CA2367124C - A thrombin blood fraction for use in a medical procedure - Google Patents

Abstract

The subject invention relate s to the use of thrombin in a medical procedure in an animal.
More specifically, the subject invention relates to such use of thrombin wherein the thrombin is a thrombin blood fraction, as defined hereinbelow.

Description

WO 94/00566 PCf/GB93/Ot323 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, 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 .. . . ....r.._. .~ uv-~. very 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:

(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 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

1~V0 94/005G6 PCT/GB93/01323 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.

WO 9d/00566 PCTIGB93/01323 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 .~ 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.

WO 94!00566 PCTlGB9310t323 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 .

i WO 94!00566 P.CT/GB93/01323 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, WO 94/00566 PCTIG893/Ot323 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.

CVO 94/00566 PCfJGB93/OI323 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;

WO 94!00566 PCC/GB93/01323 ~ 13 -(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 WO 94!00566 PCTlGB93l01323 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) 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 around a stirring rod or collection of the fibrin onto glass beads.
4'.1.3. PREPARATION OF THROMBIhI BLOOD

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 plasma by passing the plasma through a column that 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 WO 94/00566 PC1"/GB93/Ot3Z3 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 WO 9a/oo566 PCT/GB93/oi323 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.

WO 94/00566 PCTIG$93/01323 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 .

fYO 94/00566 PCTlG893/01323 - 21. -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 WO 94!00566 PCT/GB93/01323 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.

-z3-4.3. 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 CVO 94/00566 PCflGB93/01323 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.

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 , _ 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 6 PCf/GB93/01323 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 WO 94/00566 PCfIGB93/01323 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

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 .

WO 94!00566 PCT/GB93/01323 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).

«'O 94!00566 PGT/GB93/01323 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 WO 94/00566 pCTIG893/OI323 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 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 i 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 WO 94/00566 PCTlGB93/OI323 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.

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.

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.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. Use of a thrombin blood fraction comprising:
(a) a thrombin concentration of from 1 NIH unit/m1 to 2,000 NIH units /m1;
and (b) a specific activity of thrombin of from 1 NIH unit/mg of blood protein to 200 NIH units/mg of blood protein;
wherein said thrombin blood fraction contains less than 50% activity of antithrombin III in normal plasma per unit volume, for use in the treatment of blood loss in an individual.
2. The use of claim 1 wherein said individual animal is a mammal.
3. The use of claim 2 wherein said mammal is a human.
4. The use of claim 3 wherein said thrombin blood fraction is substantially free of fibrinogen and fibrin.
5. The use of claim 3 wherein said thrombin blood fraction is prepared from 10 ml to about 50 ml of whole blood.
5. The use of claim 3 wherein said thrombin concentration is from about 100 NIH

units/ml to about 800 NIH units/ml.
7. The use of claim 3 wherein said specific activity is from about 5 NIH units/mg of blood protein to about 100 NIH units/mg at blood protein.
8. The use of claim 7 wherein said specific activity is from about 5 NIH units/mg of blood protein to about 50 NIH units/mg of blood protein.
9. The use of claim 1 wherein said thrombin blood fraction is prepared by means of the preparation of a euglobulin blood fraction, wherein said preparation comprises the steps of:
(a) separating plasma from whole blood;
and (b) lowering the ionic strength and pH of the plasma to precipitate the euglobulin blood fraction.
10. The use of claim 1 wherein said thrombin blood fraction is blood plasma wherein said blood plasma is substantially free of active antithrombin III.
11. The use of claim 1 for the formation of a fibrin clot.
12. The use of claim 11 wherein said fibrin clot is formed by means of a fibrin sealant and one component of said fibrin sealant is said thrombin blood fraction and a second component of said fibrin sealant is fibrinogen.
13. The use of claim 1 to staunch oozing hemorrhages or hemorrhages in hollow organs.
14. The use of claim 1 wherein said thrombin blood traction is a component of a composition comprising platelet derived factors released from platelets and said thrombin blood fraction, wherein said platelet derived factors are released from said platelets by said thrombin blood fraction.
15. The use of claim 1 for the autologous transplantation of keratinocytes or fibroblasts.
16. Use of a thrombin blood fraction comprising:
(a) a thrombin concentration of from 1 NIH unit/ml to 2,000 NIH units/ml;
and (b) a specific activity of thrombin of from 1 NIH unit/mg of blood, protein to 240 NIH units/mg of blood protein;
wherein said thrombin blood fraction contains less than 50% activity of antithrombin III normal plasma per unit volume, for the manufacture of a medicament for use in the treatment of blood loss.
17. The use of claim 1 wherein said thrombin blood fraction is autologous.

18. The use of claim 1 wherein said thrombin blood fraction is used as a sealant.
19. The use of claim 14 for the treatment of damaged live animal tissue.
20. A two component fibrin sealant suitable for use in a medical procedure on a human or animal, the sealant comprising a fibrinogen fraction in which the fibrinogen is recovered only from said human or animal, and a thrombin blood fraction having a specific activity of thrombin of from 1 NIH to 200 NIH units per mg. of total blood protein and a thrombin concentration of from 1 NIH
unit/ml to 2000 NIH units/ml also recovered only from said same human or animal, said thrombin blood fraction being substantially free of active antithrombin III.
21. A fibrin sealant according to claim 20, suitable for use in a medical procedure on a human, the sealant comprising a fibrinogen fraction in which the fibrinogen is recovered only from said human, and a thrombin blood fraction also recovered only from said same human.
22. A fibrin sealant according to claim 20 or claim 21, wherein said thrombin blood fraction is prepared by subjecting donor plasma from said human or animal to precipitate to form a fibrinogen-containing precipitate and a supernatant, and converting the fibrinogen in the precipitate to fibrin.

23. A fibrin sealant according to any of claims 20 to 22, wherein said, thrombin blood fraction has a specific activity of thrombin of from 5 NIH units to 100 NIH units per mg. of total blood protein.
24. A fibrin sealant according to any of claims 20 to 23, wherein said thrombin blood fraction has a specific activity of thrombin of from 5 NIH to 50 NIH units per mg. of total blood protein.
25. A method for preparing a fibrin sealant suitable for use in a medical procedure on a human or animal, the fibrin sealant comprising a fibrinogen fraction and a thrombin blood fraction having a specific activity of thrombin of from 1 NIH
unit to 200 NIH units per mg. of total blood protein and a thrombin concentration of from 1 NIH unit/ml to 2000 NIH units/ml, which is substantially free of active antithrombin III, the method comprising the steps of recovering the fibrinogen and the thrombin blood fraction from the human or animal so that the fibrin sealant is autologous.
26. The method according to claim 25, wherein the step of recovering the thrombin blood fraction comprises the steps of:
diluting whole blood to an ionic strength of less than 100 millimolar;
separating plasma from the whole blood;
lowering the pH of the plasma to precipitate a prothrombin blood fraction:
separating the prothrombin blood from the plasma; and converting the prothrombin in the prothrombin blood fraction to thrombin to prepare the thrombin blood fraction.
27. The method according to claim 25 or 26, further comprising the step of substantially removing any fibrinogen which may be contained in, or formed in, the thrombin fraction.
2a. The method according to any of claims 25 to 27, wherein fibrin sealant is suitable for use in a medical procedure on a human and the fibrinogen and the thrombin blood fraction are recovered from the human.
29. The method according to any of claims 25 to 28, wherein said thrombin blood fraction has a specific activity of thrombin of from about 5 NIH
units to about 100 NIH units per mg. of total blood protein.
30. The method according to any of claims 25 to 29. wherein said thrombin blood fraction has a specific activity of thrombin of from 5 NIH units to 50 NIH units per mg. of total blood protein.