Note: Descriptions are shown in the official language in which they were submitted.
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WO 94/00566 PCT/GB93/01323
A THROMBIN BLOOD FRACTION FOR USE IN A MEDICAL PROCEDURE.
1. FIELb OF TAE xNVEN,~,~ON
The subject invention relates to the use of
thrombin in a medical procedure in an animal. More
srecif ically, the subject invention relates to such
utie of thrombin wherein the thrombin is a thrombin
blood fraction, as defined hereinbelow.
2. B_ACICGROUND OF THE INVENTION
One mechar. ~m for hemostasis, i.e.,
prevention of blood loss, of an aniatal is the
formation of a blood clot. Clot formation, i.e.,
blood coagulation, occurs by means of a complex
cascade of reactions with the final steps being the
conversion of fibrinogen by throabin, 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. Jackson, Arin. Rev.
Biochem., 49:765-811 (1980) and. B. Furie and B.C.
Furie, Cell, 53:505-518 (1988).
Thrombin, which is a proteolytic enzyme, is
derived from prothrombin. Prothrambin is converted to
thrombin by calcium arid prothrombinase.
Prathrombinase is formed through a cascade of
reactions that begins with the proteins factor XI and
factor XII.
A fibrin sealant is a biological adhesive
80 whose effect imitates the final stages of coagulation,
thereby resulting in a fibrin clot. Conventional
fibrin sealants generally consist of concentrated
human fibrinogen, bovine aprotinin and factor XIII, as
the first component and bovine thrombin and calcium
s5 chloride as the second component. Application is
generally carried out with a double-barrelled syringe,
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which permits simultaneous application of both
components to the site where one wants to form the
fibrin clot. Aprotinin is a t:ibrinolytic inhibitor
added to promote stability of fibrin sealants.
The fibrinogen component of the Fibrin
sealant is prepared from pooled human plasma. The '
fibrinogen can be concentrated from the human plasma
~by cryaprecipitation and precipitation using various
reagents, a.g., polyethylene glycol, ether, ethanol,
io ammonium sulfate or glycine. For an excellent review.
of ffibrin sealants, sae M. Brennan, Blood Reviews,
5:240-244 (1991); J.W. Gibble and P.N. Ness,
Transfusion, 30:741-?4? (1990); H. Matras, J. Oral
Maxillofac Surg., 43:605-611 (1985) and R. Lerner and
N. $inur, J. of Surgical Rese3,rCh, 48:165-181 (1990).
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
2o fibrin sealant is extracted from the patients own
blood. The use of an autologous fibrin sealant is
preferred because it eliminates the risk of
transmission of blood-transmitted infections, e.g.,
hepatitis B, non A, non B 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, 99:974-976
30 (1989) and A. Dresdale et al., The Annals of Thoracic
Surgery, 40:385-387 (1985).
An infection can be transmitted by a fibrin
sealant not only by oceans of the fibrinogen but also
by means of the bovine aprotini.n and the bovine
35 thrombin component. Bovine thrombin has been known to
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carry the infectious agent bovine spongiform encephalitis
(BSFs) and other viruses pathogenic to mammals.
F~rthertnore, bovine thrombin is a potential antigen,
which can cause immunvlogical reactions in humans. Thus,
the use of bovine thrombin could reswlt in the recipient
of the bovine thrombin being adversely affected. See
D_M. Taylor, J. of Hospital Infection, 18(Supplement
A):141-146 (1991), S.B. Prusiner et al., Cornell Vet, 81
No. z: 85-96 (1991) and D. Matthews, J. Roy. Soc. Health, CIitGI)~
3 - 5 ( February 19 91 ) .
Accordingly, there is the need for a sealant
that utilizes thrombin that can be delivered to a patient
without the risk of viral contaminatiozi yr other adverse
affects.
20
3. SUN>rlARY OF THE INVENTION
The subject invention relates to a composition
for use in a medical procedure in an animal, which
composition is a thrombin containing blood fraction which
comprises:
(a) a thrombin concentration of frcm about 1
NIH unit/ml to about 2;, 000 NIFi units/ml,
and
(b) a specific activity of thrombin of from
~''~ 25 about 1 NIH unit/mg of blood protein to
about 200 NIH 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
30 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 whale
blood;
(c) lowering the pZi of said plasma to
precipitate a prothrombin blood
fraction; and
(d) separating said prothrombin blood
l0 fraction from said plasma.
The prothrombin bland fraction is then
redissolved and converted to thrombin, i.e., the
thrombin blood fraction is farmed, which can then be
utilized in a medical procedure in an animal, e.g., as
a component of a fibrin sealant.
4. DETAILED DESCRI TION OF THE INVENTION
The subject invention relates to the use of
a thrombin blood fraction, as defined hereinbelow, in
20 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, or other mammals that have an adequate blood
volume to prepa=e 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 and rapidly,
e.g., in less than about one or two hours, and is
30 believed to be as efficacious as a highly pure
thrombin preparation.
The thrombin blood fraction can be prepared
from whole blood. It is preferred that the whole
blood be obtained from a single individual animal.
'35 Also, it is pref erred that the thrombin blood fraction
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WO 94/00566 PCTlGB93/OI323
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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 thrombin
blood fraction in a medical procedure. rn 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
1o 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
is small volumes of whole blood are required, especially
since it is also preferred to prepare a thrombin blood
fraction for a single use. Also, it is preferred to
prepare the fraction within several hours of the time
of use. It is preferred that ~:rom about 10 ml to
2o about SO ml, more preferably from about 10 ml to about
30 ml and most preferably from about l0 ml to about 20
ml of whole blood be utilized to prepare the thrombin
blood fraction of the subject invention.
The thrombin blood fraction of the subject
25 invention has a thrombin concentration of from about 1
NIH unit to about 2,000 NIIi units, preferably from
.. about 100 NIH units to about 800 NIH units and most
preferably from about 100 NIH units to about 500 NIH
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
35 fraction.
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The thrombin concentration of the thrombin
blood fraction can be determined by measuring the
coagulation time of a standard fibrinogen solution
of ter addition of the thrombin blood fraction in a
5 suitable diluted form. As a reference, standard
thrombin solutions, containing from 2 to 15 NIH
units/ml, can be utilised.
The thrombin blood fraction of the subject
invention has a specific activity of thrombin 4f from
1D 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. Sucb lower
specific activities of the thrombin blood fraction are
~5 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
20 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 subject
invention is quite low relative to thrombin
25 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,
United States Patent Na. 5,15x.,355 discloses a
30 thrombin preparation with a specific activity of
greater than 1,000 NIH units/mg. of total protein.
Although the specific aCtivit~~ of the thrombin blood
fraction of the subject is low, never the less it is
believed that the blood fraction is efficacious for
35 use in medical procedures.
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WO 94100566 PCT/GB93/01323
The specific activity of the thrombin blood
fraction of the subject invention can be calculated by
measuring the thrombin concentration (NIH units/ml)
and by dividing that number by the protein
concentration (mgjml) measured by any standard protein
assay, e.g., W absorbance.
The thrombin blood fraction is also
substantially free of active antithrombin ILI. For
the purpose of the subject invention, "substantially
1o free of active antithrombin III" means that the
thrombin blood fraction per unit volume contains an
amount of active antithrombin III that is less than
about SO% by activity of antithrombin III in normal
plasma per unit volume. It is preferred that such
percentage be less than about 30%, more preferably
less than about 10% and most preferably less than
about 5%. It is essential that the antithromhin III
either be removed from or in-~tivatad in the thrombin
blood fraction. Otherwise, antithrombin III will
prevent the conversion of the ps:athrombin in the blood
fraction to thrombin and/or inactivate the thrombin
that is farmed.
It is preferred that t:he thrombin blood
fraction also be substantially free of fibrinogen and
fibrin. For the purpose of the subject invention,
substantially free of fibrinogen and fibrin means that
-. the thrombin blood fraction per unit volume contains
an amount of fibrinogen plus fibrin that is less than
about 1% by weight of fibrinogen in normal plasma per
3o unit volume. It is preferred 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 biaod fraction impracticable. The fibrinogen
itself pan be removed from the thrombin blood fraction
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or the fibrinogen can be removed as the fibrin clot,
thereby, of course, also removing the fibrin.
However, the fibrinogen need not be removed if, for
example, the thrombin is inactivated as described in
PCT publication No. W091j09641.
The thrombin blood Fraction can be utilized
immediately after it is prepared. If the fraction is
not utilized iiamediately after its preparation, the
fraction can be stored. Storage of the fraction
io 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 farm will be stable f or a period
of months. When the fraction is held at 4°C, it is
~5 stable for at least a period of days.
If the fraction is frozen, the fraction must
be thawed at the time of use. tf 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, far example, a solution,
suspension, emulsion or solid, with a solution being
preferred. Thus, for example, such fraction can be a
25 liquid, gel, paste or salve. Also, of course, the
fraction can be in the form of a. granule.
If the thrombin blood ;raction is in a solid
form, then the concentration of the thrombir. blood
fraction can be deterained by dissolving it in a
30 solution and then measuring the thrombin
concentration. If the res~:lting thro:~bi~
concentration is from about 1 NIH unitJ:~l to about
2,000 NIH units/ml, then the thrombin blood fraction
is within the scope of the subject invention.
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4.1. METHOD FOR PREPA,R.ATION OF THE
T~iROMHIN.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. W091/17778.
4.1.1. METHOD FOR PREPARATION OF THE THROMBIN
BLOOD FRACTION BY FORMATION OF THE
EUGLOB IN FRACTION FROM PLASMA
Whale blood can be withdrawn from an
individual animal, e.g.,.a hunan, and preferably in
the presence of an anticoagulant. Any anticoagulant
:5 can be utili2ed 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 formatian~of
thrombin, with citrate being preferred.
=o The plasma, which contains the prothrombin,
is then separated from the whole blood. Any
separation technique can be utilized, for example,
sedimentation, centrifugation or filtration.
Centrifugation can be carried out at about 1,500 to
25 about 3,000 g. far about 10 r~inutas. The supernatant,
which contains the plasma, can be re~aoved 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. for about 20
30 minutes er l,OdO g. for about.2 to 3 ainutes. The
thrombin blood traction of the subject invention will
then contain groc.2h Factors, which are released from
the platelets during the conversion o~ prothrombin to
thrombin.
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The plasma 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,
5 preferably less than about 50 millimolar and :host
preferably from about 20 to about 40 millimolar 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
10 the plasma: distilled water or acid of from about 1:5
to about 1:50, with 1:10 being pref erred.
This treatment, i.e., the lowering of the
ionic strength and pH of the plasma, results in the
formation of a precipitate that is generally referred
L5 to as the °euglobulin fraction." The euglobulin
fraction captains the prothron~bin, 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 the plasma, the
ionic strength can be lowered by dialyzing the plasma
by placing the plasma in a dialysis bag, Which is then
placed in distilled water. The dialysis permits the
ions to diffuse out of the plasma, thereby lowering
25 the ionic strength. A suitable dialysis bag is
composed of cellulose nitrate. Also, the ionic
- strength can be lowered by diafiltration or occlusion
chromatography.
The euglobulin fraction can be prepared as
3o described in A. Quick, Production of Thrombin From
Precipitate Obtained by Acidification of Diluted
Plasma, Am. J. Phys3.ol., ,~,:i14-118 (1955) and R.
Biggs and R.G. Macfarlane, Human Blood Coagulation,
pages 3?5-376, Blackwells Scientif is Publications,
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Oxford, 3rd Edition (1962).
The excess fluid can then be separated from
the euglobulin fraction by, for example,
centrifugation, filtration or sedimentation.
Centrifugation can be carried at about 1,500 g. for
about 2 to about 5 minutes.
The prothrombin of the euglobulin fraction
is then redissolved and converted to thrombin, thereby
to 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 pref erably less than ( about 10% ) the original
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.
Nonlimiting examples of suitable alkaline buffers
include sodium hydroxide, potassium hydroxide, calcium
2o hydroxide, bicarbonate buff ers such as sodium
bicarbonate and potassium bicarbonate, salts of acetic
acid and salts of sulfuric acid. Preferred alkaline
buffers include: Sodium carbonatejbicarbonate pH 7.0,
Sodium bicarbonate/NaOH pH 7.0, 1.5M Glycine/NaOH pH
6.5-?.5, Bis hydroxyethylaminaethane sulphonic acid
fBES) pH 7.5, Hydroxyethylpiperazine propane sulphonic
acid (EPPS) pH 7.5, Tricine pH 7.5, Morpholino propane
sulphonic acid (MOPS) pH 7.0, Trishydroxymethyl
aminoethane sulphonic acid (TES) pH 7.o and
3o Cyclohexylaminoethane sulphonic acid (CHES) pH ~.0;
with Sodium carbonate/bicarbonate pH 6.5 - 7.5 Bis
hydroxethylaminoethane sulphonic acid (BES) pH ~.5,
Fiydroxyethylpigerazine propane sulphonic acid fEPPS)
pH 7.5 and Trishydroxymethyl aminoethane sulphonic
acid (TES) pH 7.5 being most preferred.
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CVO 94/00566 PCTlGB93l0I323
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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, far
S 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 5O
millimolar calcium chloride ib sufficient.
Also, rather than adding a source of calcium
ions, prothrombin activating enzymes from snake venams
can be utilized. For example, snake venom-grom Eccis
carinatus or the Australian Tiger snake can be
utilized.
As the thrombin forms, it converts the
2o 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 collecting the fibrin onto
glass beads.
4.1.2. METHOD FOR PREPARATION OF THROMBIN BLOOD
FR~C'~,ION BY DILUTIhIG WHOLE BLOOD
In a preferred embodiment, the thrombin
blood fraction can be prepared by initially preparing
3o a Prothrombin blood fraction from whole blood
comprising:
(a) diluting whole bland to an ionic
strength of less than about iQQ
millimolar;
3s (b) separating plasma from said whole
blood;
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13
(c) lowering the pH of said plasma to
precipitate a prothrombin blood
fraction; and
(d) separating said prathrombin blood
fraction from said plasma.
The prathrombin blood fraction is then
redissolved and converted to thrombin, i.e., the
thrombin blood Fraction of the subject invention is
formed, which can then be utilized in a medical
lo pracedure in an animal, e.g., as a component of a
fibrin sealant. This method provides a thrombin blood
fraction of the subject invention, which can be
prepared in anly about 45 minutes:
specifically, whole 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 milliaiolar, preferably less than about 50
millimolar and preferably to from about 20 to about 40
2o millimolar. It should be noted that since the whole
blood is diluted immediately, there is no need for the
use of an anticoagulant. Any physiologically
acceptable solution at physiological osmotic 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.
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 fractivn~ is then acidif led,
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
5 should be lowered to from about 4.5 to about 6 and
preferably 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.
1o Centrifugation can be carried out at about 1,500 g fvr
about 2 to about 5 minutes. This prothrombin blood
fraction contains prothrombin, fibrinogen ahd many
other blood proteins, but does not contain
antithrombin III. The prothrombin blood fraction is
15 then redissolved and the prothrombin is converted to
thrombin. Any physiologically acceptable solution,
e.g., saline, can be utilized to redissolve the
prothrombin fraction. Furthermore, only a small
volume of solution is required to redissolve the
2o 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
ZS preferably to about b.5 to about 7.5. Nonlimiting
examples of suitable alkaline buffers include sodium
hydroxide, potassium hydroxide, calcium hydroxide,
bicarbonate buffers such as sodium bicarbonate and
potassium bicarbonate, tri-metal salts of citric acid,
30 salts of acetic acid and salts of sulfuric acid.
Preferred alkaline buffers include: Sodium
carbanate/bicarbonate pH 5-8, Sodium bicarbonate/NaOH
pH 6-8, Glycine/NaOH pH 6-8, Bis
hydroxyethylaminoethane sulphanic acid (BES) pH 6-8,
35 Hydraxyethylpiperazine propane sulphonic acid (EPPS)
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pH 6-8, Tricine pH 6-e, Morpholino propane sulphonic
acid (MAPS) pH 6-8, Trishydroxymethyl aminoethane
sulphonic acid (TES) pH 6-8 and Cyclohexylaminoethane
sulphonic acid (CHES) pH 6-e; with Sodium
5 carbonatejbicarbonate pH 6-8 B:~s
hydroxethylaminoethane sulphonic acid (BES) pH 6-8,
Hydroxyethylpiperazine propane sulphonic acid (EPPS)
pH 6-8 and Trishydroxymethyl aminoethane sulphonic
acid (TES) pH 6-8 being most preferred.
10 Calcium is added to the neutral solution in
order to convert the prothrambin to thrombin. Of
course the calcium can be part of the alkaline buffer.
Calcium can be added in the form of farm of, for
example, calcium chloride. The amount of calcium
t5 added should be sufficient to convert an amount of
prothrambin to thrombin that is sufficient for the
intended medical use. Furthermore, the reaction
should be permitted to occur for a period of time
sufficient to convert enough of the prothrornbin to
20 thrombin that is sufficient f nr the intended medical
use. Generally, from about 5 millimolar to about 50
millimolar calcium chloride is sufficient. It has
been observed that at about 25 minutes of react-ion
sufficient amounts of thrombin are formed for most
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 venoms
can be utilized . For example, snake venom from Eccis
30 carinatus or the Australian Tiger snake can be
utilized.
As the thrombin forms, it converts the
fibrinogen to fibrin,.which forms a fibrin clot. It
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 THROMBIN BLOOD
FRACTION BY REMOVING OR INACTIVATING
ANTITHROMBIN III FROM hLASMA
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
10 anticoagulant. 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 citrate being
preferred.
15 The lasma, which contains the
p prothrombin,
is then separated from the whole blood. Any
separation technique can be utilized, for example,
sedimentation, centrifugation or filtration.
Centrifugation can be carried out at about 3,000 g.
20 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 antithrombin III; otherwise
antithromhin III would prevent 'the conversion of
prothrombin to thrombin. Antithrombin III can be
inactivated by any technique. :for example, by the
addition of 0.05 m1 of 5 mol./liter FiCl per l.0 ml
30 plasma. After about 10 to about 20 minutes of
incubation, the plasma can be neutralized with 0.05 ml
of 5 mol./liter NaOfi 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 III.
The plasma fraction, which contains
prothrombin, is then treated to convert prothrombin to
thrombin. This can be carried out by, for 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.Jliter per ml of plasma. Also, rather than adding
a source of calcium ions, prothrombin activating
to enzymes froze snake venoms can be utilized. For
example, snake venom from Ecci~~ carinatus or the
Australian Tiger snake.
As the thrombin f arms, 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 collection of the fibrin onto
glass beads.
The resulting plasma is a thrombin blood
2o fraction of the subject invention.
4.1.4. METHOD FOR THE PREPAFL~1TION OF A
TXROMBIN BLOOD FRACTION BY DILUTING
WITH AN ACID SOLUTION DIRECTLY
8y the known process for preparing thrombin
f ram plasma, plasma is diluted in the ratio 1:10 with
water, whereafter a pH-reducing acid is added, such as
acetic acid, with the result that 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
3o 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, whereagter a pH-value-increasing
ss agent, such as sodium carbonate, is added until the
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pH-value is about 7.9. When the precipitate has been
dissolved, calcium chloride is added and pauses the
conversion of the prothrombin into thrombin by a
conventional, so-called coagulation cascade. The
5 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 subject invention is
to that the diluting step -is performed directly with a
diluted acid solution, e.g., 0.4% 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 far increasing
15 pH, and the calcium chloride being added at the same
time to the precipitate as a m:'txture, whereby said
precipitate is dissolved and then coagulates while
forming fibrin, and by the thrombin then being
removed.
2o 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 performed in a
container with a relatively las:ge flat precipitation
25 surface. A quick and complete dissolution is thereby
ensured of the precipitate containing prothrombin and
w fibrinogen. The complete dissolution is important
before the presence of calcium chloride causes the
conversion of prcthrombin to thrombin. A too early
3o thrombin formation leads to fibrin formation and the
ffibrin will bind the added f lurid, whereby the
dissolution of the precipitate stops and cannot
continue until said fluid has again been passed out of
the fibrin. The relatively small precipitate with a
35 relatively large surface area relative to the amount
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causes a complete dissolving of said precipitate by
addition of the combined mixture of physiological
solution, with the agent for increasing pFi, and
calcium chloride before the thrombin has been formed
through said coagulation cascade and starts the
formation of fibrin.
Moreover according to the invention, the
mixture may be admixed with a plasminogen catalyst,
such as streptokinase, before being added to the
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 advantageously be set to
increase the pH-value to 6.5, whereby the resulting
active thrombin is found to obtain the best keeping
qualities as the thrombin has a tendency to become
inactive on standing, which is usually the case in
connection with enzymes. In addition, the dissolved
precipitate can he transferred to a flexible material
before the coagulation starts, said flexible material
presenting a relatively large surface upon which the
fibrin resulting from the coagulation can be
deposited, whereafter the thrombin may be pressed out
of the fibrin by the flexible material being subjected
to a compaction. Exactly the obtained complete
dissolving of the precipitate prior to formation 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
3o that the fibrin is deposited across a particularly
large surface, the compaction of which facilitates the
separation of thrombin from fibrin.
It is particularly preferred when the
flexible material is a sponge with open pores.
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The flexible material can be placed in 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 tv
the invention suitably be performed at about 1,500 g
for about 5 min; which also accelerates the preparing
to of thrombin.
Thus, the subject invention comprises a
process f or preparing thrombin from human blood
plasma, whereby the blood plasma is diluted to about
20 to 17%.with water or another ion-intensity-reducing
is fluid and an acid to reduce pH to about 5.0 to 5.3,
whereafter the iaixture is centrifuged, and whereby the
precipitate resulting from the centrifuging is admixed
a physiological solution and ca.lciura chloride, said
physiological solution increasing pH to about 6 to 8,
20 characterized by the diluting step being performed
directly caith a diluted acid solution, by the
centrifuging step being performed in a container with
a relatively large plane precipitation surface, by the
physiological solution, with the agent for increasing
25 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 ffibrin, and by the thrombin then being removed
from the ffibrin.
3d
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4.2. ACCELERATION OF THROMBIN FORMATION
BY CONTACTING BLOOD OR PLASMA WITH
A SU1~FACE THAT ACTIVATES BLOOD
COAGULATION FACTORS XI AND XII
Another aspect of the subject invention is
the preparation of a thrombin blood fraction wherein
whole blood or plasma is contacted with 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 preferred, can
10 provide such activation. Nonlimiting examples of
glass surfaces are glass beads, glass wool, glass
filters and glass capillary tubes.
The whole blood or plasma should contact the
surface fvr a period of time sufficient to activate
15 such blood coagulation factor.., e.g., far about 5 to
about 10 minutes. If the whale blood or plasma has
not been treated with an a:....ic:oagulan~, then the whole
blood or plasma should be in contact with such a
surface for not more than about 60 seconds, preferably
20 less than about 30 seconds and more preferably about
seconds. Without the anticoagulant, and if the
contact of the whole blood ar plasma crith the surface
is too long, then fibrin ciats will fore prematurely.
It is preferred that plasma be exposed to a
surface of about 4 cm2 to about 60 cm=, preferably from
about 10 cm to about 30 cmZ and most preferably about
20 cmz of such surface f or each milliliter of plasma.
Also, it is preferred that whale blood be exposed to a
surface of from about 2 cm= to about 3o cm', preferably
3o from about 5 cm= to about 15 ctr~2 and most preferably
about 10 cm' of such surface for each milliliter of
whole blood.
It is believed that the contacting of the
plasma or whale blood with a surface that can activate
factors XI and XII,accelerates the conversion time of
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prothrombin to thrombin. Thus, a thrombin blood
fraction can be prepared in an extremely short period
of time.
This activation of factors XI and XII can be
utilized to accelerate the preparation of any thrombin
blood fraction ,regardless of how the fraction is made
and regardless of its purity and specific activity,
However, it is essential that the thrombin blood
fraction be substantially 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 prothroiabin to
thrombin. Otherwise, antithrombin III will prevent
the conversion of the prothrombin to thrombin andjor
Z5 inactivate the thrombin. It is also preferred that
the thrombin blood fraction be substantially free of
fibrinogen and fibrin. Far example, with respect to
the activation when the thrombin blood fraction is
prepared by means of the euglobulin fraction as
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
i5 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,
the whole blood is discharged from the syringe and the
thrombin blood fraction of the subject invention is
then prepared, as described above.
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_ 23 _
4.3. THE USES OF TfiE THROMBIN BLOOD
FRACTIOft OF TH~ SUBJECT INVE~1TION
The thrombin blood fraction of the subject
invention can be utilized in any medical procedure,
known or to be developed, in an animal, including
vetinary praceduras. Any species of animal is
suitable, but, of course, humans are preferzed.
The thrombin blood fraction can be employed
as a component of a fibrin sealant or can be employed
1Q alone just as conventional thrombin preparations have
been eiaployed. The thrombin blood fraction is
utilized by contacting the desired site of the animal
with the thrombin blood fraction. Far the purpose of
the subject invention, the "desired site" is that
location in or an an animal where one desires to form
a fibrin clot. What or where the desired site is
depends on the use of the thrombin blood fraction of
the subject invention.
The use of the thrombin blood fraction as a
2p component of a fibrin sealant, can be utilized for
connecting tissues or organs, stopping bleeding,
healing wounds, sealing a surgical wound, use in
vascular surgery include providing hemostasis for
stitch hole bleeding of distal coronary. artery
anastomoses; left ventricular suture lines; aortotomy
and cannulation sites; diffuse epimyocardial bleeding
seen in reoperations; and oozing,from venous bleeding
sites, e.g. at atrial, caval, or right ventricular
levels. The subject invention is also useful for
sealing of dacran artery grafts prior to grafting,
sealing tissues outside the body, producing fibrin
rafts for cell growth, stopping bleeding from damaged
spleens (thereby savinq the organ), livers, and other
parenchymatous organs; sealing tracheal and bronchial
ss anastamoses and air leaks or lacera-ions of the lung,
sealing bronchial stumps, bronchial fistulas and
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esophageal fistulas; for sutureless seamless healing
("Zipper~' technique), and embalization in vascular
radiology of intracerebral AVM's, liver AVM's,
angiadysplasia of colon, esophageal varices, "pumping"
GI bleeders~secondary to peptic ulcers, etc. The
subject invention is further useful f or providing
hemostasis in corneal transplants, nosebleeds, post
tonsillectoaties, teeth extractions and other
applications. See G.F. Gestring and R. Leaner,
:0 Vascular Surgery, 294-304, Sept./Oct. 1983.
The thrombin 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
treatment of damaged live animal tissue by utilizing
the fraction to activate the release of the materials,
i.e., plated-derived factors, from platelets, wherein
such materials can be utilized to heal damaged tissue.
See United States Patent No. 5,155,938. The thrombin
2a blood fraction can also be utilized to assist in the
cell culture growth of keratinocytes and to assist in
the autologous transplantation of keratinocytes, or
any other skin-derived cells, e.g., fibroblasts. See
V. Ronfard et al., Burns 17:183-184 (1991); H. Hroly,
Canadian Patent No. 2,018,020 and J. Hunyadi et al.,
J. Dermatol. Surg. Oncol. 1:75-?8.(1988).
Also, the thro~ubin blood Fraction can be
placed on a solid support, e.g., bandage, suture,
prosthesis., or dressing, that will be in contact with
3o the desired site. Such support is then placed ir,
contact with the desired site until, Eor example, ~he
fibrin clot corms.
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The dosage of the thrombin blood fraction
depends an its particular use, but the dosage should
be an effective amount for the composition to perform
its intended use. Generally, it is believed that from
5 about 0.5 ml to about 5 ml of the thrombin blood
fraction is sufficient. However, depending on 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 fibrin sealant, then the fibrin
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
15 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 simultaneous
contacting of the desired site.. Also, the
2o compositions of the double-barrelled syringe can be
sprayed onto the desired site. See H.B. Kram et al.,
The American Surgeon, 57:381 (1991). Also, if the
blood fraction is employed as a component of a fibrin
sealant, then autologous fibrinogen caz be utilized,
25 thereby rendering the entire fibrin sealant
autologous. Also, if the thrombin blood fraction is
.. employed alone, then the fraction can be applied to
the desired site with a single-barrelled syringe.
It should also be noted that the thrombin
30 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 clot. The
amount of calcium ions should be the same as that
35 utilized in conventional fibrin sealants. However,
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26
since the thrombin blood fraction may contain a source
of calcium ions already due to the conversion of
prothrombin to thrombin, an additional source of
calcium ions may not be required. But, if more
calcium, is needed to form the fibrin clot than to form
thrombin, then, as an option, excess calcium from what
is requixed to form thrombin can be utilized so that
no additional calcium need be added when the thrombin
blood fraction is utilized in t:he medical procedure,
e.g., as a component of a fibrin sealant.
5. EXA.MP ES
EXAMPLE I
Preparation of a Composition Containing a
=5 Thrombin Blood Fraction Obtained Frvm 17 ml
of Fresh Blood From a Human Aduit Donor
A puncture of the vein of a human was
performed by a needle and 17 ml of brood was drawn
into an empty 30 ml syringe. Immediately after
ZO drawing the blood, it was transferred to a 50 ml test
tube containing 34 ml of a so7.ution 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, 40 ml of
25 the supernatant plasma/glucose: solution was removed by
a syringe, and transferred to a new 50 ml test tube.
By means of 1.07 ml of~a 2.8$ citric acid
solution; the pH in the plasma/glucose solution was
lowered tQ 5.2 and after a period of 10 minutes at
3a room temperature, the solution was 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 NaHC03 and 8 gram/L of NaCl. This
35 precipitate contains fibrinogen, prothrambin and ether
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_ 27
blood proteins, but is substantially tree of
antithrombin III. The pH of this dissolved
prothrombin containing euglobu,lin solution was 7.35.
Activation of the prothrombin was performed
by the addition of 0.027 m1 of a solution containing
CaCl2, 0.f 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
time after 10 20 30 60 120 27
n M
Ca-addition min min mA ain min hour
NIH u~ml 0 180 3~8 540 600 737
ZO EXAMPLE II
Preparation of a Composition containing a
Thrombin Blood Fraction Obtained From 17 ml
of Fresh Glass Activated 8load 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 captaining 20 grams of glass beads witr: a
30 diameter of approximately 2 mm: The total surface
area of the beads was approximately 230 cmZ and,
therefore, the surface area wa:5 about 11.5 cm~ per m2
of whole blood.
Immediately after drawing the blood, the
ss syringe was turned gently for 10 to 15 seconds before
17 ml of the blood was transferred to a 50 ml test
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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, 40 ml of
s the supernatant plasma/glucose solution was removed by
a syringe, and transferred to another 50 ml test tube. "
By means of 1.07 ml 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
1o centrifuged at is°C at 1,500 x g for 5 minutes.
After centrifugation the supernatant was
drained off and the precipitate was dissolved in 4.424
ml of a solution containing 14 mmole/L of NaIiC~3 and 8
gram/L of NaCl. This precipitate contains
15 prothrombin, fibrinogen and other blood proteins, but
is substantially free of antithrombin III. The pH of
this dissolved prothrambin containing euglobulin
solution was 7.35.
Activation of the prothrombin was performed
20 by the addition of a 0.027 ml of a solution containing
CaClz, 0.5 mole/L. From 4 to 9 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
25 samples ware 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 min hour
NIH u/ml 130 444 560 720 695 880
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 unita/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 detectableythrombin activity.
EXAMPLE III
Preparation of a Composition Containing a
Thrombin Fraction Obtained From 17 ml of
Fresh Blood From Human Adult Donors,
Characterized by Low Specific Thrombin
Actiyity
In 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
z5 min
to
2 hours
after
dissclution~of
the
euglobulin
fraction.
Nll~i
u/ml
donor pH -280 15 min 30 min 60 min 2 hr
~
JH 6.96 25.8 142 483 661 779
HJS 6.35 26.7 0 !56 942 1,238
LK 6.98 19.5 30 249 430 562
ICN 6.?1 31.5 0 142 ~ 67.6 725
I
(E-280 is a measurement of the total protein
concentration in mg/ml).
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Table IV
8 Specific
activity
of thrombin
measured
from
15 min to
2 hours
after dissolution
of the
euglobulin
traction.
~IIH u/mg
protein
donor 15 min 30 min 6o min 2 hr
JH 5.5 19 26 30
HJS 0 2 .1 3 5 ~ 4 6
LK 1.5 13 22 29
10 ~ 0 4.5 20 23
Thus, the preparation of a thrombin blood
traction as described in Example I results in a
thrombin blood fraction of a concentration and
1g specific activity of the subject invention.
EXAMPLE IV
Preparation of Thrombin From Whole Blood
Using the Device of w091/I7778
The device described in W091/17?78 was
used for the preparation of thrombin. Bef ore the
blood was introduced into the de~rice, 40 n1 ef a 5.5~
glucose solution was introduced into the first chamber
(14) through the filter (43) mounted on the tubing
(39).
Blood, 10 ml, was collected from human
donors into a syringe and irunediately 'hereafter
transferred through the tubing (=!9) into the glucose
solution. The device was placed in a centrifuge and
centrifuged f or 5 min at 1,500 x g. after
centrifugation, the plasma/glucose solution was
transferred into the second chamber ( 3 0 ) c,rit:~ the red
cells remaining in the chamber (1.:). Through the
sterile filter in tube (0'0) 0.6 ml of 2.8% citric acid
33
solution was introduced into the' plasrra/glucose
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- 31 -
solution. After 5 to 10 min, the device was placed in
a centrifuge and centrifuged rot 5 min at 1,500 x g.
After centrifugation the supernatant was transferred
to the first chamber (14) and the precipitate, the
euglobulin fraction, remained in the second chamber
(30). Through the sterile filter in tube (60) 0,85 ml
of a solution containing 7.5 mrR NaIiC03, 52 mM NaCl and
30 mM CaClZ was introduced into the second chamber
(30). The euglobulin precipitate was dissolved within
1-2 minutes, and transferred to the syringe (51)
connected to the second chamber (30). The syringe
contained a polyurethane sponge; facilitating the
removal of the formed fibrin. Thrombin concentrations
were measured after 30 min to 22 hours. The results
~5 are recited in Table V.
Table V
~ throabin
concentration
NiH
u/m1
Zo Donor plasma 30 min 1 hour 2 hour 2Z hour
dilution
RH-A 11.9% I86 1240 231 279
RFi-H 12.3% 54 7.30 130 ~ 132
,
RH-C 11.7$ ~ 1?8 , 7.86 180 192
~
25 ~'D 12.5$ ~ 104 92 123
I ~ 120
I
EXAMPLE v
Preparation of Thr o:abin and Fibrinogen arc.;.
whole Blood Using a Device System riade From
Two Inter-Connected Devices of
30 W091/17778, and the Use of Thrombin and
Fibrinogen in a Fibrin Glue
The device system: consists of two devices as
described in W091/17778. The two tubings (39)
3s from the too devices wets connected to the sate
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- 3 2 ~-
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% salut~.on,
1.0 was,introduced into the ffirst 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 first
chamber (14) of the thrombin device (hereafter named
i5 Device-T) through the filter (43) mounted on the
tubing (39).
Blood, 45 ml was collected through the
cannula into the first chamber (14) in Device-F
containing the citrate solution, and 1.7 ml was
20 collected into the first chamber (14) in the Device-T
containing the glucose solution. After collection of
the blood, the separator-system was disconnected from
the donor, and the tubing (39) was sealed close~to
inlet (38). Both devices Were placed in a centrifuge
25 and centrifuged for 10 min at 1,500 x g.
The separated plasma in Device-F was
. transferred to the second chamber,(30) and 2.5 ml of a
96% ethanol solution was introduced into the second
chamber (30) through the sterile filter in tube (60).
30 The device was now placed into a ice-water bath f or 20
minutes to reduce the temperature in the
plasma/ethanol solution to approximately 0 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 2,500 x g.
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The supernatant serum was transferred to the first
chamber (14), and the solid fibrinogen was dissolved
by incubation for 5 minutes at 37°C. The dissolved
solution was transf erred to the sterile syringe (51).
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
io the sterile filter in tube (60) 1.2 ml of a 2.8%
citric acid solution was introduced into the
plasma/glucose solution. After 5 to i0 min the device
was placed in a centrifuge and centrifuged for 5 min
at 1,500 x g. After centrifugation, the supernatant
was transferred to the first chamber (14) and the
precipitate, the euglohulin fraction, remained in the
second chamber (30). Through the sterile filter in
the tube (60), 0.85 ml of a solution containing 7.5 mM
NaHC03, 52 mM NaCI and 30 mM Ca~Cl~ was introduced into
2o the second chamber (30). The ~euglobulin precipitate
was dissolved within Z-2 minutes, and the fibrin
formed during the activation o:f prothrombin to
thrombin was collected onto 5 glass beads placed in
the second chamber (30). After 15 minutes the
thrombin solution was transferred to the syringe (51)
connected to the second chamber (30). Thrombin
w concentration was measured to be 24a-340-372 NIA u/m1
after 15-30-60 minutes, respectively:
The two syringes containing the fibrinogen
and the thrombin were used as a double barrelled
syringe. The two solutions ware expelled from the
syringes and formed immediately a firm fibrin clot.
3S
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_ 34 ..
EXAMPLE VI
54 ml of 0.04% HAc were added to 6 1n1 of
plasma. This mixture was placed in a flat-bottomed
container of the type known from the above
PCT/DK91/00131. The container is of a circular cross
section with an inner diameter of 4.5 am. The
pli-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
factors, inter olio prothrombin, as a precipitate by a
centrifuging. The centrifuging was performed at 1,500
g f or 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 f laid, an 0.75 ml ac;ueous
solution of 0.9% NaCl, 0.03% Na:C03 and 25 mM CaClz was
added to the precipitate. After dissolving of the
precipitate, the solution was sucked into a 2.5 ml
syringe containing a polyurethane sponge. The
2o formation of fibrin did not start until about 1 to 2
min. after the precipitate had become completely
dissolved, and accordingly more: than enough time for
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 syr.i:nge, the fibrin
remaining depositing on the large surface of the
sponge.
The dissolved precipitate had a pH-value of
6.5. Other amounts of NazCO3 or another base or a
buffer system can be used provided the pH-value is
between 6.0 and 7.5, 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 02367105 2002-O1-25
CVO 94/00566 PCT/GB93/01323
-- 3 5
The thrombin was expelled From the syringe
after 30 min, and a concentration of 256 NIFi units per
ml was obtained. The thrombin concentration increased
by time, but after 30 to 60 min a sufficient amount of
thrombin was obtained for a conventional use in a
Fibrin sealant.
The preparation of thrombin was in the
present Example produced from 12 ml os autalogous
blood and was terminated over a period of 45 to 60
:0 min, i.e., almost simultaneously with the termination
of the preparation of fibrinogen. The produced amount
of thrombin was sufficient for being used in
combination with fibrinogen produced from 45 ml of
autolagous blood in the manner described in
t5 W091/I???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 of a plasminogen catalyst,
2o such as streptokinase, urokinase or t-PA (tissue
plasminogen catalyst) optional:ty admixed With a
physiological solution.
30
