrombin. Thus, a thrombin blood
fraction can be prepared in an extremely short period
o;e time .
This activation of factors XI and XII can be
utilized to accelerate the preparation, of any thrombin
blood fraction, regardless of how the traction is made
and regardless of its purity and specif i c activity .
However, it is essential that the thrombiz~ blood
fraction be substahtially free of active antithrombin
III. It is essential that the antithrombin III either
be removed from or inactivated in the thrombin blood
fraction prior to converting the prothrombin to
thrombin. Otherwise, antithrambin III will prevent
the conversion of the pzvthrombin tv thrombin and/or
is inactivate the thrombin. It is also pr8ferred that
the thrombin blood fraction be substantially free of
fibrinogen and fibrin. For example, with respect to
the activation when the thrombin blood fraction is
prepared by means of the euglobulin fractior~ as
Zo described in Section 4 ,1.1. , the contacting of the
plasma should be carried out immediately prior to the
dilution of the plasma. When a thrombin blood
fraction is prepared by diluting .whole blood, as
described in Section .4 .1. 2 . , the contacting of the
25 whole blood should be carried out immediately prior to
the dilution of the whole blood. For example, this
.. contacting step can be carried out by withdrawing
whole blood into a syringe that contains , for example,
glass beads. After not more than about 60 seconds,
30 the whole blood is discharged from the syringe and the
thrombin blood fraction oP the subject iriventian is
tben prepared, as described above.
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4.3. THE USFS OF TEE TEi~tolCB=N HZOOD
FRACTIQN of ,~$ S~yHJL~G,~,' INVEHT~,g~j
The t?~rombia blood fractior~ of 'tee sub j act
inv~asstiorr rexs be utilised is any mndi.oal proaedura,
known an to be davelop~ed, in a.t~ animal, iticludfng
vetinary procedures. Jlny species of animal is
suitable, bet, of course, humans are preferred.
2bo tt~ombitn blood Inaction can be emplQyad
as a ea~mponent of a fibrin sealant or cari ha ea~playad
to alone just as coaventiariti thrombin preparations have
bans employed. Tho thrombin blood ~xacti.on is
utilised by aontacti.ag the desired site e~f the st~imal
with the thrombin blood fraction. Far the parpos,e _o.f
the subject ~inventi.va, the "desired siteN is tdat
location in-or an an animal whsre vr~ desires 'Co fcrm
a sa.br~.n clot. What or where the desired site is
depends on the uas of the throtabin blood lractiort of
the subject invGat3oa.
The use of the thrombin blood fraction as a
so component of a fibrit~t sealant, can be utilized tr~r
' connecting tissues or organs, stopping bleeding,
heeling wounds, sealing a surgical woussd, use is
vaaaular s~ttgsi~~r include provid3.ng homoeetasia t4r
stitch hole bleeding of distal coronary . artery
Z5 anastomaaesp left ventricular suture 13.:sea; aortozomy
and aannulntion sites; di!luse epi:ayccardial bleeding
seen irr reoperatl.ons; arnd ooaing ,from Vesnous blend.inq
sites, e.g. at atxial, canal, or r~.ght ventriculax
levels. The se~.bjaot invention is also usotul for
so sealing of decree artery grafts prior t~ graleing, .
ssaliae~ tisauss outside the body, prodnc3.~ tibrit~
rafts fox ael,l Qrowth, stopping blending tree damaged
~cplaens (tlnersby saving the organ) , liVe~xs, and oeh~ar
parenchymatoua orgsaa; sealing tracheal and bronchia,
se ana,stwa°ces arid air lex~ks or lacara:.fons or tae lung,
scaling bronchial atumpa, hroachi~al viatulas at~d
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esophageal fistulas; for sutureless seamless healing
( "tipper" technique) , and emboliaation in vascular
radiology of intracerebral AVM's, liver AVM's:,
angiodysplasia of colon, esophageal vazices, ''pumping"
GI bleeders secondary to peptic ulcers, etc. The
sub~eat invention is further useful for providizig
hemostasis in corneal transplants, nosebleeds, pest
tonsillectomies, teeth extractions and other
applications. See G.F. Gestring and R. Leaner,
lo Vascular Surgery, 294-304, Sept. JOct. 1983.
The throiahin blood fraction of the subject
invention can be employed alone to staunch oozing
hemorrhages or hemorrhages in hollow organs . The
thrombin blood fraction can also be utilized in the
l5 treatzaent of damaged live animal tissue by utilizing
the fraction to activate the release of the materials ,
l. e. , plated-derived factors, from platelets, wherein
such materials can he utilized to heal damaged tissue.
See United States Patent No. 5,165, 938. The thrombin
2o blood fraction can also be utilized to assist in the
cell culture growth of keratinocytes and to assist in
the autologous transplantation of keratinecytes, or
any other skin-derived cells, e.g. , fibroblasts. See
V. Ronfard et al. , Burns 17:181-38~ (1991) ; H. Hro3y,
25 Canadian Patent Ho. 2, Oi8, 020 and S. Hunyadi et al. ,
J. Dera~atol . Surg. oncol . 1:'75-?8. ( 1988 ) .
Also, the thrombin blood fraction can be
placed on a solid support, e.g., bandage, suture,
prosthesis., or dressing, that wi:.l be in contact :rich
3o the desired site. Such support is then placed in
contact with the desired site Lntil, for example, ~he
fibrin clot Eorms.
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The dosage of the thrombin blood fraction
depends on its particular use, but the dosage show id
be an effective amount for the composition to perform
its intended use. Generally, it is believed that from
about 0.5 ml to about 5 ml of the thrombin blood
fraction is sufficient. However, depending an the
use, the dosage can range from about 0.05 ml to about
40 ml.
If the thrombin blood fraction is utilized
1o as a component of a f ibriw sealant, then the fibr in
sealant can be applied to the desired site ..with, for
example, a double-barrelled syringe. The
double-barrelled syringe can be Y-shaped, thereby
permitting the mixing of fibrinogen and the thrombin
blood fraction immediately prior to the contacting
step. Also, rather than a Y-shaped double-.barrelled
syringe a double-barrelled syringe with two openings
can be utilized. This permits the siaultaneous
contacting of the desired site. Also, the
zo compositions of the double-barrelled syringe can be
sprayed onto the desired site. See H.B. Kram et a l.,
The American Surgeon, 5:381 (1991) . Also, if the
blood fraction is employed as a component of a fibrin
sealant, then autologous fibrinogen can be utilized,
'thereby rendering the entire fibrin sealant
autologaus. Also, if the thrombin blood fraction is
employed alone, then the fractibn can be applied to
the desired site with a single-barrelled syringe.
It should also be noted that the thromb in
3o blood fraction of the subject invention can further
comprise a source of calcium ions, e.g., calcium
chloride. The source of calcium ions assists in the
conversion of fibrinogen to the fibrin c lot. The
amount of calcium ions should be the same as that
utilized in- conventional fibrin sealants . However ,
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_ 26 v
since the thrombin blood fraction may contain a source
of calcium ions already due to the corwersion of
prothrombin to thrombin, an additional source of
calcium ions may not be required. Hut, if more
calcium, is needed to form the fibrin clot than to form
thrombin, then, as an option, excess calcium froxo what
is required to form thrombin can be utilized so ti~at
no additional calcium need be added when the thrombin
blood fraction is utilized in the medical procedure,
1o e. g. , as a component of a f fibrin sealant:
5.
EXAMPLE I
Preparation of a Composition Containing a
=5 Thrombin Blood Fraction Obtained From 17 ml
of Fresh Blood From a Human Adult Donor
A puncture of the vein of a human was
performed by a needle and 17 ml cf blood was drawn
into an empty 30 ml syringe. Immediately after
20 :awing the blood, ft was transferred to a 50 ml test
tube containing 34 ml of a solution containing 5 . 5 %
glucose. The 50 ml test tube was placed in a
centrifuge and centrifuged for 5 minutes at 1, 500 x g
at room temperature: After centrifugation, 4o ml of
Z5 the supernatant plasma/glucose solution was removed by
a syrznge, and transferred to a new 50 m1 test tube.
By means of 1.07 ml of'a 2.8~ citric acid
solution, the pH in the plasma/glucosg solution was
lowered to 5.2 and after a period of 10 minutes at
30 room temperature, the solution was centrifuged at I8~C
at 1,500 x g for 5 minutes. After centrifugation, the
supernatant was drained off and the precipitate was
dissolved in 0.424 ml of a solution containing 14
mmole/L of NaHC03 and 8 gram/L of NaCl. This
precipitate contains fibrinogen, prothrombin and other
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blood proteins, but is substantially free of
antithrambin III. The pH of this dissolved
prothrombin containing euglobulin solution was 7.35.
Activation of the prathrombin was performed
s by the addition of o.027 ml of a solution containing
CaClz, 0.5 mole/L. From 12 to 17 minutes after the
addition of the CaClZ, the fibrinogen in the solution
started to coagulate, and the fibrin thus formed ~~was
removed by means of a polystyrene spatula. Small
to samples were removed at different intervals and the
thrombin concentration was measured. The results from
the example are shown in Table I.
Table I
15 '
time after 10 20 30 60 120 2 7
,
Ca-addition min min min min min hour
NIH u/ml 0 180 368 540 600 737
2a E~LE II
Preparation of a Composition Containing a
Thrombin Blood Fraction obtained From i7 m1
of Fresh Glass Activated Blood From a Human
Adult Donor
25 In this experiment, the donor and the day
for the performance was the same as used in Example I.
A puncture of the vein. of a human was performed by a
needle and 20 ml of blood was dratan into a 30 ml
syringe containing 20 grams of glass beads witt: a
3o diameter of approximately 2 mm: The total surface
area of the beads was approximately 23o cmz and,
therefore, the surface area was about 11.5 cm= per ml
of whole blood.
Immediately after drawing the blood, the
3.5 syringe was turned gently for 10 to 15 seconds before
17 ml of the blood was transferred to a 5 O ml test
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tube containing 34 ml of a solution containing 5. S%
glucose. The 50 ml test tube was placed in a
centrifuge and centrifuged for 5 minutes at 1, 500 x g
at room temperature. After centrifugation, 40 ml of
s the supernatant plasma/glucose solutiori was re~aoved by
a syringe, and transferred to another 50 ml test tube.
By means of 1.07 m1 of a 2.8% citric acid solution,
the pH was lowered to 5.2 and after a period of 10
minutes at room temperature, the solution Was
io centrifuged at 18~C at 1, 500 x g for 5 minutes.
After centrifugation the supernatant was
drained off and the precipitate was dissolved in 0.424
ml of a solution containing 14 mmole/L of NaFiC03 acrd 8
gram/L of NaCl. This precipitate contair~s
15 prothrombin, fibrinogen and other blood proteins, hut
is substantially free of antithrombin ==Z . The pH of
this dissolved prothrombin containing euglobulin
solution was 7.35.
Activation of the prothrombiri was performed
20 by the addition of a 0.027 ml of a solution containing
CaCh, 0.5 mole/L. From 4 to 9 minutes after the
addition of the CaCl2, the fibrinogen in the solution
started to coagulate, and the fibrin thus formed was
removed by means of a polystyrene spatula. Small
ZS samples were removed at different intervals and the
thrombin concentration was measured. The results are
shown in table II: .
Table II
30
time after 10 20 30 60 120 27
Ca--addition min min min min n~in hour
NIH u/ml 130 444 560 920 695 880
I
as Thus, from Table II it is readily apparent
that the glass activation accelerates the time
.
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required for the conversion of the prothrombin to
thrombin. For example, at only 10 minutes after the
addition of a source of calcium ions, 130 .NIH units/ml
of thrombin activity was measured. In contrast,
without glass activation, as in Example I, at 10
minutes after the addition of a source of calcium
ions, there was no detectable thrombin activity.
EXAMPLE III
i0 ' Preparation of a Composition Containing a
Thrombin Fraction Obtained From I7 m1 of
Fresh Blood From Human Adult Donors,
Characterized by Low Specific Thrombin
Activity
=n four experiments, performed as described
in Example I, the thrombin concentration and the
specific activity of thrombin were measured. The
results are given in Tables III and Iv where the pH
value is the pH in the dissolved euglobulin fractions.
Table III
A thrombin
measured
from
15 mire
to
2 hours
after
dissolution
~of the
euglobulin
fraction.
NIH u/ml
donor pH E-280 15 min 30 min 60 min 2' hr
JH 6.96 25.8 142 483 661 ??9
HJS 6.35- 26.7 0 56 942 I, 238
LK 6.98 19.5 30 249 430 562
KN 6.21 31.5 O 142 616 725
( E-280 is a measurement of the total protein
concentration in mg/ml).
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Table IV
8 specific
activity
of thrombin
iaeasured
from
15 min to
2 hours
after dissolution
of the
euglobulin
fraction.
NIH u/mg
protein
donor 15 min 30 min 6o min 2 hr
JH 5.5 19 26 30
HJS 0 2.1 35 ~ 46
LK 1.5 13 22 29
ZO ~I 0 4.5 20 23
Thus, the preparation of a thra~nbin blood
fraction as described in Examtle ~ results in a
thrombin blood fraction of a concentration and
specif is activity of .the subj ect invention .
EX~.MPLE IV
Preparation of Thrombin From WYiole Blood
Using the Device of W093./I7778
The device described in. W091/17778 was
used for the preparation of thrombin. Before the
blood was introduced into the device, 40 z~l of a 5. S~
glucose solution was introduced into the f first chamber
(14) through the filter (43) counted on the tubing
(39) .
Blood, l0 gal; was collected from human
donors into a syringe and immediately the=softer
transferred through the tubing ( 3 9 ) into the glucose .
solution. The device was placed in a cen-tr iEuge and
centrifuged for 5 min at 1, 500 x g. after
centrifugation, the plasma/glucose solution was
transferred into the second chamber (30) wlt:~ tie red
cells remaining in the chamber ( 14 ) . Thr ough the
sterile filter in tube (60) 0.6 ml of 2.8~ citric acid
3~
solution was introduced into the' plastra/g lucose
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solution. After 5 to 10 min, the device was planed in
a centrifuge and centrifuged sor 5 min at 1,500 x g.
After centrifugation the supernatant was tzansferred
to the first chamber ( 14 ) and the precipitate, the
euglabulin fraction, remained in the second chamber
(30). Through the sterile filter in tube (60) 4.85 ml
of a solution containing 7.5 mM NaliC03, 52 mM NaCI and
3 0 mM CaCl2 was introduced into the second chamber
( 30) . The euglobulin precipitate was dissolved within
:4 1-2 minutes, and transferred to the syringe (51)
connected tc the second chamber ( 30 ) . The syringe
contained a polyurethane sponge facilitating the
removal of the formed fibri:~. Thrombin concentrations
were measured after 30 min to 22 hours. The results
:5 are recited ~in Table' V.
Table V
.- ~ thrombin
concentration
HIH.
a/ml
2 Donor plasma 3 o min 1 hour 2 hour 22 hour
o dilution.
.
RFi-A I1.9~ 186 240 231 279
RH-B 12.3% 54 130 130 ~ I32
~
RH-C 11.7% 178 ~ 180 192
186
25 ~-0 12.5% I 104 - 120 92 123
~
EXAMPLE V
Preparation of Th~o:abin and Fibrinogen FZ'C.T.
Whole Blood Using a Device System riade F~ or,
Two Inter-Connected Devices of
30 W091/17778, and the Use of Thrombin and
Fibrinogen in a Fibrin Glue
The device syster: consists of two devices as
described in W091/17778. The Two tubings (39?
35 from the tuo devices were connected to the same
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- 32 -
cannula (40) by means of a three-way connector. In
the device used for the thrombin precipitation 5 glass
beads, 3 mm in diameter, were placed in the second
chamber (30) . The syringe in the thrombin device was
s changed from being a 3 ml syringe in the fibrinogen
device to a 1 ml syringe. '
Before the blood was collected from the .
donor, citrate and glucose solutions were filled into
the two devices . Citrate, 5 ml of a 3 . 8 % solution ,
i0 was, introduced into the first chamber ( 14 ) of the
fibrinogen device (hereafter named Device-F~ through
the filter (43) mounted on the tubing ( 39 ) . Glucose,
34 ml of a 5 .5% solution was introduced into the f first
chamber ( 14 ) of the thrombin device ( hereafter nam8d
15 Device-T) through the filter ( 4 3 ) mounted an the
tubing (39).
Blood, 45 ml was collected through the
cannula into the f first chamber ( 14 ) in Device-F
containing the citrate solution, and 17 ml Was
2 O collected into the first chamber ( 14 ) in the Devi ce-T
containing the glucose solution. After collection of
the blood, the separator-system Was disconnected from
the donor, and the tubing ( 3 9 ) was sealed close ~to
inlet (38) . Both devices were placed in a centrifuge
25 and centrifuged fcr IO min at 1,500 x g.
The separated plasma in Device-F was
transf erred to the second chamber . ( 3 0 ) and z . 5 ml o f a
96% ethanol solution was introduced into the second
chamber (30) through the sterile filter in tube ( 60 ) .
30 the device was new placed into a ice-water bath far 2o
minutes to reduce the temperature in the
plasma/ethanol solution to approximately O to 4°C. At
this temperature, 85% of the fibrinogen in plasma Was
precipitated. The device was now placed in a
35 centrifuge and centrifuged for 5 min at 1 , 500 x g.
a 7
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The supernatant serum was transferred to the first
chamber (IA) , and the solid fibrinogen was dissolved
by incubation f or 5 minutes at 3 7 ° C . The dissolved
solution was transferred to the sterile syringe ( 51) .
s The concentration of fibrinogen was measured to be 31
mg/ml.
The separated plasma/glucose in Device-T was
transferred into the second chamber (30 ) with the red
cells remaining in the first chamber ( 14 ) . Through
the sterile filter in tube (60) 1.2 ml of a 2.8%
citric acid solution was introduced into the
plasma/glueose solution. After 5 tv 10 min the device
was placed in a centrifuge and centrifuged fox 5 min
at 1, 500 x g. After centrifugation, the supernatant
s5 was transferred to the first chamber (1 4) and the
precipitate, the euglobulin fraction, remained in the
second chamber (30) . Through the sterile filter in
the tube (60) , 085 ml of a solution containing 7. 5 mM
NaHCO3, 52 mM NaCi and 30 mM CaCl2 was introduced into
2 o the second chamber ( 3 0 ) . The euglobulin precipitate
.was dissolved within 1-2 minutes, and the fibrin
formed during the activation of prothrombinto
thrombin was collected onto 5 glass beads planed in
the second chamber ( 3 0 ) . After 15 minus es the
25 thrombin solution was transferred to the syringe ( 51)
connected to the second chamber (30): Thrombin
concentration was measured to be 248-340-372 NIH u/ml
after 15-30-so minutes, respectively.
The two syringes containing the f fibrinogen
30 and the thrombin were used as a double barrelled
syringe. The two solutions were expelled from the
syringes and formed Immediately a firm (fibrin clot.
CA 02367124 2002-O1-25
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34 _
EXAMPhE VI
54 m1 of 0. 04% HAc were added to 6 ml of
plasma. This mixture was placed in a flat-bottomed
container of the type known from the above
PCT/DK91/o0131. The container is of a circular cross
section with an inner diameter of 4:5 cm. The
pH-value was 5.3. The pH-value and the relatively low
concentration of ions in the provided mixture ensure
the following precipitation of the coagulation
to f actors, inter alia prothrombin, as a precipitate by a
centrifuging. The centrifuging was performed at 1, 500
g fvr 5 min. Thus the centrifuging was relatively
quickly terminated, which is due to the relatively
short falling height and large precipitation surface.
After removal of excess fluid, an 0.?5 ml aqueous
solution of 0.9% NaCl, 0.03% Na.:C03 and 25 mM CaCl= was
added to the precipitate. After dissolving of the
precipitate, the solution was sucked into a Z.5 lal
syringe containing a polyurethane sponge. The
2o formation of fibrin dil not start until about 1 to 2
min. after the precipitate had become completely
dissolved, and accordingly more than enough time f or
the sucking procedure. After termination of the
formation of fibrin in the syringe, the: thrombin
solution could be expelled by squeezing the sponge by
means of the piston of the syringe, the fibrin
remaining depositing on the large surface of the
sponge.
The dissolved precipitate had a pH-value of
6.5. Other amounts of Na,C~3 or another base or a
buffer system can be used provided the pH-value is
between 6: o and ?.5, but an optimum balance between
the keeping qualities of the thrombin and the capacity
of the thrombin to accelerate the coagulation process
is found at 6.5.
CA 02367124 2002-O1-25
w0 9x/00566 PCTlCB93/Ot323
-~ 3 5 -
The thrombin was expelled from the syringe
after 30 min, and a concentration of 256 NIH units psr
ml was obtained: The thrombin concentration increased
by time, but after 30 to 60 min a sufficient amount og
thrombin was obtained for a conventional use in a
Fibrin sealaht.
The preparation of thrombin was in the
present Example produced from 12 ml of autalogous
blood and was terminated over a period of 45 to 60
to min, i:e., almost simultaneously with the termination
of the preparation of fibrinogen. The produced axuount
of thrombin was sufficient for being used in
combination with fibrinogen produced from 45 ~n1 of
autologous blood in the manner described in
W091/1???8.
The fibrin formed during the thrombin
preparation has always a tendency to bind the
thrombin. The release of this thrombin can, however,
be promoted by the addition og a plasminogen catalyst,
2 o such as streptakinase , urokinase or t-PA ( tissue
plasminogen catalyst) optionally adaixed with a
physiological solution.
30
